Adeno-associated virus (AAV) adsorbent and method for purifying AAV using the adsorbent

Abstract

An object of the present invention is to provide an insoluble carrier capable of purifying adeno-associated virus (AAV) with high efficiency.SOLUTION: The adsorbent contains an insoluble carrier and an adeno-associated virus (AAV) -binding protein immobilized on the carrier, wherein the insoluble carrier has pores with a diameter of 150 nm or more and 1000 nm or less.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to an adeno-associated virus (AAV) adsorbent and a method for purifying AAV using the adsorbent. [Background technology]

[0002] In recent years, in the field of biopharmaceuticals, such as antibody drugs, remarkable progress has been made in the expression technology of target substances such as proteins, which has led to a demand for improved productivity in purification processes using chromatography, etc. In particular, there is a growing demand for highly accurate separation and purification methods that can be used for viral vectors and virus-like particles, which have large molecular sizes.

[0003] Generally, in purification using a column packed with porous particles, the larger the pore size, the less the back pressure of the column increases, enabling highly efficient purification under high linear velocity conditions. However, the larger the pore size, the smaller the surface area of ​​the porous particles, and the fewer functional groups there are on the particle surface, which raises concerns about a decrease in the amount of adsorption of the target substance.

[0004] Adeno-associated viruses (AAVs) are 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, muscle, and nerve cells. They are 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, AAVs are attracting attention as vectors for gene transfer aimed at treating congenital genetic diseases.

[0005] Patent Document 1 discloses a method for easily purifying AAV to a high degree of purity, which involves affinity chromatography using an AAV adsorbent containing an insoluble carrier and an AAV-binding protein (a protein having binding activity to AAV) immobilized on the carrier.

[0006] However, there have been no reports on improving the efficiency of AAV purification using insoluble supports with large pore sizes, and no investigations have been conducted into purification under high linear velocity conditions. [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 objective of the present invention is to provide an insoluble carrier that enables highly efficient purification of adeno-associated virus (AAV). [Means for solving the problem]

[0009] As a result of extensive investigations to solve the above problems, the present inventors have discovered purification conditions that enable highly efficient purification of adeno-associated virus (AAV) even when an insoluble carrier with a large pore size is used, and have completed the present invention. That is, the present invention encompasses the following aspects.

[0010] [1] An adeno-associated virus (AAV) adsorbent comprising an insoluble carrier and an AAV-binding protein immobilized on the carrier, The adsorbent, wherein the insoluble carrier has pores with diameters of 150 nm or more and 1000 nm or less.

[0011] [2] The adsorbent according to [1] above, wherein the AAV-binding protein is a polypeptide selected from any one of the following (i) to (iii) or a polypeptide comprising at least a heavy chain antibody variable region capable of binding to AAV: (i) a polypeptide containing at least the amino acid residues from the serine residue at position 312 to the aspartic acid residue at position 500 in the amino acid sequence set forth in SEQ ID NO: 1; (ii) a polypeptide comprising at least the amino acid residues from the serine residue at position 312 to the aspartic acid residue at position 500 of the amino acid sequence set forth in SEQ ID NO: 1, wherein the amino acid residues at positions 312 to 500 have undergone any one or more of substitution, deletion, insertion, and addition of one or more amino acid residues at one or more positions, and has AAV-binding activity; (iii) A polypeptide comprising at least the amino acid residues from the serine residue at position 312 to the aspartic acid residue 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 entire amino acid sequence consisting of the amino acid residues at positions 312 to 500, and wherein the polypeptide has AAV-binding activity.

[0012] [3] A method for purifying or analyzing AAV, comprising: applying a sample containing AAV to a column packed with the AAV adsorbent to adsorb the AAV to the adsorbent; applying an eluate to the column to elute the AAV adsorbed to the adsorbent, The AAV adsorbent is the adsorbent according to [1] or [2] above, The method, wherein the linear velocity when the sample is applied to the column is 1200 cm / h or more and 3500 cm / h or less.

[0013] [4] The adsorbent according to any one of [1] to [3] above, wherein the AAV-binding protein is a polypeptide selected from the following (iv) to (vi): (iv) A polypeptide having an amino acid sequence from the serine residue at position 312 to the aspartic acid residue at position 500 of the amino acid sequence set forth in SEQ ID NO: 1, which contains one or more amino acid substitutions selected from the following (1) to (29): (1) The amino acid residue corresponding to the 317th valine residue in SEQ ID NO: 1 is substituted with an aspartic acid residue (2) The amino acid residue corresponding to the asparagine residue at position 324 of SEQ ID NO: 1 is substituted with a histidine residue (3) The amino acid residue corresponding to the 326th valine residue of SEQ ID NO: 1 is substituted with an alanine residue (4) The amino acid residue corresponding to the asparagine residue at position 329 of SEQ ID NO: 1 is substituted with a lysine residue (5) The amino acid residue corresponding to the alanine residue at position 330 of SEQ ID NO: 1 is substituted with a valine residue (6) The amino acid residue corresponding to the 334th glutamine residue of SEQ ID NO: 1 is substituted with a leucine residue (7) The amino acid residue corresponding to the 335th glutamic acid residue of SEQ ID NO: 1 is substituted with a valine residue (8) The amino acid residue corresponding to the threonine residue at position 341 of SEQ ID NO: 1 is substituted with an alanine residue (9) The amino acid residue corresponding to the tyrosine residue at position 342 of SEQ ID NO: 1 is substituted with a serine residue (10) The amino acid residue corresponding to the lysine residue at position 362 of SEQ ID NO: 1 is substituted with a glutamic acid residue (11) The amino acid residue corresponding to the isoleucine residue at position 366 of SEQ ID NO: 1 is substituted with a phenylalanine residue (12) The amino acid residue corresponding to the lysine residue at position 371 of SEQ ID NO: 1 is substituted with an asparagine residue (13) The amino acid residue corresponding to the phenylalanine residue at position 379 of SEQ ID NO: 1 is substituted with a tyrosine residue (14) The amino acid residue corresponding to the lysine residue at position 380 of SEQ ID NO: 1 is substituted with an arginine residue (15) The amino acid residue corresponding to the 381st valine residue of SEQ ID NO: 1 is substituted with an alanine residue (16) The amino acid residue corresponding to the isoleucine residue at position 382 of SEQ ID NO: 1 is substituted with a valine residue (17) The amino acid residue corresponding to the 390th glycine residue of SEQ ID NO: 1 is substituted with a serine residue (18) The amino acid residue corresponding to the 394th valine residue of SEQ ID NO: 1 is substituted with an alanine residue (19) The amino acid residue corresponding to the lysine residue at position 399 of SEQ ID NO: 1 is substituted with a glutamic acid residue (20) The amino acid residue corresponding to the 401st glutamic acid residue of SEQ ID NO: 1 is substituted with a glycine residue (21) The amino acid residue corresponding to the 426th threonine residue of SEQ ID NO: 1 is substituted with an alanine residue (22) The amino acid residue corresponding to the lysine residue at position 455 of SEQ ID NO: 1 is substituted with an arginine residue (23) The amino acid residue corresponding to the 461st alanine residue of SEQ ID NO: 1 is substituted with a proline residue (24) The amino acid residue corresponding to the lysine residue at position 467 of SEQ ID NO: 1 is substituted with an asparagine residue (25) The amino acid residue corresponding to the serine residue at position 476 of SEQ ID NO: 1 is substituted with an arginine residue (26) The amino acid residue corresponding to the serine residue at position 482 of SEQ ID NO: 1 is substituted with a threonine residue (27) The amino acid residue corresponding to the asparagine residue at position 487 of SEQ ID NO: 1 is substituted with an aspartic acid residue (28) The amino acid residue corresponding to the asparagine residue at position 492 of SEQ ID NO: 1 is substituted with an aspartic acid residue (29) the amino acid residue corresponding to the lysine residue at position 497 of SEQ ID NO: 1 is replaced with a glutamic acid residue; (v) a polypeptide having an amino acid sequence comprising amino acid residues from the serine residue at position 312 to the aspartic acid residue at position 500 of the amino acid sequence set forth in SEQ ID NO: 1, which contains one or more amino acid substitutions selected from (1) to (29), and further including, in addition to the amino acid substitutions, any one or more of substitutions, deletions, insertions, and additions of one or more amino acid residues at one or more positions, and which has AAV-binding activity; (vi) A polypeptide having an amino acid sequence from the serine residue at position 312 to the aspartic acid residue at position 500 of the amino acid sequence set forth in SEQ ID NO: 1, which has an identity of 70% or more to the entire amino acid sequence containing one or more amino acid substitutions selected from any of (1) to (29), in which the amino acid sequence maintains the amino acid substitutions, and which has AAV-binding activity. [Effects of the Invention]

[0014] According to the present invention, it is possible to provide an insoluble carrier that can purify adeno-associated virus (AAV) highly efficiently in a short time, and an AAV purification method using the carrier. [Brief explanation of the drawings]

[0015] [Figure 1] This figure compares the amount of adeno-associated virus (AAV) adsorbed to insoluble carriers packed in a column depending on the diameter (average pore size) of the pores in the insoluble carriers. The amount of AAV adsorbed is expressed as a relative value, with the amount of AAV adsorbed by particle 1 being set to 1. DETAILED DESCRIPTION OF THE INVENTION

[0016] The present invention will be described in detail below.

[0017] In addition, regarding numerical ranges, the numbers on either side of "~" are considered to be included in the numerical range.

[0018] The AAV adsorbent used in an embodiment of the present invention is an AAV adsorbent comprising an insoluble carrier and an adeno-associated virus (AAV) binding protein immobilized on the carrier. Examples of the insoluble carrier include inorganic carriers such as glass beads and silica gel, organic carriers made of synthetic polymers such as cross-linked polyvinyl alcohol, cross-linked polyacrylate, cross-linked polyacrylamide, and cross-linked polystyrene, and polysaccharides such as crystalline cellulose, cross-linked cellulose, cross-linked agarose, and cross-linked dextran, and composite carriers obtained by combining these.

[0019] Hydrophilic carriers are particularly preferred because they have relatively little nonspecific adsorption and are expected to improve the purification purity of AAV. Examples of hydrophilic carriers include polysaccharides such as cellulose, chitosan, and dextran, polyvinyl alcohol, saponified ethylene-vinyl acetate copolymer, polyacrylamide, polyacrylic acid, polymethacrylic acid, polymethyl methacrylate, polyacrylic acid-grafted polyethylene, and polyacrylamide-grafted polyethylene.

[0020] The insoluble carrier is preferably a porous particle or a porous membrane. When the insoluble carrier is a porous particle or a porous membrane, the surface area is increased, and the processing capacity per unit time can be increased.

[0021] When the insoluble carrier is a porous particle or a porous membrane, the specific surface area is not particularly limited, but the specific surface area is preferably 1 m 2 / g~500m 2 / g, more preferably 1m 2 / g~100m 2 / g, more preferably 1m 2 / g~50m 2 / g, and even more preferably 1m 2 / g~45m 2 / g. If the specific surface area is within this range, the amount of antibody adsorption is improved. The specific surface area of ​​the insoluble carrier can be measured by mercury intrusion porosimetry using a mercury porosimeter.

[0022] The particle size of the insoluble carrier is not particularly limited, but is preferably 1500 μm or less, more preferably 1000 μm or less, even more preferably 300 μm or less, and even more preferably 100 μm or less. The particle size of the insoluble carrier is preferably 0.5 μm or more, more preferably 1 μm or more, and even more preferably 50 μm or more. If the particle size is within this range, the pressure loss when packed into a column and passed through the column is small, the liquid passing rate can be increased, the treatment efficiency is improved, and the adsorption capacity is improved. The particle size of the porous particles can be measured by known methods, for example, by using a particle size distribution measuring device.

[0023] When the insoluble carrier is a porous particle or a porous membrane, the pore size is not particularly limited, but is preferably 1000 nm or less, more preferably 900 nm or less, and even more preferably 800 nm or less. The pore size is preferably 150 nm or more, more preferably 200 nm or more, even more preferably 300 nm or more, and even more preferably 500 nm or more. When the pore size is within this range, the pressure loss when the carrier is packed into a column and a liquid is passed through it is small, the liquid passage rate can be increased, and the polymer can enter the pores, improving the processing efficiency. The pore size can be measured by a known method, for example, using a mercury porosimeter.

[0024] As used herein, an "insoluble carrier having a large pore size" refers to an insoluble carrier having a pore size of 150 nm or more, preferably 200 nm or more, more preferably 350 nm or more, and even more preferably 500 nm or more.

[0025] The pores of the insoluble carrier may be closed pores or interconnected pores, and the pores may include both closed pores and interconnected pores.

[0026] Commercially available insoluble carriers include, but are not limited to, the Sepharose series (manufactured by Cytiva), which is an agarose-based carrier; the Cellufine series (manufactured by JNC), which is a cellulose-based crosslinked carrier; the Sephacryl series (manufactured by Cytiva), which is a crosslinked polymer of allyl dextran and N,N'-methylenebisacrylamide; and the TOYOPEARL (registered trademark) HW series (manufactured by Tosoh Corporation), which is an acrylate-based carrier.

[0027] The AAV-binding protein used in embodiments of the present invention is not particularly limited as long as it is a polypeptide that can bind to AAV, and examples include laminin receptors such as integrins, anti-AAV antibodies, and AAV receptors (AAVR).

[0028] When the AAV-binding protein is an anti-AAV antibody, a preferred embodiment is a polypeptide comprising at least a heavy chain antibody variable region (VHH) capable of binding to AAV. When the AAV-binding protein is AAVR, a preferred embodiment is a protein comprising at least the amino acid residues from serine (S) at position 312 to aspartic acid (D) at position 500, which correspond to extracellular domain 1 (PKD1) and domain 2 (PKD2) in the amino acid sequence of the native AAV-binding protein KIAA0319L (UniProt No. Q8IZA0) set forth in SEQ ID NO: 1, with the proviso that amino acid substitutions have occurred at specific positions within the amino acid residues at positions 312 to 500.

[0029] In particular, the AAV binding protein may be selected from any of the following (i) to (iii): (i) a protein 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 protein 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 proviso that one or more of the following occurs in one or more positions of the amino acid residues from positions 312 to 500: substitution, deletion, insertion, and addition of one or more amino acid residues, and the protein has AAV-binding activity; (iii) A protein 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 protein has 70% or more identity to the entire amino acid sequence consisting of amino acid residues at positions 312 to 500, and wherein the protein has AAV-binding activity.

[0030] Furthermore, the AAV binding protein herein may be a protein having an amino acid sequence with a "specific mutation" in the amino acid sequence shown in SEQ ID NO: 1. Furthermore, the AAV binding protein may be, for example, a protein having a "specific mutation" in the amino acid sequence shown in SEQ ID NO: 1, and further having one or more activities of substitution, deletion, insertion, and addition of one or several amino acid residues at one or several positions.

[0031] In particular, the AAV binding protein may be a protein selected from any of the following (iv) to (vi): (iv) A protein having an amino acid sequence from serine at position 312 to aspartic acid at position 500 of the amino acid sequence set forth in SEQ ID NO: 1, in which the following amino acid substitutions (1) to (29) occur, and which has AAV-binding activity: (1) The valine residue (V) at position 317 of SEQ ID NO: 1 (position 7 of SEQ ID NO: 4) is replaced with an aspartic acid residue (D) (2) The asparagine residue (N) at position 324 of SEQ ID NO: 1 (position 14 of SEQ ID NO: 4) is substituted with a histidine residue (H). (3) A substitution of the valine residue (V) at position 326 of SEQ ID NO: 1 (position 16 of SEQ ID NO: 4) with an alanine residue (A) (4) The asparagine residue (N) at position 329 of SEQ ID NO: 1 (position 19 of SEQ ID NO: 4) is substituted with a lysine residue (K). (5) The alanine residue (A) at position 330 of SEQ ID NO: 1 (position 20 of SEQ ID NO: 4) is replaced with a valine residue (V). (6) The glutamine residue (Q) at position 334 of SEQ ID NO: 1 (position 24 of SEQ ID NO: 4) is replaced with a leucine residue (L). (7) The glutamic acid residue (E) at position 335 of SEQ ID NO: 1 (position 25 of SEQ ID NO: 4) is replaced with a valine residue (V). (8) The threonine residue (T) at position 341 of SEQ ID NO: 1 (position 31 of SEQ ID NO: 4) is substituted with an alanine residue (A). (9) The tyrosine residue (Y) at position 342 of SEQ ID NO: 1 (position 32 of SEQ ID NO: 4) is substituted with a serine residue (S). (10) The lysine residue (K) at position 362 of SEQ ID NO: 1 (position 52 of SEQ ID NO: 4) is substituted with a glutamic acid residue (E). (11) The isoleucine residue (I) at position 366 of SEQ ID NO: 1 (position 56 of SEQ ID NO: 4) is substituted with a phenylalanine residue (F) (12) The lysine residue (K) at position 371 of SEQ ID NO: 1 (position 61 of SEQ ID NO: 4) is substituted with an asparagine residue (N). (13) The phenylalanine residue (F) at position 379 of SEQ ID NO: 1 (position 69 of SEQ ID NO: 4) is replaced with a tyrosine residue (Y). (14) The lysine residue (K) at position 380 of SEQ ID NO: 1 (position 70 of SEQ ID NO: 4) is substituted with an arginine residue (R). (15) A valine residue (V) at position 381 of SEQ ID NO: 1 (position 71 of SEQ ID NO: 4) is substituted with an alanine residue (A). (16) The isoleucine residue (I) at position 382 of SEQ ID NO: 1 (position 72 of SEQ ID NO: 4) is substituted with a valine residue (V) (17) The glycine residue (G) at position 390 of SEQ ID NO: 1 (position 80 of SEQ ID NO: 4) is substituted with a serine residue (S). (18) The valine residue (V) at position 394 of SEQ ID NO: 1 (position 84 of SEQ ID NO: 4) is substituted with an alanine residue (A). (19) The lysine residue (K) at position 399 of SEQ ID NO: 1 (position 89 of SEQ ID NO: 4) is substituted with a glutamic acid residue (E). (20) The glutamic acid residue (E) at position 401 of SEQ ID NO: 1 (position 91 of SEQ ID NO: 4) is replaced with a glycine residue (G). (21) The threonine residue (T) at position 426 of SEQ ID NO: 1 (position 116 of SEQ ID NO: 4) is substituted with an alanine residue (A). (22) The lysine residue (K) at position 455 of SEQ ID NO: 1 (position 145 of SEQ ID NO: 4) is substituted with an arginine residue (R) (23) The alanine residue (A) at position 461 of SEQ ID NO: 1 (position 151 of SEQ ID NO: 4) is substituted with a proline residue (P) (24) The lysine residue (K) at position 467 of SEQ ID NO: 1 (position 157 of SEQ ID NO: 4) is substituted with an asparagine residue (N). (25) The serine residue (S) at position 476 of SEQ ID NO: 1 (position 166 of SEQ ID NO: 4) is substituted with an arginine residue (R) (26) The serine residue (S) at position 482 of SEQ ID NO: 1 (position 172 of SEQ ID NO: 4) is substituted with a threonine residue (T). (27) The asparagine residue (N) at position 487 of SEQ ID NO: 1 (position 177 of SEQ ID NO: 4) is substituted with an aspartic acid residue (D). (28) The asparagine residue (N) at position 492 of SEQ ID NO: 1 (position 182 of SEQ ID NO: 4) is substituted with an aspartic acid residue (D). (29) The lysine residue (K) at position 497 of SEQ ID NO: 1 (position 187 of SEQ ID NO: 4) is replaced with a glutamic acid residue (E); (v) a protein comprising 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, wherein at least one of the amino acid substitutions (1) to (29) has occurred in the amino acid residues at positions 312 to 500, and further comprising one or more substitutions, deletions, insertions, and additions of one or more amino acid residues at one or more positions other than the amino acid substitutions set forth in (1) to (29), and having AAV-binding activity; (vi) A protein comprising an amino acid sequence 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, which has an identity of 70% or more to the entire amino acid sequence in which at least one of the amino acid substitutions (1) to (29) has occurred, and which contains the amino acid sequence in which the at least one amino acid substitution remains, and which has AAV-binding activity.

[0032] In (ii) and (v), "one or several" varies depending on the position of the amino acid substitution in the three-dimensional structure of the AAV-binding protein and the type of amino acid residue, but, for example, means any of 1 to 50, 1 to 40, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 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.

[0033] Examples of the substitutions, deletions, insertions and additions described in (ii) and (v) above include the amino acid residue substitutions disclosed in WO2021 / 106882 and WO2023 / 140197.

[0034] The "substitution of one or several amino acid residues" in (ii) and (v) above may include not only the amino acid substitution at the specific position described above, but also conservative substitutions between amino acids with similar physical and / or chemical properties. In the case of conservative substitutions, it is generally known to those skilled in the art that protein function is maintained between substituted and unsubstituted amino acids. Examples of conservative substitutions include substitutions between glycine and alanine, between serine and threonine, or between glutamic acid and aspartic acid (Protein Structure and Function, Medical Science International, 9, 2005). Another example of the amino acid substitution is a substitution for monomerizing the AAV binding protein of this embodiment. Specifically, an amino acid substitution such as substituting a cysteine ​​residue, which is likely to form a higher-order structure, with a serine or methionine residue is used.

[0035] Furthermore, the "substitution, deletion, insertion, and addition of one or several amino acid residues" in (ii) and (v) above also includes naturally occurring mutations (mutants or variants) due to differences in the origin or species of the AAV-binding protein.

[0036] The identity of the amino acid sequences in (iii) and (vi) above may be 70% or more, but may be higher (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more). "Amino acid sequence identity" refers to identity with respect to the entire amino acid sequence. "Identity" between amino acid sequences refers to the proportion of amino acid residues in those amino acid sequences that have the same type of amino acid (Experimental Medicine, 31(3), Yodosha). Amino acid sequence identity can be determined using alignment programs such as BLAST (Basic Local Alignment Search Tool) or FASTA.

[0037] The AAV-binding protein of this embodiment may further have an oligopeptide attached to its N-terminus or C-terminus that is useful for separating the protein from a solution containing contaminants. Examples of such oligopeptides include polyhistidine, polylysine, polyarginine, polyglutamic acid, and polyaspartic acid. Furthermore, a cysteine-containing oligopeptide that is useful for immobilizing the AAV-binding protein on a solid phase such as a chromatographic support may further be attached to the N-terminus or C-terminus of the AAV-binding protein.

[0038] The length of the oligopeptide added to the N- or C-terminus of the AAV-binding protein is not particularly limited, as long as it does not impair the AAV-binding ability or stability of the AAV-binding protein of this embodiment. When adding the oligopeptide to the AAV-binding protein, a polynucleotide encoding the oligopeptide may be prepared and then added to the N- or C-terminus of the AAV-binding protein by genetic engineering using methods well known to those skilled in the art, or a chemically synthesized oligopeptide may be chemically linked to the N- or C-terminus of the AAV-binding protein.

[0039] Furthermore, the AAV-binding protein of this embodiment may contain an additional sequence at the N-terminus, C-terminus, or both. The additional sequence is not particularly limited as long as it has AAV-binding activity. Examples of the additional sequence include a peptide tag, a signal peptide (also referred to as a signal sequence), and a protease recognition sequence. The additional sequence may be a single amino acid sequence, or a combination of two or more amino acid sequences.

[0040] Specific examples of peptide tags include His tags, FLAG tags, GST tags, Myc tags, maltose binding protein (MBP), cellulose binding protein (CBP), thioredoxin (TRX), green fluorescent protein (GFP), horseradish peroxidase (HRP), alkaline phosphatase (ALP), and antibody Fc regions. Examples of His tags include 6xHis tags. Peptide tags can be used, for example, to detect and purify expressed AAV-binding proteins.

[0041] Specific examples of signal peptides include signal peptides that direct protein secretion into the periplasm, such as PelB, OmpA, DsbA, DsbC, MalE, and TorT (Japanese Patent Application Laid-Open No. 2011-097898). It is also possible to omit the addition of a signal peptide to the N-terminus. This is preferable because it improves protein homogeneity during protein preparation.

[0042] The AAV-binding protein of this embodiment can be used, for example, for purifying or analyzing AAV. When used for this purpose, there are no particular limitations on the AAV to be bound, and it may be a naturally occurring AAV or an 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 share characteristics (cell tropism and infectivity) of two or more of these serotypes.

[0043] The AAV-binding protein of this embodiment can be used, for example, by immobilizing it on an insoluble carrier. That is, specifically, AAV purification or analysis can be performed using, for example, an AAV adsorbent comprising an insoluble carrier and an AAV-binding protein immobilized on the insoluble carrier. In this specification, an AAV adsorbent comprising an insoluble carrier and an AAV-binding protein immobilized on the insoluble carrier is also referred to as the AAV adsorbent of the present invention. Note that AAV purification is not limited to the purification of AAV from a solution containing contaminants, but also includes the purification of AAV based on its structure, properties, activity, etc.

[0044] The AAV-binding protein of this embodiment can be immobilized on an insoluble carrier, for example, via a covalent bond. Specifically, the AAV-binding protein can be immobilized on an insoluble carrier by covalently binding the AAV-binding protein to the insoluble carrier via an active group possessed by the insoluble carrier. The immobilization of the AAV-binding protein on an insoluble carrier can be carried out, for example, based on the disclosure in WO2021 / 106882.

[0045] The AAV adsorbents of the present invention can be packed into a column and used for purifying or analyzing AAV. Specifically, AAV can be purified or analyzed by adding a solution containing AAV to a column packed with the AAV adsorbent of the present invention (hereinafter also referred to simply as the "column of the present invention") to adsorb the AAV to the adsorbent, and then eluting the AAV adsorbed to the adsorbent. That is, the present invention provides a method for purifying or analyzing AAV, comprising, for example, the steps of adding a solution containing AAV to the column of the present invention to adsorb the AAV to the adsorbent, and eluting the AAV adsorbed to the adsorbent.

[0046] Purification of AAV using the AAV adsorbent of the present invention can yield, for example, purified AAV. That is, in one embodiment, the method for purifying AAV can be a method for producing AAV, specifically, a method for producing purified AAV. AAV can be obtained, for example, as an elution fraction containing AAV. That is, the eluted fraction containing AAV can be separated and collected. Separation of the AAV fraction can be performed, for example, by conventional methods. Methods for separating the AAV fraction include replacing the collection container at regular intervals or at regular volumes, changing the collection container to match the shape of the chromatogram of the eluate, and separating fractions using an automated fraction collector such as an autosampler. Furthermore, AAV can also be recovered from the AAV-containing fraction. Recovery of AAV from the AAV-containing fraction can be performed, for example, by known methods used for protein purification.

[0047] The linear velocity at which a sample containing AAV is applied to the column of the present invention is not particularly limited, but is preferably within the range of 1200 cm / h to 3500 cm / h, more preferably 1200 cm / h to 3000 cm / h, and even more preferably 1400 cm / h to 3000 cm / h. A linear velocity within this range improves the adsorption capacity. As used herein, linear velocity refers to the velocity at which a liquid passes through the cross section of the column and may be calculated using the following formula:

[0048] [Math 1] Linear velocity (cm / h) = flow velocity (cm 3 / h) / Column cross-sectional area (cm2 ) The separation performance of this embodiment is expressed by dynamic binding capacity (DBC). DBC can be determined by adding a standard substance solution of known concentration to a column, monitoring the absorbance of the eluate, and determining the amount of substance added at the point when 10% of the absorbance of the added sample breaks through (10% breakthrough time), and may be calculated using the following formula:

[0049] [Equation 2] DBC (mg / mL) = 10% breakthrough time (min) × flow rate (mL / min) × antibody concentration (mg / mL) / column volume (mL) [Example]

[0050] EXAMPLES Hereinafter, examples will be shown to explain the present invention in more detail, but the present invention is not limited to these examples.

[0051] Example 1 Preparation of insoluble carrier The insoluble carriers (porous particles) used in the following examples were prepared by radical polymerization of hydrophilic vinyl monomers. The diameters (average particle diameters) of the resulting carriers were measured using a particle size distribution analyzer MT3000II (MicrotracBEL), and the pore diameters (average pore diameters) and specific surface areas of the carriers were measured by mercury intrusion using a mercury porosimeter POREMASTER GT (Quantachrome Instruments).

[0052] The average particle size and average pore size of the prepared insoluble carrier, as well as the specific surface area per gram of the carrier, are shown in Table 1.

[0053] [Table 1]

[0054] Example 2 Purification of adeno-associated virus (AAV) contained in a sample (1) Preparation of AAV vectors The AAV vector used as the AAV in this example was prepared by the following method.

[0055] (1-1) A nucleotide sequence (SEQ ID NO: 3) was designed in which a restriction enzyme EcoRI recognition sequence (GAATCC) 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.

[0056] (1-2) 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).

[0057] (1-3) The pUC-EGFP obtained in (1-2) was digested with the restriction enzymes EcoRI and BamHI and then ligated to the expression vector pAAV-CMV (manufactured by Takara Bio Inc.) which had been previously digested with the restriction enzymes EcoRI and BamHI. The ligation product (designated pAAV-EGFP) was used to transform the Escherichia coli JM109 strain.

[0058] (1-4) The transformants obtained in (1-3) were cultured overnight at 37°C with shaking in a 5-L baffled flask containing 1 L of 2xYT 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. pAAV-EGFP was prepared in large quantities from the collected cells using a Plasmid Mega Kit (Qiagen).

[0059] (1-5) The Escherichia coli JM109 strain was transformed with a plasmid (hereinafter also referred to as "pRC8 Vector") containing a polynucleotide encoding the capsid of serotype 8 (AAV8) and the pHelper Vector (manufactured by Takara Bio Inc.) The resulting transformant was used in the same manner as in (1-4) to prepare large quantities of the pRC8 Vector and pHelper.

[0060] (1-6) HEK293T cells were cultured in ten T-225 flasks (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) containing 45 mL of D-MEM medium (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) containing 10% (v / v) bovine serum. The pAAV-EGFP prepared in (1-4), the pRC8 Vector prepared in (1-5), pHelper, and polyethyleneimine (manufactured by Polysciences) complex were added for gene transfer, and the cells were cultured statically for 3 days under conditions of 5% (v / v) carbon dioxide and 37°C. After culture, the cells were detached by centrifugation and collected. The cells obtained from each of the five T-225 flasks were stored frozen at -80°C.

[0061] (1-7) The frozen cells obtained in (1-6) were thawed and suspended in 10 mL of 20 mmol / L Tris-HCl buffer (pH 7.4) containing 150 mmol / L sodium chloride, 4 mmol / L magnesium chloride, and 0.01% (w / v) Tween® 20. A 1 / 2000 volume of Benzonase (Merck Millipore) was added, and the mixture was left to stand at 37°C for 1 hour. The mixture was then centrifuged at 13,000 × g and 4°C for 10 minutes to obtain a supernatant. Ammonium sulfate was added to the resulting supernatant to 15% saturation, and the mixture was centrifuged again under the same conditions. The resulting supernatant was passed through a 0.45 μm pore size filter to remove any floating material.

[0062] (1-8) The supernatant, from which the suspended matter had been removed, was applied to a 7 mL POROS AAVX column (manufactured by Thermo Fisher Scientific) that had been equilibrated in advance with 20 mmol / L Tris-HCl buffer (pH 8.0) containing 500 mmol / L sodium chloride (hereinafter also referred to as "equilibration solution A").

[0063] (1-9) After washing with equilibration solution A, the column was eluted with 100 mmol / L acetate buffer (pH 2.5) containing 500 mmol / L sodium chloride. The resulting eluate was neutralized by adding 1 / 4 volume of 1 mol / L Tris-HCl buffer (pH 8.5) containing 20 mmol / L magnesium chloride to obtain a solution of the AAV vector AAV8-EGFP (diameter 20 to 30 nm). The diameter of the AAV8-EGFP was measured by dynamic light scattering using a Zetasizer (Malvern Panalytical).

[0064] (2) Preparation of AAV-binding proteins (2-1) Escherichia coli strain BL21(DE3) was transformed with a plasmid containing a polynucleotide encoding an AAV-binding protein consisting of the amino acid sequence set forth in SEQ ID NO: 4. The resulting transformant was inoculated into 20 mL of 2xYT liquid medium containing 50 μg / mL kanamycin and precultured overnight at 37°C under aerobic shaking. In SEQ ID NO: 4, the AAV-binding protein AVR29c is located at the second serine (S) through the 190th aspartic acid (D), and the six C-terminal histidine (H) residues form a histidine tag. AVR29c is a polypeptide of KIAA0319L (UniProt No. Q8IZA0) in which the following 29 amino acid substitutions have occurred in the amino acid residues 312 to 500 of SEQ ID NO: 1, which correspond to extracellular domain 1 (PKD1) and domain 2 (PKD2): The valine residue (V) at position 317 of SEQ ID NO: 1 (position 7 of SEQ ID NO: 4) is substituted with an aspartic acid residue (D). The asparagine residue (N) at position 324 of SEQ ID NO: 1 (position 14 of SEQ ID NO: 4) is substituted with a histidine residue (H). The valine residue (V) at position 326 of SEQ ID NO: 1 (position 16 of SEQ ID NO: 4) is replaced with an alanine residue (A). The asparagine residue (N) at position 329 of SEQ ID NO: 1 (position 19 of SEQ ID NO: 4) is substituted with a lysine residue (K). The alanine residue (A) at position 330 of SEQ ID NO: 1 (position 20 of SEQ ID NO: 4) is substituted with a valine residue (V). The glutamine residue (Q) at position 334 of SEQ ID NO: 1 (position 24 of SEQ ID NO: 4) is substituted with a leucine residue (L). The glutamic acid residue (E) at position 335 of SEQ ID NO: 1 (position 25 of SEQ ID NO: 4) is substituted with a valine residue (V). The threonine residue (T) at position 341 of SEQ ID NO: 1 (position 31 of SEQ ID NO: 4) is substituted with an alanine residue (A). The tyrosine residue (Y) at position 342 of SEQ ID NO: 1 (position 32 of SEQ ID NO: 4) is substituted with a serine residue (S). The lysine residue (K) at position 362 of SEQ ID NO: 1 (position 52 of SEQ ID NO: 4) is substituted with a glutamic acid residue (E). The isoleucine residue (I) at position 366 of SEQ ID NO: 1 (position 56 of SEQ ID NO: 4) is substituted with a phenylalanine residue (F) The lysine residue (K) at position 371 of SEQ ID NO: 1 (position 61 of SEQ ID NO: 4) is substituted with an asparagine residue (N). The phenylalanine residue (F) at position 379 of SEQ ID NO: 1 (position 69 of SEQ ID NO: 4) is replaced with a tyrosine residue (Y). The lysine residue (K) at position 380 of SEQ ID NO: 1 (position 70 of SEQ ID NO: 4) is substituted with an arginine residue (R). The valine residue (V) at position 381 of SEQ ID NO: 1 (position 71 of SEQ ID NO: 4) is substituted with an alanine residue (A). The isoleucine residue (I) at position 382 of SEQ ID NO: 1 (position 72 of SEQ ID NO: 4) is substituted with a valine residue (V) The glycine residue (G) at position 390 of SEQ ID NO: 1 (position 80 of SEQ ID NO: 4) is substituted with a serine residue (S). The valine residue (V) at position 394 of SEQ ID NO: 1 (position 84 of SEQ ID NO: 4) is substituted with an alanine residue (A). The lysine residue (K) at position 399 of SEQ ID NO: 1 (position 89 of SEQ ID NO: 4) is substituted with a glutamic acid residue (E). The glutamic acid residue (E) at position 401 of SEQ ID NO: 1 (position 91 of SEQ ID NO: 4) is replaced with a glycine residue (G) The threonine residue (T) at position 426 of SEQ ID NO: 1 (position 116 of SEQ ID NO: 4) is substituted with an alanine residue (A). The lysine residue (K) at position 455 of SEQ ID NO: 1 (position 145 of SEQ ID NO: 4) is substituted with an arginine residue (R). The alanine residue (A) at position 461 of SEQ ID NO: 1 (position 151 of SEQ ID NO: 4) is substituted with a proline residue (P) The lysine residue (K) at position 467 of SEQ ID NO: 1 (position 157 of SEQ ID NO: 4) is substituted with an asparagine residue (N). The serine residue (S) at position 476 of SEQ ID NO: 1 (position 166 of SEQ ID NO: 4) is substituted with an arginine residue (R). The serine residue (S) at position 482 of SEQ ID NO: 1 (position 172 of SEQ ID NO: 4) is substituted with a threonine residue (T). The asparagine residue (N) at position 487 of SEQ ID NO: 1 (position 177 of SEQ ID NO: 4) is substituted with an aspartic acid residue (D). The asparagine residue (N) at position 492 of SEQ ID NO: 1 (position 182 of SEQ ID NO: 4) is substituted with an aspartic acid residue (D). The lysine residue (K) at position 497 of SEQ ID NO: 1 (position 187 of SEQ ID NO: 4) is substituted with a glutamic acid residue (E). (2-2) 20 mL of the preculture solution from (2-1) was inoculated into 1000 mL of 2xYT 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.

[0065] (2-3) 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 mmol / L, and the mixture was subsequently cultured overnight at 25°C under aerobic shaking.

[0066] (2-4) After the culture was completed, the culture solution was centrifuged at 4°C and 8000 rpm for 20 minutes to collect the bacterial cells.

[0067] (2-5) The bacterial cells collected in (2-4) were suspended in 20 mmol / L Tris-HCl buffer (pH 7.4) containing 150 mmol / L sodium chloride and 20 mmol / L imidazole (hereinafter also referred to as "equilibration solution B") at a concentration of 5 mL / 1 g (bacterial cells), and then disrupted using an ultrasonic generator (Insonator 201M, Kubota Shoji Co., Ltd.) at an output of approximately 150 W for approximately 10 minutes at 8°C. The disrupted bacterial cell solution was centrifuged twice at 8,000 rpm for 20 minutes at 4°C, and the supernatant was collected.

[0068] (2-6) The supernatant obtained in (2-5) was applied to an XK26 / 20 column (Cytiva) packed with 20 mL of Ni Sepharose 6 Fast Flow (Cytiva) that had been previously equilibrated with equilibration solution B. After washing with equilibration solution B, the column was eluted with 20 mmol / L Tris-HCl buffer (pH 7.4) containing 500 mmol / L imidazole and 150 mmol / L sodium chloride.

[0069] (2-7) The eluate obtained in (2-6) was dialyzed against 20 mmol / L Tris buffer (pH 7.4) containing 150 mmol / L sodium chloride to prepare the amount of AAV binding protein (SEQ ID NO: 4) required for producing an AAV adsorbent.

[0070] (3) Preparation of AVR column (3-1) Carriers were prepared by modifying the surfaces of the insoluble carriers (particles 1 to 4) prepared in Example 1 with iodoacetamide groups.

[0071] (3-2) To 3.5 g of the iodoacetamide group-modified insoluble support prepared in (3-1), 28 mg of the AAV binding protein (SEQ ID NO: 4) prepared in (2) and TCEP (Tris(2-CarboxyEthyl)Phosphine) as a reducing agent at a final concentration of 0.1 mmol / L were added, and the mixture was allowed to react by shaking at pH 7.4 and 4°C for 15 hours. This produced an insoluble support onto which the AAV binding protein was immobilized as a ligand (hereinafter also referred to as "AAV adsorbent").

[0072] (3-3) 0.58 mL of the AAV adsorbent prepared in (3-2) was packed into an empty stainless steel column (diameter 4.6 mm, length 35 mm, manufactured by Tosoh Corporation) to prepare a column (hereinafter also referred to as "AVR column").

[0073] (4) AAV adsorption onto the AVR column (4-1) The AVR column prepared in (3) was connected to an AKTA avant (Cytiva) and equilibrated with 50 mmol / L sodium acetate buffer (pH 6.0) containing 15 mmol / L glycine and 10 mmol / L magnesium chloride. The AAV8-EGFP solution obtained in (1) was then loaded at a linear velocity of 1800 cm / h.

[0074] (4-2) The eluate from the AVR column was monitored by absorbance at 280 nm. When the concentration of the eluate exceeded 10% of the concentration of the applied AAV8-EGFP solution, the application of the solution was stopped, and the concentration of AAV8-EGFP in the eluate at that time was calculated as the adsorption amount.

[0075] The results are shown in Figure 1. The amount of AAV (AAV8-EGFP in this example) adsorption is expressed as a relative value, with the result for particle 1 (average pore diameter 70 nm) set to 1. It can be seen that particles 2 (average pore diameter 210 nm), particle 3 (average pore diameter 370 nm), and particle 4 (average pore diameter 780 nm), which have smaller specific surface areas per particle (Table 1) than particle 1, adsorbed greater amounts of AAV. These results demonstrate that in an AAV adsorbent containing an insoluble carrier and an AAV-binding protein immobilized on the carrier, AAV can be more efficiently adsorbed when the insoluble carrier is made into porous particles with pores having diameters of 150 nm to 1000 nm.

Claims

1. An adeno-associated virus (AAV) adsorbent comprising an insoluble carrier and an AAV binding protein immobilized on the carrier, The adsorbent, wherein the insoluble carrier has pores with diameters of 150 nm or more and 1000 nm or less.

2. The adsorbent according to claim 1, wherein the AAV-binding protein is a polypeptide selected from any one of the following (i) to (iii) or a polypeptide comprising at least a heavy chain antibody variable region capable of binding to AAV: (i) a polypeptide containing at least the amino acid residues from the serine residue at position 312 to the aspartic acid residue at position 500 in the amino acid sequence set forth in SEQ ID NO: 1; (ii) a polypeptide comprising at least the amino acid residues from the serine residue at position 312 to the aspartic acid residue at position 500 of the amino acid sequence set forth in SEQ ID NO: 1, wherein the amino acid residues at positions 312 to 500 have any one or more of substitution, deletion, insertion, and addition of one or more amino acid residues at one or more positions, and has AAV-binding activity; (iii) A polypeptide comprising at least the amino acid residues from the serine residue at position 312 to the aspartic acid residue at position 500 of the amino acid sequence set forth in SEQ ID NO: 1, wherein the polypeptide has an identity of 70% or more to the entire amino acid sequence consisting of the amino acid residues at positions 312 to 500, and wherein the polypeptide has AAV-binding activity.

3. 1. A method for purifying or analyzing AAV, comprising: applying a sample containing AAV to a column packed with the AAV adsorbent to adsorb the AAV to the adsorbent; applying an eluate to the column to elute the AAV adsorbed to the adsorbent; The AAV adsorbent is the adsorbent of claim 1 or 2, The method, wherein the linear velocity when the sample is applied to the column is 1200 cm / h or more and 3500 cm / h or less.

4. The adsorbent according to claim 1, wherein the AAV-binding protein is a polypeptide selected from any one of the following (iv) to (vi) or a polypeptide comprising at least a heavy chain antibody variable region capable of binding to AAV: (iv) a polypeptide having an amino acid sequence from the serine residue at position 312 to the aspartic acid residue at position 500 of the amino acid sequence set forth in SEQ ID NO: 1, which contains one or more amino acid substitutions selected from any of the following (1) to (29): (1) The amino acid residue corresponding to the 317th valine residue in SEQ ID NO: 1 is substituted with an aspartic acid residue (2) The amino acid residue corresponding to the asparagine residue at position 324 of SEQ ID NO: 1 is substituted with a histidine residue (3) The amino acid residue corresponding to the 326th valine residue in SEQ ID NO: 1 is substituted with an alanine residue. (4) The amino acid residue corresponding to the asparagine residue at position 329 of SEQ ID NO: 1 is substituted with a lysine residue. (5) The amino acid residue corresponding to the alanine residue at position 330 of SEQ ID NO: 1 is substituted with a valine residue. (6) The amino acid residue corresponding to the glutamine residue at position 334 of SEQ ID NO: 1 is substituted with a leucine residue (7) The amino acid residue corresponding to the glutamic acid residue at position 335 of SEQ ID NO: 1 is substituted with a valine residue. (8) The amino acid residue corresponding to the threonine residue at position 341 of SEQ ID NO: 1 is substituted with an alanine residue. (9) The amino acid residue corresponding to the tyrosine residue at position 342 of SEQ ID NO: 1 is substituted with a serine residue (10) The amino acid residue corresponding to the lysine residue at position 362 of SEQ ID NO: 1 is substituted with a glutamic acid residue (11) The amino acid residue corresponding to the isoleucine residue at position 366 of SEQ ID NO: 1 is substituted with a phenylalanine residue (12) The amino acid residue corresponding to the lysine residue at position 371 of SEQ ID NO: 1 is substituted with an asparagine residue. (13) The amino acid residue corresponding to the phenylalanine residue at position 379 of SEQ ID NO: 1 is substituted with a tyrosine residue (14) The amino acid residue corresponding to the lysine residue at position 380 of SEQ ID NO: 1 is substituted with an arginine residue (15) The amino acid residue corresponding to the 381st valine residue of SEQ ID NO: 1 is substituted with an alanine residue (16) The amino acid residue corresponding to the isoleucine residue at position 382 of SEQ ID NO: 1 is substituted with a valine residue (17) The amino acid residue corresponding to the glycine residue at position 390 of SEQ ID NO: 1 is substituted with a serine residue (18) The amino acid residue corresponding to the 394th valine residue of SEQ ID NO: 1 is substituted with an alanine residue (19) The amino acid residue corresponding to the lysine residue at position 399 of SEQ ID NO: 1 is substituted with a glutamic acid residue (20) The amino acid residue corresponding to the 401st glutamic acid residue of SEQ ID NO: 1 is substituted with a glycine residue (21) The amino acid residue corresponding to the 426th threonine residue of SEQ ID NO: 1 is substituted with an alanine residue (22) The amino acid residue corresponding to the lysine residue at position 455 of SEQ ID NO: 1 is substituted with an arginine residue (23) The amino acid residue corresponding to the alanine residue at position 461 of SEQ ID NO: 1 is substituted with a proline residue (24) The amino acid residue corresponding to the lysine residue at position 467 of SEQ ID NO: 1 is substituted with an asparagine residue (25) The amino acid residue corresponding to the serine residue at position 476 of SEQ ID NO: 1 is substituted with an arginine residue (26) The amino acid residue corresponding to the serine residue at position 482 of SEQ ID NO: 1 is substituted with a threonine residue (27) The amino acid residue corresponding to the asparagine residue at position 487 of SEQ ID NO: 1 is substituted with an aspartic acid residue (28) The amino acid residue corresponding to the asparagine residue at position 492 of SEQ ID NO: 1 is substituted with an aspartic acid residue (29) the amino acid residue corresponding to the lysine residue at position 497 of SEQ ID NO: 1 is substituted with a glutamic acid residue; (v) a polypeptide having an amino acid sequence comprising amino acid residues from the serine residue at position 312 to the aspartic acid residue at position 500 of the amino acid sequence set forth in SEQ ID NO: 1, which contains one or more amino acid substitutions selected from any of (1) to (29), and further including, in addition to the amino acid substitutions, any one or more of substitutions, deletions, insertions, and additions of one or more amino acid residues at one or more positions, and which has AAV-binding activity; (vi) A polypeptide having an amino acid sequence from the 312th serine residue to the 500th aspartic acid residue in the amino acid sequence set forth in SEQ ID NO: 1, which has an identity of 70% or more to the entire amino acid sequence containing one or more amino acid substitutions selected from (1) to (29), with the proviso that the amino acid substitutions are maintained, and which has AAV-binding activity.

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