Methods for analyzing adeno-associated viruses

Abstract

To provide a method for analyzing an adeno-associated virus (AAV) included in a sample with high sensitivity.SOLUTION: The foregoing problem is solved by adding, to an AAV included in a sample, AAV-binding protein and calcium ions, and performing an analysis on the basis of the binding affinity between the AAV and the AAV-binding protein.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention relates to a method for analyzing adeno-associated virus (AAV) contained in a sample, and in particular to a method for analyzing the AAV based on its binding affinity to an AAV-binding protein. [Background technology]

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

[0003] In nature, AAV lacks the ability to replicate autonomously and depends on helper viruses such as adenoviruses and herpesviruses for replication. In the presence of the helper virus, the AAV genome replicates in the host cell, forming complete AAV particles containing the AAV genome, which are then released from the host cell. On the other hand, in the absence of the helper virus, the AAV genome is maintained in an episome or integrated into the host chromosome (latent state).

[0004] AAV is capable of infecting cells of a wide range of species, including humans, and can also infect non-dividing cells that have completed differentiation, such as blood cells, muscles, and nerve cells. It is also non-pathogenic to humans, meaning there is little risk of side effects. Furthermore, the viral particles are physically and chemically stable. For these reasons, AAV is attracting attention as a potential vector for gene transfer aimed at treating congenital genetic diseases.

[0005] Recombinant AAV vectors (hereafter simply referred to as AAV vectors) are typically produced by introducing nucleic acids encoding elements essential for AAV particle formation into cells to generate cells capable of producing AAV (hereafter also referred to as AAV-producing cells), and then culturing the 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 formulations.

[0006] One method for analyzing AAV vectors is affinity chromatography, which uses an adsorbent comprising an insoluble carrier and an AAV-binding protein immobilized on the carrier, and can analyze the vector from a sample containing the AAV vector in the presence of contaminants (Patent Document 1). However, the method described in Patent Document 1 is insufficient for highly sensitive analysis of AAV vectors contained in a sample. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] WO2021 / 106882 issue Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is to provide a method for analyzing adeno-associated viruses contained in a sample with high sensitivity. [Means for solving the problem]

[0009] As a result of extensive research to solve the above-mentioned problems, the present inventors discovered that the above-mentioned problems can be solved by adding calcium ions to a solution containing adeno-associated virus (AAV) and an AAV-binding protein and analyzing the solution, thereby completing the present invention.

[0010] That is, the present invention includes the following aspects: [1] A method for analyzing adeno-associated virus (AAV) contained in a sample based on the binding affinity with an AAV-binding protein, the method comprising adding calcium ions to a solution containing the AAV and the AAV-binding protein and then performing the analysis.

[0011] [2] The method according to [1], wherein the AAV-binding protein is in the form of an adsorbent comprising an insoluble carrier and the AAV-binding protein immobilized on the carrier.

[0012] [3] The method described in [2], comprising the steps of: adsorbing AAV contained in a sample to an adsorbent comprising an insoluble carrier and an AAV-binding protein immobilized on the carrier; washing the adsorbent with a washing solution containing calcium ions; and eluting the AAV adsorbed to the adsorbent with an elution solution containing calcium ions.

[0013] [4] The method according to any one of [1] to [3], wherein the AAV binding protein is a polypeptide selected from any one of the following (i) to (iii): (i) a polypeptide containing at least the amino acid residues from serine at position 312 to aspartic acid at position 500 in the amino acid sequence set forth in SEQ ID NO: 1; (ii) A polypeptide having an amino acid sequence containing 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 the amino acid sequence contains substitution, deletion, insertion, or addition of one or several amino acid residues at one or several positions within the amino acid residues at positions 312 to 500, and having 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, wherein the polypeptide has 70% or more identity to the amino acid sequence consisting of the amino acid residues at positions 312 to 500, and wherein the polypeptide has AAV-binding activity.

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

[0015] In the present invention, the AAV-binding protein is not particularly limited as long as it is a polypeptide capable of binding to AAV, and examples thereof include laminin receptors such as integrins, anti-AAV antibodies, and AAV receptors (AAVRs). When the AAV-binding protein is AAVR, preferred embodiments include polypeptides shown in any of (i) to (iii) below. (i) a polypeptide containing at least the amino acid residues from serine at position 312 to aspartic acid at position 500 in the amino acid sequence set forth in SEQ ID NO: 1; (ii) A polypeptide having an amino acid sequence containing 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 the amino acid sequence contains substitution, deletion, insertion, or addition of one or several amino acid residues at one or several positions within the amino acid residues at positions 312 to 500, and having 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, wherein the polypeptide has 70% or more identity to the amino acid sequence consisting of the amino acid residues at positions 312 to 500, and wherein the polypeptide 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 one embodiment of AAVR, and the amino acid residues from serine (Ser) at position 312 to aspartic acid (Asp) at position 500 in the amino acid sequence set forth in SEQ ID NO: 1 correspond to the extracellular domain 1 (PKD1) and domain 2 (PKD2) of KIAA0319L.

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

[0018] Examples of (ii) include a polypeptide containing at least the amino acid residues from serine at position 25 to aspartic acid at position 213 in the amino acid sequence set forth in SEQ ID NO: 4, and the AAV-binding protein disclosed in WO 2021 / 106882. Examples of the substitution, deletion, insertion, or addition described in (ii) include the amino acid residue substitutions disclosed in WO 2021 / 106882.

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

[0020] The "substitution of one or several amino acid residues" in (ii) above may include not only the amino acid substitution at the specific position described above, but also conservative substitutions in which amino acids with similar physical and / or chemical properties are substituted. Those skilled in the art know that conservative substitutions generally maintain protein function between substituted and unsubstituted amino acids. 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, 9, 2005). Furthermore, the "substitution, deletion, insertion, or addition of one or several amino acid residues" in (ii) above also includes naturally occurring mutations (mutants or variants) due to differences in the origin of AAVR or differences in species.

[0021] The homology of the amino acid sequences in (iii) above may be 70% or more, but may be even higher (e.g., 80% or more, 85% or more, 90% or more, or 95% or more). As used herein, "homology" may refer to similarity or identity, and may particularly refer to identity. "Amino acid sequence homology" refers to homology across the entire amino acid sequence. "Identity" between amino acid sequences refers to the proportion of amino acid residues of the same type in those amino acid sequences (Experimental Medicine, 31(3), Yodosha). "Similarity" between amino acid sequences refers to the sum of the proportion of amino acid residues of the same type and the proportion of amino acid residues with similar side chain properties in those amino acid sequences (Experimental Medicine, 31(3), Yodosha). Amino acid sequence homology can be determined using alignment programs such as BLAST (Basic Local Alignment Search Tool) and FASTA.

[0022] The present invention is characterized in that, when AAV contained in a sample is analyzed based on the binding affinity with an AAV-binding protein, calcium ions are further added to a solution containing AAV and the AAV-binding protein. The addition of calcium ions improves the binding affinity of the AAV-binding protein to AAV, thereby enabling high-sensitivity analysis of AAV contained in the sample. The amount of calcium ions added is preferably 0.03 mM to 100 mM, and more preferably 0.3 mM to 70 mM, in terms of final concentration. The calcium ions can be added, for example, by adding a water-soluble calcium salt, such as calcium chloride or calcium nitrate.

[0023] The AAV-binding protein may be in a free form (dispersed in a solution) or in the form of an adsorbent immobilized on an insoluble carrier; however, the latter form is preferred in that it allows for simple and highly sensitive analysis of AAV contained in a sample by using B / F (Bound / Free) separation or the like.

[0024] The insoluble carrier is not particularly limited in shape or material, as long as it is insoluble in the sample containing AAV and the solution used for analysis. Examples of shapes include plates, films, fibers, particles, membranes, and hollow fibers. Among these, porous or non-porous granular, monolithic, membranous, or fibrous materials used as chromatography carriers are preferred because they allow for simple and highly sensitive analysis of AAV contained in samples. Below, we will explain in detail examples of using the aforementioned chromatography carriers as insoluble carriers.

[0025] Examples of materials for chromatography supports include supports made from polysaccharides such as agarose, alginate (alginate salts), carrageenan, chitin, cellulose, dextrin, dextran, and starch; supports made from synthetic polymers such as polyvinyl alcohol, polymethacrylate, poly(2-hydroxyethyl methacrylate), and polyurethane; and supports made from ceramics such as silica. Among these, supports made from polysaccharides and synthetic polymers are preferred as chromatography supports. Examples of preferred supports include polymethacrylate gels with introduced hydroxy groups, such as Toyopearl (manufactured by Tosoh Corporation), agarose gels such as Sepharose (manufactured by Cytiva), and cellulose gels such as Cellufine (manufactured by JNC). It is preferable that the chromatography support be shaped so that it can be packed into a column.

[0026] The AAV-binding protein can be immobilized on a chromatographic support, for example, via a covalent bond. Specifically, for example, the AAV-binding protein can be immobilized on the chromatographic support by covalently bonding the protein to the chromatographic support via an active group possessed by the chromatographic support, thereby producing the adsorbent used in the present invention (hereinafter, the adsorbent obtained by this method will also be referred to as an "AVR-immobilized gel"). Examples of the active group include an N-hydroxysuccinimide (NHS)-activated ester group, an epoxy group, a carboxy group, a maleimide group, a haloacetyl group, a tresyl group, a formyl group, and a haloacetamide group. For example, a commercially available chromatographic support having an active group may be used as is, or an active group may be introduced into the chromatographic support. Examples of commercially available chromatography supports having active groups include TOYOPEARL AF-Epoxy-650M and TOYOPEARL AF-Tresyl-650M (both manufactured by Tosoh Corporation), HiTrap NHS-activated HP Columns, NHS-activated Sepharose 4 Fast Flow, and Epoxy-activated Sepharose 6B (all manufactured by Cytiva), and SulfoLink Coupling Resin (manufactured by Thermo Fisher Scientific).

[0027] An example of a method for introducing active groups onto the surface of a chromatography support is to react one of a compound having two or more active sites with a hydroxy group, epoxy group, carboxy group, amino group, etc. present on the surface of the support.

[0028] Examples of compounds that introduce epoxy groups into hydroxyl or amino groups present on the surface of a chromatography support 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).

[0029] Examples of compounds that introduce carboxy groups into epoxy groups present on the surface of a chromatographic support include 2-mercaptoacetic acid, 3-mercaptopropionic acid, 4-mercaptobutyric acid, 6-mercaptobutyric acid, glycine, 3-aminopropionic acid, 4-aminobutyric acid, and 6-aminohexanoic acid.

[0030] Compounds that introduce maleimide groups into hydroxy groups, epoxy groups, carboxy groups, and amino groups present on the surface of a chromatographic support 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-(maleimido)phenyl isocyanate, 2-maleimidoacetic acid, 3-maleimidopropionic acid, and 4-(4-N-maleimidophenyl)acetic acid hydrazide. Examples include propionic acid, 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.

[0031] Furthermore, examples of compounds that introduce haloacetyl groups into hydroxy groups or amino groups present on the surface of a chromatographic support 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-(iodoacetamido)acetic acid-N-hydroxysuccinimide ester, 3-(bromoacetamido)propionic acid-N-hydroxysuccinimide ester, and 4-(iodoacetyl)aminobenzoic acid-N-hydroxysuccinimide ester.

[0032] Another example of a method for introducing active groups onto the surface of a chromatography support is to react hydroxyl or amino groups present on the support surface with an ω-alkenyl alkane glycidyl ether, followed by activation 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.

[0033] Another example of a method for introducing active groups onto the surface of a chromatography support is to use a condensing agent and an additive to introduce active groups into carboxy groups present on the support surface. Condensing agents include 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), dicyclohexylcarbodiamide, and carbonyldiimidazole. Additives include NHS, 4-nitrophenol, and 1-hydroxybenzotriazole.

[0034] Immobilization of an AAV-binding protein on a chromatography support 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 depending on 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 or higher and 50°C or lower, and preferably 10°C or higher and 35°C or lower.

[0035] The analytical method of the present invention may be appropriately selected depending on the type of AAV-binding protein used (e.g., free protein, adsorbent, etc.), and examples include ELISA (Enzyme-Linked Immunosorbent Assay), SPR (Surface Plasmon Resonance), and affinity chromatography using an AVR-immobilized gel prepared by the above-mentioned method. Among these, affinity chromatography is preferred because it allows for highly sensitive and simple analysis of AAV. The analytical method of the present invention using affinity chromatography is described in detail below.

[0036] The analytical method of the present invention using affinity chromatography includes the steps of adsorbing AAV contained in a sample to an AVR-immobilized gel prepared by the method described above (hereinafter simply referred to as the "adsorption step"), washing the gel with a washing solution containing calcium ions (hereinafter simply referred to as the "washing step"), and eluting the AAV adsorbed to the gel with an elution solution containing calcium ions (hereinafter simply referred to as the "elution step"). Packing the AVR-immobilized gel into a column (hereinafter also referred to as an "AVR column") is preferred because it simplifies these steps. The following describes in detail the example of using an AVR column.

[0037] A sample containing AAV can be applied to an AVR column using a liquid delivery device such as a pump. Herein, adding a liquid to a column is also referred to as "delivering a liquid to the column." The AAV-containing sample may be solvent-exchanged with an appropriate buffer solution before application to the AVR column. Furthermore, the AVR column may be equilibrated with an appropriate buffer solution (equilibration solution) before application of the AAV-containing sample to the AVR column (i.e., before the adsorption step). This equilibration is expected to enable, for example, more sensitive analysis of AAV. Examples of buffer solutions used for solvent exchange and equilibration include phosphate buffer, acetate buffer, succinate buffer, citrate buffer, Tris buffer, HEPES buffer, and MES buffer, all of which have buffering capacity in the neutral range (referring to a pH range of 4.0 to 9.0 in this specification). Such buffer solutions may further contain an inorganic salt, such as sodium chloride, at a concentration of 10 mM to 600 mM. The buffer solution used for solvent exchange and the equilibration solution may or may not be the same.

[0038] The AAV to be analyzed in the present invention may be either naturally occurring AAV or artificially produced AAV. Examples of naturally occurring AAV include serotype 1 (AAV1), serotype 2 (AAV2), serotype 3 (AAV3), serotype 4 (AAV4), serotype 5 (AAV5), serotype 6 (AAV6), serotype 7 (AAV7), serotype 8 (AAV8), serotype 9 (AAV9), serotype 10 (AAV10), serotype 11 (AAV11), serotype 12 (AAV12), and serotype 13 (AAV13). Artificially produced AAV include AAVrh8, AAVrh10, and chimeric AAVs that share characteristics (cell tropism and infectivity) of two or more of these serotypes. [Effects of the Invention]

[0039] The present invention is characterized in that analysis of adeno-associated virus (AAV) contained in a sample is performed by adding an AAV-binding protein and calcium ions to the AAV, and based on the binding affinity between the AAV and the AAV-binding protein, thereby enabling highly sensitive detection of AAV contained in a sample. [Brief explanation of the drawings]

[0040] [Figure 1] FIG. 1 shows the results of ELISA measurement of the change in binding affinity between adeno-associated virus (AAV) and AAV-binding protein due to the addition of calcium ions. [Figure 2] This figure shows the difference in the chromatographic patterns of each AAV serotype due to the addition of calcium ions to the wash solution and eluate. (a) shows the results for AAV2-EGFP, (b) for AAV5-EGFP, (c) for AAV8-EGFP, and (d) for AAV9 virus-like particles (VLP9). The dotted line shows the results without calcium ions, and the solid line shows the results with calcium ions added to a final concentration of 1 mM. [Figure 3] 1 shows the results of analyzing AAV contained in the culture supernatant and cell lysate of AAV-expressing cells after adding calcium ions in Example 10. (a) shows the results for the culture supernatant of cells that do not express AAV (Nega), (b) shows the results for the culture supernatant of AAV-expressing cells, and (c) shows the results for the cell lysate of AAV-expressing cells. [Figure 4] 1 shows the results of analysis of an AAV-containing sample after adding calcium ions in Example 11. (a) is a Western blot photograph of the applied AAV-containing sample, (b) is the chromatographic pattern of the sample, and (c) is a silver-stained photograph of the flow-through fraction. [Example]

[0041] EXAMPLES The present invention will be described in detail below using examples and comparative examples, but the present invention is not limited to these examples.

[0042] Example 1 Preparation of Adeno-Associated Virus (AAV) Vectors (1) A nucleotide sequence (SEQ ID NO: 3) was designed in which a restriction enzyme EcoRI recognition sequence (GAATTC) was added to the 5' end of a polynucleotide encoding EGFP (Enhanced Green Fluorescent Protein) consisting of the amino acid sequence set forth in SEQ ID NO: 2, and a stop codon (TAG) and a BamHI recognition sequence (GGATTC) were added to the 3' end.

[0043] (2) A polynucleotide consisting of the sequence set forth in SEQ ID NO: 3 was totally synthesized and cloned into a plasmid (commissioned to FASMAC, named pUC-EGFP). Escherichia coli JM109 strain was transformed with pUC-EGFP, and the resulting transformant was cultured. pUC-EGFP was extracted from the culture medium using a QIAprep Spin Miniprep kit (Qiagen).

[0044] (3) The pUC-EGFP obtained in (2) was digested with the restriction enzymes EcoRI and BamHI, and then ligated into the expression vector pAAV-CMV (Takara Bio Inc.) which had been previously digested with the restriction enzymes EcoRI and BamHI. The ligation product was used to transform the Escherichia coli JM109 strain.

[0045] (4) The transformant obtained in (3) was cultured overnight at 37°C with shaking in a 5-L baffled flask containing 1 L of 2YT medium (1.6% (w / v) Tryptone, 1% (w / v) Yeast Extract, 0.5% (w / v) sodium chloride) containing 100 μg / mL carbenicillin. After the culture was completed, the cells were collected by centrifugation. The EGFP-expressing vector pAAV-EGFP was prepared in large quantities from the collected cells using a Plasmid Mega Kit (Qiagen).

[0046] (5) The Escherichia coli JM109 strain was transformed with a plasmid containing a polynucleotide encoding the capsid of any of serotypes 2 (AAV2), 5 (AAV5), 8 (AAV8), and 9 (AAV9) (collectively referred to as "pRCX Vector") and pHelper Vector (Takara Bio Inc.). The resulting transformants were used in the same manner as in (4) to prepare large quantities of pRCX Vector and pHelper.

[0047] (6) HEK293T cells were cultured in eight Cellstack cell culture surface-treated 5-chamber (Corning) plates containing 500 mL of D-MEM medium (Fujifilm Wako Pure Chemical Industries, Ltd.) containing 5% (v / v) bovine serum. Gene transfection was performed by adding the pAAV-EGFP prepared in (4), the pRCX Vector and pHelper prepared in (5), and a complex of polyethyleneimine (Polysciences), and the cells were cultured statically for 3 days under conditions of 5% (v / v) carbon dioxide and 37°C.

[0048] (7) To the culture medium obtained in (6), Triton X-100 (Sigma) (final concentration 0.1%) and Benzonase (Merck Millipore) (final concentration 1 U / mL) were added to a final concentration of 0.1%, and the medium was incubated at 37°C for 3 hours. After centrifugation, the supernatant was collected and concentrated using a 300 kD-cutoff membrane cassette for a tangential flow system (Pall). The supernatant was then replaced with 20 mM Tris-HCl buffer (pH 8.0) containing 0.5 M sodium chloride (hereinafter referred to as "equilibration solution A"). The resulting concentrate was passed through a 0.22 μm pore filter to remove suspended matter.

[0049] (8) The solution from which the suspended matter had been removed was applied to a 7 mL AVB Sepharose column (manufactured by Cytiva) or a POROS AAVX column (manufactured by Thermo Fisher Scientific).

[0050] (9) After washing with equilibration solution A, the column was eluted with 0.1 M acetate buffer (pH 2.5) containing 0.5 M sodium chloride. The resulting eluted fraction containing the AAV vector was neutralized with 1 / 4 volume of 1 M Tris-HCl buffer (pH 8.5) containing 20 mM magnesium chloride to obtain solutions of the AAV vectors of each serotype: AAV2-EGFP, AAV5-EGFP, and AAV8-EGFP (hereinafter collectively referred to as "AAVX-EGFP").

[0051] (10) The AAVX-EGFP concentration in the solution obtained in (9) was quantified by qPCR using the AAVpro Titration Kit (Takara Bio Inc.).

[0052] As a result, the concentration of AAV in the AAVX-EGFP solution was 1.7 × 10 13 cp / mL (cp indicates the number of AAV particles; the same applies below), and the AAV5-EGFP solution was 2.5 × 10 13 cp / mL, and the AAV8-EGFP solution was 3.5 x 10 13 cp / mL.

[0053] In addition, the AAVX-EGFP solution was subjected to SDS-PAGE and silver stained using the Pierce Silver Stain Kit (Thermo Fisher Scientific) to confirm the purity of the AAV vector contained in the solution. As a result, only bands corresponding to the three types of outer capsid proteins (VP1, VP2, and VP3) that make up the AAV vector were observed in all solutions, confirming that there was no problem with purity.

[0054] Example 2: Preparation of VLPs AAV2 and AAV9 virus-like particles, VLP2 and VLP9, were produced by the following method.

[0055] (1) HEK293T cells were cultured in eight Cellstack cell culture surface-treated 5-chamber (Corning) plates containing 500 mL of D-MEM medium (Fujifilm Wako Pure Chemical Industries, Ltd.) containing 5% (v / v) bovine serum. Gene transfer was performed by adding the pRCX Vector prepared in Example 1(5), pHelper, and polyethyleneimine (Polysciences) complex, and the cells were cultured statically for 3 days at 37°C in 5% (v / v) carbon dioxide.

[0056] (2) To the culture medium obtained in (1), Triton X-100 (Sigma) (final concentration 0.1%) and Benzonase (Merck Millipore) (final concentration 1 U / mL) were added to a final concentration of 0.1%, and the medium was incubated at 37°C for 3 hours. After centrifugation, the supernatant was collected and concentrated using a 300 kD-cutoff membrane cassette for a tangential flow system (Pall). The supernatant was then replaced with 20 mM Tris-HCl buffer (pH 8.0) containing 0.5 M sodium chloride (hereinafter referred to as "equilibration solution A"). The resulting concentrate was passed through a 0.22 μm pore filter to remove suspended matter.

[0057] (3) The solution from which the suspended matter had been removed was applied to a 7 mL AVB Sepharose column (manufactured by Cytiva) or a POROS AAVX column (manufactured by Thermo Fisher Scientific).

[0058] (4) After washing with equilibration solution A, the column was eluted with 0.1 M acetate buffer (pH 2.5) containing 0.5 M sodium chloride. The resulting eluted fraction containing VLPs was neutralized with 1 / 4 volume of 1 M Tris-HCl buffer (pH 8.5) containing 20 mM magnesium chloride to obtain solutions of VLP2 and VLP9, which are VLPs of each serotype (hereinafter collectively referred to as "VLPs").

[0059] (5) The VLP concentration in the solution obtained in (4) was measured by DLS.

[0060] As a result, the concentration of VLP2 in the VLP solution was 3.1 × 1013 cp / mL (cp indicates the number of particles; the same applies below), and the VLP9 solution was 8.0 × 10 13 cp / mL.

[0061] In addition, the purity of the AAV vectors contained in the VLP solutions was confirmed by subjecting the VLP solutions to SDS-PAGE and silver staining using the Pierce Silver Stain Kit (Thermo Fisher Scientific). As a result, only bands corresponding to the three outer coat proteins (VP1, VP2, and VP3) that make up the VLPs were observed in all solutions, confirming that there were no problems with purity.

[0062] Example 3: Production of VLPs using suspension cells VLP8, a virus-like particle of AAV8, was cultured as follows.

[0063] (1) Viral Production Cells (Thermo Fisher Scientific) were cultured in a 250 mL Erlenmeyer flask (Corning) containing 60 mL of Viral Production Medium (Thermo Fisher Scientific). AAV-MAX Enhancer was added to the culture. Gene transfer was then performed by adding the pRCX Vector and pHelper prepared in Example 1(5) and a complex of AAV-MAX Transfection Reagent, Viral Plex Complexation, and AAV-MAX Transfection Booster (all manufactured by Thermo Fisher Scientific). The cells were then cultured at 8% (v / v) carbon dioxide and 37°C for 3 days with shaking at 120 rpm.

[0064] (2) A portion of the culture medium obtained in (1) was centrifuged at 8000 × g for 10 minutes at 4°C to precipitate the cells, and the supernatant was collected and used as a culture supernatant sample. One-tenth the volume of AAV-MAX Lysis Buffer (Thermo Fisher Scientific) was added to the remaining culture medium, and Benzonase (Merck Millipore) was added to a final concentration of 1 U / mL. Magnesium chloride was then added to a final concentration of 2 mM. The mixture was shaken at 120 rpm for 2 hours at 37°C, and then centrifuged at 8000 × g for 10 minutes at 4°C. The supernatant was used as a cell lysate sample. Both samples were passed through a 0.22 μm pore size filter to remove floating material.

[0065] Example 4 Preparation of AAV-binding proteins (part 1) (1) Escherichia coli BL21(DE3) strain was transformed with the plasmid pET-AVRwild, which contains a polynucleotide (SEQ ID NO: 7) encoding a polypeptide (SEQ ID NO: 6) containing the amino acid sequence from serine at position 312 to aspartic acid at position 500 (hereinafter also referred to as "AVRwild"), which corresponds to the region of the amino acid sequence of the AAV receptor KIAA0319L set forth in SEQ ID NO: 1, corresponding to extracellular domain 1 (PKD1) and domain 2 (PKD2). The resulting transformant capable of expressing the AAV-binding protein AVRwild was inoculated into 3 mL of 2YT liquid medium containing 50 μg / mL kanamycin and pre-cultured overnight at 37°C under aerobic shaking. In SEQ ID NO: 6, the portion from the 1st methionine (Met) to the 22nd alanine (Ala) is the PelB signal peptide, the portion from the 25th serine (Ser) to the 213th aspartic acid (Asp) is the AAV binding protein AVRwild (a polypeptide consisting of amino acid residues 312 to 500 of SEQ ID NO: 1), and the portion from the 220th cysteine ​​(Cys) to the 226th glycine (Gly) is a cysteine ​​tag sequence used as a tag for immobilization.

[0066] (2) 2 mL of the preculture solution from (1) was inoculated into 200 mL of 2YT liquid medium containing 50 μg / mL of kanamycin in a 1 L baffled flask, and the mixture was cultured aerobically at 37°C with shaking.

[0067] (3) After 2.0 hours of culturing, the mixture was cooled on ice, IPTG (IsoPropyl-β-D-ThioGalactopyranoside) was added to a final concentration of 0.1 mM, and the mixture was then cultured aerobically with shaking at 37°C for 3 hours.

[0068] (4) After the cultivation was completed, the culture medium was centrifuged at 4°C and 8000 rpm for 20 minutes to collect the bacterial cells.

[0069] (5) The 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 (cells), and then disrupted using an ultrasonic generator (Insonator 201M [Kubota Shoji Co., Ltd.]) at 8°C for approximately 10 minutes at an output of approximately 150 W. The disrupted cell solution was centrifuged twice at 8000 rpm for 20 minutes at 4°C, and the supernatant was recovered from each suspension.

[0070] (6) The supernatant obtained in (5) was applied to an XK26 / 20 column (manufactured by Cytiva) packed with 50 mL of Ni Sepharose 6 Fast Flow (manufactured by Cytiva) that had been equilibrated with Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride and 20 mM imidazole (hereinafter also referred to as "equilibration solution B"). After washing with equilibration 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.

[0071] (7) Each eluate obtained in (6) was dialyzed against 20 mM Tris buffer (pH 7.4) containing 150 mM sodium chloride to prepare the amount of AVRwild required for producing the AAV adsorbent.

[0072] Example 5 Preparation of AAV-binding proteins (part 2) The AAV binding protein AVR10s was prepared in the same manner as in Example 4, except that a transformant capable of expressing AVR10s was used, which was obtained by transforming the Escherichia coli BL21(DE3) strain with the plasmid pET-AVR10s containing a polynucleotide (SEQ ID NO: 5) encoding a polypeptide containing the AAV binding protein AVR10s consisting of the amino acid sequence set forth in SEQ ID NO: 4. In SEQ ID NO: 4, the PelB signal peptide is located from methionine (Met) at position 1 to alanine (Ala) at position 22, the AAV binding protein AVR10s is located from serine (Ser) at position 25 to aspartic acid (Asp) at position 213, and the cysteine ​​tag sequence, which is a tag for immobilization, is located from cysteine ​​(Cys) at position 220 to glycine (Gly) at position 226. Furthermore, AVR10s is a polypeptide in which the amino acid substitutions shown in (I) to (X) below have occurred relative to AVRwild (amino acid residues from serine at position 312 to aspartic acid at position 500 in SEQ ID NO: 1). (I) Valine at position 317 of SEQ ID NO: 1 (position 30 of SEQ ID NO: 4) is substituted with aspartic acid (II) Tyrosine at position 342 of SEQ ID NO: 1 (position 55 of SEQ ID NO: 4) is substituted with serine (III) Lysine at position 362 of SEQ ID NO: 1 (position 75 of SEQ ID NO: 4) is substituted with glutamic acid (IV) Lysine at position 371 of SEQ ID NO: 1 (position 84 of SEQ ID NO: 4) is substituted with asparagine (V) Valine at position 381 of SEQ ID NO: 1 (position 94 of SEQ ID NO: 4) is substituted with alanine (VI) Isoleucine at position 382 of SEQ ID NO: 1 (position 95 of SEQ ID NO: 4) is substituted with valine (VII) Glycine at position 390 of SEQ ID NO: 1 (position 103 of SEQ ID NO: 4) is substituted with serine (VIII) Lysine at position 399 of SEQ ID NO: 1 (position 112 of SEQ ID NO: 4) is substituted with glutamic acid (IX) Serine at position 476 of SEQ ID NO: 1 (position 189 of SEQ ID NO: 4) is substituted with arginine (X) Asparagine at position 487 of SEQ ID NO: 1 (position 200 of SEQ ID NO: 4) is substituted with aspartic acid Example 6 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 with iodoacetamide groups was prepared by chemically modifying the hydroxyl groups on the polymer surface.

[0073] (2) To 7 g (wet weight) of the carrier prepared in (1), 14 mg of AVRwild prepared in Example 4 or AVR10s prepared in Example 5 and TCEP (Tris(2-CarboxyEthyl)Phosphine) at a final concentration of 0.3 mM as a reducing agent were added, and the mixture was allowed to react by shaking at pH 8.1 and 25°C for 3 hours. This resulted in the preparation of an AVRwild-immobilized gel, which is an AAV adsorbent in which AVRwild is immobilized, and an AVR10s-immobilized gel, which is an AAV adsorbent in which AVR10s is immobilized.

[0074] Example 7: Evaluation of the effect of calcium ion addition on the binding affinity between AAV and AAV-binding protein (part 1) The effect of calcium ion addition on the binding affinity between AAV and AAV-binding proteins was evaluated using the ELISA method described below.

[0075] (1) The AAV virus-like particles (VLP2 or VLP9) prepared in Example 2 were diluted 200-fold with 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride, added to a 96-well microplate (Thermo Fisher Scientific) at 100 μL / well, and immobilized (at 4°C for 18 hours).

[0076] (2) After immobilization, the membrane was blocked with 20 mM Tris-HCl buffer (pH 7.4) containing 2% (w / v) skim milk (Becton Dickinson) and 150 mM sodium chloride.

[0077] (3) After washing with a washing buffer (20 mM Tris-HCl buffer (pH 7.4) containing 0.05% [w / v] Tween 20 (trade name) and 150 mM sodium chloride), the AAV binding protein AVR10s (Example 5) prepared at 0.01 mg / mL in 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride with or without 10 mM calcium chloride was added to react AVR10s with VLP2 or VLP9 (30°C, 1 hour).

[0078] (4) After the reaction was completed, the plate was washed with the washing buffer, and 100 μL / well of Anti-6His antibody (manufactured by Bethyl Laboratories) diluted to 100 ng / mL was added.

[0079] (5) After incubation at 30°C for 1 hour, the plate was washed with the washing buffer and TMB Peroxidase Substrate (KPL) was added at 50 μL / well. Color development was stopped by adding 50 μL / well of 1 M phosphoric acid, and the absorbance at 450 nm was measured using a microplate reader (Tecan).

[0080] The results are shown in Figure 1. For both VLP2 and VLP9, the addition of calcium chloride (calcium ions) to a final concentration of 10 mM increased the absorbance at 450 nm, indicating improved binding affinity with AVR10s.

[0081] Example 8: Evaluation of the effect of calcium ion addition on the binding affinity between AAV and AAV-binding protein (part 2) The effect of calcium ion addition on the binding affinity between AAV and AAV-binding proteins was evaluated using the SPR (surface plasmon resonance) method described below.

[0082] (1) AAV (VLP2 [Example 2] or AAV5-EGFP [Example 1]) was immobilized on a CM5 sensor chip (manufactured by Cytiva) using the amine coupling method.

[0083] (2) AAV binding protein (AVRwild [Example 4] or AVR10s [Example 5]) was diluted (concentration: 8 concentrations ranging from 0 μM to 5 μM) with 20 mM HEPES buffer (pH 7.4) containing 150 mM sodium chloride with or without 10 mM calcium chloride.

[0084] (3) The diluted solution in (2) was subjected to SPR measurement using a Biacore 8K (manufactured by Cytiva) according to the following method. The running buffer used in this measurement was the same as that used to dilute the AAV-binding protein. (3-1) The AAV-binding protein solutions prepared in (2) were injected at various concentrations at a flow rate of 30 μL / min for 6 minutes, allowing the AAV-binding protein to bind to the AAV-immobilized sensor chip prepared in (1). (3-2) AAV-binding proteins bound to the sensor chip were dissociated by injecting 10 mM glycine-hydrochloric acid buffer (pH 3.0) at a flow rate of 30 μL / min for 30 seconds. (3-3) The obtained results were analyzed using the software provided with the Biacore 8K.

[0085] The results are shown in Table 1. When any of the AAVs (VLP2 and AAV5) and any of the AAV binding proteins (AAVwild and AVR10s) were used, the addition of calcium chloride (calcium ions) to a final concentration of 10 mM resulted in a K D The value decreased, indicating that the binding affinity between AAV and the AAV-binding protein improved.

[0086] [Table 1]

[0087] Example 9 Chromatography using an AAVR-immobilized column (Part 1) (1) 1.25 mL of the AVR10s immobilized gel prepared in Example 6 was packed into an empty stainless steel column (φ4.6 mm×75 mm, manufactured by Tosoh Corporation) to prepare a column (designated AVR10s column).

[0088] (2) The AAVR column prepared in (1) was connected to an HPLC M40A (Shimadzu Corporation) and equilibrated with a mixture of 99.99% 10 mM sodium acetate buffer (pH 5.0) containing 150 mM sodium chloride (hereinafter also referred to as "eluent A") and 0.01% 1 M calcium chloride (hereinafter also referred to as "eluent B") (final concentration of calcium ions: 1 mM).

[0089] (3) The column equilibrated in (2) was charged with the AAVX-EGFP solution ((a) AAV2-EGFP solution, (b) AAV5-EGFP solution, and (c) AAV8-EGFP solution) obtained in Example 1 and the (d) VLP9 solution (concentration: 4.0 × 10) obtained in Example 2. 13 0.01 mL of either 0.01 cp / mL was applied.

[0090] (4) After washing for 10 minutes at a flow rate of 0.5 mL / min with a mixture of 99.99% eluent A and 0.01% eluent B, the column was pumped through a linear gradient of 0% to 99.99% eluent C over 50 minutes using 10 mM glycine buffer (pH 2.0) containing 150 mM sodium chloride (hereinafter referred to as "eluent C") and eluent A (flow rate 0.5 mL / min). After the linear gradient, a mixture of 0.01% eluent B and 99.99% eluent C was pumped for 10 minutes, and then a mixture of 0.01% eluent B and 99.99% eluent A was pumped through the AVR10s column for re-equilibration.

[0091] (5) Each AAVX-EGFP or VLP9 eluted from the AVR10s column was detected by fluorescence intensity at 350 nm with excitation light at 280 nm.

[0092] Comparative Example 1 Chromatography using an AAVR-immobilized column (Part 2) AAV analysis was carried out using an AVR10s column in the same manner as in Example 7, except that eluent B was not used (i.e., calcium ions were not added).

[0093] The chromatographic patterns obtained in Example 9 and Comparative Example 1 are shown together in Figure 2. By adding calcium ions to a final concentration of 1 mM to the wash solution and elution solution, the peaks became sharper than when calcium ions were not added, indicating that AAV could be detected with higher sensitivity.

[0094] Example 10 Chromatography using an AAVR-immobilized column (Part 3) (1) The AVR10s column prepared in Example 9(1) was connected to an HPLC M40A (Shimadzu Corporation) and equilibrated with 10 mM glycine buffer (pH 4.5) containing 50 mM calcium chloride and 15 mM sodium acetate (hereinafter also referred to as "eluent D").

[0095] (2) To the column equilibrated in (1), 0.2 mL of either (a) the culture supernatant of HEK293F (before transfection), (b) the culture supernatant of VLP8-expressing HEK293F prepared in Example 3, or (c) the cell lysate was applied.

[0096] (3) After washing with eluent D at a flow rate of 0.5 mL / min for 10 minutes, eluent D was pumped through the AVR10s column at a flow rate of 0.5 mL / min, using a 10 mM glycine buffer (pH 2.5) containing 50 mM calcium chloride and 15 mM sodium acetate (hereinafter also referred to as "eluent E") and eluent D, so that the concentration of eluent E increased from 0% to 100% over 50 minutes. After the linear gradient was completed, eluent E was pumped for 5 minutes, and then eluent D was pumped through the AVR10s column again to re-equilibrate.

[0097] (4) VLP8 eluted from the AVR10s column was detected by fluorescence intensity at 350 nm in response to excitation light at 280 nm.

[0098] The resulting chromatographic pattern is shown in Figure 3. By adding calcium ions to the eluent, It is clear that AAV contained in the culture supernatant and cell extract of AAV-expressing cells can be analyzed.

[0099] Example 11 Chromatography using an AAVR-immobilized column (Part 4) (1) The AVR10s column prepared in Example 9(1) was connected to an HPLC M40A (Shimadzu Corporation) and equilibrated with 10 mM glycine buffer (pH 4.5) containing 50 mM calcium chloride and 10 mM sodium acetate (hereinafter also referred to as "eluent F").

[0100] (2) 0.01 mL of the elution fraction (the Western blot image shown in Figure 4(a)) obtained from an externally obtained AAV culture medium in accordance with Example 1 on a POROS AAVX column (manufactured by Thermo Fisher Scientific) was applied to the AVR10s column equilibrated in (1).

[0101] (3) After washing with eluent F for 10 minutes at a flow rate of 0.5 mL / min, 10 mM glycine buffer (pH 2.5) containing 50 mM calcium chloride and 10 mM sodium acetate (hereinafter also referred to as "eluent G") and eluent F were pumped onto the AVR10s column to achieve a linear gradient of eluent G concentration from 0% to 100% over 50 minutes. After the linear gradient was completed, eluent E was pumped for 5 minutes, and then eluent F was pumped onto the AVR10s column again to re-equilibrate.

[0102] (4) AAV eluted from the AVR10s column was detected by fluorescence intensity at 350 nm in response to excitation light at 280 nm.

[0103] The resulting chromatographic pattern is shown in Figure 4(b). By adding calcium ions to the wash and eluent, peaks corresponding to heterogeneous AAV were confirmed (Figure 4(b)). In addition, a silver-stained photograph of the flow-through fraction of the chromatographic pattern (Figure 4(c)) confirmed bands corresponding to contaminants.

Claims

1. A method for analyzing adeno-associated virus (AAV) contained in a sample based on binding affinity to an AAV-binding protein, comprising: The AAV binding protein is a polypeptide selected from any one of the following (i) to (iii): (i) a polypeptide containing at least the amino acid residues from serine at position 312 to aspartic acid at position 500 in the amino acid sequence set forth in SEQ ID NO: 1; (ii) A polypeptide having an amino acid sequence containing 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, with the proviso that the amino acid sequence contains a substitution, deletion, insertion, or addition of 1 to 10 amino acid residues in the amino acid residues at positions 312 to 500, and having AAV-binding activity; (iii) a polypeptide comprising at least the amino acid residues from the serine at position 312 to the aspartic acid at position 500 of the amino acid sequence set forth in SEQ ID NO: 1, and having 90% or more homology to the amino acid sequence consisting of the amino acid residues at positions 312 to 500, and having AAV-binding activity; The method further comprises adding calcium ions to a solution containing the AAV and the AAV-binding protein and then analyzing the solution.

2. The method according to claim 1, wherein the AAV binding protein is in the form of an adsorbent comprising an insoluble carrier and the AAV binding protein immobilized on the carrier.

3. The method according to claim 2, comprising the steps of: adsorbing AAV contained in a sample to an adsorbent comprising an insoluble carrier and an AAV-binding protein immobilized on the carrier; washing the adsorbent with a washing solution containing calcium ions; and eluting the AAV adsorbed to the adsorbent with an elution solution containing calcium ions.

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