Method for stabilizing adeno-associated virus binding proteins

Abstract

To provide a method for improving the stability of adeno-associated virus (AAV) binding protein, particularly its thermal stability.SOLUTION: The foregoing problem is solved by adding calcium ions to a solution including adeno-associated virus (AAV) binding protein.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for stabilizing an adeno-associated virus (AAV) binding protein, and in particular to a method for improving the heat stability of 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 recovering and purifying AAV vectors is an affinity chromatography method based on the binding affinity for AAV, which uses an adsorbent comprising an insoluble carrier and an AAV-binding protein immobilized on the carrier, and can recover and purify the vector from a sample containing the AAV vector in the presence of contaminants (Patent Document 1). However, when attempting to recover and purify AAV vectors in large quantities using the method described in Patent Document 1, there was a problem with the stability of the AAV-binding protein used as the ligand protein for the adsorbent. [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 improving the stability, particularly heat stability, of adeno-associated virus binding proteins. [Means for solving the problem]

[0009] As a result of intensive 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 an adeno-associated virus (AAV) binding protein, and thus completed the present invention.

[0010] That is, the present invention includes the following aspects: [1] A method for improving the heat stability of an AAV binding protein, the method comprising further adding calcium ions to a solution containing the protein.

[0011] [2] The method according to [1], 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.

[0012] The present invention will be described in detail below.

[0013] In the present invention, AAV 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). Examples of artificially produced AAV include AAVrh8, AAVrh10, and chimeric AAVs that have characteristics (cell tropism and infectivity) of two or more of these serotypes.

[0014] 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.

[0015] 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.

[0016] 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.

[0017] 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: 2, 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.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] The present invention is characterized in that calcium ions are further added to a solution containing an AAV-binding protein to improve the heat stability of the protein. The amount of calcium ions added is preferably 0.03 mM to 100 mM in final concentration, more preferably 0.3 mM to 70 mM in final concentration, and even more preferably 3 mM to 30 mM in final concentration. Calcium ions can be added, for example, by adding a water-soluble calcium salt. Examples of water-soluble calcium salts include calcium chloride and calcium nitrate. [Effects of the Invention]

[0022] The present invention is characterized by improving the heat stability of an adeno-associated virus (AAV)-binding protein by further adding calcium ions to a solution containing the protein. The AAV-binding protein stabilized by the present invention can be used to separate AAV from a solution based on its binding affinity to the AAV, enabling large-scale recovery and purification of AAV. [Brief explanation of the drawings]

[0023] [Figure 1] 1 shows the results of evaluating the heat resistance of adeno-associated virus (AAV) binding proteins with the addition of metal ions using Optim 2 (product name). (a) shows the results when calcium ions were added (Example 3), and (b) shows the results when magnesium ions were added (Comparative Example 1). [Figure 2] This shows the results of ELISA measurement of the change in binding affinity between AAV and AAV-binding proteins due to the addition of calcium ions. [Example]

[0024] The present invention will be described in detail below using examples and comparative examples, but the present invention is not limited to these examples. In addition, the reference examples do not constitute the present invention.

[0025] Example 1 Preparation of AAV binding proteins (1) E. coli BL21(DE3) was transformed with the plasmid pET-AVR10s, which contains a polynucleotide (SEQ ID NO: 3) encoding a polypeptide containing the AAV-binding protein AVR10s, consisting of the amino acid sequence set forth in SEQ ID NO: 2. The resulting transformant capable of expressing AVR10s was inoculated into 3 mL of 2YT liquid medium containing 50 μg / mL kanamycin and precultured overnight at 37°C under aerobic shaking. In SEQ ID NO: 2, the PelB signal peptide is located from methionine (Met) 1 to alanine (Ala) 22, the AAV-binding protein AVR10s is located from serine (Ser) 25 to aspartic acid (Asp) 213, and the cysteine ​​tag sequence, which is used as a tag for immobilization, is located from cysteine ​​(Cys) 220 to glycine (Gly) 226. Furthermore, AVR10s is a polypeptide in which the amino acid substitutions shown in (I) to (X) below have occurred in the amino acid residues from serine 312 to aspartic acid 500 of SEQ ID NO: 1, which is the region corresponding to extracellular domain 1 and domain 2 of KIAA0319L (SEQ ID NO: 1). (I) Valine at position 317 of SEQ ID NO: 1 (position 30 of SEQ ID NO: 2) is substituted with aspartic acid (II) Tyrosine at position 342 of SEQ ID NO: 1 (position 55 of SEQ ID NO: 2) is substituted with serine (III) Lysine at position 362 of SEQ ID NO: 1 (position 75 of SEQ ID NO: 2) is substituted with glutamic acid (IV) Lysine at position 371 of SEQ ID NO: 1 (position 84 of SEQ ID NO: 2) is substituted with asparagine (V) Valine at position 381 of SEQ ID NO: 1 (position 94 of SEQ ID NO: 2) is substituted with alanine (VI) Isoleucine at position 382 of SEQ ID NO: 1 (position 95 of SEQ ID NO: 2) is substituted with valine (VII) Glycine at position 390 of SEQ ID NO: 1 (position 103 of SEQ ID NO: 2) is substituted with serine (VIII) Lysine at position 399 of SEQ ID NO: 1 (position 112 of SEQ ID NO: 2) is substituted with glutamic acid (IX) Serine at position 476 of SEQ ID NO: 1 (position 189 of SEQ ID NO: 2) is substituted with arginine (X) Asparagine at position 487 of SEQ ID NO: 1 (position 200 of SEQ ID NO: 2) is substituted with aspartic acid (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.

[0026] (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.

[0027] (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.

[0028] (5) The bacterial cells collected in (4) were suspended in 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride and 20 mM imidazole (hereinafter also referred to as "Buffer A") at 5 mL / 1 g (bacterial 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 bacterial cell solution was centrifuged twice at 8000 rpm for 20 minutes at 4°C, and the supernatant was collected.

[0029] (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 previously equilibrated with buffer A. After washing with buffer A, the column was eluted with 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride and 0.5 M imidazole.

[0030] (7) The eluate obtained in (6) was dialyzed against 20 mM Tris buffer (pH 7.4) containing 150 mM sodium chloride to prepare AVR10s.

[0031] Example 2: Effect of calcium ion addition on the heat resistance of AAV binding proteins (part 1) The effect of calcium ion addition on the heat resistance of AAV binding proteins was evaluated using differential scanning calorimetry (DSC).

[0032] (1) AVR10s obtained in Example 1 was prepared at a concentration of 1 mg / mL using 20 mM HEPES (4-(2-HydroxyEthyl)-1-PiperazineEthaneSulfonic acid) buffer (pH 7.4) containing 150 mM sodium chloride with or without the addition of calcium chloride to a final concentration of 10 mM.

[0033] (2) The solution containing AVR10s prepared in (1) was used to measure the denaturation temperature Tm (°C) of AVR10s due to temperature increase using a DSC manufactured by Malvern Panalytical, and the stability of the protein was evaluated.

[0034] As a result, the denaturation temperature Tm (°C) was 62°C when calcium chloride was added at a final concentration of 10 mM, and 48.7°C when calcium chloride was not added. These results demonstrate that the thermal stability of the AAV-binding protein is improved by further adding calcium ions to the solution containing the protein.

[0035] Example 3: Effect of calcium ion addition on the heat resistance of AAV binding proteins (part 2) In Example 2, the heat resistance of the AAV binding protein was evaluated by DSC, but in this example, the evaluation was performed using Optim 2 (manufactured by Avacta).

[0036] (1) The solution containing the AAV-binding protein obtained in Example 1 was adjusted to a concentration of 0.1 mg / mL using 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride (hereinafter also referred to as "Buffer B") to which calcium chloride was added at a final concentration of 0.1 mM, 1 mM, or 10 mM.

[0037] (2) The solution containing AVR10s prepared in (1) was used to obtain a denaturation curve of AVR10s due to temperature increase using Optim 2 manufactured by Avacta.

[0038] (3) As a negative control, an AAV-binding protein solution (without calcium chloride added) prepared at 0.1 mg / mL in buffer B and buffer B itself were measured in the same manner as in (2), and denaturation curves were obtained.

[0039] Comparative Example 1: Effect of magnesium ion addition on the heat resistance of AAV binding proteins A denaturation curve was obtained in the same manner as in Example 3, except that magnesium chloride was used instead of calcium chloride.

[0040] The denaturation curves obtained in Example 3 and Comparative Example 1 are shown in Figure 1. It can be seen that adding calcium ions (calcium chloride) to a solution containing an AAV-binding protein improves the thermal stability of the protein. Furthermore, it can be seen that increasing the amount of calcium ions (calcium chloride) added improves the thermal stability of the AAV-binding protein in a concentration-dependent manner (Example 3, Figure 1(a)).

[0041] On the other hand, when magnesium ions (magnesium chloride) were added to a solution containing an AAV-binding protein (Comparative Example 1), the thermal stability of the protein was only slightly improved when added to a final concentration of 10 mM, and no improvement in thermal stability due to the addition of magnesium ions was confirmed (Comparative Example 1, Figure 1(b)).

[0042] Reference Example 1: Preparation of VLPs (virus-like particles) (1) Escherichia coli JM109 strain was transformed with a plasmid containing a polynucleotide encoding the capsid of serotype 2 (AAV2) or serotype 9 (AAV9) (hereinafter collectively referred to as "pRCX Vector") and pHelper Vector (Takara Bio).

[0043] (2) The resulting transformants were 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. Using a Plasmid Mega Kit (Qiagen), EGFP-expressing vectors pRCX Vector and pHelper were prepared in large quantities from the collected cells.

[0044] (3) 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 (2), pHelper, and polyethyleneimine (Polysciences) complex, and the cells were cultured statically for 3 days at 37°C in 5% (v / v) carbon dioxide.

[0045] (4) Triton X-100 (Sigma-Aldrich) and Benzonase (Merck Millipore) (final concentration 1 U / mL) were added to the culture medium obtained in (3) to a final concentration of 0.1% (v / v), and the medium was left to stand at 37°C for 3 hours and then centrifuged.

[0046] (5) The supernatant was collected and concentrated using a 300 kD cutoff membrane cassette for a tangential flow system (Pall Corporation). The supernatant was then replaced with 20 mM Tris-HCl buffer (pH 8.0) containing 0.5 M sodium chloride (hereinafter also referred to as "Buffer C"). The resulting concentrate was passed through a 0.22 μm pore size filter to remove suspended matter.

[0047] (6) 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).

[0048] (7) After washing with buffer C, 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").

[0049] (8) The concentration of VLPs in the solution obtained in (7) was measured by DLS (dynamic light scattering). The solution was subjected to SDS-PAGE (SDS polyacrylamide gel electrophoresis), and the purity of VLPs in the solution was confirmed by silver staining using a Pierce Silver Stain Kit (Thermo Fisher Scientific).

[0050] The concentration of VLP in the solution was measured, and the VLP2 solution was 3.1 × 10 13 cp / mL (cp indicates the number of particles; the same applies below), and the VLP9 solution was 8.0 × 10 13 Furthermore, when purity was confirmed by SDS-PAGE and silver staining, only bands corresponding to the three outer coat proteins (VP1, VP2, and VP3) that make up VLP were observed in all solutions, confirming that there were no problems with purity.

[0051] Reference Example 2: Evaluation of the effect of calcium ion addition on the binding affinity between AAV and AAV-binding proteins The effect of calcium ion addition on the binding affinity between AAV and AAV-binding proteins was evaluated using the ELISA method described below.

[0052] (1) The AAV virus-like particles (VLP2 or VLP9) prepared in Reference Example 1 were diluted 200-fold with buffer B, and then added to a 96-well microplate (Thermo Fisher Scientific) at 100 μL / well and immobilized (at 4°C for 18 hours).

[0053] (2) After immobilization, the cells were blocked with 2% (w / v) skim milk (Becton Dickinson) and buffer B.

[0054] (3) After washing with a washing buffer (Buffer B containing 0.05% [w / v] Tween 20 (trade name)), the AAV binding protein AVR10s, prepared at a concentration of 0.01 mg / mL in Buffer B containing 10 mM calcium chloride or not, was added to react AVR10s with VLP2 or VLP9 (at 30°C for 1 hour).

[0055] (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.

[0056] (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).

[0057] The results are shown in Figure 2. For both VLP2 and VLP9, the addition of calcium ions (calcium chloride) to a final concentration of 10 mM increased the absorbance at 450 nm, indicating improved binding affinity with AVR10s. These results demonstrate that adding calcium ions to a solution containing an AAV-binding protein does not reduce the binding affinity between the protein and AAV.

Claims

1. A method for improving the thermal stability of an adeno-associated virus (AAV) binding protein selected from any one of the following (i) to (iii), comprising the step of contacting a solution containing the protein with calcium ions: (i) A polypeptide comprising the amino acid residues from the 312th serine to the 500th aspartic acid in the amino acid sequence set forth in SEQ ID NO: 1, with the amino acid substitutions (I) to (X) for the amino acid residues: (I) The amino acid residue corresponding to the 317th valine residue in SEQ ID NO: 1 is substituted with an aspartic acid residue (II) The amino acid residue corresponding to the tyrosine residue at position 342 of SEQ ID NO: 1 is substituted with a serine residue. (III) The amino acid residue corresponding to the lysine residue at position 362 of SEQ ID NO: 1 is substituted with a glutamic acid residue (IV) The amino acid residue corresponding to the lysine residue at position 371 of SEQ ID NO: 1 is substituted with an asparagine residue. (V) the amino acid residue corresponding to the 381st valine residue of SEQ ID NO: 1 is substituted with an alanine residue (VI) The amino acid residue corresponding to the isoleucine residue at position 382 of SEQ ID NO: 1 is substituted with a valine residue. (VII) The amino acid residue corresponding to the glycine residue at position 390 of SEQ ID NO: 1 is substituted with a serine residue (VIII) The amino acid residue corresponding to the lysine residue at position 399 of SEQ ID NO: 1 is substituted with a glutamic acid residue (IX) The amino acid residue corresponding to the serine residue at position 476 of SEQ ID NO: 1 is substituted with an arginine residue. (X) an amino acid residue corresponding to the asparagine residue at position 487 of SEQ ID NO: 1 is substituted with an aspartic acid residue; (ii) 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, with the amino acid substitutions shown in (I) to (X) above for the amino acid residues at positions 312 to 500, and further comprising substitutions, deletions, insertions, or additions of 1 to 10 amino acid residues, and having AAV-binding activity; (iii) A polypeptide having 90% or more identity to an amino acid sequence 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, provided that the amino acid residues at positions 312 to 500 have any of the amino acid substitutions (I) to (X) set forth above, and having AAV-binding activity.

2. A solution comprising an adeno-associated virus (AAV) binding protein selected from any one of the following (i) to (iii) and calcium ions: (i) A polypeptide comprising the amino acid residues from the 312th serine to the 500th aspartic acid in the amino acid sequence set forth in SEQ ID NO: 1, with the amino acid substitutions (I) to (X) for the amino acid residues: (I) The amino acid residue corresponding to the 317th valine residue in SEQ ID NO: 1 is substituted with an aspartic acid residue (II) The amino acid residue corresponding to the tyrosine residue at position 342 of SEQ ID NO: 1 is substituted with a serine residue. (III) The amino acid residue corresponding to the lysine residue at position 362 of SEQ ID NO: 1 is substituted with a glutamic acid residue (IV) The amino acid residue corresponding to the lysine residue at position 371 of SEQ ID NO: 1 is substituted with an asparagine residue. (V) the amino acid residue corresponding to the 381st valine residue of SEQ ID NO: 1 is substituted with an alanine residue (VI) The amino acid residue corresponding to the isoleucine residue at position 382 of SEQ ID NO: 1 is substituted with a valine residue. (VII) The amino acid residue corresponding to the glycine residue at position 390 of SEQ ID NO: 1 is substituted with a serine residue (VIII) The amino acid residue corresponding to the lysine residue at position 399 of SEQ ID NO: 1 is substituted with a glutamic acid residue (IX) The amino acid residue corresponding to the serine residue at position 476 of SEQ ID NO: 1 is substituted with an arginine residue. (X) an amino acid residue corresponding to the asparagine residue at position 487 of SEQ ID NO: 1 is substituted with an aspartic acid residue; (ii) 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, with the amino acid substitutions shown in (I) to (X) above for the amino acid residues at positions 312 to 500, and further comprising substitutions, deletions, insertions, or additions of 1 to 10 amino acid residues, and having AAV-binding activity; (iii) A polypeptide having 90% or more identity to an amino acid sequence 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, provided that the amino acid residues at positions 312 to 500 have any of the amino acid substitutions (I) to (X) set forth above, and having AAV-binding activity.

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