Brain tropism AAV mutants

Abstract

The present invention provides a nucleic acid encoding an adeno-associated virus (AAV) capsid protein mutant comprising a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 4.

Description

Technical Field

[0001] This invention relates to an adeno-associated virus (AAV) capsid protein mutant that exhibits high tropism for the brain and low tropism for the lungs, nucleic acid encoding the capsid protein mutant, AAV particles containing the capsid protein mutant, and a method for generating gene transduced cells using the particles. Background Technology

[0002] AAV is a virus with a 4.7 kb linear single-stranded DNA genome containing open reading frames of two genes, rep and cap. The rep gene encodes four proteins essential for genome replication (Rep78, Rep68, Rep52, and Rep40). The cap gene encodes three capsid proteins (VP1, VP2, and VP3) used to assemble the viral capsid, as well as an assembly activation protein (AAP). AAV replication in nature depends on the presence of helper viruses, such as adenoviruses or herpesviruses. In the absence of helper viruses, the AAV genome is maintained in a free form or integrated into the host chromosome, thus allowing AAV to exist in a latent state. More than one hundred AAV serotypes and clades have been identified (Non-Patent Document 1). In particular, progress has been made in the development of AAV2-based vectors for gene delivery.

[0003] In 1989, AAV2-based gene delivery vector systems were first developed. AAV-based vectors have been found to have many advantages. Because wild-type AAV is non-pathogenic and not associated with any known diseases, AAV-based vectors are considered extremely safe. Furthermore, AAV exhibits high gene transduction efficiency.

[0004] The administration of AAV particles enables long-term and stable gene transduction to various target organs and cells. To date, high-efficiency gene transduction into skeletal muscle, liver (hepatocytes), heart (cardiomyocytes), nerve cells, pancreatic cells, and islet cells has been reported. Furthermore, AAV has been used in human clinical trials. On the other hand, attempts have been made to alter the cell tropism of AAV by modifying the AAV capsid protein and to prevent the clearance of AAV particles by neutralizing antibodies. For example, AAV capsids exhibiting tropism for specific organs and cells (such as glial cells, airway epithelial cells, coronary artery endothelial cells, and lungs) and AAV capsids exhibiting tropism for tumor cells (such as glioblastoma cells, melanoma cells, lung cancer cells, and breast cancer cells) have been generated (Non-Patent Document 2).

[0005] In recent years, it has been shown that AAV9 vectors and their modified vectors can cross the blood-brain barrier to deliver target genes to the brain; and it has been reported that in mice, intravascular administration of AAV9 vectors and their modified vectors can introduce genes into nerve cells (neurons) throughout the brain.

[0006] For example, WO2015 / 038958 (Patent Document 1) discloses an AAV vector containing a specific amino acid sequence. Among them, the vector with the amino acid sequence TLAVPFK inserted at position 588 of the AAV9 capsid protein is named AAV-PHP.B. AAV-PHP.B demonstrates that, upon intravenous administration, it delivers genes to the entire CNS with at least 40 times the efficiency of AAV9 (Non-Patent Document 3).

[0007] Furthermore, AAV-PHP.eB containing the amino acid sequence DGTLAVPFK inserted at position 588 of the AAV9 capsid protein has been shown to reduce the viral load required for transduction in most CNS cells (Non-Patent Document 5). In addition, systemic and intravitreal delivery of AAV-PHP.eB has been shown to produce high transduction efficiency in retinal ganglion cells and horizontal cells (Non-Patent Document 6).

[0008] Furthermore, WO2015 / 158749 (Patent Document 2) discloses a capsid protein for AAV vectors containing a specific amino acid sequence that specifically binds to brain and / or spinal cord cells. It has been shown that inserting a concordant sequence (XXGXXWX) consisting of seven amino acids at position 588 of the AAV2 capsid protein can produce an AAV vector specific to brain microvascular endothelial cells (Non-Patent Document 4).

[0009] In addition, WO2020 / 218419 (Patent Document 3) discloses an AAV capsid protein mutant that contains a specific amino acid sequence and exhibits brain tropism.

[0010] However, since AAV vectors are usually administered systemically, gene therapy targeting brain diseases requires AAV vectors with higher brain tropism.

[0011] Existing technical documents Patent documents Patent document 1: WO2015 / 038958 Patent document 2: WO2015 / 158749 Patent document 3: WO2020 / 218419 Non-patent documents Non-patent document 1: Gao et al., J. Virology, Vol. 78, pp. 6381-6388, 2004 Non-Patent Document 2: Adachi K. et al., Gene Ther. Regul., Vol. 5, pp. 31-55, 2010 Non-Patent Document 3: Nat Biotechnol. Feb 2016;34(2): 204-9 Non-Patent Document 4: EMBO Mol. Med., June 1, 2016; 8(6): 609-25 Non-Patent Document 5: Nat. Neurosci. Aug 2017;20(8): 1172-1179 Non-Patent Document 6: Mol. Ther. Methods Clin. Dev. 28 March 2022; 25:236-249. Summary of the Invention

[0012] The problem the invention aims to solve The present invention aims to provide an AAV capsid protein mutant that exhibits high tropism for the brain and low tropism for the lungs, and to provide a method for effectively introducing the gene into the brain.

[0013] Solution to the problem The inventors have made in-depth efforts to solve the above-mentioned problems, and have thus produced an AAV capsid protein mutant comprising a peptide containing an amino acid sequence selected from SEQ ID NO: 1-4. This completes the present invention.

[0014] This invention generally relates to: [1] A nucleic acid encoding a mutant of an adeno-associated virus (AAV) capsid protein, the mutant comprising a peptide containing an amino acid sequence selected from SEQ ID NO: 1-4; [2] According to the nucleic acid in [1], the AAV capsid protein is derived from AAV2; [3] According to the nucleic acid in [2], the peptide is located between amino acid number 588 and amino acid number 589 in VP1 of AAV2; [4] A recombinant DNA comprising nucleic acids according to any one of [1] to [3]; [5] A cell containing nucleic acids according to any one of [1] to [3]; [6] An AAV particle comprising an AAV capsid protein mutant comprising a peptide comprising an amino acid sequence selected from SEQ ID NO:1-4; [7] According to [6], AAV particles, wherein the AAV capsid protein is derived from AAV2; [8] According to [7], the AAV particles, wherein the peptide is located between amino acid number 588 and amino acid number 589 in VP1 of AAV2; [9] A method for generating gene-transduced cells, the method comprising the steps of: contacting cells with AAV particles containing an AAV capsid protein mutant, the mutant comprising a peptide containing an amino acid sequence selected from SEQ ID NO: 1-4;

[10] According to the method of [9], wherein the AAV capsid protein is derived from AAV2;

[11] According to the method of

[10] , the peptide is located between amino acid number 588 and amino acid number 589 in VP1 of AAV2;

[12] A method for producing AAV particles, the method comprising using a cell according to [5] as a host.

[0015] The effects of the invention According to the present invention, a gene transduction system for transducing genes into the brain is provided. The AAV particles of the present invention have high cell tropism for the brain and low cell tropism for the lungs, and therefore genes transduced by the AAV particles can be strongly expressed in the brain. Attached Figure Description

[0016] [ Figure 1 ] Figure 1 A method for generating a nucleic acid construct that enables a capsid protein to contain a random peptide is shown.

[0017] [ Figure 2 ] Figure 2 The frequency of AAV vector occurrence in each tissue is shown in Example 4.

[0018] [ Figure 3 ] Figure 3 The results of the luciferase activity measurement in Example 5 are shown.

[0019] [ Figure 4 ] Figure 4 The fluorescence microscopy measurements of AcGFP in Example 6 are shown. Detailed Implementation

[0020] As used herein, "adeno-associated virus" refers to a small virus belonging to the genus Dependovirus within the family Parvoviridae, capable of infecting primates and other mammals, including humans. Hereinafter, adeno-associated virus is abbreviated as AAV. AAV has a non-enveloped, regular icosahedral capsid (capsid) and linear single-stranded DNA within it. As used herein, unless otherwise stated, AAV includes wild-type viruses and their derivatives, and includes all serotypes and clades of AAV.

[0021] As used herein, “vector” refers to a molecule or related molecule that mediates the delivery of polynucleotides into cells and contains or is associated with polynucleotides. Unless otherwise stated, examples of vectors include vector DNA (such as plasmid vectors and phage vectors), viral vector particles, liposomes, and other mediators used for gene delivery.

[0022] As used in this article, "capsid protein" refers to the protein encoded by the cap gene, which is present in the AAV genome and constitutes the AAV capsid. The wild-type AAV genome encodes three capsid proteins: VP1, VP2, and VP3. As used in this article, capsid proteins include VP1, VP2, and VP3. Capsid proteins are sometimes also referred to as "Cap proteins."

[0023] (1) Nucleic acid encoding AAV capsid protein mutant The present invention provides a nucleic acid-encoding AAV capsid protein mutant, the mutant comprising a peptide containing an amino acid sequence selected from SEQ ID NO: 1-4. Preferably, the present invention provides a nucleic acid-encoding AAV capsid protein mutant comprising a peptide containing the amino acid sequence of SEQ ID NO: 1.

[0024] The AAV capsid protein mutant encoded by the nucleic acid of the present invention can be prepared by inserting a peptide into the AAV capsid protein of any wild-type AAV, such as AAV1 (AAV1), AAV2 (AAV2), AAV3 (AAV3A, AAV3B, etc.), AAV4 (AAV4), AAV5 (AAV5), AAV6 (AAV6), AAV7 (AAV7), AAV8 (AAV8), AAV9 (AAV9), AAV10 (AAV10), AAV11 (AAV11), avian AAV, bovine AAV, canine AAV, equine AAV, or sheep AAV, or by replacing a portion of the amino acid sequence of the AAV capsid protein with a peptide (in other words, by making the AAV capsid protein contain a peptide). In the present invention, the capsid protein of AAV2 is preferably used. The amino acid sequence of the VP1 capsid protein of wild-type AAV2 is shown in SEQ ID NO: 9 (GenBank: AAC03780.1).

[0025] The AAV capsid protein mutant encoded by the nucleic acid of the present invention can be a protein containing one or more amino acid substitutions, deletions, insertions, and / or additions, as well as the peptides mentioned above, in the wild-type AAV capsid protein. "A protein containing one or more amino acid substitutions, deletions, insertions, and / or additions, as well as the peptides mentioned above" retains the characteristics of the original protein, such as the cell tropism, capsid formation ability, and capsid protein function (e.g., protecting the viral genome, uncoating after entering the host cell) conferred by the aforementioned peptides. For example, in the present invention, an AAV2 capsid protein in which asparagine at position 587 is replaced by glutamine is used. The amino acid sequence of the AAV2 VP1 capsid protein containing glutamine at position 587 is shown in SEQ ID NO:10.

[0026] In addition, a spacer region can be added to the N-terminus and / or C-terminus of the peptide to be included in the AAV capsid protein. The spacer region preferably consists of 1 to 5 amino acid residues. There are no particular limitations on the amino acid residues constituting the spacer region. For example, the spacer region may contain amino acids selected from glycine, alanine, and serine. A single glycine amino acid residue is suitable as a spacer region added to the N-terminus, while a single alanine amino acid residue is suitable as a spacer region added to the C-terminus.

[0027] As the AAV capsid protein used to contain the peptide, AAV VP1, VP2, or VP3 can be used. Only one of VP1, VP2, and VP3 can contain the peptide, or all of VP1, VP2, and VP3 can contain the peptide. Furthermore, two capsid proteins (such as VP1 and VP2, VP2 and VP3, or VP1 and VP3) can contain the peptide. VP1 to VP3 are encoded by the cap gene region in the AAV genome. In one embodiment of the invention, the region common to VP1 to VP3 contains the peptide so that mutations can be introduced into all VP1 to VP3. In another embodiment of the invention, the gene encoding VP1, VP2, or VP3 is prepared separately from the AAV cap gene region, and the mutation is introduced into that gene. In this case, treatment can be performed to suppress the expression of the wild-type capsid protein corresponding to the capsid protein encoded by the gene with the introduced mutation from the AAV cap gene region.

[0028] When using AAV2 VP1, the AAV capsid protein mutant encoded by the nucleic acid of the present invention preferably contains a peptide between amino acid numbers 588 and 589. Amino acid number 588 of AAV2 VP1 is arginine. Amino acid number 589 of AAV2 VP1 is glutamine. Amino acid number 588 of AAV2 VP1 corresponds to amino acid number 451 of AAV2 VP2 and amino acid number 386 of AAV2 VP3. When using capsid proteins from other AAV serotypes and clades besides AAV2 as the AAV capsid protein, the AAV capsid protein contains a peptide between the amino acids corresponding to amino acid numbers 588 and 589 of AAV2 VP1. Those skilled in the art can readily identify the amino acid corresponding to amino acid number 588 of AAV2 VP1 in the capsid proteins of other AAV serotypes and clades besides AAV2. For example, see the VP1 amino acid sequence alignment shown in Gao et al., Proc. Natl. Acad. Sci. USA, Vol. 99, No. 18, pp. 11854-11859, 2002. For example, amino acid number 588 of AAV2 VP1 corresponds to amino acid number 589 of AAV1, amino acid number 590 of AAV7, and amino acid number 591 of AAV8.

[0029] In this invention, it is preferred to use an AAV capsid protein mutant containing a spacer region and ETGHGYV (SEQ ID NO: 1) between amino acid positions 588 and 589 of the AAV2 VP1 capsid protein (e.g., SEQ ID NO: 12).

[0030] The nucleic acid of this invention can be operatively ligated to a suitable control sequence. Examples of control sequences include promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites (IRES), and enhancers. Examples of promoter sequences include inducible promoter sequences and constitutive promoter sequences. The control sequence can be an endogenous or exogenous sequence of AAV derived from the capsid protein, a natural sequence, or a designed sequence. This invention also includes such recombinant DNA capable of expressing AAV capsid protein mutants.

[0031] The recombinant DNA of the present invention can be used to deliver the nucleic acids of the present invention into cells in vitro, ex vivo, or in vivo, and confer the ability of cells to express AAV capsid protein mutants. Subsequently, the cells delivered with the nucleic acids of the present invention can be used to generate AAV particles. The recombinant DNA is particularly useful for delivering or introducing the nucleic acids of the present invention into animal cells, preferably mammalian cells.

[0032] In this invention, the recombinant DNA of the invention can be prepared by using DNA as a vector to retain the nucleic acids of the invention. For example, plasmid DNA, bacteriophage DNA, transposons, coplasmal DNA, episomal DNA, or viral genome can be used.

[0033] (2) Cells containing the nucleic acid of the present invention The present invention also provides cells comprising the nucleic acids of the present invention, particularly isolated host cells containing the recombinant DNA as described in (1) above. For example, the isolated cells are cell lines maintained in vitro. As explained below, the host cells of the present invention can be used to produce the AAV particles of the present invention. When the host cells of the present invention are used to produce AAV particles, the host cells may be referred to as “packaging cells” or “production cells.” The host cells of the present invention may contain the recombinant DNA of the present invention as described in (1) above, integrated into the genome, or the recombinant DNA may be retained in the cell to transiently express an AAV capsid protein mutant.

[0034] The recombinant DNA of the present invention can be introduced into host cells by known methods. For example, electroporation, calcium phosphate precipitation, direct microinjection into cells, liposome-mediated gene transfection, or nucleic acid delivery using a high-speed particle gun can be used. When a viral vector is used, an infection method suitable for that vector can be selected. By using such established techniques, the recombinant DNA of the present invention can be stably introduced into the chromosome of a host cell or transiently introduced into the cytoplasm of a host cell. For stable transformation, a selection marker (e.g., a known selection marker, such as a neomycin resistance gene (encoding neomycin phosphotransferase) or a hygromycin B resistance gene (encoding aminoglycoside phosphotransferase (APH))) can be ligated to the recombinant DNA of the present invention.

[0035] A variety of cells can be used as host cells, such as mammalian cells, including mouse cells and primate cells (e.g., human cells) or insect cells. Suitable examples of mammalian cells include, but are not limited to, primary cells and cell lines. Examples of suitable cell lines include 293 cells, COS cells, HeLa cells, Vero cells, 3T3 mouse fibroblasts, C3H10T1 / 2 fibroblasts, CHO cells, and their derivatives.

[0036] (3) AAV particles containing a mutant of the AAV capsid protein encoded by the nucleic acid of the present invention. The AAV particles of the present invention are AAV particles containing a mutant of the AAV capsid protein, the mutant comprising a peptide containing an amino acid sequence selected from SEQ ID NO: 1 to SEQ ID NO: 4, the mutant being encoded by the nucleic acid described in (1) above. The AAV particles of the present invention can be produced by the host cells described in (2) above. The AAV particles of the present invention are tropistic to the brain and can be used to introduce genes into the brain. The AAV particles of the present invention are also tropistic to the spinal cord. Since the brain and spinal cord are collectively referred to as the central nervous system, it can also be said that the AAV particles of the present invention are tropistic to the central nervous system. The brain and spinal cord contain cells such as neurons and glial cells (microglia, oligodendrocytes, astrocytes). Genes introduced by the AAV particles of the present invention are strongly expressed in the tissues, organs, and cells mentioned above.

[0037] To produce AAV particles, cells containing some of the elements necessary for AAV particle production can be used as packaging cells. The first element is the recombinant AAV vector genome (also called the expression vector), which can be replicated in the host cell and packaged into AAV particles. The recombinant AAV vector genome contains a target heteropolynucleotide and AAV inverted terminal repeat (ITR) sequences located on each side (i.e., the 5' and 3' sides) of the target heteropolynucleotide. The target heteropolynucleotide may have an expression control sequence. The nucleotide sequence of the ITR sequence is known. For example, see Human Gene Therapy, Vol. 5, pp. 793-801, 1994 for the AAV2-ITR sequence. As the AAV ITR sequence, an ITR sequence derived from any of the various AAV serotypes (including AAV1, AAV2, AAV3, AAV4, AAV5, AAV7, etc.) can be used. The ITR sequence used in this invention may be derived from wild-type AAV or may be altered by nucleotide insertion, deletion, or substitution. The ITR sequence enables the replication of the recombinant AAV vector genome in the presence of the Rep protein and allows the recombinant AAV vector genome to be incorporated into the capsid particles during AAV particle formation.

[0038] The target heteropolynucleotides that can be accommodated within the AAV particles of the present invention are typically smaller than about 5 kilobits (kb). For example, the target heteropolynucleotide may be a gene encoding a target protein lacking or missing from the receptor, a gene encoding a protein having desired biological or therapeutic activity (e.g., antimicrobial, antiviral, or antitumor activity), a desired nucleotide sequence encoding RNA that inhibits or reduces the production of harmful or unwanted proteins, or a nucleotide sequence encoding an antigen protein. The target heteropolynucleotide may be appropriately selected according to the purpose.

[0039] In one embodiment of the invention, the recombinant AAV vector genome lacks the cap gene region and / or the rep gene region. In this embodiment, the AAV particles packaging the recombinant AAV vector genome do not replicate independently in infected cells to re-form AAV particles.

[0040] The second essential element for generating AAV particles is a construct that provides the protein encoded in wild-type AAV. This construct encodes an AAV-derived gene that provides the AAV gene product required for AAV particle formation. In other words, the construct contains one or two major AAV ORFs, namely the coding regions of the rep gene region and the cap gene region. To generate the AAV particles of the present invention, at least a nucleic acid encoding an AAV capsid protein mutant is used as the cap gene, which comprises a peptide containing an amino acid sequence selected from SEQ ID NO: 1 to SEQ ID NO: 4. The host cells of the present invention described above, capable of expressing this mutant, can be used to generate AAV particles. The AAV particles have a shell composed of a plurality of capsid proteins. All capsid proteins may be mutants, or some capsid proteins may be mutants while the remainder may be wild-type capsid proteins. The AAV particles of the present invention may contain one or more capsid protein mutants.

[0041] The AAV rep gene is contained within the coding region of the rep gene and includes genes encoding the replication proteins Rep78, Rep68, Rep52, and Rep40. These Rep expression products exhibit multiple functions, including recognizing, binding to, and nicking AAV genomic DNA replication origins, DNA helicase activity, and regulating transcription from AAV-derived promoters.

[0042] The third essential element for AAV particle production is the helper virus function (also known as the auxiliary function) for AAV replication. Adenoviruses are typically used to introduce the helper function. However, other viruses, such as herpes simplex virus type 1 or 2 and vaccinia virus, can also be used. When a virus is used, the host cell is infected with the virus acting as the helper virus. For example, since only the expression of early adenovirus genes is required for AAV particle packaging, an adenovirus that does not exhibit late gene expression can be used. Adenovirus mutants lacking late gene expression (e.g., ts100K or ts149 adenovirus variants) can also be used. Nucleic acid constructs providing the helper virus function can also be prepared by using nucleic acids necessary for the helper virus function isolated from the helper virus, and then introduced into the host cell. Constructs providing the helper virus function contain nucleotide sequences that provide one or more helper virus functions and are provided to the host cell in the form of plasmids, bacteriophages, transposons, granules, or other viruses.

[0043] To generate AAV particles, the following steps are performed: (a) introducing the first element (the recombinant AAV vector genome) into the host cell; (b) introducing the second element (a construct providing AAV helper functions) into the host cell; and (c) introducing the third element (helper viral functions) into the host cell. These steps can be performed simultaneously or sequentially. The order of steps (a) through (c) can be arbitrary. After the first through third elements are introduced into the host cell, the rep gene expression product is excised and the recombinant vector genome is replicated. The expressed capsid protein forms a capsid, and the recombinant vector genome is packaged within the capsid to generate AAV particles. When the host cell expresses an AAV capsid protein mutant, the resulting AAV particle's shell contains that AAV capsid protein mutant.

[0044] AAV particles can be isolated and purified from culture supernatants or host cell lysates using various purification methods, such as CsCl density gradient centrifugation. For example, when using a virus in step (c) above, a step can be added to separate AAV particles from helper viruses based on their size. AAV particles can also be separated from helper viruses based on differences in affinity for heparin. Furthermore, any remaining helper viruses can be inactivated using known methods. For example, adenovirus can be inactivated by heating at approximately 60°C for, for example, 20 minutes or longer. Because AAV particles are very heat-stable, the above treatment is effective for selectively removing adenovirus used as helper viruses.

[0045] (4) Method for generating gene transduction cells of the present invention For gene therapy or other purposes, the AAV particles of the present invention obtained by means of (3) above are used to deliver the target heterologous polynucleotide to cells. AAV particles are typically introduced into cells in vivo or in vitro. For in vitro introduction, the AAV particles are brought into contact with cells obtained from a living organism. The cells can then be administered to a living organism. To administer the cells to a living organism, the cells can be formulated into a pharmaceutical composition, and various techniques can be used, such as intramuscular, intravenous, subcutaneous, and intraperitoneal administration. For in vivo transduction, the AAV particles are formulated into a pharmaceutical composition and are typically administered parenterally (e.g., via intramuscular, subcutaneous, intratumoral, transdermal, or intraspinal routes). Pharmaceutical compositions containing AAV particles may contain a pharmaceutically acceptable carrier and, as needed, other drugs, agents, stabilizers, carriers, adjuvants, diluents, etc.

[0046] Pharmaceutical compositions comprising the AAV particles of the present invention can be used to deliver desired polynucleotides (e.g., polynucleotides effective in preventing or treating diseases) to cells, tissues, or organs to which the AAV particles are attracted. In other words, the pharmaceutical compositions can be used as preventative or therapeutic agents for diseases. Furthermore, one aspect of the invention also provides a method for preventing or treating diseases, comprising using the pharmaceutical composition.

[0047] Example In the following description, the invention will be explained with reference to the embodiments, but the invention is not particularly limited to these embodiments.

[0048] Example 1: Preparation of recombinant plasmids (1) pRC2-insertion Multiple plasmids containing different amino acid sequences inserted between amino acid numbers 588 and 589 of the AAV2 Cap protein were prepared by modifying plasmid pRC2 (manufactured by TakaraBio Inc.) which contains sequences encoding the AAV2 Rep and AAV2 Cap proteins. Figure 1 The nucleotide sequences before and after positions 1756 to 1773 in the modified AAV2 cap gene, and the amino acid sequences before and after positions 586 to 591 in the modified AAV2 Cap protein, are shown. The resulting plasmids are collectively referred to as pRC2-insertes. The plasmid names and inserted amino acid sequences (excluding spacer regions) contained in the pRC2-insertes are shown in the first and second columns of Tables 1 to 3, respectively.

[0049] (2) pAAV-CAG-AcGFP-BC Oligomeric DNA (SEQ ID NO: 5: taaggatccgcacnnnnnnnnnnnnnnnctggggatccacgggtggcat, where n represents a, t, g, or c) containing a random 15-nucleotide sequence (hereinafter referred to as the barcode sequence) was synthesized. The oligomeric DNA, primers (SEQ ID NO: 6), and the Klenow fragment (3'→5' exo-) (manufactured by NEB) were mixed and reacted at 37°C for 1 hour and 30 minutes. This reaction produced double-stranded DNA from the oligomeric DNA used as a template. The double-stranded DNA was purified using NucleoSpin (registered trademark) Gel and PCR Clean-up (manufactured by Machley-Nagel) and then digested with the restriction enzyme BamHI (manufactured by Takara Bio Inc.). A DNA ligation kit was used.<Mighty Mix> (Manufactured by Takara Bio Inc.), double-stranded DNA is inserted downstream of the stop codon of the AcGFP gene in pAAV-CAG-AcGFP, which has been pre-digested with BamHI. The resulting plasmids are collectively referred to as pAAV-CAG-AcGFP-BC. After cloning the plasmids contained in pAAV-CAG-AcGFP-BC, the barcode sequences contained in each plasmid are identified by Sanger sequencing. The names of the plasmids contained in pAAV-CAG-AcGFP-BC are shown in the third column of Tables 1 to 3.

[0050] Example 2: Generation of AAV vector (1) Seeding of 293T cells 293T cells were suspended in DMEM (manufactured by Sigma) containing 10% FBS and 50 μg / ml streptomycin sulfate solution, and sputtered at 3 × 10⁻⁶ ppm. 6 293T cells / flask were seeded into approximately 90 T225 cell culture flasks (manufactured by Corning). The cells were then cultured in a CO2 incubator at 37°C for 72 hours.

[0051] (2) Plasmids were introduced into 293T cells. In each of approximately 90 culture vessels, 293T cells were transfected using PEIpro (registered trademark) (manufactured by Polyplustransfection) with a pRC2-insert plasmid, a pAAV-CAG-AcGFP-BC plasmid, and a pHelper vector (manufactured by Takara Bio Inc.). The pRC2-insert and pAAV-CAG-AcGFP-BC combinations used for transfection are shown in Tables 1 through 3. The 293T cells were cultured in a CO2 incubator at 37°C for 72 hours. 。

[0052] [Table 1] [Table 2] [Table 3] (3) Lysis of 293T cells and recovery of supernatant In each of approximately 90 culture vessels, 293T cells were suspended in a solution containing a surfactant and then lysed. Each suspension was collected in a 50 mL centrifuge tube and centrifuged, and the supernatant was collected. The collected supernatant was frozen and stored at −80°C until use.

[0053] (4) Measurement of viral genome size The amount of viral genome contained in the supernatant recovered in Example 2-(3) was measured using the AAVpro (registered trademark) titration kit Ver. 2 (manufactured by Takara Bio Inc.).

[0054] Example 3: Purification of AAV vector (1) Affinity chromatography The approximately 90 portions of supernatant that were frozen and stored in Examples 2-(3) were thawed and each was divided into 5 × 10⁻⁶ portions. 11 The mixture was mixed. The mixture was filtered using a 0.45 μm bottle-top filter (manufactured by Corning) and then loaded onto an AVB Sepharose HighPerformance (manufactured by Cytiva). The eluent was separated into 1 mL fractions, and the amount of viral genome contained in each fraction was measured using the same method as in Examples 2-(4). Protein quantification of each fraction was performed by A280 measurement. As a result, fractions rich in AAV vectors were identified.

[0055] (2) Cesium chloride density gradient centrifugation method After mixing the fractions rich in AAV carrier, cesium chloride solution was added to adjust the refractive index to 1.371. The mixture was aliquoted into 13PA ultracentrifuge tubes (manufactured by Eppendorf Himac Technologies) and centrifuged at 34,000 rpm and 21°C for 42 hours. After centrifugation, the supernatant was fractionated into 0.5 mL fractions from the top of the tube. The fractions rich in AAV carrier were identified using the same method as in Example 3-(1).

[0056] (3) Dialysis The fractions rich in AAV vectors obtained in Example 3-(2) were mixed and then subjected to a Slide-A-lyzer dialysis cartridge (manufactured by Thermo Scientific). The dialysis cartridge was immersed in 1 L of phosphate-buffered saline (PBS) and dialyzed three times at 4°C for 2 hours or longer. Next, the dialysis cartridge was immersed in 1 L of PBS containing 0.001% Pluronic F-68 (manufactured by Thermo Scientific), 5% sorbitol, and 200 mM NaCl and dialyzed at 4°C for 3 hours. The solution was recovered from the dialysis cartridge and concentrated by ultrafiltration using an Amicon (registered trademark) Ultra-4 Centrifugal Filter Unit (manufactured by Merck). The concentrated solution was filtered using a 0.22 μm filter (manufactured by Millipore) and stored at −80°C until used as an “AAV mutant barcode library”. The amount of viral genome was measured using the same method as in Example 2-(4).

[0057] Example 4: Biodistribution analysis of AAV mutants in mice using barcode sequences (1) Intravenous administration of AAV mutant barcode library to mice The AAV mutant barcode library obtained in Example 3-(3) was used at 7.5 × 10⁻⁶. 11 vg was administered to BALB / c mice via the tail vein. Four weeks after administration, brain, spinal cord, lungs, muscles, heart, and liver were harvested. Genomic DNA was extracted from these tissues using NucleoSpin (trademarked) tissue (manufactured by Machley-Nagel). RNA was also extracted from these tissues using RNeasy Plus Universal Mini (manufactured by Qiagen).

[0058] (2) cDNA synthesis Using the RNA extracted in Example 4-(1) as a template, cDNA was synthesized using the PrimeScript RT kit (manufactured by TakaraBio Inc.).

[0059] (3) Amplification and purification of barcode sequences Using the cDNA synthesized in Example 4-(2) as a template, amplicons encoding the barcode region were amplified by PCR. For the PCR, forward primers (SEQ ID NO: 7) and reverse primers (SEQ ID NO: 8) were used. PCR was performed under the following conditions: preheating at 94°C for 1 minute, followed by 30 cycles of 98°C for 10 seconds, 55°C for 15 seconds, and 72°C for 60 seconds. After electrophoresis, the amplicons were purified using NucleoSpin (registered trademark) Gel and PCR Clean-up (manufactured by MachlinerGel).

[0060] (4) Next-generation sequencing (NGS) analysis of barcode sequences The amplicon purified in Example 4-(3) was subjected to NGS analysis using Miseq (manufactured by Illumina) to determine the frequency of barcode sequences in each tissue. Furthermore, the gene transfer efficiency in each tissue was calculated as GFP / GAPDH using qPCR, using genomic DNA extracted in Example 4-(1) as a template. In qPCR, the GFP sequence was amplified using the TransgeneDetection Primer Set for Real Time (Mouse) GFP-1 primer (manufactured by Takara Bio Inc.), and the GAPDH sequence was amplified using the Mouse Housekeeping Gene Primer Set (manufactured by Takara Bio Inc.). The frequency of barcode sequences obtained from NGS analysis was normalized using the gene transfer efficiency, thereby calculating the frequency of AAV vectors in each tissue. Figure 2 ).

[0061] like Figure 2 As shown, AAV vectors containing ETGAWV (SEQ ID NO: 3), ETGYV (SEQ ID NO: 1), ETGGFV (SEQ ID NO: 2), and ETGWA (SEQ ID NO: 4) exhibit high infectivity to the brain and spinal cord. In particular, AAV vectors containing ETGAWV (SEQ ID NO: 3), ETGYV (SEQ ID NO: 1), and ETGGFV (SEQ ID NO: 2) show high tropism to the brain and spinal cord.

[0062] Example 5: Evaluation of AAV vectors with specific amino acid sequences (1) Preparation of pRC2Km-intercalated material particles Multiple plasmids encoding the Cap protein were prepared by modifying the kanamycin resistance plasmid pRC2Km (manufactured by Takara Bio Inc.) containing sequences encoding the AAV2 Rep and AAV2 Cap proteins. The Cap proteins contained various amino acid sequences inserted between amino acid numbers 588 and 589 of the AAV2 Cap protein. The resulting pRC2Km-insertion plasmids are as follows: pRC2Km-ETGHGWV, pRC2Km-ETAHGWV, pRC2Km-ETGAGWV, pRC2Km-ETGHGAV, pRC2Km-ETGHGYV, pRC2Km-ETGHGFV, and pRC2Km-ETGHGHV.

[0063] (2) Preparation and purification of AAV vector Using PEIpro (manufactured by Polyplus), 293T cells were transfected with pAAV-CAG-fLuc (manufactured by Takara Bio Inc.), pHelper vector (manufactured by TakaraBio Inc.), and pRC2Km-insertion material obtained in Example 5-(1). As a control, 293T cells were transfected with pRC2-mi342 vector (manufactured by Takara Bio Inc.) or pRC9 vector (manufactured by TakaraBio Inc.) instead of pRC2Km-insertion material. The transfected 293T cells were cultured in a CO2 incubator at 37°C for 72 hours. The various AAV vectors produced were collected and purified using the same methods as in Example 2-(3). For affinity purification of the AAV9 vector, POROS CaptureSelect™ AAV9 Affinity Resin (manufactured by Thermo) was used.

[0064] (3) AAV vector infection via tail vein injection in mice The AAV carrier obtained in Example 5-(2) was used at a concentration of 1 × 10⁻⁶. 11 The dose was administered via tail vein to Balb / c mice (purchased from SLC). As a control, a solvent was administered. Thirty days post-administration, brain, lung, and liver tissues were harvested. Proteins were extracted from each tissue, and luciferase activity was then measured using the ONE-Glo Luciferase Assay System (manufactured by Promega). Furthermore, relative optical units (RLU) per mg of weight in each tissue were calculated. Figure 3 ).

[0065] like Figure 3As shown, AAV vectors containing ETGHGYV (SEQ ID NO: 1) and ETGHGFV (SEQ ID NO: 2) exhibited high infectivity in mouse brain tissue. In particular, the AAV vector containing ETGHGYV (SEQ ID NO: 1) showed high tropism in mouse brain tissue.

[0066] Even when the amino acid residue at position 587 of the AAV2 Cap protein encoded by the aforementioned pRC2Km-insertion particles was replaced with asparagine, it still yielded results similar to... Figure 3 The results shown are comparable to or better than those shown. Furthermore, when the amino acid residue at position 587 in the Cap protein containing ETGHGWV was replaced with asparagine (amino acid sequence shown in SEQ ID NO: 11), results were also obtained that are comparable to or better than those shown. Figure 3 The results shown are equivalent to or better than those shown.

[0067] Example 6: Evaluation of AAV vectors with specific amino acid sequences in the retina (1) Preparation and purification of AAV vector Using PEIpro (manufactured by Polyplus), 293T cells were transfected with pAAV-CAG-AcGFP (manufactured by Takara Bio Inc.), pHelper vector (manufactured by TakaraBio Inc.), and pRC2Km-insertion material obtained in Example 5-(1). The transfected 293T cells were cultured in a CO2 incubator at 37°C for 72 hours. The various AAV vectors produced were collected and purified using the same method as in Example 2-(3).

[0068] (2) Infection of mice with AAV vector via intravitreal administration The AAV vector containing ETGHGWV, ETGHGYV (SEQ ID NO: 1), and ETGHGFV (SEQ ID NO: 2) was loaded at 4 × 10⁻⁶. 9 A dose of vg / eye was administered intravitreally to Balb / c mice (purchased from SLC). As a control, a solvent was administered. The retina was removed 14 days post-administration. GFP fluorescence in the retina was observed under a fluorescence microscope. Figure 4 ).

[0069] like Figure 4As shown, AAV vectors containing ETGHGYV (SEQ ID NO: 1) and ETGHGFV (SEQ ID NO: 2) exhibited higher infectivity for mouse retina than vectors containing ETGHGWV. Furthermore, AAV vectors containing ETGHGYV (SEQ ID NO: 1) and ETGHGFV (SEQ ID NO: 2) showed higher infectivity for mouse retina than wild-type AAV2 vectors (data not shown).

[0070] The plasmids used to prepare the three types of AAV vectors mentioned above were each modified to replace the amino acid residue at position 587 of the encoded Cap protein with asparagine. When similar studies were conducted, results comparable to or better than those described above were obtained.

[0071] Industrial applicability The present invention provides an AAV capsid protein mutant that exhibits high tropism for the brain and low tropism for the lungs, as well as the amino acid sequence of the mutant, and provides a method for efficiently transferring the gene into the brain.

[0072] Sequence List Free Text SEQ ID NO:1: Peptide sequence of AAV capsid protein mutant ETGHGYV SEQ ID NO:2: Peptide sequence of AAV capsid protein mutant ETGHGFV SEQ ID NO:3: Peptide sequence of AAV capsid protein mutant ETGHAWV SEQ ID NO:4: Peptide sequence of AAV capsid protein mutant ETGHGWA SEQ ID NO:5: Oligomeric DNA containing 15 random nucleotides SEQ ID NO:6: Primers used for synthesizing double-stranded DNA SEQ ID NO:7: Forward primer for amplifying the coding region of random peptides SEQ ID NO:8: Reverse primer for amplifying the coding region of random peptides SEQ ID NO:9: Amino acid sequence of wild-type AAV2 VP1 capsid protein SEQ ID NO:10: Amino acid sequence of AAV2(587Q) VP1 capsid protein SEQ ID NO:11: Amino acid sequence of AAV2-ETGHGWV VP1 capsid protein SEQ ID NO:12: Amino acid sequence of AAV2-ETGHGYV VP1 capsid protein.

Claims

1. A nucleic acid encoding a mutant of an adeno-associated virus (AAV) capsid protein, said mutant comprising a peptide containing an amino acid sequence selected from SEQ ID NO: 1-4.

2. The nucleic acid according to claim 1, wherein the AAV capsid protein is derived from AAV2.

3. The nucleic acid according to claim 2, wherein the peptide is located between amino acid number 588 and amino acid number 589 in VP1 of AAV2.

4. A recombinant DNA comprising the nucleic acid according to any one of claims 1 to 3.

5. A cell comprising the nucleic acid according to any one of claims 1 to 3.

6. An AAV particle comprising an AAV capsid protein mutant, said mutant comprising a peptide comprising an amino acid sequence selected from SEQ ID NO:1-4.

7. The AAV particle according to claim 6, wherein the AAV capsid protein is derived from AAV2.

8. The AAV particle according to claim 7, wherein the peptide is located between amino acid number 588 and amino acid number 589 in VP1 of AAV2.

9. A method for generating gene-transduced cells, the method comprising the following steps: AAV particles containing a mutant AAV capsid protein are brought into contact with cells, the mutant comprising a peptide containing an amino acid sequence selected from SEQ ID NO: 1-4.

10. The method of claim 9, wherein the AAV capsid protein is derived from AAV2.

11. The method of claim 10, wherein the peptide is located between amino acid numbers 588 and 589 in VP1 of AAV2.

12. A method for producing AAV particles, the method comprising using a cell according to claim 5 as a host.

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