Brain-tropic AAV mutant

Abstract

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

Description

Brain-tropic AAV mutant

[0001] The present invention relates to an adeno-associated virus (AAV) capsid protein mutant that has high brain tropism and low lung tropism, a nucleic acid encoding the capsid protein mutant, an AAV particle containing the capsid protein mutant, and a method for producing a gene-transduced cell using the particle.

[0002] AAV is a virus whose genome is a 4.7-kb single-stranded DNA containing two open reading frames, the rep gene and the cap gene. The rep gene encodes four proteins (Rep78, Rep68, Rep52, and Rep40) required for genome replication, while the cap gene encodes three capsid proteins (VP1, VP2, and VP3) that assemble to form the viral capsid, as well as an assembly-activating protein (AAP). AAV replication in nature depends on the presence of a helper virus such as adenovirus or herpesvirus. In the absence of a helper virus, AAV maintains its genome episomally or integrates into the host chromosome, resulting in a latent state. Currently, over 100 AAV serotypes and clades have been identified (Non-Patent Document 1), and AAV2 in particular is being developed as a gene delivery vector.

[0003] The first gene delivery vector system was developed based on AAV2 in 1989. AAV-based vectors have been shown to have many advantages. Because wild-type AAV is non-pathogenic and has no etiological association with any known disease, AAV-based vectors are said to be extremely safe. In addition, AAV has the ability to transfer genes with high efficiency.

[0004] Administration of AAV particles enables long-term, stable gene transfer into various target organs and target cells. To date, highly efficient gene transfer has been demonstrated in skeletal muscle, liver (hepatocytes), heart (cardiomyocytes), neurons, pancreatic gland cells, and pancreatic islet cells. Furthermore, AAV has been used in human clinical trials. Meanwhile, attempts have been made to modify AAV capsid proteins to alter the cell tropism of AAV or to avoid the removal of AAV particles by neutralizing antibodies. For example, AAV capsids with tropism for specific organs or cells, such as glial cells, airway epithelial cells, coronary artery endothelial cells, or lungs, as well as AAV capsids with tropism for tumor cells, such as glioblastoma, melanoma, lung cancer, or breast cancer, have been produced (Non-Patent Document 2).

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

[0006] For example, WO2015 / 038958 (Patent Document 1) discloses an AAV vector containing a predetermined amino acid sequence. Among these, one in which the amino acid sequence TLAVPFK was inserted at position 588 of the AAV9 capsid protein was named AAV-PHP.B. It has been shown that AAV-PHP.B can transfer genes to the entire CNS after intravenous administration with at least 40-fold higher efficiency than AAV9 (Non-Patent Document 3).

[0007] Furthermore, AAV-PHP.eB, which contains the amino acid sequence DGTLAVPFK inserted at position 588 of the AAV9 capsid protein, has been shown to reduce the amount of virus required to transduce most CNS neurons (Non-Patent Document 5). Systemic and intravitreal delivery of AAV-PHP.eB has also been shown to result in high transduction efficiency of retinal ganglion cells and horizontal cells (Non-Patent Document 6).

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

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

[0010] However, since AAV vectors are generally administered systemically, AAV vectors with higher brain-tropism are needed for gene therapy targeting brain diseases.

[0011] International Publication No. WO2015 / 038958 International Publication No. WO2015 / 158749 International Publication No. W2020 / 218419

[0012] Gao et al., Journal of Virology, Vol. 78, pp. 6381-6388, 2004; Adachi K et al., Gene Ther. Regul., Vol. 5, pp. 31-55, 2010; Nat Biotechnol. 2016 Feb; 34(2): 204-9; EMBO Mol Med. 2016 Jun 1; 8(6): 609-25; Nat Neurosci. 2017 Aug; 20(8): 1172-1179; Mol Ther Methods Clin Dev. 2022 Mar 28; 25: 236-249

[0013] An object of the present invention is to provide an AAV capsid protein mutant that has high brain tropism and low lung tropism, and to provide a method for efficiently transferring genes into the brain.

[0014] As a result of intensive efforts to solve the above problems, the present inventors have constructed an AAV capsid protein mutant containing a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 4, thereby completing the present invention.

[0015] That is, the present invention can be summarized as follows: [1] a nucleic acid encoding an adeno-associated virus (AAV) capsid protein mutant containing a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 4; [2] the nucleic acid according to [1], wherein the AAV capsid protein is derived from AAV2; [3] the nucleic acid according to [2], wherein the peptide is contained at a position between amino acid numbers 588 and 589 of VP1 of AAV2; [4] a recombinant DNA containing the nucleic acid according to any one of [1] to [3]; [5] a cell containing the nucleic acid according to any one of [1] to [3]; [6] an AAV particle containing an AAV capsid protein mutant containing a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 4; [7] the AAV particle according to [6], wherein the AAV capsid protein is derived from AAV2; [8] The AAV particle according to [7], which contains the peptide at a position between amino acid numbers 588 and 589 of VP1 of AAV2; [9] A method for producing a transfected cell, which comprises a step of contacting a cell with AAV particles containing an AAV capsid protein mutant containing a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 4;

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

[11] The method according to

[10] , which contains the peptide at a position between amino acid numbers 588 and 589 of VP1 of AAV2;

[12] A method for producing an AAV particle, which uses the cell according to [5] as a host.

[0016] The present invention provides a gene transfer system useful for gene transfer to the brain. The AAV particles of the present invention have high cell tropism for the brain but low cell tropism for the lung, and therefore can achieve strong expression of the transferred gene in the brain.

[0017] Fig. 1 shows a method for preparing a nucleic acid construct in which a random peptide is contained in a capsid protein. Fig. 2 shows the frequency of AAV vectors in each tissue in Example 4. Fig. 3 shows the results of measuring luciferase activity in Example 5. Fig. 4 shows the results of measuring AcGFP under a fluorescent microscope in Example 6.

[0018] As used herein, "adeno-associated virus" refers to a small virus belonging to the genus Dependovirus in the family Parvoviridae that infects primates, including humans, and other mammals. Hereinafter, Adeno Associated Virus will be abbreviated as AAV. AAV has a non-enveloped, regular icosahedral outer shell (capsid) containing a single strand of DNA. As used herein, AAV includes wild-type viruses and their derivatives, and includes all serotypes and clades unless otherwise specified.

[0019] As used herein, the term "vector" refers to a molecule or molecular assembly that contains a polynucleotide or that can be associated with a polynucleotide and used to mediate delivery of the polynucleotide into a cell. Examples of vectors include vector DNA such as a plasmid vector or a phage vector, a viral vector particle, a liposome, and other gene delivery vehicles, all of which are included in the term "vector" unless otherwise specified.

[0020] As used herein, "capsid protein" refers to a protein encoded by the cap gene present in the AAV genome and constituting the AAV capsid. The wild-type AAV genome encodes three types of capsid proteins, VP1, VP2, and VP3. As used herein, VP1, VP2, and VP3 are all included in the capsid protein. Capsid proteins are sometimes referred to as "Cap proteins."

[0021] (1) Nucleic Acid Encoding an AAV Capsid Protein Mutant The nucleic acid of the present invention is a nucleic acid encoding an AAV capsid protein mutant containing a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 4. Preferably, the nucleic acid of the present invention is a nucleic acid encoding an AAV capsid protein mutant containing a peptide comprising the amino acid sequence of SEQ ID NO: 1.

[0022] The AAV capsid protein mutants encoded by the nucleic acids of the present invention can be prepared by inserting the above-mentioned peptide into any wild-type AAV capsid protein, such as AAV type 1 (AAV1), AAV type 2 (AAV2), AAV type 3 (AAV3A, AAV3B, etc.), AAV type 4 (AAV4), AAV type 5 (AAV5), AAV type 6 (AAV6), AAV type 7 (AAV7), AAV type 8 (AAV8), AAV type 9 (AAV9), AAV type 10 (AAV10), AAV type 11 (AAV11), avian AAV, bovine AAV, canine AAV, equine AAV, or ovine AAV, or by substituting the above-mentioned peptide for a portion of the amino acid sequence of the AAV capsid protein (i.e., by incorporating the above-mentioned peptide into the AAV capsid protein). In the present invention, the capsid protein of AAV2 is particularly 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).

[0023] The AAV capsid protein mutant encoded by the nucleic acid of the present invention may be a protein in which one to several or more amino acids have been substituted, deleted, inserted, and / or added to the wild-type AAV capsid protein in addition to retaining the above-mentioned peptide. The "protein in which one to several or more amino acids have been substituted, deleted, inserted, and / or added in addition to retaining the above-mentioned peptide" is a protein that maintains the properties of the original protein, such as the cell tropism, capsid formation ability, and capsid protein functions (e.g., protection of the viral genome, uncoating after host cell entry) of the AAV capsid protein mutant conferred by the above-mentioned peptide. In the present invention, for example, an AAV2 capsid protein in which asparagine at position 587 is substituted with glutamine can be used, without particular limitation. The amino acid sequence of AAV2 VP1 capsid protein in which glutamine is at position 587 is shown in SEQ ID NO: 10.

[0024] Furthermore, in the present invention, a spacer may be added to the N-terminus and / or C-terminus of the peptide contained in the AAV capsid protein. A sequence consisting of 1 to 5 amino acid residues is preferred as the spacer. There are no particular limitations on the amino acids constituting the spacer, and for example, amino acids selected from the group consisting of glycine, alanine, and serine can be used. A single amino acid residue of glycine is preferred as the spacer added to the N-terminus, and a single amino acid residue of alanine is preferred as the spacer added to the C-terminus.

[0025] The AAV capsid protein into which the peptide is to be incorporated can be any of AAV VP1, VP2, and VP3. The peptide may be incorporated only in VP1, only in VP2, or only in VP3, or may be incorporated in all capsid proteins VP1 to VP3. Furthermore, the peptide may be incorporated in two types of capsid proteins, such as a combination of VP1 and VP2, VP2 and VP3, or VP1 and VP3. VP1 to VP3 are encoded in the cap gene region of the AAV genome. In one embodiment of the present invention, the peptide can be incorporated in a region common to VP1 to VP3, thereby introducing a mutation into all of VP1 to VP3. In another embodiment, a gene encoding VP1, VP2, or VP3 can be prepared separately from the AAV cap gene region, and a mutation can be introduced into this gene. In this case, treatment may be carried out so that the wild-type capsid protein corresponding to the capsid protein encoded by the gene into which the mutation has been introduced is not expressed from the cap gene region of AAV.

[0026] In the AAV capsid protein mutant encoded by the nucleic acid of the present invention, the position at which the peptide is to be contained is preferably between amino acid positions 588 and 589 in AAV2 VP1. Amino acid position 588 in AAV2 VP1 is arginine, and amino acid position 589 in AAV2 VP1 is glutamine. Amino acid position 588 in AAV2 VP1 corresponds to amino acid position 451 in AAV2 VP2 and amino acid position 386 in AAV2 VP3. When a capsid protein derived from an AAV of a serotype or clade other than AAV2 is used as the AAV capsid protein, the peptide is preferably contained in the AAV capsid protein at a position between the amino acids corresponding to amino acid positions 588 and 589 in AAV2 VP1. Those skilled in the art can easily identify the amino acid in the capsid protein of AAV of a serotype or clade other than AAV2 that corresponds to amino acid 588 of AAV2 VP1. For example, reference can be made to the alignment of VP1 amino acid sequences described in Gao et al., Proc. Natl. Acad. Sci. USA, Vol. 99, No. 18, pp. 11854-11859, 2002. For example, amino acid 588 of AAV2 VP1 corresponds to amino acid 589 in AAV1, amino acid 590 in AAV7, and amino acid 591 in AAV8.

[0027] In the present invention, an AAV capsid protein mutant (e.g., SEQ ID NO: 12) containing a spacer ETGHGYV (SEQ ID NO: 1) between amino acid positions 588 and 589 of the AAV2 VP1 capsid protein can be preferably used.

[0028] The nucleic acid of the present invention may be operably linked to an appropriate control sequence. Control sequences include promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, replication origins, internal ribosome entry sites (IRES), enhancers, etc. Promoter sequences include inducible promoter sequences and constitutive promoter sequences. Control sequences may be native or foreign to the AAV from which the capsid protein is derived, and may be natural or artificially designed sequences. Such recombinant DNAs capable of expressing AAV capsid protein mutants are also encompassed by the present invention.

[0029] The recombinant DNA of the present invention is useful for delivering the nucleic acid of the present invention to cells in vitro, ex vivo, and in vivo, and for conferring the ability of the cells to express AAV capsid protein mutants. The cells into which the nucleic acid of the present invention has been delivered are then useful for producing AAV particles. The recombinant DNA can be particularly used to deliver or introduce the nucleic acid of the present invention into animal cells, more preferably mammalian cells.

[0030] In the present invention, the recombinant DNA of the present invention can be produced by carrying the nucleic acid of the present invention in DNA used as a vector. For example, plasmid DNA, phage DNA, transposon, cosmid DNA, episomal DNA, and virus genome can be used.

[0031] (2) Cells Comprising the Nucleic Acid of the Present Invention The present invention also provides cells comprising the nucleic acid of the present invention. Specifically, it provides isolated host cells comprising the recombinant DNA of (1) above. The isolated cells are, for example, cell lines maintained in vitro. The host cells of the present invention are useful for producing AAV particles of the present invention, as described below. When the host cells of the present invention are used to produce AAV particles, they may be referred to as "packaging cells" or "producer cells." The host cells of the present invention may have the recombinant DNA of the present invention described in (1) above integrated into their genome, or may retain the recombinant DNA within the cells so as to transiently express the AAV capsid protein mutant.

[0032] 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 transfer, and nucleic acid delivery using high-velocity particle bombardment can be used. When using a viral vector, an infection method appropriate for the vector can be selected. Using such established techniques, the recombinant DNA of the present invention can be stably introduced into the chromosome of the host cell or transiently introduced into the cytoplasm of the host cell. For stable transformation, well-known selectable markers, such as the neomycin resistance gene (encoding neomycin phosphotransferase) and the hygromycin B resistance gene (encoding aminoglycoside phosphotransferase (APH)), can be linked to the recombinant DNA of the present invention.

[0033] A variety of host cells can be used, including mammalian cells, including mouse cells and primate cells (e.g., human cells), insect cells, etc. Suitable mammalian cells include, but are not limited to, primary cells and cell lines, such as 293 cells, COS cells, HeLa cells, Vero cells, 3T3 mouse fibroblasts, C3H10T1 / 2 fibroblasts, CHO cells, and cells derived therefrom.

[0034] (3) AAV particles containing AAV capsid protein mutants encoded by the nucleic acids of the present invention. The AAV particles of the present invention are AAV particles containing AAV capsid protein mutants containing a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 4, which is 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 tropic to the brain and are useful for gene transfer into the brain. The AAV particles of the present invention are also tropic to the spinal cord. Since the brain and spinal cord are collectively referred to as the central nervous system, the AAV particles of the present invention can also be said to be tropic to the central nervous system. The brain and spinal cord contain cells such as neurons and glial cells (microglia, oligodendrocytes, and astrocytes). The genes transferred by the AAV particles of the present invention are strongly expressed in the tissues, organs, and cells.

[0035] AAV particles can be produced using packaging cells that contain several components necessary for AAV particle production. The first component is a recombinant AAV vector genome (also called an expression vector) that can be replicated and packaged into AAV particles in a host cell. The recombinant AAV vector genome contains a desired heterologous polynucleotide flanked on both sides, i.e., on the 5' and 3' sides, by AAV inverted terminal repeat (ITR) sequences. The desired heterologous polynucleotide may have a regulatory sequence for its expression. The nucleotide sequences of ITR sequences are known; for example, the ITR sequence of AAV2 can be found in Kotin R. M et al., Human Gene Therapy, Vol. 5, pp. 793-801, 1994. Furthermore, ITR sequences derived from any of various AAV serotypes, such as AAV1, AAV2, AAV3, AAV4, AAV5, and AAV7, can be used as ITR sequences. The ITR sequences used in the present invention may be derived from wild-type AAV, or may be modified by nucleotide insertion, deletion, or substitution. The ITR sequences enable replication of the recombinant AAV vector genome in the presence of Rep proteins and enable integration into capsid particles during AAV particle formation.

[0036] The desired heterologous polynucleotide that can be loaded onto the AAV particles of the present invention is generally less than about 5 kilobases (kb) in size and can be appropriately designed depending on the purpose, such as a gene encoding a desired protein that is defective or lost in the recipient, a gene encoding a protein having a desired biological or therapeutic effect (e.g., antibacterial, antiviral, or antitumor effect), a desired nucleotide sequence encoding RNA that inhibits or reduces the production of harmful or undesirable proteins, or a nucleotide sequence encoding an antigenic protein.

[0037] In one embodiment of the present invention, the vector genome of the recombinant AAV lacks the cap gene region and / or the rep gene region, and AAV particles packaged with the vector genome of the recombinant AAV of this embodiment do not replicate independently into AAV particles again in infected cells.

[0038] The second component required for the production of AAV particles is a construct providing proteins encoded by wild-type AAV. This construct encodes AAV-derived genes that provide AAV gene products necessary for AAV particle formation. That is, it contains one or both of the coding regions of the rep and cap gene regions, which are the major AAV ORFs. To produce the AAV particles of the present invention, a nucleic acid encoding an AAV capsid protein mutant containing a peptide comprising at least an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 4 in the Sequence Listing is used as the cap gene. The host cells of the present invention described in (2) above, which are capable of expressing this mutant, can be used to produce AAV particles. AAV particles have an outer shell composed of multiple capsid proteins; all of the capsid proteins may be mutants, or some of them may be mutants and the rest may be wild-type capsid proteins. Furthermore, the capsid protein mutants contained in the AAV particles of the present invention may be one type of mutant or multiple types of mutants.

[0039] The AAV rep genes are contained in the rep gene coding region and include genes encoding the replication proteins Rep78, Rep68, Rep52, and Rep40. These Rep expression products have been shown to have many functions, including recognition, binding, and nicking of the AAV genomic DNA replication origin, DNA helicase activity, and modulation of transcription of AAV-derived promoters.

[0040] The third component required for AAV particle production is helper virus functions (also called accessory functions) for AAV replication. Adenoviruses are commonly used to introduce helper functions, but viruses such as herpes simplex virus type 1 or type 2, vaccinia virus, etc. can also be used. When using viruses, host cells are infected with the virus as a helper virus. For example, since only the expression of adenovirus early genes is required for packaging AAV particles, adenoviruses that do not exhibit late gene expression can also be used. Adenovirus mutants that are deficient in late gene expression (e.g., ts100K or ts149 adenovirus mutants) can be used. Alternatively, nucleic acids required for helper virus function isolated from a helper virus can be used to create a nucleic acid construct providing helper virus function and then introduced into host cells. The construct providing helper virus function contains nucleotide sequences providing one or more helper virus functions and is provided to host cells in the form of a plasmid, phage, transposon, cosmid, or other virus.

[0041] To produce AAV particles, the following steps are performed: (a) introducing the first component, a recombinant AAV vector genome, into a host cell; (b) introducing the second component, a construct providing AAV helper functions, into a host cell; and (c) introducing the third component, a helper virus function, into a host cell. These steps may be performed simultaneously or sequentially. Steps (a) to (c) may be performed in any order. By introducing the first to third components into a host cell, the expression product of the rep gene excises and replicates the recombinant AAV vector genome. The expressed capsid protein forms a capsid, and the recombinant AAV vector genome is packaged into the capsid to produce AAV particles. When the host cell expresses an AAV capsid protein mutant, the outer shell of the produced AAV particles contains the AAV capsid protein mutant.

[0042] AAV particles can be isolated and purified from host cell culture supernatants or cell lysates using various purification methods, such as CsCl density gradient centrifugation. When a virus is used in step (c), a step of separating AAV particles and helper virus based on their size may be added. AAV particles can also be separated from helper virus based on their difference in affinity for heparin. Furthermore, remaining helper virus can be inactivated using known methods. For example, adenovirus can be inactivated by heating at a temperature of about 60°C for, for example, 20 minutes or more. Because AAV particles are extremely heat-stable, this treatment is effective for selectively removing adenovirus used as a helper virus.

[0043] (4) Method for Producing Genetically Transduced Cells of the Present Invention The AAV particles of the present invention obtained by (3) above are used to deliver a desired heterologous polynucleotide to cells for gene therapy or other purposes. Generally, AAV particles are introduced into cells either in vivo or in vitro. In vitro introduction is achieved by contacting AAV particles with cells obtained from a living organism. These cells can also be administered to a living organism. When administering cells to a living organism, the cells can be formulated as a pharmaceutical composition and administered using various techniques, such as intramuscular, intravenous, subcutaneous, and intraperitoneal administration. For in vivo transduction, AAV particles are formulated as a pharmaceutical composition and generally administered parenterally (e.g., by intramuscular, subcutaneous, intratumoral, transdermal, or intrathecal routes). Pharmaceutical compositions containing AAV particles may contain a pharmaceutically acceptable carrier, and, if necessary, other drugs, pharmaceuticals, stabilizers, buffers, carriers, adjuvants, diluents, etc.

[0044] The pharmaceutical composition comprising the AAV particles of the present invention can be used to deliver a desired polynucleotide, such as a polynucleotide effective for the prevention or treatment of a disease, to cells, tissues, or organs targeted by the AAV particles. That is, the pharmaceutical composition is useful as a preventive or therapeutic agent for a disease. Furthermore, a method for preventing or treating a disease using this pharmaceutical composition is also an aspect of the present invention.

[0045] The present invention will be described below with reference to examples, but the present invention is not particularly limited to these examples.

[0046] Example 1 Preparation of Recombinant Plasmids (1) pRC2-Insert Plasmid pRC2 (Takara Bio Inc.), which contains sequences encoding the AAV2 Rep protein and the AAV2 Cap protein, was modified to prepare multiple plasmids with various amino acid sequences inserted between amino acid positions 588 and 589 of the AAV2 Cap protein. The nucleotide sequence of the AAV2 cap gene before and after modification at positions 1756 to 1773 and the amino acid sequence of the AAV2 Cap protein before and after modification at positions 586 to 591 are shown in Figure 1. The multiple plasmids thus obtained were collectively named pRC2-Insert. The names of the plasmids contained in pRC2-Insert are shown in the first column of Tables 1 to 3, and the inserted amino acid sequences (excluding spacers) are shown in the second column of Tables 1 to 3, respectively.

[0047] (2) pAAV-CAG-AcGFP-BC An oligo DNA (SEQ ID NO: 5: taaggatccgcacnnnnnnnnnnnnnnnctggggatccacgggtggcat (n represents a, t, g, or c)) containing a random 15-nucleotide sequence (hereinafter referred to as the barcode sequence) was artificially synthesized. This oligo DNA, a primer (SEQ ID NO: 6), and Klenow Fragment (3'→5' exo-) (NEB) were mixed and reacted at 37°C for 1 hour and 30 minutes. This reaction produced double-stranded DNA using the oligo DNA as a template. The double-stranded DNA was purified using NucleoSpin® Gel and PCR Clean-up (Machrei-Nagel) and then digested with the restriction enzyme BamHI (Takara Bio). The double-stranded DNA was inserted downstream of the termination codon of the AcGFP gene in pAAV-CAG-AcGFP, which had been previously digested with BamHI, using the DNA Ligation Kit <Mighty Mix> (Takara Bio Inc.). The multiple plasmids thus obtained were collectively named pAAV-CAG-AcGFP-BC. After cloning the plasmids contained in pAAV-CAG-AcGFP-BC, the barcode sequence contained in each plasmid was 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.

[0048] Example 2 Production of AAV Vector (1) Seeding of 293T Cells 293T cells were suspended in DMEM (Sigma) containing 10% FBS and 50 μg / ml streptomycin sulfate solution, and seeded in approximately 90 T225 flask cell culture vessels (Corning) at 3 × 10 ６ 293T cells were seeded at 100 cells / flask. ２ The cells were cultured in an incubator for 72 hours.

[0049] (2) Plasmid introduction into 293T cells In each of approximately 90 culture vessels, one plasmid from pRC2-Insert, one plasmid from pAAV-CAG-AcGFP-BC, and pHelper Vector (manufactured by Takara Bio Inc.) were transfected into 293T cells using PEIpro (registered trademark) (manufactured by Polyplus transfection). The combinations of pRC2-Insert and pAAV-CAG-AcGFP-BC used during transfection are shown in Tables 1 to 3. The 293T cells were cultured in a CO atmosphere at 37°C. ２ The cells were cultured in an incubator for 72 hours.

[0050]

[0051]

[0052]

[0053] (3) Solubilization 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 solubilized. Each suspension was collected in a 50 mL centrifuge tube and centrifuged, after which the supernatant was recovered. The recovered supernatant was frozen and stored at -80°C until use.

[0054] (4) Measurement of viral genome amount The viral genome amount contained in the supernatant recovered in Example 2-(3) was measured using AAVpro (registered trademark) Titration Kit Ver. 2 (manufactured by Takara Bio Inc.).

[0055] Example 3 Purification of AAV Vector (1) Affinity Chromatography Approximately 90 supernatants that had been frozen and stored in Example 2-(3) were thawed and each was purified by 5x10 １１The supernatants of each fraction were mixed. The mixture was filtered using a 0.45 μm bottle-top filter (Corning) and then loaded onto AVB Sepharose High Performance (Cytiva). 1 mL of the eluate was collected, and the amount of viral genome contained in each fraction was measured using the same method as in Example 2-(4). Each fraction was also subjected to protein quantification by A280 measurement. As a result, the fractions containing a large amount of AAV vector were determined.

[0056] (2) Cesium chloride density gradient centrifugation After mixing the fractions containing large amounts of AAV vectors, a cesium chloride solution was added to adjust the refractive index to 1.371. The solution was dispensed into 13PA ultracentrifuge tubes (Eppendorf Himac Technologies) and centrifuged at 34,000 rpm and 21°C for 42 hours. After centrifugation, 0.5 mL of the solution was dispensed from the top of the tube. The fractions containing large amounts of AAV vectors were determined using the same method as in Example 3-(1).

[0057] (3) Dialysis The fractions containing large amounts of AAV vectors in Example 3-(2) were mixed and then subjected to a Slide-A-lyzer dialysis cassette (Thermo Scientific). The dialysis cassette was immersed in 1 L of phosphate-buffered saline (PBS), and dialysis was performed three times for 2 hours or more at 4°C. Subsequently, the dialysis cassette was immersed in 1 L of PBS containing 0.001% Pluronic F-68 (Thermo Scientific), 5% sorbitol, and 200 mM NaCl, and dialysis was performed for 3 hours at 4°C. The solution recovered from the dialysis cassette was concentrated by ultrafiltration using an Amicon® Ultra-4 Centrifugal Filter Unit (Merck). The concentrated solution was filtered using a 0.22 μm filter (Millipore) and stored at −80° C. until use as the “AAV mutant barcode library.” The viral genome amount was measured using the same method as in Example 2-(4).

[0058] Example 4 Analysis of the biodistribution 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) contained 7.5 × 10 １１ The drug was administered at 100 mg / kg via the tail vein of BALB / c mice. Four weeks after administration, the brain, spinal cord, lung, muscle, heart, and liver were collected. Genomic DNA was extracted from these tissues using NucleoSpin® tissue (Machlery-Nagel). RNA was also extracted from these tissues using RNeasy Plus Universal Mini (Qiagen).

[0059] (2) cDNA Synthesis Using the RNA extracted in Example 4-(1) as a template, cDNA was synthesized using PrimeScript RT reagent kit (manufactured by Takara Bio Inc.).

[0060] (3) Amplification and Purification of Barcode Sequence Using the cDNA synthesized in Example 4-(2) as a template, an amplicon encoding the barcode region was amplified by PCR. In the PCR, a forward primer (SEQ ID NO: 7) and a reverse primer (SEQ ID NO: 8) were used as primers. The 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. The amplicon was subjected to electrophoresis and then purified using NucleoSpin® Gel and PCR Clean-up (Machliner Gel).

[0061] (4) Next-generation sequencing (NGS) analysis of barcode sequences The amplicons purified in Example 4-(3) were subjected to NGS analysis using Miseq (Illumina) to determine the frequency of barcode sequences in each tissue. Furthermore, the gene transfer efficiency between tissues was calculated as GFP / GAPDH by qPCR using the genomic DNA extracted in Example 4-(1) as a template. In the qPCR, the Transgene Detection Primer set for real time (mouse) GFP-1 primer (Takara Bio Inc.) was used to amplify the GFP sequence, and the Mouse Housekeeping Gene Primer Set (Takara Bio Inc.) was used to amplify the GAPDH sequence. The gene transfer efficiency was used to normalize the frequency of appearance of the barcode sequence obtained by NGS analysis, and the frequency of appearance of the AAV vector in each tissue was calculated (Figure 2).

[0062] As shown in Figure 2, AAV vectors containing ETGHAWV (SEQ ID NO: 3), ETGHGYV (SEQ ID NO: 1), ETGHGFV (SEQ ID NO: 2), and ETGHGWA (SEQ ID NO: 4) showed high infectivity to the brain and spinal cord. In particular, AAV vectors containing ETGHAWV (SEQ ID NO: 3), ETGHGYV (SEQ ID NO: 1), and ETGHGFV (SEQ ID NO: 2) showed high tropism to the brain and spinal cord.

[0063] Example 5 Evaluation of AAV Vectors Having Specific Amino Acid Sequences (1) Preparation of pRC2Km-Insert Plasmids A kanamycin-resistant plasmid pRC2Km (Takara Bio Inc.) containing sequences encoding the AAV2 Rep protein and the AAV2 Cap protein was modified in the same manner as in Example 1 to prepare multiple plasmids encoding Cap proteins in which various amino acid sequences were inserted between amino acid positions 588 and 589 of the AAV2 Cap protein. The finally obtained pRC2Km-Insert plasmids are shown below. pRC2Km-ETGHGWV, pRC2Km-ETAHGWV, pRC2Km-ETGAGWV, pRC2Km-ETGHGAV, pRC2Km-ETGHGYV, pRC2Km-ETGHGFV, and pRC2Km-ETGHGHV.

[0064] (2) Preparation and purification of AAV vector pAAV-CAG-fLuc (Takara Bio Inc.), pHelper Vector (Takara Bio Inc.), and the pRC2Km-Insert plasmid obtained in Example 5-(1) were transfected into 293T cells using PEIpro (Polyplus). As a control, pRC2-mi342 Vector (Takara Bio Inc.) or pRC9 Vector (Takara Bio Inc.) was used instead of the pRC2Km-Insert plasmid. The transfected 293T cells were incubated at 37°C in a CO atmosphere. ２ The cells were cultured in an incubator for 72 hours. The various AAV vectors produced were collected and then purified using the same method as in Example 2-(3). For affinity purification of the AAV9 vector, POROS CaptureSelect™ AAV9 Affinity Resin (Thermo) was used.

[0065] (3) AAV Vector Infection by Intravenous Administration to Mouse Tail Vein The AAV vector obtained in Example 5-(2) was 1 x 10 １１The drug was administered via the tail vein of Balb / c mice (SLC) at a dose of 1000 mg / mouse. Vehicle was administered as a control. Thirty days after administration, the brain, lung, and liver were collected. Protein was extracted from each tissue, and luciferase activity was measured using the ONE-Glo Luciferase Assay System (Promega). Furthermore, the relative light intensity (RLU) per mg of tissue was calculated (Figure 3).

[0066] As shown in Figure 3, the AAV vectors containing ETGHGYV (SEQ ID NO: 1) and ETGHGFV (SEQ ID NO: 2) showed high infectivity in mouse brain tissue. In particular, the AAV vector containing ETGHGYV (SEQ ID NO: 1) showed high tropism for mouse brain tissue.

[0067] Even when the amino acid residue at position 587 of the AAV2 Cap protein in the Cap protein encoded by each of the above pRC2Km-Insert plasmids was substituted with asparagine, results equivalent to or better than those shown in Figure 3 were obtained. Furthermore, when the Cap protein of ETGHGWV was substituted with asparagine at position 587 (amino acid sequence shown in SEQ ID NO: 11), results equivalent to or better than those shown in Figure 3 were obtained.

[0068] Example 6 Evaluation of AAV vectors having specific amino acid sequences in the retina (1) Preparation and purification of AAV vectors pAAV-CAG-AcGFP (Takara Bio Inc.), pHelper Vector (Takara Bio Inc.), and the pRC2Km-Insert plasmid obtained in Example 5-(1) were transfected into 293T cells using PEIpro (Polyplus). The transfected 293T cells were incubated in a CO atmosphere at 37°C. ２ The cells were cultured in an incubator for 72 hours. The various AAV vectors produced were collected and purified using the same method as in Example 2-(3).

[0069] (2) AAV Vector Infection by Intravitreal Injection into Mice AAV vectors carrying ETGHGWV, ETGHGYV (SEQ ID NO: 1), and ETGHGFV (SEQ ID NO: 2) were administered intravitreously to Balb / c mice (SLC) at 4 × 10 ９ The compound was administered intravitreally to mice at a dose of 100 mg / eye. A vehicle was administered as a control. Retinas were harvested 14 days after administration. GFP fluorescence in the retina was observed under a fluorescence microscope (Figure 4).

[0070] As shown in Figure 4, the AAV vectors containing ETGHGYV (SEQ ID NO: 1) and ETGHGFV (SEQ ID NO: 2) showed higher infectivity in mouse retina than the vector containing ETGHGWV. Furthermore, these AAV vectors containing ETGHGYV (SEQ ID NO: 1) and ETGHGFV (SEQ ID NO: 2) showed higher infectivity in mouse retina than the wild-type AAV2 vector (Data Not Shown).

[0071] The plasmids used to prepare the above three AAV vectors were each modified to replace the amino acid residue at position 587 of the encoded Cap protein with asparagine, and similar studies were performed. As a result, the results were comparable to or better than those described above.

[0072] The present invention provides an AAV capsid protein mutant or amino acid sequence that has high brain tropism and low lung tropism, and provides a method for efficiently transferring genes into the brain.

[0073] SEQ ID NO:1: Peptide sequence ETGHGYV for AAV capsid protein mutant SEQ ID NO:2: Peptide sequence ETGHGFV for AAV capsid protein mutant SEQ ID NO:3: Peptide sequence ETGHAWV for AAV capsid protein mutant SEQ ID NO:4: Peptide sequence ETGHGWA for AAV capsid protein mutant SEQ ID NO:5: Oligo DNA containing random 15 nucleotides SEQ ID NO:6: Primer for synthesizing double strand DNA SEQ ID NO:7: Forward primer for amplification of random peptide coding region SEQ ID NO:8: Reverse primer for amplification of random peptide coding region 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 an adeno-associated virus (AAV) capsid protein mutant that contains a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:

4.

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

3. The nucleic acid of claim 2, wherein said peptide is contained at a position between amino acid number 588 and amino acid number 589 of 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 containing a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:

4.

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

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

9. A method for producing a gene-transduced cell, comprising the step of contacting a cell with an AAV particle comprising an AAV capsid protein mutant containing a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:

4.

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

11. The method of claim 10, wherein said peptide is contained at a position between amino acid number 588 and amino acid number 589 of VP1 of AAV2.

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

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