AAV capsid protein variants targeting central nervous system and uses thereof

Abstract

The present invention relates to an adeno-associated virus (AAV) capsid protein variant having an amino acid mutation at the 328th to 331st amino acid residues of the AAV capsid protein relative to the 328th to 331st amino acid residues of the parent AAV capsid protein, for example, an amino acid sequence as shown in SEQ ID NO: 4 to replace the 328th to 331st amino acid residues of the parent AAV capsid protein wherein the amino acid position is numbered according to SEQ ID NO: 5. The invention also relates to an isolated nucleic acid encoding the AAV capsid protein variant, a nucleic acid vector comprising the same, and a host cell. The present invention also relates to a recombinant adeno-associated virus (rAAV) vector comprising the capsid protein variant, a pharmaceutical composition comprising the rAAV vector, and uses thereof in treatment or prevention of central nervous system diseases.

Description

Technical Field

[0001] The present invention relates to the field of gene therapy of recombinant adeno-associated virus vectors. Specifically, the present invention relates to adeno-associated virus (AAV) capsid protein variants, isolated nucleic acid molecules encoding the same, recombinant adeno-associated virus vectors having the AAV capsid protein variants and uses thereof in treating central nervous system (CNS) diseases. Background Art

[0002] Gene therapy is a highly promising strategy for treating intractable diseases such as hereditary diseases. It treats intractable diseases by replacing pathogenic genes with functional genes or expressing functional proteins. Safe and efficient delivery of therapeutic genes and gene editing tools to specific cells and organs in the body is crucial for gene therapy.

[0003] Recombinant adeno-associated virus (rAAV) is the preferred viral vector for gene therapy through in vivo gene replacement and gene editing in basic research and clinical research. In the prior art, rAAV vectors are usually injected directly into the brain to deliver functional genes to the brain parenchyma to treat central nervous system diseases. However, this delivery method makes the transduction range of rAAV vectors extremely limited, limiting the treatment of many neurodegenerative diseases such as Alzheimer's disease. In addition, each direct injection of rAAV vectors into the brain requires general anesthesia and cranial surgery, which may cause serious side effects (Saraiva J, et al. (2016), J Control Release, 241: 94–109. https: / / doi.org / 10.1016 / j.jconrel.2016.09.011). Therefore, non-invasive treatment methods such as intravascular injection of rAAV vectors have become important alternatives.

[0004] The capsid protein of rAAV determines the immunogenicity of rAAV vector and its tropism to cells and tissues to a large extent. rAAV with natural capsid protein is usually isolated in large quantities in the liver after intravascular injection. This isolation limits the transduction efficiency and therapeutic effect of rAAV on other organs such as the central nervous system (Murrey D.A., Naughton BJ, Duncan FJ et al. (2014), Hum. Gene Ther. Clin. Dev. 25, 72–84). If rAAV with natural capsid protein is to be used to achieve therapeutic effects in the central nervous system, it is necessary to inject an extremely high viral dose (about 2E+14 vg / kg) of rAAV, which leads to easy induction of body immunity or tissue toxicity. The low transduction efficiency and limited tissue tropism of rAAV vectors limit their application as effective gene therapy vectors for central nervous system diseases.

[0005] Therefore, there is a need in the art for adeno-associated virus (AAV) capsid protein variants having tropism for central nervous system cells, and recombinant adeno-associated virus vectors having the capsid protein variants for delivering target genes to central nervous system cells of a subject with improved transduction efficiency.

[0006] At present, the most studied mutation region of the capsid protein of rAAV is the region near position 588 of the VP protein. For example, Drew H. Bryant et al. (Nature Biotechnology, 2021; https: / / doi.org / 10.1038 / s41587-020-00793-4) used machine deep learning to design highly diverse adeno-associated virus 2 (AAV2) capsid protein variants, and predicted different capsid activities for variants of amino acid residues 561–588 of the capsid protein sequence through a deep neural network model. The field also needs to mutate other regions on the rAAV capsid protein to obtain recombinant adeno-associated virus vectors with high transduction efficiency for cells in the central nervous system of subjects, thereby expanding the application potential of rAAV. Summary of the invention

[0007] The present invention provides an adeno-associated virus capsid mutant having a novel mutation site. The AAV virus vector having the capsid mutant can deliver a target gene to cells constituting an adult brain (eg, neuronal cells) in a subject's central nervous system with improved transduction efficiency.

[0008] Therefore, in a first aspect, the present invention provides an adeno-associated virus (AAV) capsid protein variant having an amino acid mutation at positions 328 to 331 of a parent AAV capsid protein, for example, the amino acid sequence at positions 328 to 331 of the AAV capsid protein is the amino acid sequence shown in SEQ ID NO:4, to replace the amino acid residues 328-331 of the parent AAV capsid protein, wherein the amino acid positions are numbered according to SEQ ID NO:5.

[0009] In some embodiments, the capsid protein variants of the present invention confer increased transduction of central nervous system cells to AAV virus particles comprising the capsid protein, compared to the parent AAV capsid protein, preferably, increased transduction of cells (e.g., neurons and / or glial cells) in the hippocampus, cortex, and thalamus regions.

[0010] In some embodiments, the capsid protein variant of the present invention is a parent AAV capsid protein whose amino acid sequence from position 328 to position 331 is replaced with an amino acid, for example, replaced with the amino acid sequence shown in SEQ ID NO: 4, wherein the parent AAV capsid protein is an AAV9 capsid protein, which comprises or consists of the following amino acid sequence:

[0011] (a) an amino acid sequence of amino acids 203 to 736 of SEQ ID NO:5, or an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical thereto;

[0012] (b) an amino acid sequence of amino acids 137 to 736 of SEQ ID NO:5, or an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical thereto; or

[0013] (c) the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical thereto.

[0014] In some embodiments, the capsid protein variant of the present invention is a parent AAV capsid protein whose amino acid sequence from position 328 to position 331 is replaced with an amino acid, for example, replaced with the amino acid sequence shown in SEQ ID NO:4, wherein the parent AAV capsid protein is a chimeric capsid protein formed by splicing parts of capsid proteins of different AAV serotypes, for example, an AAV5 / AAV9 chimeric capsid protein, an AAV2 / AAV9 chimeric capsid protein.

[0015] In some embodiments, the capsid protein variant of the present invention is a VP1, VP2 or VP3 capsid protein variant, for example, the capsid protein variant:

[0016] (a) comprising the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and the amino acid sequence from positions 328 to 331 is the amino acid sequence set forth in SEQ ID NO:4;

[0017] (b) encoded by a nucleic acid comprising SEQ ID NO: 2 or a nucleotide sequence that differs from the nucleotide sequence of SEQ ID NO: 2 due to the degeneracy of the genetic code;

[0018] (c) is derived from a transcript encoding the amino acid sequence of (a) or comprising the nucleotide sequence of (b).

[0019] In a second aspect, the present invention provides an isolated nucleic acid, wherein the nucleic acid comprises a polynucleotide sequence encoding the AAV capsid protein variant described in the present invention.

[0020] In some embodiments, the nucleic acid according to the present invention comprises a polynucleotide encoding SEQ ID NO: 3, or a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto and having an amino acid substitution at position 328 to position 331 (e.g., replaced with an amino acid sequence as shown in SEQ ID NO: 4).

[0021] In some embodiments, the nucleic acid according to the present invention is capable of transcribing and expressing at least one or any two or all three AAV capsid proteins selected from the following: VP1, VP2 and VP3. In this article, the expression "capable of transcribing and expressing" with respect to nucleic acid means that when the nucleic acid is placed under the control of a suitable promoter, it can be transcribed and expressed in a suitable cell into which the nucleic acid is introduced to produce the gene product mentioned.

[0022] In a third aspect, the present invention provides a nucleic acid vector, e.g., a nucleic acid expression vector, comprising the nucleic acid of the present invention. In some embodiments, the vector further comprises a nucleic acid encoding an AAV REP protein. In some embodiments, the vector is a plasmid.

[0023] In a fourth aspect, the present invention provides an isolated host cell comprising the nucleic acid of the present invention, optionally wherein the nucleic acid is stably transfected into the host cell, preferably, the host cell further comprises a nucleic acid encoding an AAV REP protein and / or a nucleic acid encoding a helper virus function.

[0024] In some embodiments, the host cell is a recombinant adeno-associated virus vector (rAAV) production cell. In some embodiments, the nucleic acid stably transfects the production cell. In other embodiments, the production cell further comprises a nucleic acid encoding an AAV REP protein (especially an AAV9 or AAV2 REP protein). In other embodiments, the production cell further comprises a nucleic acid capable of trans-providing the helper virus functions required for AAV replication and assembly (e.g., E1a and E1b helper genes from adenovirus, and / or E4, E2a and VA helper genes). In other embodiments, the production cell further comprises an rAAV genome carrying a heterologous nucleic acid.

[0025] In a fifth aspect, the present invention provides the use and method of using a nucleic acid according to the present invention, a vector according to the present invention, or a host cell or a production cell according to the present invention encoding an AAV capsid protein variant according to the present invention for preparing a recombinant adeno-associated virus (rAAV) vector. In some embodiments, the use or method comprises:

[0026] (i) providing a production cell according to the invention;

[0027] (ii) culturing the cells under conditions that allow packaging to produce rAAV virions comprising the recombinant AAV genome; and

[0028] (iii) harvesting the cultured host cells or the culture medium to collect the produced recombinant AAV viral vector.

[0029] In a sixth aspect, the present invention provides a recombinant adeno-associated virus (rAAV) vector comprising a capsid protein variant according to the present invention or produced using a production cell of the present invention comprising a nucleic acid according to the present invention.

[0030] In some embodiments, rAAV vectors comprising a capsid protein variant according to the present invention exhibit increased transduction of central nervous system cells, e.g., increased transduction of cells (e.g., neurons and / or glial cells) in the hippocampus, cortex, and thalamus regions, relative to rAAV vectors comprising wild-type AAV capsid proteins or relative to reference rAAV vectors comprising parental capsid proteins.

[0031] In some embodiments, the rAAV vector according to the present invention comprises in its genome:

[0032] a. 5' and 3' AAV inverted terminal repeat (ITR) sequences, and

[0033] b. an expression construct comprising a heterologous nucleic acid located between the 5' and 3' ITRs, wherein the heterologous nucleic acid encodes a gene product of interest;

[0034] Preferably, the expression construct comprises the following elements functionally linked to each other in the transcriptional direction:

[0035] - promoter,

[0036] - a heterologous nucleic acid,

[0037] - Transcription terminator.

[0038] In some embodiments, the heterologous nucleic acid encodes a target gene product for gene replacement, gene suppression or gene editing. Preferably, the target gene product is a protein or RNA.

[0039] In a seventh aspect, the present invention provides a pharmaceutical composition comprising a recombinant AAV viral vector according to the present invention and a pharmaceutically acceptable carrier.

[0040] In an eighth aspect, the present invention provides a method for delivering a heterologous nucleic acid to a subject in need thereof, wherein the method comprises administering to the subject a recombinant AAV viral vector according to the present invention or a pharmaceutical composition according to the present invention, for example, by intravenous injection, for example, for the treatment or prevention of a central nervous system disease.

[0041] In a ninth aspect, the present invention provides the use of the recombinant AAV viral vector according to the present invention for preparing a pharmaceutical composition for treating or preventing a central nervous system disease, preferably, the pharmaceutical composition is administered by intravenous injection. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Combined with the following Figure 1 When reading together, the preferred embodiments of the present invention described in detail below will be better understood. For the purpose of illustrating the present invention, the drawings show the currently preferred embodiments. However, it should be understood that the present invention is not limited to the precise arrangement and means of the embodiments shown in the drawings.

[0043] Figure 1 The schematic diagram shows the pRDAV-CMV-EGFP-Cap9 backbone plasmid (Construct 1). ITR: AAV inverted terminal repeat (ITR); CMV promoter: cytomegalovirus promoter; EGFP: gene encoding enhanced green fluorescent protein; shortpA: short polyadenylation signal; p40: p40 promoter from AAV9; CAP9: gene encoding capsid protein of wild-type AAV9 serotype.

[0044] Figure 2 The electrophoresis diagram of each packaged recombinant AAV virus (using CAPX numbering) obtained is shown, and the leftmost lane is a molecular weight marker.

[0045] Figure 3Figure 2 shows EGFP fluorescence transduction in the hippocampus of mouse brains after infection of mice with AAV virus harboring the CapX-584 capsid variant via tail vein injection relative to control rAAV9 virus.

[0046] Figure 4 Shown is the change in mean fluorescence intensity in the hippocampus of mouse brains after infection of mice with AAV virus harboring the CapX-584 capsid variant via tail vein injection relative to control rAAV9 virus.

[0047] Figure 5 The figure shows the EGFP fluorescence transduction in the cortical area and thalamic nuclei of the mouse brain after the mice were infected with AAV virus with CapX-584 capsid variant by tail vein injection, relative to the control rAAV9 virus. The dotted line box represents the cortical area, and the solid line box represents the thalamic nuclei.

[0048] Figure 6 Figure 2 shows the count of EGFP-positive cells in the cerebral cortex and thalamic nuclei of mice infected by tail vein injection with AAV virus harboring the CapX-584 capsid variant relative to control rAAV9 virus. DETAILED DESCRIPTION

[0049] Unless defined otherwise hereinafter, all technical and scientific terms used in this specification have the same meaning as those of ordinary skill in the art to which the present invention belongs. All publications, patent applications, patents and other references mentioned herein are fully incorporated by reference. In addition, the materials, methods and examples described herein are only illustrative and are not intended to be restrictive. Other features, purposes and advantages of the present invention will be apparent from this specification and the accompanying drawings and from the appended claims.

[0050] I. Definitions

[0051] As used herein, the term "about" when used in conjunction with a numerical value is intended to encompass numerical values ​​within a range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value. The term is also intended to encompass numerical values ​​within the range of ±1%, ±0.5%, or ±0.1% of the specified numerical value.

[0052] Herein, the expression "and / or" is used to indicate any one of the related listed items, or any and all possible combinations of a plurality of the related listed items.

[0053] In this article, the term "comprising" or "including" means including the mentioned elements, integers or steps, or groups of elements, integers or steps, but does not exclude any other elements, integers or steps, or other groups of elements, integers or steps. In this article, when the term "comprising" or "including" is used, unless otherwise indicated, the situation consisting of the mentioned elements, integers or steps is also covered. For example, when referring to a polypeptide / protein "comprising" a specific sequence, it is also intended to cover polypeptides / proteins consisting of the specific sequence.

[0054] As used herein, the terms "AAV capsid protein variant", "AAV variant capsid protein" and "AAV capsid variant" refer to an AAV capsid protein having at least one modification (including deletion, insertion and / or substitution) in the amino acid sequence relative to the parent AAV capsid protein. The variant AAV capsid protein may have at least 90%, at least 95%, at least 98%, at least 99% identity to the amino acid sequence of the parent capsid protein.

[0055] Herein, "parent AAV capsid protein" or "parent capsid protein" refers to an AAV capsid protein used as a template for introducing capsid protein mutations at positions 328-331 according to the present invention. The parent of the variant AAV capsid protein may be a naturally occurring or "wild-type" AAV capsid protein; it may also be a non-wild-type capsid protein, for example, a capsid protein into which one or more (e.g., no more than 10 or 5, 4, 3, 2, 1) amino acid modifications are introduced into the natural AAV capsid protein; a chimeric capsid protein formed by splicing capsid proteins of different AAV serotypes, for example, an AAV5 / AAV9 chimeric capsid protein, an AAV2 / AAV9 chimeric capsid protein. It should be understood that in the case where the parent capsid protein is a non-natural capsid protein, the parent capsid protein does not contain the capsid protein mutations at positions 328-331 according to the present invention. In some embodiments, the parent capsid protein is a native AAV9 serotype capsid protein, for example, a VP1 protein having the amino acid sequence of SEQ ID NO: 5, or a corresponding VP2 or VP3 protein having an N-terminal truncation.

[0056] In this article, when referring to the amino acid position in the AAV capsid protein or its segment, it refers to the amino acid position numbered according to SEQ ID NO:5. The amino acid position corresponding to the amino acid position of SEQ ID NO:5 in the sequence of the capsid protein can be determined by comparing the amino acid sequence of the AAV capsid protein mentioned with SEQ ID NO:5. For example, when referring to position 328 of the capsid protein, it refers to the amino acid residue at position 328 of SEQ ID NO:5, or the amino acid residue that appears at the corresponding position of other capsid proteins after comparison. It should be understood that in the present disclosure, when the AAV capsid protein variant involves amino acid substitutions in certain specific amino acid position segments, the alignment of the AAV capsid protein variant with SEQ ID NO:5 in the segment can be visually detected, and if necessary, a gap is introduced in one or both of the two compared sequences to ensure that after the introduction of the gap, the segment of the AAV capsid protein and the corresponding segment of SEQ ID NO:5 are in the corresponding position region in the comparison and have the corresponding amino acid residue number. Sequence alignments to determine amino acid positions can be performed using the Basic Local Alignment Search Tool available from https: / / blast.ncbi.nlm.nih.gov / Blast.cgi using default parameters.

[0057] In this article, "AAV virus particle" and "AAV virus particle" are used interchangeably to refer to a complete virus particle comprising an AAV capsid and an AAV nucleic acid genome (including a wild-type AAV genome and a recombinant AAV genome) packaged in the capsid. In this regard, the AAV nucleic acid molecule chain packaged into any one AAV virus particle can be a sense (e.g., "positive") chain or an "antisense" chain, and these two chains have equal infectivity.

[0058] Herein, the terms "recombinant AAV vector" and "recombinant AAV virions" and "recombinant AAV virus particles" are used interchangeably to refer to non-wild-type recombinant AAV virus particles that can serve as a carrier of a target nucleic acid. Typically, the viral vector comprises a capsid and a viral genome packaged therein, and preferably the viral genome comprises a target nucleic acid to be delivered to a target cell or tissue inserted therein. Herein, "recombinant" may be abbreviated as "r", for example, recombinant AAV may be referred to as rAAV. Therefore, herein, the terms "recombinant AAV virions" and "recombinant AAV vectors" may also be used interchangeably with "rAAV virions", "rAAV virus particles" or "rAAV" or "rAAV vectors". Typically, recombinant AAV vectors are infectious but replication-defective.

[0059] Herein, for the purpose of the present disclosure, the rAAV capsid may be from or derived from various adeno-associated virus serotypes, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and AAV13. For example, the rAAV may have a viral protein capsid composed of an AAV capsid protein from the serotype, a chimeric capsid protein formed by splicing at least two AAV capsid protein portions of the serotype (e.g., AAV5 / AAV9 chimeric capsid protein, AAV2 / AAV9 chimeric capsid protein), and / or variants thereof.

[0060] The term "vector genome (vg)" refers to the nucleic acid sequence packaged in the rAAV capsid to form the rAAV vector. The vector genome may contain regulatory sequences that direct the expression of functional target proteins.

[0061] Herein, for the purposes of this disclosure, the viral genome packaged in the rAAV vector is recombinant, that is, an AAV genomic DNA with genetic modification relative to wild-type AAV genomic DNA. In order to produce recombinant AAV virions that can deliver the nucleic acid of interest to tissues or cells, it is generally only necessary to retain the reverse terminal repeat (ITR) cis element in the genome, and the remaining sequences required for viral packaging can be provided in trans. Therefore, typically, rAAV can have one or more AAV wild-type genes that are completely or partially deleted, for example, rep and / or cap genes that are completely or partially deleted, and are thus replication-defective; but functional flanking ITR sequences necessary for the rescue, replication and packaging of AAV virions are retained. More typically, the recombinant AAV viral genome packaged in the rAAV virion can retain only functional ITR sequences, and preferably comprises or consists of one or more exogenous nucleotide sequences located between two AAV ITR sequences. It should be understood that, with respect to rAAV, the functional ITR sequence can be, but not necessarily, a wild-type nucleotide sequence, which can be changed, for example, by insertion, deletion or substitution of nucleotides, as long as it still provides the functions required for rescue, replication and packaging. Therefore, in this article, the rAAV vector is a viral vector that at least contains the functional ITR required for viral replication and packaging in cis. It should also be understood that the recombinant AAV viral genome can contain two or more (e.g., three) ITR sequences, and these ITR sequences can be the same or different.

[0062] As used herein, the terms AAV "inverted terminal repeat" or "ITR" are used interchangeably to refer to the functional inverted terminal repeat cis-acting elements from the AAV viral genome and encompass wild-type ITR sequences and variant ITR sequences.

[0063] The wild-type ITR of the natural AAV virus contains a Rep protein binding site (RBS) and a terminal resolution site trs (terminal resolution site) in the sequence. It can be bound and recognized by the Rep protein and produce an incision at the trs, and can form a unique "T"-shaped secondary structure, which plays an important role in the life cycle of the AAV virus. The earliest isolated AAV virus, AAV2, has a "reverse terminal repeat sequence" (ITR) with a length of 145bp and a palindromic-hairpin structure located at both ends of the genome. Afterwards, different ITR sequences were found in AAV viruses of various serotypes, but they can all form a hairpin structure and have a Rep binding site. Recombinant AAV viral vectors based on these wild-type ITR sequences are generally single-stranded AAV vectors (ssAAV).

[0064] Relative to the wild-type ITR sequence, the variant ITR can be, for example, a non-natural ITR sequence comprising one or more nucleotide deletions, substitutions, and / or additions, and / or truncations, but still functional, that is, capable of being used to produce rAAV viral vectors. It has been found that, unlike the above-mentioned ssAAV, by modifying the ITR, deleting the trs sequence and optionally the D sequence in the ITR sequence on one side of the AAV virus, the packaged recombinant AAV viral vector can carry self-complementary genomic DNA, thereby producing a virus called scAAV (self-complementary AAV). The packaging capacity of the scAAV viral vector is half that of the ssAAV viral vector, about 2.2kb-2.5kb, but the transduction efficiency is higher after infecting cells. See, for example, Self-complementary AAV Vectors; Advances and Applications, https: / / doi.org / 10.1038 / mt.2008.171. Such variant ITR sequences that can be used to produce scAAV viruses are also referred to herein as ΔITR. The present disclosure contemplates not only ssAAV vectors generated by combining two wild-type ITRs, but also scAAV vectors generated by combining a ΔITR sequence with a wild-type ITR.

[0065] As those skilled in the art will appreciate, the viral capsid protein and viral genome ITR sequence of the recombinant adeno-associated virus may be from the same or different AAV virus serotypes.

[0066] As used herein, the term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced, including the progeny of such cells. Examples of host cells include, but are not limited to, microorganisms, yeast cells, insect cells, and mammalian cells. In some embodiments, the host cell is a mammalian cell. Depending on the context in which the term is used, the host cell may be an in vitro, ex vivo, or in vivo cell. In other embodiments, the host cell is a production cell for producing an rAAV vector according to the present invention, for example, a "HEK293 cell" or a "293 cell", or a cell line derived from the cell.

[0067] The term "transduction efficiency" refers to the percentage of cells that are transduced with at least one AAV genome. 6 cells were exposed to AAV virus, and 0.5×10 6 If 10 cells contain at least one copy of the AAV genome, the transduction efficiency is 50%. The method for determining the transduction efficiency is, for example, using flow cytometry. For example, when the AAV genome contains a polynucleotide encoding green fluorescent protein (GFP), GFP + The percentage of cells is a measure of transduction efficiency.

[0068] Herein, in some embodiments, the term "producer cell" or "producer cell line" refers to a cell population that can grow and divide continuously or for a long time in vitro. It is known in the art that during the storage or passage of such a clonal cell population, the karyotype may undergo spontaneous or induced changes, and thus may not be completely identical to the ancestral cell or culture, but still maintain the desired characteristics of the original producer cell. Such producer cells or cell lines are encompassed within the scope of the present invention.

[0069] As used herein, the terms "exogenous" or "heterologous" are used interchangeably to describe a nucleic acid or protein and refer to a nucleic acid or protein that is exogenous or heterologous relative to a virus, host cell, subject, or other organism containing the nucleic acid or protein, or relative to a flanking nucleic acid or polypeptide to which it is attached, i.e., the nucleic acid or protein is present in a non-natural state in the virus, host cell, subject, or organism, or is non-naturally attached to the flanking nucleic acid or polypeptide. For example, a nucleic acid that is introduced into a particular viral genome or host cell or subject by recombinant techniques so as to be attached to a sequence that is not naturally attached thereto or is in a non-natural chromosomal or cellular location or state is heterologous relative to the viral genome or host cell or subject. Thus, a nucleic acid that is inserted into the same host cell as the cell from which it is derived or into the same organism as the organism from which it is derived, but is present in a non-natural state, e.g., in a different copy number or under the control of different regulatory elements, is exogenous or heterologous.

[0070] In this article, "isolated" nucleic acid refers to a nucleic acid molecule that is artificially synthesized or separated from at least some components of the natural environment containing it. For example, an isolated nucleic acid can be part of a larger nucleic acid, or part of a carrier or material composition, or can be contained in a cell and still be "isolated" if the larger nucleic acid, carrier, material composition or specific cell is not the natural environment of the nucleic acid. In some embodiments, the "isolated" nucleic acid is enriched at least about 10 times, 100 times, 1000 times, 10,000 times or more relative to the starting material.

[0071] As used herein, the term "functional connection" is also referred to as "operably linked", meaning that the specified components are in a relationship that allows them to function in an intended manner. For example, a promoter or enhancer sequence is operably linked to a coding sequence if it stimulates or regulates transcription of the coding sequence in a suitable host cell or other expression system. In general, promoters that are operably linked to a transcribable sequence are continuous with the transcribable sequence, i.e., they are cis-acting. However, some transcriptional regulatory sequences (such as enhancers) do not need to be physically adjacent to or in close proximity to the coding sequence whose transcription they enhance.

[0072] In this article, the term sequence "identity" is used to describe the sequence structure similarity between two amino acid sequences or polynucleotide sequences. To determine the identity percentage of two amino acid sequences or two nucleic acid sequences, the sequences can be compared for the best comparison purpose (for example, a room can be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for the best comparison or a non-homologous sequence can be abandoned for the purpose of comparison). In a preferred embodiment, for the purpose of comparison, the length of the compared reference sequence is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, or 100% of the length of the reference sequence. Subsequently, the amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are compared. When the position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, the molecules are identical at this position.

[0073] Mathematical algorithms can be used to compare sequences and calculate percent identity between two sequences. In a preferred embodiment, the Needlema and Wunsch ((1970) J. Mol. Biol. 48: 444-453) algorithm (available at http: / / www.gcg.com) that has been integrated into the GAP program of the GCG software package is used, using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6 or 4 and a length weight of 1, 2, 3, 4, 5 or 6 to determine the percent identity between two amino acid sequences. In another preferred embodiment, the GAP program in the GCG software package is used (available at http: / / www.gcg.com) to determine the percent identity between two nucleotide sequences using the NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70 or 80 and a length weight of 1, 2, 3, 4, 5 or 6. A particularly preferred parameter set (and the one that should be used unless otherwise stated) is the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0074] The percent identity between two amino acid or nucleotide sequences can also be determined using the algorithm of E. Meyers and W. Miller, ((1989) CABIOS, 4: 11-17), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weighted remainder table, a gap length penalty of 12, a gap penalty of 4).

[0075] As used herein, the term "conservative" amino acid or nucleotide changes refers to neutral or nearly neutral amino acid or nucleotide changes that result in the protein or nucleic acid molecule containing the amino acid or nucleotide changes substantially maintaining the original function. For example, a conservative amino acid substitution is a substitution or replacement of an amino acid with a different amino acid having a similar biochemical property (e.g., charge, hydrophobicity, and size) in the side chain. Such conservatively modified variants are additional to and do not exclude polymorphic variants, interspecies homologs, and alleles. The following 8 groups contain amino acids that are conservative substitutions for each other: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine ​​(C), methionine (M) (see, e.g., Creighton, Proteins (1984)). Those skilled in the art can easily detect the conservative nature of amino acid or nucleotide changes in a particular polypeptide sequence or nucleotide sequence by conventional technical means, such as functional assays.

[0076] As used herein, "individual" or "subject" refers to a mammal. Examples of mammals include, but are not limited to, humans, non-human primates (e.g., cynomolgus monkeys, rhesus monkeys), rodents, and other mammals, e.g., cattle, pigs, horses, dogs. As used herein, mammals include individuals at all stages of development (including embryonic and fetal stages).

[0077] Central nervous system diseases refer to diseases related to the central nervous system, including, for example, cerebral hemorrhage and cerebral thrombosis (also known as "stroke"); tumors of the brain and central nervous system, for example, acoustic neuroma, meningioma, glioblastoma, central nervous system lymphoma, medulloblastoma, etc.; neurodegenerative diseases, for example, Alzheimer's disease, Parkinson's disease, multiple sclerosis, Huntington's disease, amyotrophic lateral sclerosis (also known as "Lou Gehrig's disease"); epilepsy; mental illnesses, for example, depression, bipolar disorder, autism and various substance addictions, etc.

[0078] As used herein, the term "treatment" refers to a clinical intervention intended to alter the natural course of a disease in the individual being treated. Desired therapeutic effects include, but are not limited to, preventing the onset or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of progression of the disease, ameliorating or relieving the disease state, and alleviating or improving prognosis. The term "treatment" also encompasses modification or improvement of at least one physical parameter, including physical parameters that may not be discernible by the patient.

[0079] As used herein, the term "prevention" refers to preventing or delaying the onset or development or progression of a disease or condition. As used herein, "prevention" generally refers to hospital intervention performed before at least one symptom of a disease occurs.

[0080] Various aspects of the invention are described below.

[0081] II. Capsid protein variants

[0082] Adeno-associated virus (AAV) is a non-pathogenic parvovirus consisting of a 4.7 kb single-stranded DNA genome within a non-enveloped capsid. The genome contains three open reading frames (ORFs) flanked by inverted terminal repeats (ITRs) that serve as the origin of viral replication and packaging signals. The rep ORF encodes four nonstructural proteins that play a role in viral replication, transcriptional regulation, site-specific integration, and virion assembly. The cap ORF encodes three structural proteins (VP1-3) that assemble to form the viral capsid.

[0083] The AAV viral capsid is composed of a total of about 60 VP monomers from three VP capsid proteins, arranged in an icosahedral structure, in which the molar ratio of the three capsid proteins VP1:VP2:VP3 is "optimal" at about 1:1:10. The three capsid proteins VP1, VP2, and VP3 are all encoded by the cap gene of AAV and produced from the same transcript, regulated by the p40 promoter. The three VP proteins have a common C-terminal sequence, but different N-terminal start sites; VP2 and VP3 produced by alternative splicing are two truncated forms of VP1. As an example, the capsid protein sequence of AAV9 is shown below, in which the VP1-specific amino acid sequence is shown in black and bold (VP1 unique region). The amino acid sequence shared by VP1 and VP2 ("VP1 / VP2 common region") is underlined and italicized; the entire VP3 is contained in VP1 and VP2, and is an amino acid shared by all three capsid proteins ("common VP3 region"), which is bold and italicized below.

[0084]

[0085]

[0086] VP1 and VP2 are mainly located in the nucleus, while the monomeric VP3 is distributed in both the nucleus and the cytoplasm. The outer surface of the assembled AAV capsid is composed of the VP3 sequence (including VP3 and the C-termini of VP1 and VP2), and the N-termini of VP1 and VP2 are located inside the capsid.

[0087] The VP has nine protruding loops called variable regions (VRs). VRs vary among AAV serotypes and are responsible for serotype-specific differences in receptor binding. Due to their exposed position and their function in receptor binding, the VRs forming the protruding loops are ideal locations for capsid modifications aimed at redirecting or expanding AAV tropism (i.e., cell surface targeting).

[0088] In order to develop new cell / tissue-targeted AAV capsid protein variants, the inventors screened AAV capsid protein variants. By inserting a random sequence into the cap ORF at the amino acid position (position 328-331) in the VR-II loop, and constructing an AAV transfer plasmid containing a reporter gene and the cap ORF to couple the genotype and phenotype of the AAV capsid variant with reporter gene expression, a plasmid library was generated. Subsequently, through capsid variant screening and tissue distribution detection in animals, AAV capsid protein variants that can more efficiently and / or more specifically transduce central nervous system cells (e.g., hippocampal, cortical and thalamic regional cells (e.g., neurons and / or glial cells)) under intravenous administration were identified. Thus, the inventors established new capsid protein variants of the present invention.

[0089] Therefore, in one aspect, the present invention provides AAV capsid protein variants. In one embodiment, the AAV capsid protein variant according to the present invention is derived from a parent AAV capsid protein, which is subjected to amino acid substitutions at amino acid positions 328 to 331 of the parent capsid protein, for example, replaced with the amino acid sequence shown in SEQ ID NO:4 to replace the original 328-331 residues (according to the AAV9 VP1 numbering of SEQ ID NO:5). The capsid protein modification according to the present invention does not interfere with capsid assembly and genome packaging, and allows rAAV to obtain modified central nervous system cells (e.g., hippocampal, cortical and thalamic regional cells (e.g., neurons and / or glial cells)) targeting.

[0090] In some embodiments, the parent AAV capsid protein is a capsid protein of an AAV serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 and AAV13, for example, an AAV9 serotype capsid protein. In some preferred embodiments, the parent AAV capsid protein is a chimeric capsid protein formed by splicing capsid proteins of different AAV serotypes, for example, an AAV5 / AAV9 chimeric capsid protein, an AAV2 / AAV9 chimeric capsid protein. In some embodiments, the parent AAV capsid protein comprises an AAV9 capsid protein consisting of the following amino acid sequence or an AAV9 capsid protein consisting of the following amino acid sequence:

[0091] (a) an amino acid sequence of amino acids 203 to 736 of SEQ ID NO:5;

[0092] (b) an amino acid sequence of amino acids 137 to 736 of SEQ ID NO:5;

[0093] (c) the amino acid sequence of SEQ ID NO: 5;

[0094] (d) an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to the amino acid sequence of any one of (a) to (c); or

[0095] (e) has one or more (e.g., 1, 2, 3, 4, 5, 7, 8, 9 or 10) amino acid residue substitutions, deletions and / or additions (preferably conservative amino acid substitutions) compared to the amino acid sequence of any one of (a) to (c).

[0096] In other embodiments, the AAV capsid protein variant according to the present invention is derived from a transcript encoding the following amino acid sequence, wherein the amino acid sequence is an amino acid sequence shown in SEQ ID NO: 4 at amino acid positions 328-331 of the AAV capsid protein. In this article, the expression "derived from" a transcript related to a capsid protein variant means that the transcript is capable of producing the capsid protein variant when it is under conditions suitable for its expression to produce protein, including different forms of capsid protein variants produced from the transcript by using alternative translation start sites and / or alternative splicing, such as VP1, VP2 or VP3 capsid protein variants. In some embodiments, therefore, the AAV capsid protein of the present invention derived from the transcript may be a capsid protein variant selected from VP1, VP2 and VP3. In some preferred embodiments, the AAV capsid protein variant according to the present invention is derived from a transcript encoding the amino acid sequence shown in SEQ ID NO: 3. In some preferred embodiments, the AAV capsid protein variant according to the present invention is derived from a transcript comprising the nucleotide sequence shown in SEQ ID NO: 2.

[0097] In some embodiments, in addition to the substitutions at positions 328-331 according to the present invention, the capsid protein variants according to the present invention may or may not include amino acid substitutions (preferably conservative substitutions) at one or more other amino acid positions, for example, 2 to 5, 5 to 10, or 10 to 15 amino acid substitutions.

[0098] In some embodiments, the AAV capsid protein variants according to the present invention can be further modified, for example, to enhance or extend the delivery of the recombinant vector. Methods for constructing and screening recombinant AAV capsid protein libraries are well known in the art. See, for example, US 2005 / 0053922 and US 2009 / 0202490, the disclosures of which are incorporated herein by reference in their entirety.

[0099] In some embodiments, the variant capsid proteins disclosed herein confer increased transduction of central nervous system cells (e.g., hippocampal, cortical and thalamic regional cells (e.g., neurons and / or glial cells)) to rAAV virions comprising the corresponding parental AAV capsid protein or wild-type AAV, compared to transduction of central nervous system cells using rAAV virions comprising the corresponding parental AAV capsid protein or wild-type AAV. For example, relative to AAV virions comprising parental AAV capsid proteins or wild-type AAV, variant capsid proteins of the present invention cause central nervous system cells (e.g., hippocampal, cortical and thalamic regional cells (e.g., neurons and / or glial cells)) to take up more AAV virions. In some embodiments, AAV viral particles comprising a variant capsid protein of the present invention preferentially transduce central nervous system cells (e.g., cells in the hippocampus, cortex, and thalamus regions (e.g., neurons and / or glial cells)), for example, the transduction efficiency of central nervous system cells (e.g., cells in the hippocampus, cortex, and thalamus regions (e.g., neurons and / or glial cells)) is increased by 1.5 times, 2 times, 2.5 times, 3 times, or more compared to other tissue cells.

[0100] The transduction efficiency of the AAV viral particles to be tested to cells of the central nervous system (e.g., cells in the hippocampus, cortex, and thalamus regions (e.g., neurons and / or glial cells)) can be assessed in vitro or in vivo using a variety of methods known in the art for measuring gene expression, such as an increase in transduction efficiency, transduced cell type selection, transduction preference, etc. For example, rAAV genomes containing a reporter gene (e.g., a fluorescent protein under the control of a constitutive promoter) can be packaged using AAV capsid proteins, and the transduction efficiency and / or transduced cell type selectivity / preference can be assessed in an in vitro cell-based assay (e.g., after transfection of a target cell or a group of target cells) or in an animal model-based assay by detecting the expression of the reporter gene (e.g., fluorescence microscopy).

[0101] III. rAAV production

[0102] In some aspects, the present invention provides the use of nucleic acids encoding the AAV capsid protein variants of the present invention, vectors and host cells comprising the same in preparing rAAV vectors.

[0103] The general principles of rAAV production are reviewed in, for example, Carter, 1992, Current Opinions in Biotechnology, 1533-539; and Muzyczka, 1992, CUM Topics in Microbial. and Immunol., 158: 97-129). Various methods are described in the following documents: WO 95 / 13365; WO 95 / 13392; WO 96 / 17947; PCT / US98 / 18600; WO 97 / 09441; WO 97 / 08298; WO 97 / 21825; WO 97 / 06243; WO 99 / 11764; US 5,786,211; US ​​5,871,982; and US 6,258,595. These documents, especially those related to rAAV production, are incorporated herein by reference in their entirety.

[0104] It has been confirmed that the two inverted terminal repeat sequences (ITR) in the AAV genome are cis-acting elements necessary for AAV integration, replication, rescue and packaging. The two open reading frames located between the ITR sequences in the wild-type AAV genome, called Rep and Cap, encode four Rep proteins (Rep78, Rep68, Rep52 and Rep40) and three Cap proteins (VP1, VP2 and VP3) involved in AAV replication and packaging, as well as one assembly activation protein (AAP). The transcription of Rep78 and Rep68 is controlled by the p5 promoter, the transcription of Rep52 and Rep40 is initiated by the p19 promoter, and the p40 promoter regulates the transcription of Cap proteins VP1, VP2 and VP3.

[0105] Generally, to produce rAAV virions, it is necessary to retain the cis-acting element ITR in the rAAV genome, but the REP and CAP proteins required for replication and packaging can be provided in trans. Therefore, typically, a vector (e.g., a plasmid) carrying an rAAV genome that has deleted all or part of the rep and / or cap genes can be used to transfect a suitable host cell (e.g., a suitable packaging cell) that can provide the deleted AAV function (e.g., rep and / or cap proteins) in trans to produce infectious rAAV virions.

[0106] Herein, the nucleic acid vector carrying the rAAV genome to be transferred into the target cell is also referred to as an "AAV transfer vector", which can be in the form of various nucleic acid vectors suitable for gene transfer, for example, a plasmid. Herein, the AAV functions that are missing from the rAAV genome and need to be provided in trans to assist the rAAV genome in packaging into productive AAV virus particles are also referred to as "AAV auxiliary functions", such as the rep and / or cap genes deleted from the rAAV genome, or nucleic acids encoding AAV rep and / or cap expression products or functional variants thereof. In some embodiments, the AAV auxiliary function can be provided by a vector encoding the auxiliary function, for example, in the form of a plasmid, phage, transposon, cosmid, virus or virus particle. Typically, such a vector provides transient expression of AAV rep and / or cap nucleic acids, but lacks AAV ITR, and therefore can neither replicate nor package itself. In other embodiments, the AAV auxiliary function can be provided by cells or cell lines that stably express the auxiliary function.

[0107] Adeno-associated virus (AAV) is a non-enveloped virus of the Parvoviridae family, belonging to the genus Dependoparvovirus, and requires the presence of a helper virus to replicate and assemble to produce infectious virions. A virus that allows AAV (e.g., wild-type AAV) to be replicated and packaged by mammalian cells is also referred to herein as an "AAV helper virus." Various helper viruses for AAV are known in the art, including adenovirus, herpes virus, and poxvirus. In this article, the functions required for AAV replication and packaging encoded by the helper virus genome are also referred to herein as "helper virus functions," for example, E1a and E1b helper genes from adenovirus, and E4, E2a, and VA helper genes. Helper virus functions can be provided in the form of plasmids or other vectors, and / or in the form of stable cell lines expressing the helper genes. In addition, as will be appreciated by those skilled in the art, AAV helper functions (rep / cap genes) and helper virus functions (e.g., E4, E2a, and VA helper genes) can be provided on a single or separate vector. In some aspects, helper virus functions can also be provided by wild-type adenovirus. For example, a cell line containing and stably expressing the AAV Rep and Cap proteins can be transduced with wild-type adenovirus and an AAV transfer vector, thereby producing infectious rAAV viral particles.

[0108] In some aspects, the technology for producing rAAV particles involves providing the rAAV genome to be packaged, rep and cap genes, and helper virus functions to the cells. Such technology is well known in the art. For example, rAAV production cells (or packaging cells) comprising the following components can be produced: rAAV genome, AAV rep and cap genes separated from the rAAV genome (i.e., not in the rAAV genome), and helper virus functions. Afterwards, the production cells can be used to produce the infectious rAAV particles of interest.

[0109] In some aspects, therefore, the present invention provides a production cell (or packaging cell) that can be used to prepare a recombinant adeno-associated virus (rAAV) vector, which comprises a cap gene encoding a capsid protein variant of the present invention, and optionally further comprises a nucleic acid encoding an AAV rep gene and / or a nucleic acid encoding a helper virus function. In some embodiments, the production cell also comprises an rAAV genome. In some embodiments, the rAAV genome comprises the cap gene or the rep gene. In other embodiments, the rAAV genome does not comprise the cap gene and the rep gene.

[0110] In other aspects, the present invention provides a method for preparing a recombinant adeno-associated virus (rAAV) vector. In some embodiments, the method comprises:

[0111] (i) providing a production cell according to the invention;

[0112] (ii) culturing the cells under conditions that allow packaging to produce rAAV virions comprising the recombinant AAV genome; and

[0113] (iii) harvesting the cultured host cells or the culture medium to collect the produced recombinant AAV viral vector.

[0114] In some embodiments, the production cell comprises a nucleic acid according to the invention encoding an AAV capsid protein variant according to the invention. In some embodiments, the nucleic acid according to the invention comprises:

[0115] (a) encoding SEQ ID NO:3, or a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% identity thereto and having the amino acid sequence from positions 328 to 331 as shown in SEQ ID NO:4; or

[0116] (b) the nucleotide sequence of SEQ ID NO:2, or a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto and encodes the amino acid sequence at positions 328 to 331 as shown in SEQ ID NO:4.

[0117] In some embodiments, the nucleic acid according to the present invention is capable of transcribing and expressing at least one, or any two, or all three AAV capsid proteins selected from the group consisting of: VP1, VP2, and VP3.

[0118] In some embodiments, the nucleic acid according to the present invention is introduced into the production cell in the form of a nucleic acid vector. In some embodiments, the nucleic acid vector further comprises a nucleic acid encoding an AAV REP protein. In some embodiments, the nucleic acid vector is a plasmid.

[0119] In some embodiments, the nucleic acid according to the present invention is stably transfected and integrated into the genome of the producer cell.

[0120] In other embodiments, the production cell further comprises (e.g., stably integrated) a nucleic acid encoding an AAV REP protein (particularly an AAV9 or AAV2 REP protein). In other embodiments, the production cell further comprises (e.g., stably integrated or introduced via a vector) a nucleic acid capable of providing helper virus functions in trans (e.g., E1a and E1b helper genes, and / or E4, E2a and VA helper genes from adenovirus).

[0121] In some embodiments, step (i) of the method according to the present invention comprises: introducing a recombinant AAV genome comprising a heterologous nucleic acid into a production cell according to any of the above embodiments. In some embodiments, the rAAV genome to be introduced into the host cell lacks AAV rep and cap DNA. In some embodiments, the rAAV genome to be introduced into the host cell comprises cap DNA encoding a capsid protein variant of the present invention, but lacks AAV rep DNA. In some embodiments, the rAAV genome is introduced into the host cell via an AAV transfer vector.

[0122] In some embodiments, the rAAV production method according to the present invention further comprises: a step of generating a production cell (or cell line). In some embodiments, the production cell (or cell line) expresses one or more of the following components necessary for rAAV genome packaging: an rAAV genome lacking AAV rep and cap genes, an AAV rep gene separated from the rAAV genome, and a cap gene encoding a capsid protein variant according to the present invention. Afterwards, the production cell can be infected with a helper virus (such as an adenovirus) to produce infectious rAAV virus particles.

[0123] In some preferred embodiments, the rAAV production method according to the present invention comprises: co-transfection with three plasmids to produce production cells (or cell lines), wherein the three plasmids are: a plasmid (transfer plasmid) providing an rAAV genome containing heterologous nucleic acid and flanking ITRs, a plasmid providing AAV auxiliary functions (cap and / or rep proteins), and a plasmid providing auxiliary virus (such as adenovirus or herpes simplex virus) functions. In other embodiments, the present invention also contemplates the use of adenovirus or baculovirus to replace the above-mentioned plasmids to introduce AAV auxiliary functions and auxiliary virus functions.

[0124] IV. rAAV vectors

[0125] In some aspects, the present invention provides a viral vector, which is a man-made recombinant viral particle having an rAAV genome packaged in a viral capsid composed of a capsid protein variant of the present invention. In some embodiments, the rAAV genome according to the present invention is a ssAAV genome or a scAAV genome.

[0126] In some embodiments, the recombinant AAV vector according to the present invention comprises in its genome:

[0127] a. AAV inverted terminal repeat (ITR) sequences, and

[0128] b. an expression construct comprising a heterologous nucleic acid, wherein the heterologous nucleic acid encodes a gene product of interest,

[0129] Preferably, the expression construct comprises the following elements functionally linked to each other in the transcriptional direction:

[0130] - promoter,

[0131] - a heterologous nucleic acid encoding a gene product of interest,

[0132] - Transcription terminator.

[0133] In some embodiments, the genome lacks AAV rep and cap DNA, ie, AAV rep and cap DNA are not present in the genome.

[0134] In some embodiments, the rAAV genome disclosed herein comprises two AAV ITRs flanking the expression construct. In some embodiments, the ITR sequences flanking the expression construct in the rAAV genome are native, variant or modified AAV ITR sequences. In some embodiments, at least one ITR sequence is a native, variant or modified AAV ITR sequence. In some embodiments, both ITR sequences are native, variant or modified AAV ITR sequences. In some embodiments, one of the two flanking ITRs is a modified AAV ITR sequence that allows the generation of a self-complementary genome, and the other ITR is a native AAV ITR sequence.

[0135] In the rAAV genome according to the present invention, the AAV ITR may be derived from or may be derived from any AAV serotype, including but not limited to AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, and AAV-13. The genomic sequences of various serotypes of AAV, as well as the natural inverted terminal repeats (ITRs), can be found in the literature or in public databases such as GenBank. See, e.g., GenBank Accession Nos. NC_002077 (AAV1), AF063497 (AAV1), NC_001401 (AAV2), AF043303 (AAV2), NC_001729 (AAV3), NC_001829 (AAV4), U89790 (AAV4), NC_006152 (AAV5), AF513851 (AAV7), AF513852 (AAV8), NC_006261 (AAV8), and AY530579 (AAV9). Publications describing AAV include Srivistava et al. (1983) J. Virol. 45:555; Chiorini et al. (1998) J. Virol. 71:6823; Chiorini et al. (1999) J. Virol. 73:1309; Bantel-Schaal et al. (1999) J. Virol. 73:939; Xiao et al. (1999) J. Virol. 73:3994; Muramatsu et al. (1996) Virol. 221:208; Shade et al. (1986) J. Virol. 58:921; Gao et al. (2002) Proc. Nat. Acad. Sci. USA 99:11854; Moris et al. (2004) Virology 33:375-383; International Patent Publication Nos. WO2018 / 222503A1, WO2012 / 145601A2, WO2000 / 028061A2, WO1999 / 61601A2, and WO1998 / 11244A2; U.S. Patent Application Nos. 15 / 782,980 and 15 / 433,322; and U.S. Patent No. 10,036,016 , 9,790,472, 9,737,618, 9,434,928, 9,233,131, 8,906,675, 7,790,449, 7,906,111, 7,718,424, 7,259,151, 7,198,951, 7,105,345, 6,962,815, 6,984,517 and 6,156,303.

[0136] In some embodiments, the heterologous nucleic acid contained in the rAAV genome is effectively linked to transcription control DNA, particularly promoter DNA, enhancer DNA and polyadenylation signal sequence DNA that are functional in the target cell to form an expression construct. The expression construct may also include intron sequences to facilitate the processing of RNA transcripts when expressed in mammalian cells.

[0137] The heterologous nucleic acid contained in the rAAV according to the present invention is not particularly limited. In some embodiments, the heterologous nucleic acid encodes a gene product to be expressed in a target tissue / cell (e.g., one or more central nervous system cells (e.g., hippocampal, cortical and thalamic regional cells (e.g., neurons and / or glial cells)). In some embodiments, the expression of the gene product in the target cell / tissue will be beneficial to the treatment or prevention of a disease or disorder (e.g., a central nervous system disease) associated with a genetic defect of the tissue. In other embodiments, the heterologous nucleic acid comprises a polynucleotide encoding a marker or reporter protein. In some embodiments, the heterologous nucleic acid encodes a target gene product for gene replacement, gene suppression or gene editing, preferably, the target gene product is a protein or RNA.

[0138] Gene replacement is the most "direct gene therapy method, which generally involves introducing a nucleic acid encoding a functional protein into a host cell and expressing it in the host cell to compensate for the lack of the functional protein caused by a defective gene in the host cell. As those skilled in the art will appreciate, depending on the size and properties of the functional protein, a full-length functional protein can be expressed by introducing a nucleic acid molecule encoding the protein into a host cell, or by introducing multiple nucleic acids encoding a portion of the protein into the same host cell. In some embodiments, the rAAV according to the present invention comprises an expression construct, wherein the expression construct comprises at least one heterologous nucleic acid for gene replacement operably linked to a promoter. In some embodiments, the at least one heterologous nucleic acid encodes a therapeutic protein.

[0139] Gene suppression can be achieved by RNA interference (RNAi) means such as antisense nucleic acids, siRNA or microRNA, to inhibit the expression or translation of defective genes in host cells, thereby treating or preventing functional disorders caused by the defective genes. In some embodiments, the rAAV according to the present invention comprises an expression construct, wherein the expression construct comprises at least one heterologous nucleic acid for gene suppression operably linked to a promoter. In some embodiments, the at least one heterologous nucleic acid, after being introduced into the host cell for expression, will result in the production of siRNA or microRNA to inhibit the expression or translation of defective genes in the host cell.

[0140] Gene editing is another gene therapy method for targeted repair of gene defects, including but not limited to ZFN, TALEN and CRISP / Cas editing. In some embodiments, the rAAV according to the present invention comprises an expression construct, wherein the expression construct comprises at least one heterologous nucleic acid for gene editing (especially CRISP / Cas9 editing) operably linked to a promoter. In some embodiments, the expression of the heterologous nucleic acid in the target cell / tissue will target the repair of the gene defect in the cell / tissue.

[0141] Without wishing to be bound by any theory, CNS diseases can be treated after expression of heterologous nucleic acids in rAAV according to the present invention. Non-limiting examples of CNS diseases and exemplary proteins or genes that can be targeted for each CNS disease are listed below:

[0142] - Stroke, exemplary proteins that can be targeted are caspase-3, Beclin1, Ask1, PAR1, HIF1α, PUMA and / or any of the genes described in Fukuda, AM and Badaut, J. (2013) Genes (Basel) 4:435-456;

[0143] - Huntington's disease, an exemplary targetable protein is mutant huntingtin (mHTT);

[0144] - Epilepsy, exemplary targetable proteins are SCN1A, NMDAR, ADK and / or any of the genes described in Boison, D. (2010) Epilepsia 51: 1659-1668;

[0145] - Parkinson's disease, an exemplary protein that can be targeted is alpha-synuclein;

[0146] - Amyotrophic lateral sclerosis, an exemplary targetable protein is SOD1;

[0147] - Alzheimer's disease, exemplary proteins that can be targeted are tau, amyloid precursor protein;

[0148] - Frontotemporal dementia, where an exemplary targetable protein is tau;

[0149] - amyotrophic lateral sclerosis, exemplary proteins that can be targeted are neurotrophic factors;

[0150] - Brain cancer, exemplary targetable genes are mutated or overexpressed oncogenes implicated in brain cancer

[0151] Lysosomal storage diseases. The promoter used to be connected to the heterologous nucleic acid in the expression construct is not particularly limited. In some embodiments of the rAAV vector disclosed herein, the heterologous nucleic acid encoding the gene product is effectively connected to a constitutive promoter. Suitable constitutive promoters include, for example, cytomegalovirus (CMV) promoter, CMV early enhancer / chicken β-actin (CBA) promoter / rabbit β-globin intron (CAG) 7, human elongation factor 1α promoter (EF1α), human phosphoglycerate kinase promoter (PGK), mitochondrial heavy chain promoter, ubiquitin promoter. In other embodiments, the heterologous nucleic acid encoding the gene product is effectively connected to an inducible promoter. In some cases, the heterologous nucleic acid encoding the gene product is effectively connected to a tissue-specific or cell type-specific regulatory element. Exemplary tissue-specific regulatory elements include, but are not limited to, the following tissue-specific promoters: neuronal such as neuron-specific enolase (NSE) promoter (Andersen et al., Cell. Mol. Neurobiol., 13: 503-15 (1993)), neurofilament light chain gene promoter (Piccioli et al., Proc. Natl. Acad. Sci. USA, 88: 5611-5 (1991)) and neuron-specific vgf gene promoter (Piccioli et al., Neuron, 15: 373-84 (1995)). In some embodiments, the tissue-specific promoter is a promoter of a gene selected from the group consisting of neuronal nuclear antigen (Neuronal Nuclear Antigen, NeuN), glial fibrillary acid protein (GFAP), adenomatous polyposis coli (APC) and ionized calcium binding adapter molecule 1 (Iba-1). Other suitable tissue-specific promoters will be apparent to those skilled in the art.

[0152] In addition to the promoter and the heterologous nucleic acid, other regulatory sequences may be included in the expression construct as needed. The terms "regulatory sequence" and "expression control sequence" are used interchangeably herein and refer to nucleic acid sequences that induce, inhibit or otherwise control the transcription of proteins of the coding nucleic acid sequence that is effectively connected thereto. Regulatory sequences may be, for example, initiation sequences, enhancer sequences, intron sequences, and transcription terminator sequences, etc. In some embodiments, the expression construct further comprises a Kozak sequence located upstream (i.e., 5') of the heterologous nucleic acid.

[0153] V. Pharmaceutical Compositions

[0154] In one aspect, the present invention provides a pharmaceutical composition comprising the rAAV vector of the present invention. The rAAV virions according to the present disclosure can be formulated according to known methods after purification to prepare pharmaceutically useful compositions. The compositions of the present disclosure can be formulated using techniques known in the art for administration to mammalian subjects, such as humans.

[0155] When the delivery system is formulated as a solution or suspension, the delivery system is in an acceptable carrier, such as an aqueous carrier. A variety of aqueous carriers can be used, such as water, buffered water, 0.8% saline, 0.3% glycine, hyaluronic acid, etc. These compositions can be sterilized by conventional well-known sterilization techniques, or can be aseptically filtered. The resulting aqueous solution can be packaged for use as is, or lyophilized, and the lyophilized preparation is combined with a sterile solution before administration.

[0156] The pharmaceutical composition according to the present invention may contain pharmaceutically acceptable auxiliary substances to approach physiological conditions, such as pH adjusters and buffers, tonicity adjusters, wetting agents, etc., such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc. The pharmaceutical composition according to the present invention may or may not contain a preservative.

[0157] The genomic titer of a viral vector, such as the genomic titer of a viral vector in a composition and formulation disclosed herein, can be determined using a variety of standard methods, including, for example, using PCR to determine the genomic titer of a viral vector. The genomic titer of a viral vector is determined using qPCR and ddPCR.

[0158] In a preferred embodiment, the pharmaceutical composition of the present invention is in the form of a pharmaceutical preparation formulated for intravenous delivery.

[0159] VI. Uses of viral vectors

[0160] As a gene drug, the viral vector of the present invention can be physically introduced into a subject using any of a variety of methods and delivery systems known to those skilled in the art, including but not limited to intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other non-parenteral administration, such as by injection or infusion.

[0161] AAV viral vectors having capsid protein variants of the present invention exhibit improved targeting and transduction efficiency to central nervous system cells (e.g., hippocampal, cortical and thalamic regional cells (e.g., neurons and / or glial cells)), thereby providing an effective gene delivery-based means for treating central nervous system diseases.

[0162] In some embodiments, the central nervous system disease is, for example, a lysosomal storage disease (LSD), Huntington's Disease, epilepsy, Parkinson's Disease, Alzheimer's Disease, stroke, corticobasal degeneration (CBD), corticobasal ganglionic degeneration (CBGD), frontotemporal dementia (FTD), multiple system atrophy (MSA), progressive supranuclear palsy (PSP), or brain cancer.

[0163] Example

[0164] Example 1. Construction of a backbone vector for an AAV9 capsid protein mutant library

[0165] This example uses the original plasmid (pRDAV-CMV-EGFP, basket gene preservation, which contains EcoRI and MluI restriction sites at the 3' end of the EGFP gene fragment) and the complete ORF fragment encoding the wild-type AAV9 capsid protein of SEQ ID NO: 5 (also referred to as "Cap9" in the text) to construct the pRDAV-CMV-EGFP-Cap9 backbone plasmid. The pRDAV-CMV-EGFP-Cap9 backbone plasmid is also referred to as "Construct 1" in the text, and its structure is shown in the figure Figure 1 shown.

[0166] The specific method for constructing plasmid pRDAV-CMV-EGFP-Cap9 is as follows.

[0167] Amplification of the complete ORF fragment of Cap9

[0168] Using the AAV9 complete genome fragment Rep2Cap9 sequence (GenBank: LQ502504.1) as a template, PCR amplified the Cap9 complete ORF fragment and introduced restriction sites, wherein the Cap9 complete ORF fragment refers to: starting from the P40 promoter and ending at the Cap9 termination codon TAA, with a length of 2637bp, wherein a restriction endonuclease MluI restriction site is introduced at the P40 end, and a restriction endonuclease EcoRI restriction site is introduced at the Cap9 termination codon end.

[0169] The primer sequences used in the PCR amplification are as follows:

[0170] Upstream primer (SEQ ID NO:6): 5'-ACGCGTacgtcaaaaagggtggagc-3'

[0171] Downstream primer (SEQ ID NO: 7): 5'-GAATTCttacagattacgagtcaggtatctg-3'

[0172] The PCR amplification program was as follows: 95°C pre-denaturation for 2 min, 95°C denaturation for 15 s, 56°C annealing for 15 s, 72°C extension for 30 s, repeated denaturation to extension for 35 cycles, 72°C finishing extension for 2 min, and 4°C termination. PCR products were obtained.

[0173] Generation of construct 1

[0174] The PCR product was digested with EcoRI and MluI and recovered by gel electrophoresis to obtain the complete ORF fragment of Cap9. The original plasmid pRDAV-CMV-EGFP was digested with EcoRI and MluI.

[0175] The obtained Cap9 complete ORF fragment was used as the target fragment and ligated with the linearized plasmid pRDAV-CMV-EGFP after double digestion with EcoRI and MluI, thereby obtaining the backbone plasmid pRDAV-CMV-EGFP-Cap9, i.e., construct 1 (as Figure 1 The size is 10191 bp and the sequence is shown in SEQ ID NO.1.

[0176] Example 2. Construction of AAV capsid variant virus library

[0177] In this example, the AAV capsid variant virus library was constructed using construct 1 (ie, plasmid pRDAV-CMV-EGFP-Cap9) prepared in Example 1 as the backbone. The specific method is as follows.

[0178] 2.1. Obtaining nucleic acid fragments encoding the upstream and downstream sequences of the 328th amino acid residue site in the Cap9 protein sequence

[0179] Using the original AAV9 Cap9 gene sequence on construct 1 as a template, nucleic acid fragments encoding the upstream and downstream sequences of the 328th amino acid residue site in the Cap9 protein sequence were amplified by PCR to obtain the corresponding PCR products, fragment 1 and fragment 2. The primer set sequences used are as follows:

[0180] Primer Set 1:

[0181] Forward primer 1 (SEQ ID NO: 8): cactgccacttctcaccac

[0182] Reverse Primer 1 (SEQ ID NO:9): gtccgtaacctctttgacctgaat

[0183] The program used for PCR amplification was: denaturation at 95°C for 15 s, annealing at 57°C for 30 s, and extension at 72°C for 30 s. The PCR product was subjected to gel electrophoresis and purified using a gel purification recovery kit to obtain fragment 1 with a size of ∼120 bp.

[0184] Primer set 2:

[0185] Forward Primer 2 (SEQ ID NO: 10): aggtcaaagaggttacggacNNNNNNNNNNNNaagaccatcgccaataacc Reverse Primer 2 (SEQ ID NO: 11): tgttgaaggccgttggagg

[0186] The program used for PCR amplification was: denaturation at 95°C for 15 s, annealing at 58°C for 30 s, and extension at 72°C for 15 s. The PCR product was subjected to gel electrophoresis and purified using a gel purification recovery kit to obtain fragment 2 containing a random sequence with a size of ∼1 kb.

[0187] 2.2. Homologous recombination plasmid using PCR fragments

[0188] i) Vector backbone acquisition

[0189] After double digestion of construct 1 with DraIII and BamHI, the linearized vector backbone with a size of 9.1 Kb was obtained by gel electrophoresis.

[0190] ii) Obtaining recombinant plasmid

[0191] According to the instructions of the TAKARA fusion kit (Clontech, 638948), an In-Fusion cloning reaction system was prepared, and fragments 1 and 2 were subjected to an In-Fusion reaction with the linearized backbone vector in i) above in the reaction system (incubated at 50° C. for 15 minutes) to obtain a recombinant plasmid.

[0192] 2.3. Transformation of recombinant plasmid into competent E. coli HB101 cells

[0193] 1) Add 10 μl of the recombinant plasmid prepared in Example 2.2 to the Eppendorf tube containing 100 μl of HB101 competent cells, gently tap the tube wall several times, mix thoroughly, and place on ice for 30 min;

[0194] 2) Place the Eppendorf tube in a 42°C water bath for 90 seconds;

[0195] 3) Slowly add 0.5 mL of LB medium into the Eppendorf tube and shake at 37°C, 80 rpm for 45 min;

[0196] 4) Spread the bacterial solution in the Ep tube on an LB plate containing ampicillin (0.1 g / L) and culture at 37°C overnight.

[0197] 2.4. Screening of positive clones

[0198] For the colonies grown on the LB plate cultured overnight in Example 2.3, a single colony was randomly picked and inoculated into a culture tube containing LB medium containing 0.1 g / L ampicillin, and cultured overnight at 37° C. with shaking at 200 rpm.

[0199] 2.5. Plasmid extraction

[0200] According to the instructions of the plasmid small-scale extraction kit (Shanghai Jierui Bioengineering Co., Ltd., GK2004-200), the bacterial precipitate was collected, and then the steps of bacterial lysis, column loading, washing, elution and the like were performed to collect the plasmid DNA.

[0201] The positive plasmids that have passed concentration detection and enzyme digestion identification were numbered and 10 μL were taken for sequencing. The positive plasmids were stored in a -20°C refrigerator, and single-clone bacteria were picked for preservation to obtain the AAV capsid mutant plasmid library, which is a library of transfer plasmids carrying AAV ITR and expressing variant CAP9 and green fluorescent protein eGFP.

[0202] 2.6. Packaging of AAV capsid mutant virions

[0203] Reference (Xiao X et al., Production of High-Titer Recombinant Adeno-Associated Virus Vectors in the Absence of Helper Adenovirus. J Virol. 1998; 72(3): 2224-2232), the three-plasmid packaging system was used to package and purify the recombinant virus. The packaging plasmid expressing the Rep gene of AAV, the helper plasmid and the ITR carrying AAV, and the transfer plasmid expressing the variant CAP9 and the green fluorescent protein eGFP obtained in Example 2.5, or the transfer plasmid expressing the wild-type CAP9 and the green fluorescent protein eGFP ( Figure 1 The plasmids were mixed at a molar ratio of 1:1:1 and co-transfected into HEK293 cells using the calcium phosphate method. After 72 h, the cells and culture supernatant were collected and PEG8000-NaCl&CH 3 Recombinant AAV virus was purified by Cl extraction. Figure 2The recombinant AAV virus with the CAPX number shown was dissolved in 0.01% F68-PBS, and the rAAV titer was determined by dot hybridization. After preliminary experiments, the recombinant AAV virus with the number CAPX-584 that effectively transduced neuronal cells was selected for further experiments.

[0204] The transfer plasmid used to prepare the CAPX-584 recombinant AAV virus is the CapX-584 plasmid. After sequencing, the base sequence encoding the CAP9 variant in the CapX-584 plasmid is shown in SEQ ID NO.2, and the corresponding amino acid sequence is shown in SEQ ID NO.3. By comparing with the original CAP9 sequence 328-331 sites, it can be known that there is a mutation in the CAP9 sequence 328-331 sites in the CAPX-584 recombinant AAV virus, and the amino acid sequence is shown in SEQ ID NO:4.

[0205] Example 3. Biodistribution of AAV capsid variant viruses in mice

[0206] 3.1. AAV virus injection

[0207] 6-8 weeks, 18-20g male SPF C57 mice were selected, rAAV9 virus encoding fluorescent protein eFGP and having wild-type CAP9 capsid protein was used as a control, and the AAV capsid variant virus obtained in Example 2 was used as an experimental group, with at least 3 mice in each group, and the capsid variant recombinant AAV virus CAPX-584 was injected via the tail vein at a dose of 2E13 vg / kg. After injection, the mice were euthanized 28 days later and the mouse tissues were obtained. The background EGFP fluorescence was observed for frozen sections of each tissue.

[0208] 3.2. Mouse tissue sections and immunofluorescence comparison

[0209] A. Preparation of frozen sections

[0210] 1) Tissue fixation: Fix the tissue with fresh tissue fixative for more than 24 hours, remove the tissue from the fixative, and use a scalpel to smooth the tissue at the target site.

[0211] 2) Dehydration: Place the trimmed tissue in a 15% sucrose solution and dehydrate it in a 4°C refrigerator until it sinks to the bottom. Then transfer it to a 30% sucrose solution and dehydrate it in a 4°C refrigerator until it sinks to the bottom.

[0212] 3) OCT embedding: Take out the dehydrated tissue, use filter paper to slightly absorb the surface water, place it on the embedding table with the cut surface facing up, drip OCT embedding agent around the tissue, and place the embedding table on the quick freezing table of the freezing microtome for quick freezing embedding. After the OCT turns white and hardens, it can be sliced. Fresh tissue does not need to be fixed and dehydrated for direct frozen sectioning. Use a scalpel to directly flatten the tissue of the target area, and then embed the slices with OCT embedding agent.

[0213] 4) Sectioning: Fix the embedding table on the slicer, make a rough cut first, and then start slicing after the tissue surface is trimmed flat. The slice thickness is 8-10μm. Place a clean slide flat on the cut tissue slice, then stick the tissue on the slide and write a label.

[0214] B. Observe fluorescence

[0215] The frozen sections were protected with anti-fading mounting medium (ProLong, Thermo Fischer), and the EGFP protein was observed and images were collected under a fluorescence microscope using a blue excitation band module (488 nm). The images were statistically analyzed using ImageJ software.

[0216] result

[0217] The experimental results showed that compared with the control group, after AAV with the capsid protein mutant CapX-584 infected the mouse central nervous system, the fluorescence intensity in the hippocampal cells was significantly enhanced, and the number of infected hippocampal cells increased ( Figure 3 ).

[0218] According to the fluorescence analysis results of ImageJ software, the average fluorescence intensity of the hippocampus of the brain infected with AAV with the capsid protein mutant CapX-584 was 2.3 times higher than that of AAV9 infected with the wild-type capsid protein (represented as "AAV9" in the figure), P < 0.01 ( Figure 4 ).

[0219] In addition to analyzing the hippocampal nuclei, the transduction characteristics of cells in the cortical area and thalamic nuclei were also analyzed. The results showed that compared with the control group, after AAV with the capsid protein mutant CapX-584 infected the mouse central nervous system, the EGFP-positive cell counts in the cortical area and thalamic nuclei were significantly increased ( Figure 5 ).exist Figure 5 In the figure, the dotted box is the cortex, and the solid box is the thalamic nucleus. Compared with the control group infected with AAV9 with wild-type capsid protein (denoted as "AAV9" in the figure), the EGFP-positive cell counts in the cortex and thalamic nucleus regions increased by about 4.3 times and about 2.8 times, respectively ( Figure 6 ).

[0220] The above results show that the AAV capsid protein mutant CapX-584 can significantly enhance the transduction efficiency of cells in the central nervous system and can be used as a better choice for AAV treatment of central nervous system diseases.

[0221] The exemplary embodiments of the present invention are described above, and it should be understood by those skilled in the art that these disclosures are only exemplary, and various other substitutions, adaptations and modifications can be made within the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments listed herein.

[0222] Exemplary sequences

[0223]

[0224]

[0225]

[0226]

[0227]

Claims

1. An adeno-associated virus (AAV) capsid protein variant having an amino acid mutation at amino acid residues 328 to 331 of the AAV capsid protein relative to amino acid residues 328 to 331 of the parent AAV capsid protein to replace amino acid residues 328 to 331 of the parent AAV capsid protein, wherein the amino acid positions are numbered according to SEQ ID NO:

5.

2. The AAV capsid protein variant according to claim 1, wherein Compared with the parent AAV capsid protein, the amino acid sequence from position 328 to position 331 of the AAV capsid protein variant is the amino acid sequence shown in SEQ ID NO:

4. Preferably, the capsid protein variant confers increased transduction of central nervous system cells to the AAV virus particles containing it, more preferably, increased transduction of cells in the hippocampus, cortex and thalamus regions (e.g., neurons and / or glial cells).

3. The capsid protein variant according to claim 1 or 2, wherein the parent AAV capsid protein is an AAV9 capsid protein, which comprises or consists of the following amino acid sequence: (a) an amino acid sequence of amino acids 203 to 736 of SEQ ID NO:5; (b) an amino acid sequence of amino acids 137 to 736 of SEQ ID NO: 5; or (c) the amino acid sequence of SEQ ID NO: 5; Alternatively, the parent AAV capsid protein is a chimeric capsid protein formed by partially splicing capsid proteins of different AAV serotypes, for example, an AAV5 / AAV9 chimeric capsid protein or an AAV2 / AAV9 chimeric capsid protein.

4. The capsid protein variant according to any one of claims 1 to 3, wherein the capsid protein variant is a VP1, VP2 or VP3 capsid protein variant, for example, the capsid protein variant: (a) comprising an amino acid sequence of SEQ ID NO: 3; (b) is encoded by a nucleic acid comprising SEQ ID NO: 2 or a nucleotide sequence that differs from the nucleotide sequence of SEQ ID NO: 2 due to the degeneracy of the genetic code; or (c) derived from a transcript encoding the amino acid sequence of SEQ ID NO:3 or comprising the nucleotide sequence of SEQ ID NO:

2.

5. An isolated nucleic acid, wherein the nucleic acid comprises a polynucleotide sequence encoding the AAV capsid protein variant of any one of claims 1-4.

6. A nucleic acid according to claim 5, comprising: (a) a polynucleotide encoding the amino acid sequence of SEQ ID NO: 3; or (b) the nucleotide sequence of SEQ ID NO: 2, For example, the nucleic acid transcribes and expresses at least one, any two, or all three AAV capsid proteins selected from the group consisting of: VP1, VP2, and VP3.

7. A nucleic acid vector, e.g., a nucleic acid expression vector, comprising the nucleic acid of claim 5 or 6, and optionally further comprising a polynucleotide encoding an AAV REP protein.

8. An isolated host cell comprising the nucleic acid of claim 5 or 6, optionally wherein the nucleic acid is stably transfected into the host cell, preferably, the host cell further comprises a nucleic acid encoding an AAV REP protein and / or a nucleic acid encoding a helper viral function.

9. Use of the nucleic acid according to claim 5 or 6, the nucleic acid vector according to claim 7, or the host cell according to claim 8 for preparing a recombinant adeno-associated virus (rAAV) vector.

10. A recombinant adeno-associated virus (rAAV) vector comprising the capsid protein variant according to any one of claims 1 to 5, Preferably, the rAAV exhibits increased transduction of cells of the central nervous system, more preferably increased transduction of cells (e.g., neurons and / or glial cells) in the hippocampus, cortex, and thalamus regions relative to wild-type AAV, Optionally, the rAAV vector comprises in its genome: a. 5' and 3' AAV inverted terminal repeat (ITR) sequences, and b. an expression construct comprising a heterologous nucleic acid located between the 5' and 3' ITRs, wherein the heterologous nucleic acid encodes a target gene product, Preferably, the expression construct comprises the following elements functionally linked to each other in the transcriptional direction: - promoter, - a heterologous nucleic acid, - Transcription terminator. For example, the heterologous nucleic acid encodes a target gene product for gene replacement, gene suppression or gene editing. Preferably, the target gene product is a protein or RNA.

11. A pharmaceutical composition comprising the recombinant AAV viral vector according to claim 10 and a pharmaceutically acceptable carrier.

12. A method for delivering a heterologous nucleic acid to a subject in need thereof, wherein the method comprises administering to the subject the recombinant AAV viral vector of claim 10 or the pharmaceutical composition of claim 11, e.g., by intravenous injection, e.g., for treating or preventing a central nervous system disease.

13. Use of the recombinant AAV viral vector according to claim 10 for preparing a pharmaceutical composition for treating or preventing central nervous system diseases, preferably, administering the pharmaceutical composition by intravenous injection.

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