Analytical methods for chimeric adeno-associated viruses

Abstract

To provide a method for analyzing chimera adeno-associated virus (AAV) having a plurality of serotypic characteristics while holding its structure.SOLUTION: A method for analyzing chimera AAV includes the steps of: causing an adsorbent including an insoluble carrier and a polypeptide having amino acid sequences corresponding to extracellular domain 1 and domain 2 of KIAA0316L immobilized to the carrier to adsorb chimera AAV included in a sample; and eluting the chimera AAV adsorbed to the adsorbent using an eluate.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for analyzing adeno-associated viruses (AAVs). In particular, the present invention relates to a method for analyzing chimeric AAVs that have characteristics of multiple serotypes contained in a sample. [Background technology]

[0002] Adeno-associated viruses (AAVs) are non-enveloped viruses classified in the Parvoviridae family and Dependovirus genus. The outer shell of an AAV particle is composed of three types of proteins (VP1, VP2, and VP3), with approximately 60 protein molecules mixed and assembled in a ratio of roughly VP1:VP2:VP3 = 1:1:10, forming an icosahedron shape with a diameter of 20 nm to 30 nm.

[0003] In nature, AAV lacks the ability to replicate independently, and its replication depends on helper viruses such as adenoviruses and herpesviruses. When these helper viruses are present, the AAV genome replicates within the host cell, forming complete AAV particles containing the AAV genome, which are then released from the host cell. On the other hand, when these helper viruses are absent, the AAV genome remains maintained in the episome or is integrated into the host chromosome (latent state).

[0004] AAV is attracting attention as a potential gene transfer vector for treating congenital genetic disorders because it can infect cells of a wide range of species, including humans, and can infect non-dividing cells that have completed differentiation, such as blood cells, muscle cells, and nerve cells; it is not pathogenic to humans, so there is little concern about side effects; and the viral particles are physicochemically stable.

[0005] The production of recombinant AAV vectors (hereinafter also simply referred to as AAV vectors) is typically carried out by introducing nucleic acids encoding elements essential for AAV particle formation into cells to create cells capable of producing AAV (hereinafter also referred to as AAV-producing cells), and then culturing these cells to express the elements essential for AAV particle formation. The produced AAV vectors are recovered and purified from the AAV-producing cells to obtain therapeutic AAV vector preparations.

[0006] Multiple serotypes exist for AAV vectors, each with different cell targeting and infectivity (Non-Patent Literature 1). In recent years, chimeric AAV vectors possessing characteristics of multiple serotypes have also become known. Examples include a chimeric AAV vector of serotype 1 (AAV1) and serotype 2 (AAV2) (Non-Patent Literature 2), and a chimeric AAV vector of serotype 2 (AAV2) and serotype 5 (AAV5) (Non-Patent Literature 3). Chimeric AAV vectors are useful because they can possess the properties of several serotypes and can inherit the cell targeting and infectivity characteristics of each serotype. Analyzing such vectors while maintaining their structure is essential for understanding the properties of AAV vectors.

[0007] Conventionally, methods such as transmission electron microscopy, gel filtration analysis, and dynamic light scattering have been used to analyze AAV vectors while maintaining their structure. However, while these methods can provide information such as size and shape, analyzing chimeric AAV vectors has been difficult. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Ellis B.et al.,Virol J,74,10,2013 [Non-Patent Document 2] Hauck B.et al.,Mol Ther,7,419,2003 [Non-Patent Document 3] Excoffon K.et al.,Proc Natl Acad Sci USA,106,3865,2009 [Overview of the project] [Problems that the invention aims to solve]

[0009] The object of the present invention is to provide a method for analyzing chimeric adeno-associated viruses possessing characteristics of multiple serotypes while preserving their structure. [Means for solving the problem]

[0010] As a result of diligent research, the inventors discovered that chimeric AAVs contained in a sample can be analyzed by using an adsorbent containing an insoluble carrier and an AAV-binding protein immobilized on the carrier, thus completing the present invention.

[0011] In other words, the present invention encompasses the following embodiments.

[0012] [1] A method for analyzing adeno-associated virus (AAV) contained in a sample, comprising the steps of adsorbing the AAV contained in the sample onto an adsorbent containing an insoluble carrier and an AAV-binding protein immobilized on the carrier, and eluting the AAV adsorbed onto the adsorbent using an eluate, The method wherein the sample contains at least a chimeric AAV having characteristics of multiple serotypes, and the AAV-binding protein is a polypeptide selected from any of (i) to (iii) below; (i) A polypeptide comprising at least the amino acid residues from the 312th serine to the 500th aspartic acid of the amino acid sequence described in SEQ ID NO: 1, (ii) A polypeptide having an amino acid sequence that includes at least the amino acid residues from the 312th serine to the 500th aspartic acid of the amino acid sequence described in Sequence ID No. 1, wherein the amino acid sequence includes substitution, deletion, insertion, or addition of one or more amino acid residues at one or more positions within the amino acid residues from the 312th to the 500th, and which has AAV binding activity. (iii) A polypeptide comprising at least the amino acid residues from the 312th serine to the 500th aspartic acid of the amino acid sequence described in Sequence ID No. 1, wherein it has 70% or more homology to the amino acid sequence consisting of the amino acid residues from the 312th to the 500th, and has AAV binding activity.

[0013] [2] The method according to [1], wherein the sample comprises at least a chimeric AAV having characteristics of multiple serotypes and an AAV of a single serotype.

[0014] The present invention will be described in detail below.

[0015] The AAV-binding protein, which is the ligand protein (protein immobilized on an insoluble carrier) of the adsorbent used for AAV analysis in this invention (hereinafter also referred to as "AAV adsorbent"), is a polypeptide that contains at least the extracellular domain 1 (PKD1) and domain 2 (PKD2) (amino acid residues from the 312th serine (Ser) to the 500th aspartic acid (Asp) of SEQ ID NO: 1) of KIAA0319L (UniProt accession number: Q8IZA0, SEQ ID NO: 1), which is one aspect of the AAV receptor (AAVR). Specifically, examples include polypeptides shown in any of (i) to (iii) below. (i) A polypeptide comprising at least the amino acid residues from the 312th serine to the 500th aspartic acid of the amino acid sequence described in SEQ ID NO: 1, (ii) A polypeptide having an amino acid sequence that includes at least the amino acid residues from the 312th serine to the 500th aspartic acid of the amino acid sequence described in Sequence ID No. 1, wherein the amino acid sequence includes substitution, deletion, insertion, or addition of one or more amino acid residues at one or more positions within the amino acid residues from the 312th to the 500th, and which has AAV binding activity. (iii) A polypeptide comprising at least the amino acid residues from serine at position 312 to aspartic acid at position 500 of the amino acid sequence set forth in SEQ ID NO: 1, provided that it has a homology of 70% or more with the amino acid sequence consisting of the amino acid residues from position 312 to position 500 and has AAV binding activity.

[0016] The amino acid sequence set forth in SEQ ID NO: 1 is the amino acid sequence of KIAA0319L (official database: UniProt, accession number: Q8IZA0), which is a form of AAVR, and the amino acid residues from serine (Ser) at position 312 to aspartic acid (Asp) at position 500 of the amino acid sequence set forth in SEQ ID NO: 1 correspond to the extracellular domain domains 1 (PKD1) and domain 2 (PKD2) of KIAA0319L.

[0017] The polypeptide shown in any of (i) to (iii) above only needs to contain at least the regions corresponding to PKD1 and PKD2 of KIAA0319L described above. For example, It may contain all or part of the regions corresponding to other extracellular domain domains (domain 3 (PKD3), domain 4 (PKD4) and domain 5 (PKD5)) on the C-terminal side of PKD2, or all or part of the regions corresponding to signal sequences such as the MANSC (Motif At N terminus with Seven Cysteines) domain on the N-terminal side of PKD1 or cysteine-rich regions, or all or part of the transmembrane regions and intracellular regions on the N-terminal side and / or C-terminal side of the extracellular region.

[0018] As an example of (ii) above, there are polypeptides comprising at least the amino acid residues from serine at position 25 to aspartic acid at position 213 of the amino acid sequence set forth in SEQ ID NO: 4, and AAV-binding proteins disclosed in WO2021 / 106882. Also, as an example of the substitution, deletion, insertion, or addition described in (ii) above, there is the substitution of amino acid residues disclosed in WO2021 / 106882.

[0019] In the above (ii), "one or several" varies depending on the position of amino acid substitution and the type of amino acid residue in the three-dimensional structure of AAVR. As an example, it means any one of 1 or more and 50 or less, 1 or more and 30 or less, 1 or more and 20 or less, 1 or more and 10 or less, 1 or more and 9 or less, 1 or more and 8 or less, 1 or more and 7 or less, 1 or more and 6 or less, 1 or more and 5 or less, 1 or more and 4 or less, 1 or more and 3 or less, 1 or more and 2 or less, and 1. Substitution of "one or several" amino acid residues may occur, for example, at positions other than those of amino acid residue substitutions disclosed in WO2021 / 106882 as long as it has AAV binding activity.

[0020] In addition to the amino acid substitutions at the specific positions described above, conservative substitutions in which substitutions occur between amino acids with similar physical and / or chemical properties may occur in the "substitution of one or several amino acid residues" in the above (ii). It is generally known to those skilled in the art that the function of a protein is maintained between those in which substitution has occurred and those in which substitution has not occurred in conservative substitutions. Examples of conservative substitutions include substitutions between glycine and alanine, between serine and proline, or between glutamic acid and alanine (Protein Structure and Function, Medical Science International Co., Ltd., 9, 2005). In addition, the "substitution, deletion, insertion, or addition of one or several amino acid residues" in the above (ii) includes naturally occurring mutations (mutants or variants) based on differences in the origin of AAVR, species differences, and the like.

[0021] The homology of amino acid sequences in (iii) above is sufficient if it is 70% or more, but it may have a higher homology (for example, 80% or more, 85% or more, 90% or more, or 95% or more). In this specification, "homology" may mean similarity or identity, and may particularly mean identity. "Homologous amino acid sequence" means homology to the entire amino acid sequence. "Identity" between amino acid sequences means the ratio of amino acid residues of the same type in those amino acid sequences (Experimental Medicine, 31(3), Yodosha). "Similarity" between amino acid sequences means the sum of the ratio of amino acid residues of the same type in those amino acid sequences and the ratio of amino acid residues with similar side chain properties (Experimental Medicine, 31(3), Yodosha). The homology of amino acid sequences can be determined using alignment programs such as BLAST (Basic Local Alignment Search Tool) or FASTA.

[0022] In the present invention, the insoluble carrier used for immobilizing the AAV-binding protein described above is not particularly limited as long as it is insoluble in the sample containing AAV and the solution used for analysis and purification (eluent, equilibration solution, washing solution, etc.). Examples include carriers made from polysaccharides such as agarose, alginate (alginate salt), carrageenan, chitin, cellulose, dextrin, dextran, and starch; carriers made from synthetic polymers such as polyvinyl alcohol, polymethacrylate, poly(2-hydroxyethyl methacrylate), and polyurethane; and carriers made from ceramics such as silica. Among these, carriers made from polysaccharides and carriers made from synthetic polymers are preferred as insoluble carriers. Examples of the preferred carriers include polymethacrylate gels with introduced hydroxyl groups such as Toyopal (manufactured by Tosoh Corporation), agarose gels such as Sepharose (manufactured by Cytiva Corporation), and cellulose gels such as Cellfine (manufactured by JNC Corporation). The shape of the insoluble carrier is not particularly limited, but it is preferable that it be in a shape that can be packed into a column. For example, the material may be granular, monolithic, membranous, or fibrous, and may be either porous or non-porous.

[0023] The AAV-binding protein can be immobilized on an insoluble carrier by, for example, covalent bonding. Specifically, the AAV-binding protein can be immobilized on the insoluble carrier by covalent bonding between the AAV-binding protein and the insoluble carrier via an active group present on the insoluble carrier, thereby producing the AAV adsorbent used in the present invention. Examples of the active group include N-hydroxysuccinimide (NHS) activated ester group, epoxy group, carboxyl group, maleimide group, haloacetyl group, tresyl group, formyl group, and haloacetamide group. As the insoluble carrier having an active group, for example, a commercially available insoluble carrier having an active group may be used as is, or an insoluble carrier may be used after introducing an active group. Examples of commercially available carriers containing active groups include TOYOPEARL AF-Epoxy-650M, TOYOPEARL AF-Tresyl-650M (both manufactured by Tosoh Corporation), HiTrap NHS-activated HP Columns, NHS-activated Sepharose 4 Fast Flow, Epoxy-activated Sepharose 6B (all manufactured by Cytiva Corporation), and SulfoLink Coupling Resin (manufactured by Thermo Fisher Scientific).

[0024] One example of a method for introducing active groups to a support surface is to react one of two or more active sites of a compound with hydroxyl groups, epoxy groups, carboxyl groups, amino groups, etc., present on the support surface.

[0025] Examples of compounds that introduce epoxy groups to hydroxyl groups or amino groups present on the carrier surface include epichlorohydrin, ethanediol diglycidyl ether, butanediol diglycidyl ether, hexanediol diglycidyl ether, and polyethylene glycol diglycidyl ether. Specific examples of polyethylene glycol diglycidyl ether include Denacol EX-810 (n=1), Denacol EX-811 (n=1), Denacol EX-850 (n=2), Denacol EX-851 (n=2), Denacol EX-821 (n=4), Denacol EX-830 (n=9), Denacol EX-832 (n=9), Denacol EX-841 (n=13), and Denacol EX-861 (n=22) (all manufactured by Nagase ChemteX Corporation).

[0026] Examples of compounds that introduce carboxyl groups to epoxy groups present on the support surface include 2-mercaptoacetic acid, 3-mercaptopropionic acid, 4-mercaptobutyric acid, 6-mercaptobutyric acid, glycine, 3-aminopropionic acid, 4-aminobutyric acid, and 6-aminohexanoic acid.

[0027] Compounds that introduce maleimide groups to hydroxyl groups, epoxy groups, carboxyl groups, and amino groups present on the carrier surface include N-(ε-maleimidocaproic acid)hydrazide, N-(ε-maleimidopropionic acid)hydrazide, 4-(4-N-maleimidophenyl)acetic acid hydrazide, 2-aminomaleimide, 3-aminomaleimide, 4-aminomaleimide, 6-aminomaleimide, 1-(4-aminophenyl)maleimide, 1-(3-aminophenyl)maleimide, 4-(maleimide)phenylisocyanate, 2-maleimidoacetic acid, and 3-maleimidopropionic acid Examples include 4-maleimidobutyric acid, 6-maleimidohexanoic acid, N-(α-maleimidoacetoxy)succinimide ester, (m-maleimidobenzoyl)N-hydroxysuccinimide ester, succinimidyl-4-(maleimidomethyl)cyclohexane-1-carbonyl-(6-aminohexanoic acid), succinimidyl-4-(maleimidomethyl)cyclohexane-1-carboxylic acid, (p-maleimidobenzoyl)N-hydroxysuccinimide ester, (m-maleimidobenzoyl)N-hydroxysuccinimide ester, and dextran.

[0028] Examples of compounds that introduce haloacetyl groups to hydroxyl or amino groups present on the carrier surface include chloroacetic acid, bromoacetic acid, iodoacetic acid, chloroacetic acid chloride, bromoacetic acid chloride, bromoacetic acid bromide, chloroacetic acid anhydride, bromoacetic acid anhydride, iodoacetic acid anhydride, 2-(iodoacetamide)acetic acid-N-hydroxysuccinimide, 3-(bromoacetamide)propionic acid-N-hydroxysuccinimide, and 4-(iodoacetyl)aminobenzoic acid-N-hydroxysuccinimide.

[0029] Another method for introducing active groups to the support surface involves reacting hydroxyl groups or amino groups present on the support surface with an ω-alkenyl alkane glycidyl ether, and then activating it by halogenating the ω-alkenyl moiety with a halogenating agent. Examples of ω-alkenyl alkane glycidyl ethers include allyl glycidyl ether, 3-butenyl glycidyl ether, and 4-pentenyl glycidyl ether. Examples of halogenating agents include N-chlorosuccinimide, N-bromosuccinimide, and N-iodosuccinimide.

[0030] Another method for introducing active groups to the support surface involves introducing active groups to carboxyl groups present on the support surface using a condensing agent and an additive. Examples of condensing agents include 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), dicyclohexylcarbodiamide, and carbonyldiimidazole. Examples of additives include NHS, 4-nitrophenol, and 1-hydroxybenztriazole.

[0031] Immobilization of AAV-binding proteins onto insoluble carriers can be carried out, for example, in a buffer solution. Examples of buffer solutions include acetate buffer, phosphate buffer, MES (2-MorpholinoEthaneSulfonic acid) buffer, HEPES (4-(2-HydroxyEthyl)-1-PiperazineEthaneSulfonic acid) buffer, Tris (Tris(hydroxymethyl)aminomethane) buffer, and borate buffer. The reaction temperature during immobilization can be appropriately set according to various conditions, such as the reactivity of the active group and the stability of the AAV-binding protein. The reaction temperature during immobilization may be, for example, 4°C to 50°C, and preferably 10°C to 35°C.

[0032] The present invention is characterized by an analysis method that includes the steps of adsorbing chimeric AAVs having the characteristics of multiple serotypes contained in a sample onto an AAV adsorbent prepared by the method described above (hereinafter also simply referred to as the "adsorption step"), and eluting the AAVs adsorbed onto the adsorbent using an eluent (hereinafter also simply referred to as the "elution step").

[0033] The chimeric AAVs analyzed in this invention refer to AAVs that possess characteristics of multiple serotypes. Examples of these characteristics include the cell-specificity and infectivity of each serotype. Chimeric AAVs can be obtained, for example, by mixing plasmids encoding the capsids that constitute each serotype and transfecting a host. There may be two or more types of capsids, and the ratio of the plasmids to be transfected can be arbitrarily selected. Examples of chimeric AAVs include chimeric AAVs of serotype 1 (AAV1) and serotype 2 (AAV2) (hereinafter also referred to as "AA1 / AAV2") and chimeric AAVs of AAV2 and serotype 5 (AAV5) (hereinafter also referred to as "AAV2 / AAV5").

[0034] In the present invention, the sample only needs to contain at least one of the aforementioned chimeric AAVs, and may further contain a single serotype of AAV and / or a type of chimeric AAV other than the aforementioned chimeric AAV.

[0035] When analyzing chimeric AAVs according to the present invention, it is preferable to use the AAV adsorbent in the adsorption step and the elution step in a form packed in a column (hereinafter also referred to as "AVR column"), as this simplifies these steps. The following will be a detailed explanation using an AVR column as an example.

[0036] A sample containing chimeric AAV can be added to an AVR column using, for example, a liquid delivery means such as a pump. In this specification, adding a liquid to a column is also referred to as "delivering a liquid to the column." Before adding a sample containing chimeric AAV to the AVR column, the solvent may be replaced with an appropriate buffer solution. Alternatively, the AVR column may be equilibrated using an appropriate buffer solution (equilibrium solution) before adding a sample containing chimeric AAV to the AVR column (i.e., before the adsorption step). This equilibration is expected to allow for more accurate analysis of chimeric AAV, for example. Examples of buffer solutions used for solvent replacement and equilibration include phosphate buffer, acetate buffer, succinate buffer, citrate buffer, Tris buffer, HEPES buffer, and MES buffer, which have buffering capacity in the neutral range (in this specification, the range of pH 4.0 to 9.0). Inorganic salts such as sodium chloride or calcium chloride in concentrations of 10 mM to 600 mM may be added to such buffers. The buffer solution and equilibration solution used for solvent replacement may or may not be the same. Furthermore, if components other than AAV, such as contaminants, remain on the AVR column after passing a sample containing chimeric AAV through it, these components may be removed (washed) from the AVR column before eluting the chimeric AAV adsorbed on the AAV adsorbent with the eluent (i.e., before the elution step). Components other than chimeric AAV can be removed from the AVR column, for example, by using an appropriate buffer solution as the washing solution. For this washing solution, the descriptions for buffer solutions used for solvent replacement and equilibration can be applied mutatis mutandis.

[0037] In the elution process, the delivery method of the eluate may be gradient or isocratic. However, if the sample contains multiple types of chimeric AAVs, or if it contains both chimeric AAVs and AAVs of a single serotype, it is preferable to elute using a gradient. The gradient may be changed in two or more steps (stepwise) or in a linear gradient. An example of gradient elution is a gradient that lowers the pH from the neutral region to the acidic region (a region more acidic than the neutral region). The pH of the eluate at the start should be in the neutral region, i.e., pH 4.0 to 9.0, and preferably pH 4.5 to 6.0. The acidic region of the eluate after the gradient should be pH 3.5 or lower, and preferably pH 2.0 to 2.5. There is no particular lower limit to the flow rate, and the upper limit depends on the back pressure of the insoluble carrier used in the AAV adsorbent. Typically, a flow rate of 0.5 mL / min to 2.0 mL / min is preferred. The gradient time should be such that differences in serotype can be distinguished by differences in elution time. Specifically, 5 minutes or more is preferred, more preferably 20 minutes or more, and even more preferably 45 minutes or more. [Effects of the Invention]

[0038] The present invention is characterized by a method for analyzing chimeric adeno-associated viruses (AAVs) containing characteristics of multiple serotypes in a sample, comprising the steps of adsorbing the AAV onto an adsorbent containing an insoluble carrier and an AAV-binding protein immobilized on the carrier, and eluting the AAV adsorbed onto the adsorbent using an eluate. The present invention makes it possible to analyze chimeric AAVs contained in a sample while maintaining their structure, which was not possible with conventional methods. [Brief explanation of the drawing]

[0039] [Figure 1]These figures show the chromatographic patterns obtained by analyzing single-serotype AAVs and chimeric AAVs using an AVR10s column. [a] is a summary of the results of analyzing (A) AAV1-EGFP solution, (B) AAV2-EGFP solution, and (D) AAV1 / AAV2-EGFP solution, respectively, and [b] is a summary of the results of analyzing (B) AAV2-EGFP solution, (C) AAV5-EGFP solution, and (E) AAV2 / AAV5-EGFP solution, respectively. [Figure 2] This figure shows the chromatographic patterns obtained by analyzing (B) AAV2-EGFP solution, (C) AAV5-EGFP solution, and (E) AAV2 / AAV5-EGFP solution using a gel filtration column. [Figure 3] This figure shows the particle size distribution results of the analysis of (A) AAV1-EGFP solution, (B) AAV2-EGFP solution, (C) AAV5-EGFP solution, (D) AAV1 / AAV2-EGFP solution, and (E) AAV2 / AAV5-EGFP solution using dynamic light scattering. [Figure 4] This figure shows the chromatographic patterns obtained by analyzing single-serotype AAVs, chimeric AAVs, and mixtures of AAVs with different single serotypes using an AVR10s column. [Examples]

[0040] The present invention will be described in more detail below with reference to examples and comparative examples, but the present invention is not limited to these examples.

[0041] Example 1: Preparation of an adeno-associated virus (AAV) vector (1) A nucleotide sequence (SEQ ID NO: 3) was designed by adding the restriction enzyme EcoRI recognition sequence (GAATTC) to the 5' end of a polynucleotide encoding EGFP (Enhanced Green Fluorescent Protein) consisting of the amino acid sequence described in SEQ ID NO: 2, and a stop codon (TAG) and a BamHI recognition sequence (GGATTC) to the 3' end.

[0042] (2) A polynucleotide consisting of the sequence described in Sequence ID No. 3 was totally synthesized and cloned into a plasmid (commissioned to FASMAC, named pUC-EGFP). Escherichia coli strain JM109 was transformed with pUC-EGFP, and the resulting transformants were cultured. pUC-EGFP was extracted from the culture medium using the QIAprep Spin Miniprep kit (Qiagen).

[0043] (3) The pUC-EGFP obtained in (2) was digested with restriction enzymes EcoRI and BamHI, and then ligated into the expression vector pAAV-CMV (manufactured by Takara Bio Inc.), which had been previously digested with restriction enzymes EcoRI and BamHI. The ligation product was then used to transform E. coli strain JM109.

[0044] (4) The transformants obtained in (3) were cultured overnight at 37°C with shaking in a 5L baffled flask containing 1L of 2YT medium (1.6% (w / v) Tryptone, 1% (w / v) Yeast Extract, 0.5% (w / v) Sodium Chloride) with 100 μg / mL of carbenicillin. After the culture was complete, the cells were collected by centrifugation. Using Plasmid Mega Kit (Qiagen), a large quantity of the EGFP-expressing vector pAAV-EGFP was prepared from the collected cells.

[0045] (5) Escherichia coli strain JM109 was transformed with one of the plasmids from pRC1-mi342 Vector, pRC2-mi342 Vector, and pRC5-mi342 Vector (all manufactured by Takara Bio Inc.) (hereinafter collectively referred to as "pRCX-mi342 Vector") and pHelper Vector (manufactured by Takara Bio Inc.). Using the resulting transformants, pRCX-mi342 Vector and pHelper were prepared in large quantities by performing the same procedure as in (4).

[0046] (6) HEK293T cells were cultured in eight cell stack cell culture surface-treated 5-chambers (Corning) containing 500 mL of D-MEM medium (Fujifilm Wako Pure Chemical Industries, Ltd.) with 5% (v / v) bovine serum. Gene transduction was performed by adding a complex of pAAV-EGFP prepared in (4), pRCX-mi342 Vector and pHelper prepared in (5), and polyethyleneimine (Polysciences), and the cells were cultured statically for 3 days under conditions of 5% (v / v) carbon dioxide and 37°C. The pRCX-mi342 Vector used for gene transduction was one of the following combinations (A) to (E). (A) pRC1-mi342 Vector only (B) pRC2-mi342 Vector only (C)pRC5-mi342 Vector only (D) pRC1-mi342 Vector and pRC2-mi342 Vector (E) pRC2-mi342 Vector and pRC5-mi342 Vector (7) Triton X-100 (Sigma-A) (final concentration 0.1%) and Benzonate (Merck Millipore) (final concentration 1 U / mL) were added to the culture medium obtained in (6) to a final concentration of 0.1%, and the mixture was allowed to stand at 37°C for 3 hours. After centrifugation, the supernatant was collected and concentrated using a 300 kD-cutoff tangential flow system membrane cassette (Pall Corporation), and replaced with 20 mM Tris-HCl buffer (pH 8.0) containing 0.5 M sodium chloride (hereinafter also referred to as "equilibrium solution A"). The resulting concentrate was passed through a 0.22 μm pore size filter to remove suspended solids.

[0047] (8) The solution from which suspended solids were removed was applied to a 7 mL AVB Sepharose column (Cytiva) or a POROS AAVX column (Thermo Fisher Scientific).

[0048] (9) After washing with equilibration solution A, the samples were eluted with 0.1 M acetate buffer (pH 2.5) containing 0.5 M sodium chloride. The eluted fraction containing the obtained AAV vectors was neutralized by adding 1 / 4 volume of 1 M Tris-HCl buffer (pH 8.5) containing 20 mM magnesium chloride to obtain AAV vectors for each serotype: (A) AAV1-EGFP, (B) AAV2-EGFP, and (C) AAV5-EGFP, as well as chimeric AAV vectors possessing characteristics of multiple serotypes: (D) AAV1 / AAV2-EGFP (a chimeric AAV vector of AAV1 and AAV2) and (E) AAV2 / AAV5-EGFP (a chimeric AAV vector of AAV2 and AAV5) (hereinafter collectively referred to as "AAVX-EGFP").

[0049] The AAVX-EGFP concentration in the solutions obtained in (10)(9) was quantified using a Zetasizer Ultra (Malvern). The solutions were then subjected to SDS-PAGE and silver staining using a Pierce Silver Stain Kit (Thermo Fisher Scientific) to confirm the purity of the AAV vector contained in the solutions. In all solutions, only bands corresponding to the three outer shell proteins (VP1, VP2, VP3) that constitute the AAV vector were observed, confirming that there were no issues with purity.

[0050] Example 2: Preparation of AAV-binding protein (1) The BL21(DE3) strain of Escherichia coli was transformed with plasmid pET-AVR10s, which contains a polypeptide comprising the AAV-binding protein AVR10s consisting of the amino acid sequence described in SEQ ID NO: 4, and a transformant capable of expressing AVR10s was inoculated into 3 mL of 2YT liquid medium containing 50 μg / mL kanamycin and pre-cultured overnight at 37°C with aerobic shaking. In SEQ ID NO: 4, the sequence from the 1st methionine (Met) to the 22nd alanine (Ala) is the PelB signal peptide, the sequence from the 25th serine (Ser) to the 213th aspartic acid (Asp) is the AAV-binding protein AVR10s, and the sequence from the 220th cysteine ​​(Cys) to the 226th glycine (Gly) is the cysteine ​​tag sequence, which is an immobilization tag. Furthermore, AVR10s is a polypeptide in which the extracellular domain 1 (PKD1) and domain 2 (PKD2) of the AAV receptor KIAA0319L (amino acid residues from serine at position 312 to aspartic acid at position 500 of SEQ ID NO: 1) have undergone the amino acid substitutions shown in (I) to (X) below. (I) The valine at position 317 in SEQ ID NO: 1 (position 30 in SEQ ID NO: 4) is replaced with aspartic acid. (II) The tyrosine at position 342 in SEQ ID NO: 1 (position 55 in SEQ ID NO: 4) is replaced with serine. (III) Lysine at position 362 in SEQ ID NO: 1 (position 75 in SEQ ID NO: 4) is replaced with glutamic acid. (IV) Lysine at position 371 in SEQ ID NO: 1 (position 84 in SEQ ID NO: 4) is replaced with asparagine. (V) The valine at position 381 in sequence number 1 (position 94 in sequence number 4) is replaced with alanine. (VI) The isoleucine at position 382 in sequence number 1 (position 95 in sequence number 4) is replaced with valine. (VII) The glycine at position 390 in sequence number 1 (position 103 in sequence number 4) is replaced with serine. (VIII) Lysine at position 399 in SEQ ID NO: 1 (position 112 in SEQ ID NO: 4) is replaced with glutamic acid. (IX) The 476th serine in SEQ ID NO: 1 (189th in SEQ ID NO: 4) is replaced with arginine. (X) The asparagine at position 487 in SEQ ID NO: 1 (position 200 in SEQ ID NO: 4) is replaced with aspartic acid. (2) In a 1 L baffled flask, 2 mL of the pre-culture solution from (1) was inoculated into 200 mL of 2YT liquid medium containing 50 μg / mL of kanamycin, and the culture was performed aerobically with shaking at 37°C.

[0051] (3) Two hours after the start of culture, IPTG (IsoPropyl-β-D-ThioGalactopyranoside) was added to a final concentration of 0.1 mM, and the culture was continued aerobically with shaking at 37°C for 3.0 hours.

[0052] (4) After the culturing was complete, the culture medium was centrifuged at 4°C and 8000 rpm for 20 minutes to collect the bacterial cells.

[0053] (5) The bacterial cells recovered in (4) were suspended in 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride and 20 mM imidazole at a concentration of 5 mL / 1 g (bacterial cells). The cells were then lysed using an ultrasonic generator (Insonator 201M [manufactured by Kubota Shoji Co., Ltd.]) at 8°C for approximately 10 minutes at an output of approximately 150 W. The lysate was centrifuged twice at 8000 rpm for 20 minutes at 4°C, and the supernatant was collected.

[0054] (6) The supernatant obtained in (5) was applied to an XK26 / 20 column (Cytiva) packed with 50 mL of Ni Sepharose 6 Fast Flow (Cytiva), which had been pre-equilibrated with Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride and 20 mM imidazole (hereinafter also referred to as "equilibrium solution B"). After washing with equilibrium solution B, the column was eluted with 20 mM Tris-HCl buffer (pH 7.4) containing 0.5 M imidazole and 150 mM sodium chloride.

[0055] The eluate obtained in (7)(6) was dialyzed with 20 mM Tris buffer (pH 7.4) containing 150 mM sodium chloride to prepare the amount of AVR10s protein necessary for the production of the AAV adsorbent.

[0056] Example 3: Preparation of AAV adsorbent (1) A hydrophilic vinyl polymer for separation agents (Toyopearl, manufactured by Tosoh Corporation) was prepared as an insoluble carrier, and a carrier was prepared by introducing iodoacetamide groups by chemically modifying the hydroxyl groups on the surface of the polymer.

[0057] (2) 7 g (wet weight) of the carrier prepared in (1) was mixed with 14 mg of AVR10s prepared in Example 2 and TCEP (Tris(2-CarboxyEthyl)Phosphine) at a final concentration of 0.3 mM as a reducing agent, and the mixture was reacted by shaking for 3 hours under conditions of pH 8.1 and 25°C. This prepared an AVR10s immobilized gel, which is an AAV adsorbent in which AVR10s is immobilized.

[0058] Example 4: Chimeric AAV analysis using AAV adsorbent (Part 1) (1) 1.25 mL of the AVR10s immobilized gel prepared in Example 3 was packed into a stainless steel empty column (φ4.6 mm × 75 mm, manufactured by Tosoh Corporation) to prepare a column (named AVR10s column).

[0059] (2) The AAVR column prepared in (1) was connected to an HPLC M40A (Shimadzu Corporation) and equilibrated with a 10 mM glycine buffer (pH 4.5) containing 50 mM sodium chloride and 10 mM sodium acetate (hereinafter also referred to as "equilibrium solution C").

[0060] (3) The AAVX-EGFP solutions obtained in Example 1 ((A) AAV1-EGFP solution, (B) AAV2-EGFP solution, (C) AAV5-EGFP solution, (D) AAV1 / AAV2-EGFP solution, and (E) AAV2 / AAV5-EGFP solution) were each divided into 1.0 × 10⁻¹⁰ units. 12 The solution was diluted to a concentration of cp / mL (where cp represents the number of AAV particles; the same applies hereafter).

[0061] (4) 0.1 mL of the diluted solution prepared in (3) was applied to the column equilibrated in (2).

[0062] (5) After washing with equilibration solution C at a flow rate of 0.5 mL / min for 10 minutes, the AVR10s column was infused with 10 mM glycine buffer (pH 2.0) containing 50 mM calcium chloride and 10 mM sodium acetate (hereinafter also referred to as "eluent A") and equilibration solution C, so that a linear gradient was formed in which the concentration of eluent A increased from 0% to 100% in 60 minutes (flow rate 0.5 mL / min). After the linear gradient was completed, eluent A was infused for 3 minutes, and then equilibration solution C was again infused into the AVR10s column for re-equilibrium.

[0063] (6) AAVX-EGFP eluted from the AAV adsorbent (AVR10s immobilized gel) packed into the column was detected by fluorescence intensity at 350 nm in response to excitation light at 280 nm.

[0064] The obtained chromatographic patterns are shown in Figure 1. The chromatographic patterns of all solutions showed a single peak. Furthermore, the peak elution time for (D)AAV1 / AAV2-EGFP was between the peak elution time for (A)AAV1-EGFP and the peak elution time for (B)AAV2-EGFP (Figure 1[a]), and the peak elution time for (E)AAV2 / AAV5-EGFP was between the peak elution time for (B)AAV2-EGFP and the peak elution time for (C)AAV5-EGFP (Figure 1[b]). The results shown in Figure 1 reflect the fact that AAV1 / AAV2 has properties intermediate between AAV1 and AAV2, and that AAV2 / AAV5 has properties intermediate between AAV2 and AAV5, respectively.

[0065] Comparative Example 1: AAV analysis using gel filtration column (1) A gel filtration column, TSKgel G5000PWXL (manufactured by Tosoh Corporation), was connected to an HPLC M40A (manufactured by Shimadzu Corporation) and equilibrated with 0.01% (w / v) Tween 20 (product name), 0.01% (w / v) Pluronic F-68 (product name), and 500 mM sodium acetate buffer (pH 6.0) containing 500 mM sodium chloride (hereinafter also referred to as "equilibrium solution D").

[0066] (2) Of the AAVX-EGFP solutions obtained in Example 1, (B) AAV2-EGFP solution, (C) AAV5-EGFP solution, and (E) AAV2 / AAV5-EGFP solution were each measured in 1.0 × 10⁻¹⁶ units. 11 The solution was diluted to a concentration of cp / mL.

[0067] (3) 0.2 mL of the diluted solution prepared in (2) was applied to the column equilibrated in (1).

[0068] (4) Using equilibration solution D, the solution was delivered to the AVR10s column at a flow rate of 0.5 mL / min for 22 minutes, and the AAVX-EGFP eluted from the column was detected by fluorescence intensity of 350 nm in response to excitation light of 280 nm.

[0069] The obtained chromatographic patterns are shown in Figure 2. Since the chromatographic patterns of all solutions show a single peak and the peak elution time is around 8 minutes, it can be seen that gel filtration analysis makes it difficult to analyze chimeric AAVs, which have characteristics of multiple serotypes, as having different properties from AAVs of a single serotype.

[0070] Comparative Example 2: AAV analysis using Dynamic Light Scattering (DLS) (1) AAVX-EGFP solution obtained in Example 1 ((A) AAV1-EGFP solution [Concentration: 8.7 × 10⁻¹⁰] 12 cp / mL], (B) AAV2-EGFP solution [concentration: 1.0 × 10 12 cp / mL], (C) AAV5-EGFP solution [concentration: 1.0 × 10 12 cp / mL], (D) AAV1 / AAV2-EGFP solution [concentration: 3.6 × 10 12 [cp / mL] and (E)AAV2 / AAV5-EGFP solution [Concentration: 6.9 × 10⁻¹⁰] 11 For each solution, the particle size of AAVX-EGFP was determined by measuring dynamic light scattering using a Zetasizer Ultra (Malvern Panalytical).

[0071] The obtained particle size distribution is shown in Fig. 3. Since the particle size distribution of any of the solutions has a single peak and the peak particle size is around 25 nm, it can be seen that it is difficult to analyze chimeric AAV having characteristics of multiple serotypes as properties different from those of AAV of a single serotype by DLS.

[0072] Example 5 Analysis of chimeric AAV using AAV adsorbent (Part 2) The AAVX-EGFP solution applied to the AVR10s column was 0.1 mL of any of the solutions shown in the following (α) to (η), and AAVX-EGFP was analyzed in the same manner as in Example 4 except for this. (α) AAV1-EGFP solution (concentration: 1.0×10 12 cp / mL) (β) AAV2-EGFP solution (concentration: 1.0×10 12 cp / mL) (γ) AAV1 / AAV2-EGFP solution (concentration: 1.0×10 12 cp / mL) (δ) A mixed solution of 80% of (α) and 20% of (β) (ε) A mixed solution of 60% of (α) and 40% of (β) (ζ) A mixed solution of 40% of (α) and 60% of (β) (η) A mixed solution of 20% of (α) and 80% of (β) The obtained chromatogram patterns are shown in Fig. 4. The results of (α), (β) and (γ) were single peaks as in Example 4, and the peak elution time of (γ) AAV1 / AAV2-EGFP was the time between the peak elution time of (α) AAV1-EGFP and the peak elution time of (β) AAV2-EGFP. On the other hand, in the chromatogram patterns of (δ), (ε), (ζ) and (η) (that is, a mixture of AAV1-EGFP and AAV2-EGFP), a total of two peaks corresponding to (α) AAV1-EGFP and (β) AAV2-EGFP appeared, and the peak heights approximately reflected the mixing ratio.

[0073] From these results, it can be seen that by using a column packed with AAV adsorbent, it is possible to analyze single-serotype AAVs, mixtures of different single-serotype AAVs, and chimeric AAVs based on their respective properties.

Claims

1. A method for analyzing adeno-associated virus (AAV) contained in a sample, comprising the steps of adsorbing the AAV contained in the sample onto an adsorbent containing an insoluble carrier and an AAV-binding protein immobilized on the carrier, and eluting the AAV adsorbed onto the adsorbent using an eluent, The sample contains at least a chimeric AAV having characteristics of multiple serotypes, and The AAV-binding protein is a polypeptide comprising at least the amino acid residues from the 312th serine to the 500th aspartic acid of the amino acid sequence described in Sequence ID No. 1, wherein the amino acid substitutions shown in (I) to (X) below have occurred in the amino acid residues from the 312th to the 500th, in the method described above; (I) The valine at position 317 of SEQ ID NO: 1 is replaced with aspartic acid. (II) The tyrosine at position 342 of sequence number 1 is replaced with serine. (III) Lysine at position 362 of SEQ ID NO: 1 is replaced with glutamic acid. (IV) Lysine at position 371 of Sequence ID No. 1 is replaced with asparagine. (V) The valine at position 381 of sequence number 1 is replaced with alanine. (VI) The isoleucine at position 382 of sequence number 1 is replaced with valine. (VII) The glycine at position 390 of sequence number 1 is replaced with serine. (VIII) Lysine at position 399 of Sequence ID No. 1 is replaced with glutamic acid. (IX) Serine at position 476 of sequence number 1 is replaced with arginine. (X) The 487th asparagine molecule in SEQ ID NO: 1 is replaced with aspartic acid.

2. The method according to claim 1, wherein the sample comprises at least a chimeric AAV having the characteristics of multiple serotypes and an AAV of a single serotype.

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