CN114516901B

CN114516901B - AAV vector with high affinity for nervous system and application thereof

Info

Publication number: CN114516901B
Application number: CN202210241656.7A
Authority: CN
Inventors: 程诚; 唐思琦; 吴侠; 肖啸; 杜增民; 蒋威; 郑静
Original assignee: Shanghai Mianyi Biotechnology Co ltd
Current assignee: Shanghai Mianyi Biotechnology Co ltd
Priority date: 2022-03-11
Filing date: 2022-03-11
Publication date: 2023-01-17
Anticipated expiration: 2042-03-11
Also published as: CN114516901A; WO2023169115A1; WO2023169115A9

Abstract

The invention relates to adeno-associated virus (AAV) capsid protein with high affinity in nervous system, AAV vector containing the capsid protein and application thereof. The AAV capsid protein of the invention has good affinity to the tissues and cells of the nervous system, and the AAV vector and the medicament constructed by utilizing the capsid protein have particular advantages in the aspect of treating nervous system diseases.

Description

AAV vector with high affinity for nervous system and application thereof

Technical Field

The invention belongs to the field of biotechnology. The invention relates to an AAV vector with high affinity for nervous system and application thereof. The invention also relates to AAV capsid proteins of high affinity for the nervous system, nucleic acid molecules encoding the capsid proteins, methods of delivering a gene of interest into a tissue or cell of the nervous system, genetically engineered cells and pharmaceutical compositions.

Background

Neurological disorders are a series of diseases affecting the central or peripheral nervous system, including, but not limited to, neurodegenerative diseases such as Alzheimer's Disease (AD), parkinson's Disease (PD), amyotrophic Lateral Sclerosis (ALS), and Huntington's Disease (HD), etc.; neurodevelopmental-related diseases such as mental retardation, autism Spectrum Disorder (ASD), rett syndrome, attention Deficit Hyperactivity Disorder (ADHD), and the like; chronic pain; nerve damage; and retinal diseases and the like.

Traditional drugs have had very limited efficacy against such diseases because of the difficulty in diagnosing the disease in the nervous system, the complexity of the nervous system itself, and the physiological barriers that limit the entry of drugs from the periphery into the nervous system. In fact, effective treatments for neurological diseases are rarely available, and patients with related diseases are often afflicted with long-term tolerance to immediate symptoms and complications, posing a heavy social burden. Therefore, there is an urgent need to develop new therapeutic strategies to remedy such unmet medical needs, and gene therapy is a promising new approach.

Gene therapy is a type of treatment that delivers functional DNA packaged in a vector to a target tissue and expresses a therapeutic molecule, and its good safety and effectiveness have been demonstrated in numerous clinical trials including neurological diseases. Among effective gene therapy vectors, adeno-associated virus (AAV) has been most widely used due to its advantages of high safety, low immunogenicity, abundant serotypes, and convenience in modification and screening. Both the successfully marketed gene therapy drugs LUXTURNA and ZOLGENSMA were also developed based on the AAV vector platform. Studies have shown that the tissue tropism and cellular transformation efficiency of AAV virions is largely determined by their capsid. Different capsids determine different AAV viral particles to have different tissue tropism and transformation efficiency. In the case of neurological diseases, engineering and screening AAV serotype mutants with higher affinity for the nervous system is crucial for success or failure of gene therapy.

Therefore, there is an urgent need in the art for AAV serotype mutants with high affinity for the nervous system to develop a gene therapy approach that targets the nervous system with high efficiency.

Disclosure of Invention

In order to solve the above technical problems, the present inventors have conducted extensive studies and unexpectedly found that a novel AAV capsid protein T42 (e.g., SEQ ID NO: 2) obtained by substituting, deleting or adding (e.g., substituting 2 amino acid residues) one or more amino acid residues in the amino acid sequence of AAVHH67 capsid protein (SEQ ID NO:3, which is described in, for example, WO2009137006A 2) has high affinity for tissues and cells of the nervous system. AAV vectors and drugs constructed by the capsid protein can efficiently deliver genes to tissues and cells of a nervous system, thereby realizing good treatment effect on nervous system diseases.

Accordingly, in a first aspect, the present invention provides an AAV capsid protein, wherein the AAV capsid protein is constructed by substitution, deletion or addition of one or more amino acid residues of the amino acid sequence of an AAVHH67 capsid protein; the amino acid sequence of the AAVHH67 capsid protein is shown as SEQ ID NO:3, respectively.

In a preferred embodiment, the AAV capsid protein described above is constructed by substituting the amino acid sequence of the AAVHH67 capsid protein for 2 amino acid residues; the amino acid sequence of the AAVHH67 capsid protein is shown as SEQ ID NO:3, respectively.

In a preferred embodiment, the amino acid sequence of the AAV capsid protein is identical to SEQ ID NO:2, and more preferably at least 95%, 96%, 97%, 98%, or 99% identity.

In a preferred embodiment, the AAV capsid protein comprises SEQ ID NO:2, or a pharmaceutically acceptable salt thereof.

In a preferred embodiment, the amino acid sequence of the AAV capsid protein is as set forth in SEQ ID NO:2, respectively.

In a second aspect, the invention provides the use of an AAV vector comprising an AAV capsid protein according to the first aspect, in the manufacture of a medicament for the treatment of a neurological disease.

In one embodiment, neurological diseases include, but are not limited to: neurodegenerative diseases, neurodevelopmental diseases, chronic pain, nerve injury, retinal diseases or combinations thereof.

In one embodiment, neurodegenerative diseases include, but are not limited to: alzheimer's Disease (AD), parkinson's Disease (PD), amyotrophic Lateral Sclerosis (ALS), huntington's Disease (HD).

In one embodiment, neurodevelopmental-related diseases include, but are not limited to: mental retardation, autism Spectrum Disorder (ASD), rett syndrome, attention Deficit Hyperactivity Disorder (ADHD).

In one embodiment, retinal diseases include, but are not limited to: vascular and vascular system disorders, retinal inflammation, retinal detachment, retinal degeneration and dystrophy, retinal tumors.

In a third aspect, the invention provides a nucleic acid molecule encoding an AAV capsid protein according to the first aspect.

In one embodiment, the nucleotide sequence of the above nucleic acid molecule is identical to SEQ ID NO:1 has at least 70% identity.

In a preferred embodiment, the nucleotide sequence of the above nucleic acid molecule is identical to SEQ ID NO:1 has at least 75%, 80%, 85%, 90%, 95%, or 99% identity.

In a preferred embodiment, the nucleic acid molecule comprises SEQ ID NO: 1.

In a more preferred embodiment, the nucleotide sequence of the above nucleic acid molecule is as set forth in SEQ ID NO:1 is shown.

In a fourth aspect, the present invention provides an AAV vector, wherein the AAV vector comprises: (ii) (i) an AAV capsid protein according to the first aspect; and (ii) a viral genome packaged in said AAV capsid protein.

In one embodiment, the viral genome is a native AAV genome or an artificially recombined viral genome.

In a preferred embodiment, the viral genome comprises a reporter gene.

In a preferred embodiment, the viral genome comprises an exogenous therapeutic gene.

In a preferred embodiment, the viral genome further comprises regulatory elements such as promoters, enhancers, polyas, two ITRs at either end.

In a preferred embodiment, promoters include, but are not limited to: CMV promoter, CAG promoter, UBC promoter, tetracycline promoter TRE, synapsin I promoter, camKIIa promoter, c-fos promoter, mecp2 promoter, NSE promoter, SST promoter, TH promoter, GFAP104 promoter, gfaABC1D promoter, ALDH1L1 promoter, MBP promoter, rpe65 promoter, VMD2 promoter.

In a preferred embodiment, the reporter gene includes, but is not limited to: green fluorescent protein Gene (GFP), human growth hormone gene (hGH), secretory alkaline phosphatase gene (SEAP), β -galactosidase gene (LacZ), chloramphenicol acetyl transferase gene (CAT), luciferase gene (Luciferase).

In a preferred embodiment, the exogenous therapeutic gene is a positive-regulatory therapeutic gene, which refers to: a gene encoding a protein having a therapeutic function, or a gene which can be expressed to be useful for the treatment and/or prevention of a disease.

In a preferred embodiment, positive-regulating therapeutic genes include, but are not limited to: APOE2, GRN, MECP2, TH, AADC, GBA, ASPA, TPP1, GLB1, SGSH, NAGLU, IDS, NPC1, SMN1, FXN, GAN, BDNF, GDNF, RPE65, merk, MYO7A, ABCA4, CHM, endostatin, angiostatin, CNGA3, CNGB3, RS1, ND4, or combinations thereof.

In a preferred embodiment, the exogenous therapeutic gene comprises a nucleotide sequence capable of inhibiting the expression of a negatively regulated gene, which is: genes that are highly expressed (relative to a healthy state) in a disease, or genes whose expression is suppressed can be useful for the treatment and/or prevention of a disease.

In a preferred embodiment, the nucleotide sequence capable of inhibiting the expression of a negatively regulated gene comprises: a microRNA sequence specific to the negative regulatory gene, and/or a coding sequence of an antibody specific to the negative regulatory gene.

In a preferred embodiment, negative regulatory genes include, but are not limited to: APOE4, APP, MAPT, C9orf72, HTT, SNCA, ATXN1, ATXN3, ATXN7, SOD1, TARDBP, SCN9A, SCN10A, VEGF, or a combination thereof.

In a preferred embodiment, the exogenous therapeutic gene encodes a therapeutic protein useful for treating a neurological disease.

In a preferred embodiment, the viral genome further comprises a nucleotide sequence encoding a Cas protein reaction system.

In a preferred embodiment, the Cas protein reaction system comprises: cas protein, guide RNA, and/or a target gene homologous sequence for homologous recombination repair of a target gene mutation site.

In a preferred embodiment, the Cas protein reaction system is used to repair a mutation site of a target gene in the genome of a cell of the nervous system and restore the normal function of the target gene; wherein the target gene is a positive regulatory therapeutic gene.

In a preferred embodiment, the Cas protein reaction system is used to knock-out or knock-down the expression of a target gene in a nervous system cell; wherein the target gene is a negative regulatory gene.

In a fifth aspect, the present invention provides a method of delivering a gene of interest into a tissue or cell of the nervous system, comprising: 1) Packaging a gene of interest into an AAV capsid protein according to the first aspect, forming an AAV viral particle; and 2) contacting a tissue or cell of the nervous system with the AAV viral particle.

In one embodiment, the tissues of the nervous system include, but are not limited to: brain, spinal cord, dorsal Root Ganglia (DRGs), nerve trunks, retina or combinations thereof; and/or cells of the nervous system include, but are not limited to: all cell types in the brain, spinal cord, dorsal root ganglia, neural stem, retina, such as excitatory neurons, inhibitory neurons, ganglion cells, rods, cones, microglia, astrocytes, oligodendrocytes, and muller cells.

In a sixth aspect, the present invention provides a genetically engineered cell, wherein the genetically engineered cell comprises: (i) A first nucleic acid construct comprising an exogenous therapeutic gene for a neurological disease; (ii) A second nucleic acid construct comprising a rep and a cap gene, said cap gene encoding an AAV capsid protein according to the first aspect; and (iii) a third nucleic acid construct, said third nucleic acid construct being a helper plasmid.

In one embodiment, the genetically engineered cell is a eukaryotic cell.

In one embodiment, the genetically engineered cell is selected from the group consisting of: 293T cells, HEK293 cells, sf9 cells, or BHK cells.

In one embodiment, the helper plasmid is derived from adenovirus (Ad), herpes Simplex Virus (HSV), or other helper plasmids with helper functions.

In one embodiment, the first nucleic acid construct further comprises an exogenous reporter gene.

In one embodiment, the exogenous reporter gene is selected from the group consisting of: GFP, hGH, SEAP, lacZ, CAT, luciferase genes, or combinations thereof.

In one embodiment, the first nucleic acid construct, the second nucleic acid construct, and/or the third nucleic acid construct can be transiently present in the genetically engineered cell or can be stably integrated into the genome of the genetically engineered cell.

In a seventh aspect, the present invention provides a pharmaceutical composition comprising: (ii) (i) an AAV vector according to the fourth aspect; and (ii) an excipient.

In one embodiment, component (i) comprises 0.1 to 99.9wt%, preferably 10 to 80wt%, more preferably 30 to 60wt% of the total weight of the pharmaceutical composition.

In one embodiment, excipients include, but are not limited to, salts, organics, and surfactants, or combinations thereof.

In one embodiment, excipients include, but are not limited to: solvents, dispersion media, coatings, antibacterial agents, antifungal agents, isotonic and absorption delaying agents, or combinations thereof.

In one embodiment, the excipient comprises saline, including but not limited to: buffered saline, physiological saline, phosphate buffer, citrate buffer, acetate buffer, bicarbonate buffer, sucrose solution, saline solution, polysorbate solution, or combinations thereof.

In one embodiment, excipients include, but are not limited to: a stabilizer, a preservative, a transfection facilitating agent that facilitates cellular uptake, or a combination thereof.

In one embodiment, stabilizers include, but are not limited to: sodium glutamate, glycine, EDTA, albumin (e.g., human serum albumin), or combinations thereof.

In one embodiment, preservatives include, but are not limited to: 2-phenoxyethanol, sodium benzoate, potassium sorbate, methyl hydroxybenzoate, phenol, thimerosal, an antibiotic, or a combination thereof.

In one embodiment, the transfection facilitating agent comprises calcium ions.

In one embodiment, the pharmaceutical composition is administered by brain parenchymal injection, intrathecal injection, intraventricular injection, subdural injection, intravenous injection, intravitreal injection, subretinal injection, or a combination thereof.

In one embodiment, the pharmaceutical composition is a liquid.

In one embodiment, the pharmaceutical composition is an injection, such as a brain parenchyma injection, an intrathecal injection, an intracerebroventricular injection, a subpial injection, an intravenous injection, an intravitreal injection, a subretinal injection.

In one embodiment, the AAV vector according to the fourth aspect is packaged within a genetically engineered cell according to the sixth aspect.

In an eighth aspect, the present invention provides a method for preventing and/or treating a neurological disease, comprising the steps of: administering to a subject in need thereof an effective amount of an AAV vector according to the fourth aspect or a pharmaceutical composition according to the seventh aspect.

In one embodiment, the pharmaceutical composition according to the seventh aspect is administered alone or in combination with other drugs for treating a neurological disease in a method of treating said neurological disease.

In one embodiment, the administering comprises in vivo injection.

In one embodiment, the in vivo injection mode includes, but is not limited to: brain parenchyma injection, intrathecal injection, intracerebroventricular injection, subdural injection, intravenous injection, intravitreal injection, and subretinal injection, or a combination thereof.

In one embodiment, the subject comprises a human or non-human mammal.

In one embodiment, the non-human mammal includes, but is not limited to: non-human primates, sheep, dogs, cats, horses, cattle, chickens, rats, mice, and the like.

In a ninth aspect, the invention provides a method of identifying the affinity of an AAV viral particle comprising an artificially recombined viral genome comprising a reporter gene, such as GFP, hGH, SEAP, lacZ, CAT and Luciferase, for the nervous system of a subject.

In one embodiment, the above method for identifying the affinity of an AAV viral particle for the nervous system of a subject comprises:

(1) Injecting the AAV viral particles into the nervous system of the subject in vivo to administer an effective dose of the AAV viral particles to the subject, the injection methods including, but not limited to, brain parenchymal injection, intrathecal injection, intracerebroventricular injection, subpial injection, intravenous injection, intravitreal injection, and subretinal injection, preferably brain parenchymal injection or intrathecal injection; and

(2) Samples of the nervous system of a subject are collected and the affinity of AAV viral particles for the nervous system of the subject is identified using biochemical methods including, but not limited to, immunohistochemistry, western Blot, ELISA, and quantitative PCR, preferably immunohistochemistry.

In one embodiment, the reporter gene is GFP.

Drawings

FIG. 1 shows a schematic diagram of the CMV-GFP vector structure, which comprises the following elements in order: 5'ITR, CMV promoter, GFP gene coding sequence, hGH poly (A) sequence, 3' ITR.

FIG. 2A shows a standard curve for measuring titer of virus particles of serotypes AAV9 and T42 (designated AAV9-GFP and T42-GFP, respectively) produced using CMV-GFP vectors and collected.

FIG. 2B shows the viral titers of AAV9-GFP and T42-GFP calculated on a standard curve using the results of quantitative PCR cycles of 100-fold and 1000-fold dilutions of the virus.

Figure 3A shows a summary presentation of virus injection by stereotactic injection into bilateral hippocampal regions of mouse brain at a scale of 1000 μm.

FIG. 3B shows an example of immunohistochemistry results of GFP staining of hippocampal brain of mice after AAV9-GFP (left column) and T42-GFP (right column) virus injection, on a scale of 20 μm.

Figure 3C shows a quantitative statistical plot of GFP fluorescence signal intensity after AAV9-GFP and T42-GFP virus injection into mice hippocampus to measure brain parenchymal affinity of different serotypes, where "×" indicates p <0.05.

Figure 4A shows a summary presentation of the manner of intrathecal injection and selection of DRG L5 segments in mice.

FIG. 4B shows immunohistochemistry results of GFP staining of L5 segment DRG sections of AAV9-GFP (left column) and T42-GFP (right column) viruses after intrathecal injection in mice on a scale bar of 100 μm.

Figure 4C shows a quantitative statistical plot of AAV9-GFP and T42-GFP virus for GFP fluorescence signal intensity of L5 segment DRG after intrathecal injection in mice as a measure of DRG affinity for the different serotypes, where "×" indicates p <0.01.

FIG. 5 shows the VP1 amino acid sequence of T42 (SEQ ID NO: 2).

FIG. 6 shows the nucleotide sequence encoding T42 (SEQ ID NO: 1).

Detailed Description

The present inventors have made extensive and intensive studies and, as a result, have found for the first time a novel AAV capsid protein (SEQ ID NO: 2) designated as "T42" which has a high affinity for the nervous system through a large number of alterations and screenings.

Experiments show that after the T42 capsid protein and a recombinant genome containing an exogenous reporter gene GFP are packaged into a T42-GFP viral vector (or T42-GFP viral particles), the vector is applied to a subject by a method of brain parenchyma injection or intrathecal injection, and corresponding tissue samples are collected, and the T42-GFP viral particles have strong affinity to hippocampus of the brain and dorsal root ganglia of a mouse by means of immunohistochemistry and imaging; and the affinity of the virus particles is higher than that of AAV9-GFP virus particles packaged by a traditional AAV serotype (AAV 9) which is widely used for clinical tests and even druggage at present.

Term(s) for

It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

In order that the invention may be more readily understood, certain technical and scientific terms are specifically defined below. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

As used herein, the term "about" when used in reference to a specifically recited value means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

As used herein, the term "treatment" refers to the administration of a therapeutic agent, either internally or externally, to a patient, comprising the AAV viral vectors provided herein and pharmaceutical compositions consisting thereof. The patient is suffering from one or more diseases for which the therapeutic agent has a therapeutic effect. Typically, the AAV viral vectors provided herein, and pharmaceutical compositions comprising them, are administered to a patient in an amount of a therapeutic agent effective to alleviate one or more symptoms of the disease (therapeutically effective amount).

As used herein, the term "optional" or "optionally" means that the subsequently described event or circumstance may, but need not, occur.

The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") in this context.

As used herein, the term "adeno-associated virus (AAV)" refers to a virus belonging to the genus parvoviridae-dependent parvoviruses that is capable of infecting humans and other mammals.

As used herein, the term "ITR" refers to a DNA sequence of about 145 nucleotides that mediates biological functions of replication, packaging, integration, etc. of AAV viruses. The ITRs can be from any AAV, including but not limited to AAV serotype 1 (AAV 1), AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and any other now known or later discovered AAV. The 5'ITR and 3' ITR flanking the nucleotide are not necessarily from the same AAV serotype, as long as they function as intended. Among them, the nucleotide sequence of the ITR region is known.

As used herein, the term "artificially recombinant viral genome" or "recombinant genome" refers to an artificially designed or artificially synthesized foreign DNA sequence that replaces the native AAV genome between ITRs. An AAV comprising an artificially recombined viral genome is referred to as a "recombinant AAV". Among them, recombinant AAV can fulfill different functions based on the expression of the recombinant genome contained therein.

As used herein, the term "affinity" refers to the property of a virus to favor infection and/or entry into certain types of cells or tissues.

As used herein, the term "vector" refers to a molecular tool that transports, transduces, and expresses an exogenous gene of interest contained therein in a target cell.

As used herein, "AAV viral vector," "AAV viral particle," "AAV vector" are used interchangeably and refer to an AAV viral particle that can be used to transport, transduce, and express an exogenous gene of interest contained therein in a target cell.

As used herein, the term "pharmaceutical composition" refers to a composition comprising the AAV viral vectors of the present invention and excipients, useful for treating neurological diseases.

As used herein, the term "treatment" refers to both therapeutic and prophylactic means. Subjects in need of treatment may include subjects already suffering from a neurological condition as well as subjects that may eventually suffer from the condition.

As used herein, the term "neurological disease" refers to central and/or peripheral nervous system related diseases, including but not limited to neurodegenerative diseases such as Alzheimer's Disease (AD), parkinson's Disease (PD), amyotrophic Lateral Sclerosis (ALS), and Huntington's Disease (HD); neurodevelopmental-related diseases such as mental retardation, autism Spectrum Disorder (ASD), rett syndrome, attention Deficit Hyperactivity Disorder (ADHD); chronic pain; nerve damage; and retinal diseases such as vascular and vascular system disorders, retinal inflammation, retinal detachment, retinal degeneration and dystrophy, retinal tumors, and the like.

As used herein, the term "excipient" refers to a natural or synthetic substance in a drug attached to an active ingredient, such as a solvent, dispersion medium, coating, antibacterial or antifungal agent, isotonic and absorption delaying agent, and the like. These excipients may aid in the storage and administration of the viral particles to a subject. The excipient may include any suitable ingredient, such as, but not limited to, saline. Illustrative examples of saline include, but are not limited to, buffered saline, physiological saline, phosphate buffer, citrate buffer, acetate buffer, bicarbonate buffer, sucrose solution, saline solution, and polysorbate solution.

As used herein, the terms "subject", "subject" and "subjects" are used interchangeably and include any human or non-human mammal, e.g., non-human primates, sheep, dogs, cats, horses, cows, chickens, rats, mice, and the like.

As used herein, the term "effective amount" or "effective dose" refers to an amount that produces a function or activity in a human and/or animal and is acceptable to the human and/or animal. It refers to an amount of a therapeutic agent that treats, alleviates, or prevents a target disease or condition, or that exhibits a detectable therapeutic or prophylactic effect. Therapeutic effects also include reduction of physiological symptoms. The precise effective amount for a subject will depend upon the size and health of the subject, the nature and extent of the disorder, and the therapeutic agent and/or combination of therapeutic agents selected for administration. For a given situation, the effective amount can be determined by routine experimentation.

AAV viral vectors

The genome of AAV comprises two major genes: rep genes encoding Rep proteins (Rep 76, rep 68, rep 52, and Rep 40), and cap genes encoding AAV capsid proteins (VP 1, VP2, and VP 3). As known to those skilled in the art, AAV capsid proteins contain VP1, VP2 and VP3 proteins, and VP2 and VP3 proteins undergo transcription and translation processes at the start codon inside the VP1 protein, i.e., the VP1 sequence comprises VP2 and VP3 sequences. AAV is the most common viral vector in the current gene therapy field, and has the characteristics of non-pathogenicity, low immunogenicity, abundant serotypes, durable expression of exogenous genes and the like.

In one embodiment, the invention provides the cap gene of a nervous system high affinity AAV, designated "T42". The VP1 amino acid sequence of T42 is shown as SEQ ID NO:2, respectively. It will be appreciated by those skilled in the art that, based on the rules for nucleotide coding of proteins, all can encode a polypeptide as set forth in SEQ ID NO:2 should fall within the scope of the present invention. In a preferred embodiment, the cap gene is the nucleotide sequence of VP1 encoding T42, as shown in SEQ ID NO:1 is shown.

In one embodiment, the present invention utilizes an in vivo viral injection method to screen for a nervous system high affinity AAV viral vector.

In one embodiment, the AAV viral vectors of the invention comprise an artificially recombinant viral genome comprising a transcription regulatory sequence (promoter), a gene Coding sequence (CDS) and a poly (a) sequence for maintaining messenger RNA activity and stability.

In one embodiment, the artificially recombined viral genome may also encode biological molecules with specific functions, including but not limited to proteins with functions of treating neurological diseases, micrornas (mirnas), antibodies, and guide RNAs of Cas9, wherein the genes encoding proteins with functions of treating neurological diseases include but are not limited to APOE2, GRN, MECP2, TH, AADC, GBA, ASPA, TPP1, GLB1, SGSH, NAGLU, IDS, NPC1, SMN1, FXN, GAN, BDNF, GDNF, RPE65, merk, MYO7A, ABCA4, CHM, endostatin, angiostatin, CNGA3, CNGB3, RS1, ND4, etc.; target genes for miRNA or antibody include, but are not limited to, APOE4, APP, MAPT, C9orf72, HTT, SNCA, ATXN1, ATXN3, ATXN7, SOD1, TARDBP, SCN9A, SCN10A, VEGF, and the like; target genes for Cas9 guide RNA include, but are not limited to, APOE2, GRN, MECP2, TH, AADC, GBA, ASPA, TPP1, GLB1, SGSH, NAGLU, IDS, NPC1, SMN1, FXN, GAN, BDNF, GDNF, RPE65, merk, MYO7A, ABCA4, CHM, endostatin, angiostatin, CNGA3, CNGB3, RS1, ND4, APOE4, APP, MAPT, C9orf72, HTT, SNCA, ATXN1, ATXN3, ATXN7, SOD1, TARDBP, SCN9A, SCN10A, VEGF, and the like.

In one embodiment, the strength of AAV affinity can be measured by how much of the exogenous reporter gene carried by different serotypes of AAV is expressed in a particular type of cell or tissue under the same conditions. For example, GFP can be used as an exogenous reporter gene, and the fluorescence intensity of GFP can be used as an index for measuring the affinity of AAV nervous system.

In a particular embodiment, the AAV viral vector is produced using a DNA plasmid comprising a 5'ITR, a recombinant genome and a 3' ITR, wherein the 5'ITR and the 3' ITR flank the recombinant genome respectively. AAV viral vectors can be produced by introducing the above-described DNA plasmid, a plasmid encoding AAV cap/rep gene, and a helper plasmid provided by adenovirus or herpesvirus into an appropriate host cell simultaneously using known techniques, for example, by transfection. The DNA plasmid may be expressed in a host cell and packaged into viral particles.

Pharmaceutical composition

In one embodiment, the pharmaceutical composition comprises T42 as capsid viral particles and excipients.

In one embodiment, the pharmaceutical composition can efficiently deliver the recombinant genome to the nervous system to express biological molecules with specific functions, including but not limited to proteins with functions of treating nervous system diseases, micrornas (mirnas), antibodies, and guide RNAs of Cas9, wherein genes encoding proteins with functions of treating nervous system diseases include but not limited to APOE2, GRN, MECP2, TH, AADC, GBA, ASPA, TPP1, GLB1, SGSH, NAGLU, IDS, NPC1, SMN1, FXN, GAN, BDNF, GDNF, RPE65, merk, MYO7A, ABCA4, CHM, endostatin, angiostatin, CNGA3, CNGB3, RS1, ND4, etc.; target genes for mirnas or antibodies include, but are not limited to, APOE4, APP, MAPT, C9orf72, HTT, SNCA, ATXN1, ATXN3, ATXN7, SOD1, TARDBP, SCN9A, SCN10A, VEGF, and the like; target genes for Cas9 guide RNA include, but are not limited to, APOE2, GRN, MECP2, TH, AADC, GBA, ASPA, TPP1, GLB1, SGSH, NAGLU, IDS, NPC1, SMN1, FXN, GAN, BDNF, GDNF, RPE65, merk, MYO7A, ABCA4, CHM, endostatin, angiostatin, CNGA3, CNGB3, RS1, ND4, APOE4, APP, MAPT, C9orf72, HTT, SNCA, ATXN1, ATXN3, ATXN7, SOD1, TARDBP, SCN9A, SCN10A, VEGF, and the like.

In a specific embodiment, the invention further provides methods of injecting AAV viral particles in vivo in the nervous system of a subject, including but not limited to, administering an effective dose of AAV viral particles to the subject by brain parenchymal injection, intrathecal injection, intraventricular injection, subdural injection, intravenous injection, intravitreal injection, subretinal injection, and the like. The above methods are selected based on the pathogenesis of the neurological disease and may be used in combination with each other.

Other agents useful for treating neurological diseases may also be included in or combined with the pharmaceutical compositions of the present invention.

Other drugs for treating neurological diseases include, but are not limited to: oxiracetam, edaravone, brain protein hydrolysate for injection, butylphthalide sodium chloride, ozagrel sodium, gastrodine, mecobalamin, vitamin B1, naloxone, nimodipine, metformin, polydiserazide tablet, pramipexole hydrochloride tablet, selegiline, memantine hydrochloride tablet, quetiapine fumarate tablet, risperidone, olanzapine, trazodone, sertraline, mirtazapine, alprazolam, phenobarbital, flupentixol and melitracen tablet, citalopram, buspirone, aspirin, gabapentin capsule, sodium valproate tablet, clonazepam, donepezil, rivastigmine, mannotretin capsule, nicerin tablet, betahistine, citicoline sodium tablet, amantadine tablet, and the like.

The effective amount of the pharmaceutical composition may vary with the mode of administration and the severity of the disease to be treated, among other things. The selection of a preferred effective amount can be determined by one of ordinary skill in the art based on a variety of factors (e.g., by clinical trials). Such factors include, but are not limited to: pharmacokinetic parameters of the drug such as drug tissue distribution, bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the weight of the patient, the immune status of the patient, the route of administration, and the like.

The main advantages of the present invention include:

1) Because T42 has a high affinity for the nervous system, the effect of using T42 to deliver the same exogenous gene to expression in the nervous system of a subject would be superior to traditional AAV capsids (e.g., AAV 9);

2) The therapeutic effect of T42 as a gene therapy drug for AAV capsids on nervous system diseases will be superior to gene therapy drugs comprising conventional AAV capsids (e.g., AAV 9);

3) Since T42 is not a naturally occurring AAV capsid, it is less common for a subject to be infected with AAV than with a traditional AAV capsid (e.g., AAV 9) that there are T42 neutralizing antibodies in vivo, i.e., more subjects will meet the prerequisite for the use of T42 to deliver foreign genes into the nervous system.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The present invention is not limited to the specific methods and experimental conditions described.

The experimental procedures, without specifying the specific conditions in the following examples, are generally carried out according to conventional conditions, for example according to Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations.

Experiments and materials

CMV-GFP plasmid construction

The plasmid backbone (pAAV-MCS plasmid backbone) and the GFP gene coding sequence obtained by PCR were digested at 37 ℃ for 1 hour using endonucleases ClaI and BglII to obtain the corresponding cohesive ends. After recovering the desired fragment by tapping, the fragments were ligated overnight at 16 ℃ using T4 ligase. After transformation, monoclonals were selected for culture and plasmids were extracted. The plasmid was confirmed to be correct by Sanger sequencing.

2. Quantitative PCR

AAV virus is lysed with deoxyribonuclease (Dnase I, takara 2270A, 1. Mu.L Dnase I is added to 5. Mu.L virus) at 37 ℃ for 30 minutes, centrifuged at 4500rpm for 5 minutes, and the supernatant is extracted with 1PBS was diluted 100-fold and 1000-fold, respectively. The reaction standard was a titer-determined AAV virus suspension containing GFP gene, similarly digested with Dnase I, and diluted 10-fold with 1 XPBS to prepare 1X 10 ⁵ To 1 × 10 ¹⁰ vg/mL six concentrations of standard. mu.L of the diluted virus was subjected to a quantitative PCR experiment (SYBR Green mix, reaction protocol: denaturation (95 ℃ C., 3 min.), annealing (60 ℃ C., 30 sec.), extension (72 ℃ C., 1 min.) with a cycle number of 40.

After the reaction is completed, a standard curve with the cycle number (Ct value) as the ordinate and Log (viral titer, vg/mL) as the abscissa is prepared using the standard sample, and then the corresponding viral titer (vg/mL) is calculated from the cycle number of the AAV sample to be obtained.

3. Three-dimensional positioning injection

Mice of SPF-grade male C57BL/6JGpt strain of 8 weeks old were selected for brain stereotactic injection. Mice after anesthesia were injected bilaterally in the Hippocampus (Hippocampus) with 1. Mu.L AAV (AAV 9-GFP or T42-GFP, both titers: 4X 10:. Sup. ¹² vg/mL)。

Bilateral hippocampal injection sites were AP-1.94, ML. + -. 1.5 and DV-1.80, respectively, with an injection rate of 50 nL/min.

4. Intrathecal injection

After anesthetizing the mice, the hairs around the injection site were shaved off, a microinjector was inserted into the sacral spinal cord of the mice, and 20. Mu.L of AAV virus (AAV 9-GFP or T42-GFP, both titers: 4X 10:. About. ¹² vg/mL) into the cerebrospinal fluid.

5. Immunohistochemistry

Mice were sacrificed 4 weeks after virus injection and perfused sequentially through the heart with 1 × PBS and 4% PFA solution. Brains were removed and DRG was fixed in 4% PFA solution for one week.

Followed by gradient dehydration with 15% and 30% sucrose solutions, respectively. Frozen sections of the coronal brain containing the hippocampus and DRG sections of L5 were obtained, both 20 μm thick, using the cryosectioning method. After 0.3% TritonX-100 permeabilization and 5% BSA blocking, primary antibody (anti-GFP) was incubated overnight at 4 ℃.

Finally, a secondary antibody with a fluorophore (absorption wavelength 488 nm) corresponding to the primary antigen was incubated with DAPI at room temperature for 2 hours.

6. Imaging

Brain slices including intact hippocampus were imaged with the Leica Thunder system, L5 segment DRG sections were imaged with a confocal microscope (Leica SP 8), and quantitative fluorescence analysis of GFP was performed under consistent imaging parameters without overexposure.

Example 1: CMV-GFP plasmid construction

GFP is selected as an exogenous reporter gene, and the nervous system affinity of T42 and AAV9 is compared by quantitatively analyzing the expression of AAV of serotypes T42 and AAV9 carrying GFP gene coding sequences into the nervous system. Thus, the inventors constructed CMV-GFP plasmid for providing the genomic sequence of recombinant AAV.

As shown in FIG. 1, the expression cassette sequence of the CMV-GFP plasmid contains the following elements: 5'ITR, CMV promoter, GFP gene coding sequence, hGH poly (A) sequence, 3' ITR. AAV viral vectors (AAV 9-GFP and T42-GFP) were produced by introducing a CMV-GFP plasmid, a plasmid encoding AAV cap/rep gene, and a helper plasmid simultaneously into host cell 293 cells using known transfection techniques.

Example 2: viral titers of AAV9-GFP and T42-GFP

Viral titers of AAV9-GFP and T42-GFP were determined by quantitative PCR (unit: vg/mL). Standard curves for quantification of viral titers were plotted by quantitative PCR experiments with standards at different dilution ratios (FIG. 2A, R ² ＝0.9991)。

Then, the same experimental conditions were used to test viral titers of AAV9-GFP and T42-GFP after 100-fold and 1000-fold dilution.

The results of quantitative calculation of the standard curve showed that the viral titers of AAV9-GFP and T42-GFP were substantially the same (FIG. 2B, AAV9-GFP: 4.27X 10) ¹² vg/mL；T42-GFP：4.00×10 ¹² vg/mL)。

Example 3: brain parenchymal affinity of AAV9-GFP and T42-GFP

The brain parenchymal affinity of AAV9-GFP and T42-GFP was compared by injecting an equal dose of AAV9-GFP or T42-GFP into bilateral hippocampal regions of mice using a brain stereotaxic method. As shown in fig. 3A, the injection method used can well achieve AAV gene delivery and expression localized to the hippocampal region of the brain.

Mice brains 4 weeks after virus injection were cryosectioned and immunohistochemically analyzed, and it was found that the GFP fluorescence signal was stronger in the T42-GFP group than in the AAV9-GFP group (FIG. 3B) in the same hippocampal region and under the same imaging parameters, and statistics showed that the above differences were significant (FIG. 3C, p < -0.05).

The above results indicate that T42 has a brain parenchymal affinity superior to that of the classical serotype AAV9.

Example 4: DRG affinity for AAV9-GFP and T42-GFP

The DRG affinity of T42 and AAV9 was compared using the intrathecal injection of AAV9-GFP or T42-GFP in mice. As shown in fig. 4A, a microsyringe was inserted into the sacral spinal cord segment of mice and an equal dose of AAV virus was injected into the cerebrospinal fluid. After 4 weeks of virus injection, L5 segments of DRG were removed for cryosectioning and immunohistochemical analysis.

The results indicated that the GFP fluorescence signal of the L5 DRG of the T42-GFP group was stronger than that of the AAV9-GFP group under the same imaging parameters (see FIG. 4B).

Statistics show that the differences are significant (see FIG. 4C, p- <0.01). These data demonstrate that the DRG affinity of T42 is superior to that of the classical serotype AAV9.

In conclusion, compared with the traditional AAV serotypes widely used in clinical trials even in drug preparation such as AAV9, T42 has better affinity to the nervous system, which indicates that T42 has good application prospect in gene therapy of nervous system diseases.

All publications, patent applications, patents, nucleic acid and amino acid sequences, and other references mentioned in this disclosure are incorporated by reference herein in their entirety.

While the present disclosure has been shown and described with reference to certain preferred embodiments thereof, it will be understood by those skilled in the art that the foregoing is a more detailed description of the disclosure than is possible with reference to the specific embodiments, and that no limitation to the specific embodiments of the disclosure is intended. Various changes in form and detail, including simple deductions or substitutions, may be made by those skilled in the art without departing from the spirit and scope of the present disclosure.

Sequence listing

<110> Shanghai Yuanjie Biotechnology Co., ltd

<120> AAV vector with high affinity for nervous system and application thereof

<130> PCNCNN222687G

<160> 3

<170> PatentIn version 3.5

<210> 1

<211> 2211

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> nucleotide sequence encoding T42 (SEQ ID NO: 1)

<400> 1

atggctgccg atggttatct tccagattgg ctcgaggaca acctctctga gggcattcgc 60

gagtggtggg acctgaaacc tggagccccg aaacccaaag ccaaccagca aaagcaggac 120

aacggccggg gtctggtgct tcctggctac aagtacctcg gacccttcaa cggactcgac 180

aagggggagc cggtcaacgc agcagacgcg gcggccctcg agcacgacaa ggcctacgac 240

cagcagctca aagcgggtga caatccgtac ctgcggtata accacgccga cgccgagttt 300

caggagcgtc tgcaagaaga tacgtctttt gggggcaacc tcgggcgagc agtcttccag 360

gccaaaaaga gggttctcga acctctcggt ctggttgagg aaggcgctaa gacggctcct 420

ggaaagaaac gtccggtaga gcagtcgcca caagagccag actcctcctc gggcatcggc 480

aagacaggcc agcagcccgc cagaaaaaga ctaaatttcg gtcagactgg cgactcagag 540

tcagttccag accctcaacc tctcggagaa cctccagcaa cccccgctgc tgtgggacct 600

aatacaatgg cttcaggtgg tggcgcacca atggcagaca ataacgaagg cgccgacgga 660

gtgggtaatg cctcaggaaa ttggcattgc gattccacat ggatgggcga cagagtcatc 720

accaccagca cccgcacctg ggccttgccc acctacaaca accacctcta caagcaaatc 780

tccagtgaaa ctgcaggtag taccaacgac aacacctact tcggctacag caccccctgg 840

gggtattttg actttaacag attccactgc cacttctcac cacgtgactg gcagcgactc 900

atcaacaaca attggggatt ccggcccaag agactcaact tcaaactctt caacatccaa 960

gtcaaggagg tcacgacgaa tgacggcgtt acgaccatcg ctaataacct taccagcacg 1020

gttcaagtct tctcggactc ggagtaccag ctgccgtacg tcctcggctc tgcgcaccag 1080

ggctgcctcc ctccgttccc ggcggacgtg ttcatgattc cgcagtacgg ctacctaacg 1140

ctcaacaatg gcagccaggc agtgggacgg tcatcctttt actgcctgga atatttccca 1200

tcgcagatgc tgagaacggg caataacttt accttcagct acaccttcga ggacgtgcct 1260

ttccacagca gctacgcgca cagccagagc ctggaccggc tgatgaatcc tctcatcgac 1320

cagtacctgt acttcctgaa cagaactcag aatcagtccg gaagtgccca aaacaaggac 1380

ttgctgttta gccgtgggtc tccagctggc atgtctgttc agcccaaaaa ctggctacct 1440

ggaccctgtt atcggcagca gcgcgtttct aaaacaaaaa cagacaacaa caacagcaac 1500

tttacctgga ctggtgcttc aaaatataac ctcaatgggc gtgaatccat catcaaccct 1560

ggcactgcta tggcctcaca caaagacgac aaagacaagt tctttcccat gagcggtgtc 1620

atgatttttg gaaaagagag cgccggagct tcaaacactg cattggacaa tgtcatgatt 1680

acagacgaag aggaaatcaa agccactaac cccgtggcca ccgaaagatt tgggaccgtg 1740

gcagtcaatc tccagagcag cagcacagac cctgcgaccg gagatgtgca tgttatggga 1800

gccttacctg gaatggtgtg gcaagataga gacgtgtacc tgcagggtcc catttgggcc 1860

aaaattcctc acacagatgg acactttcac ccgtctcctc ttatgggcgg ctttggactc 1920

aagaacccgc ctcctcagat cctcatcaaa aacacgcctg ttcctgcgaa tcctccggca 1980

gagttttcgg ctacaaagtt tgcttcattc atcacccagt attccacagg acaagtgagc 2040

gtggagattg aatgggagct gcagaaagaa aacagcaagc gctggaatcc cgaagtgcag 2100

tacacatcca attatgcaaa atctgccaac gttgatttca ctgtggacaa caatggactt 2160

tatactgagc ctcgccccat tggcacacgt ttcctcaccc gtcccctgta a 2211

<210> 2

<211> 736

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> VP1 amino acid sequence of T42 (SEQ ID NO: 2)

<400> 2

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro

20 25 30

Lys Ala Asn Gln Gln Lys Gln Asp Asn Gly Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ser Gly Ile Gly

145 150 155 160

Lys Thr Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro Pro

180 185 190

Ala Thr Pro Ala Ala Val Gly Pro Asn Thr Met Ala Ser Gly Gly Gly

195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ala

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Thr Trp Met Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Ser Glu Thr Ala Gly Ser Thr Asn Asp Asn Thr

260 265 270

Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe

275 280 285

His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn

290 295 300

Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln

305 310 315 320

Val Lys Glu Val Thr Thr Asn Asp Gly Val Thr Thr Ile Ala Asn Asn

325 330 335

Leu Thr Ser Thr Val Gln Val Phe Ser Asp Ser Glu Tyr Gln Leu Pro

340 345 350

Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala

355 360 365

Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly

370 375 380

Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro

385 390 395 400

Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe

405 410 415

Glu Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp

420 425 430

Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Phe Leu Asn Arg

435 440 445

Thr Gln Asn Gln Ser Gly Ser Ala Gln Asn Lys Asp Leu Leu Phe Ser

450 455 460

Arg Gly Ser Pro Ala Gly Met Ser Val Gln Pro Lys Asn Trp Leu Pro

465 470 475 480

Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Lys Thr Asp Asn

485 490 495

Asn Asn Ser Asn Phe Thr Trp Thr Gly Ala Ser Lys Tyr Asn Leu Asn

500 505 510

Gly Arg Glu Ser Ile Ile Asn Pro Gly Thr Ala Met Ala Ser His Lys

515 520 525

Asp Asp Lys Asp Lys Phe Phe Pro Met Ser Gly Val Met Ile Phe Gly

530 535 540

Lys Glu Ser Ala Gly Ala Ser Asn Thr Ala Leu Asp Asn Val Met Ile

545 550 555 560

Thr Asp Glu Glu Glu Ile Lys Ala Thr Asn Pro Val Ala Thr Glu Arg

565 570 575

Phe Gly Thr Val Ala Val Asn Leu Gln Ser Ser Ser Thr Asp Pro Ala

580 585 590

Thr Gly Asp Val His Val Met Gly Ala Leu Pro Gly Met Val Trp Gln

595 600 605

Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His

610 615 620

Thr Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu

625 630 635 640

Lys Asn Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala

645 650 655

Asn Pro Pro Ala Glu Phe Ser Ala Thr Lys Phe Ala Ser Phe Ile Thr

660 665 670

Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln

675 680 685

Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Val Gln Tyr Thr Ser Asn

690 695 700

Tyr Ala Lys Ser Ala Asn Val Asp Phe Thr Val Asp Asn Asn Gly Leu

705 710 715 720

Tyr Thr Glu Pro Arg Pro Ile Gly Thr Arg Phe Leu Thr Arg Pro Leu

725 730 735

<210> 3

<211> 735

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> HH67 capsid protein amino acid sequence (SEQ ID NO: 3)

<400> 3

Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser Glu

1 5 10 15

Gly Ile Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro Lys

20 25 30

Ala Asn Gln Gln Lys Gln Asp Asn Gly Arg Gly Leu Val Leu Pro Gly

35 40 45

Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro Val

50 55 60

Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp Gln

65 70 75 80

Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala Asp

85 90 95

Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly Asn

100 105 110

Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro Leu

115 120 125

Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg Pro

130 135 140

Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ser Gly Ile Gly Lys

145 150 155 160

Thr Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr Gly

165 170 175

Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro Pro Ala

180 185 190

Thr Pro Ala Ala Val Gly Pro Asn Thr Met Ala Ser Gly Gly Gly Ala

195 200 205

Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ala Ser

210 215 220

Gly Asn Trp His Cys Asp Ser Thr Trp Met Gly Asp Arg Val Ile Thr

225 230 235 240

Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu Tyr

245 250 255

Lys Gln Ile Ser Ser Glu Thr Ala Gly Ser Thr Asn Asp Asn Thr Tyr

260 265 270

Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His

275 280 285

Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn Trp

290 295 300

Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln Val

305 310 315 320

Lys Glu Val Thr Thr Asn Asp Gly Val Thr Thr Ile Ala Asn Asn Leu

325 330 335

Thr Ser Thr Val Gln Val Phe Ser Asp Ser Glu Tyr Gln Leu Pro Tyr

340 345 350

Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp

355 360 365

Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly Ser

370 375 380

Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro Ser

385 390 395 400

Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe Glu

405 410 415

Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp Arg

420 425 430

Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Asn Arg Thr

435 440 445

Gln Asn Gln Ser Gly Ser Ala Gln Asn Lys Asp Leu Leu Phe Ser Arg

450 455 460

Gly Ser Pro Ala Gly Met Ser Val Gln Pro Lys Asn Trp Leu Pro Gly

465 470 475 480

Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Lys Thr Asp Asn Asn

485 490 495

Asn Ser Asn Phe Thr Trp Thr Gly Ala Ser Lys Tyr Asn Leu Asn Gly

500 505 510

Arg Glu Ser Ile Ile Asn Pro Gly Thr Ala Met Ala Ser His Lys Asp

515 520 525

Asp Lys Asp Lys Phe Phe Pro Met Ser Gly Val Met Ile Phe Gly Lys

530 535 540

Glu Ser Ala Gly Ala Ser Asn Thr Ala Leu Asp Asn Val Met Ile Thr

545 550 555 560

Asp Glu Glu Glu Ile Lys Ala Thr Asn Pro Val Ala Thr Glu Arg Phe

565 570 575

Gly Thr Val Ala Val Asn Leu Gln Ser Ser Ser Thr Asp Pro Ala Thr

580 585 590

Gly Asp Val His Val Met Gly Ala Leu Pro Gly Met Val Trp Gln Asp

595 600 605

Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His Thr

610 615 620

Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu Lys

625 630 635 640

Asn Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala Asn

645 650 655

Pro Pro Ala Glu Phe Ser Ala Thr Lys Phe Ala Ser Phe Ile Thr Gln

660 665 670

Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln Lys

675 680 685

Glu Asn Ser Lys Arg Trp Asn Pro Glu Val Gln Tyr Thr Ser Asn Tyr

690 695 700

Ala Lys Ser Ala Asn Val Asp Phe Thr Val Asp Asn Asn Gly Leu Tyr

705 710 715 720

Thr Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Pro Leu

725 730 735

Claims

An AAV capsid protein, wherein the amino acid sequence of said AAV capsid protein is set forth in SEQ ID NO:2, respectively.
Use of an AAV vector in the preparation of a pharmaceutical vector for treating a neurological disease, wherein said AAV vector comprises an AAV capsid protein according to claim 1.
3. The use of claim 2, wherein the neurological condition comprises: neurodegenerative diseases, neurodevelopmental diseases, chronic pain, nerve injury, retinal diseases or combinations thereof.
4. A nucleic acid molecule, wherein the nucleic acid molecule encodes the AAV capsid protein of claim 1.
5. The nucleic acid molecule of claim 4, wherein the nucleotide sequence of the nucleic acid molecule is identical to the nucleotide sequence of SEQ ID NO:1 has at least 70% identity.
6. The nucleic acid molecule of claim 4, wherein the nucleotide sequence of the nucleic acid molecule is identical to the nucleotide sequence of SEQ ID NO:1 has 75%, 80%, 85%, 90%, 95%, or 99% identity.
7. The nucleic acid molecule of claim 4, wherein the nucleotide sequence of the nucleic acid molecule is as set forth in SEQ ID NO:1 is shown.
An AAV vector, wherein the AAV vector comprises:

(i) The AAV capsid protein of claim 1; and

(ii) A viral genome.
9. The AAV vector of claim 8, wherein the viral genome is a native AAV genome or an artificially recombined viral genome.
10. The AAV vector of claim 9, wherein the viral genome is an artificially recombined viral genome comprising a reporter gene and/or an exogenous therapeutic gene.
11. A genetically engineered cell, wherein the genetically engineered cell comprises:

(i) A first nucleic acid construct comprising an exogenous therapeutic gene for a neurological disease;

(ii) A second nucleic acid construct comprisingrepAndcapa gene ofcapA gene encoding the AAV capsid protein of claim 1; and

(iii) A third nucleic acid construct, said third nucleic acid construct being a helper plasmid.
12. A pharmaceutical composition, wherein the pharmaceutical composition comprises the following components:

(i) The AAV vector of any one of claims 8 to 10; and

(ii) And (3) an excipient.
13. The pharmaceutical composition of claim 12, wherein the pharmaceutical composition is administered by brain parenchymal injection, intrathecal injection, intraventricular injection, subpial injection, intravenous injection, intravitreal injection, subretinal injection, or a combination thereof.
14. The pharmaceutical composition of claim 12 or 13, wherein the pharmaceutical composition is an injection.