Modified recombinant adeno-associated virus (AAV) binding protein and method for purifying AAV

Abstract

This invention provides an adeno-associated virus (AAV) binding protein with improved alkali resistance. It also provides a method for purifying full AAV vectors with high purity and in a simple manner. [Solution] An AAV-binding protein is provided that contains at least the amino acid residues from the 25th serine to the 213th aspartic acid of an amino acid sequence consisting of 219 amino acids having a specific amino acid sequence, wherein a specific amino acid substitution occurs in the amino acid residues from the 25th to the 213th, and which has AAV-binding activity.

Description

Technical Field

[0001] The present disclosure relates to a protein having binding affinity for adeno-associated virus (AAV). In one aspect, the present disclosure relates to, for example, an improved recombinant AAV-binding protein with enhanced alkali resistance.

[0002] In another aspect, the present disclosure relates to a method for purifying adeno-associated virus (AAV). In a further aspect, the present disclosure relates to a method, for example, for simply purifying an AAV vector containing a gene.

Background Art

[0003] Adeno-associated virus (AAV) is a non-enveloped virus classified in the Parvoviridae family, Dependovirus genus. The AAV outer shell particles are composed of three types of proteins (VP1, VP2, and VP3), and approximately 60 protein molecules are mixed and assembled at a ratio of approximately VP1:VP2:VP3 = 1:1:10 to form an icosahedral shape with a diameter of 20 nm to 30 nm.

[0004] AAV in nature lacks the ability to grow independently, and replication depends on helper viruses such as adenovirus and herpesvirus. When the helper virus is present, the AAV genome is replicated in host cells, complete AAV particles containing the AAV genome are formed, and AAV particles are released from the host cells. On the other hand, when the helper virus is absent, the AAV genome remains in an episomal state or is integrated into the host chromosome (latent state).

[0005] AAV can infect cells of a wide range of species including humans, can also infect non-dividing cells that have completed differentiation such as blood cells, muscle, and nerve cells, has no pathogenicity to humans and thus has a low risk of side effects, and the virus particles are physically and chemically stable. Therefore, its utility value as a vector for gene transfer for the treatment of congenital genetic diseases has attracted attention.

[0006] The production of recombinant AAV vectors (hereinafter also simply referred to as AAV vectors) is typically carried out by introducing nucleic acids encoding elements essential for AAV particle formation into cells to create cells capable of producing AAV (hereinafter also referred to as AAV-producing cells), and then culturing these cells to express the elements essential for AAV particle formation. The produced AAV vectors are recovered and purified from the AAV-producing cells to obtain therapeutic AAV vector preparations.

[0007] One method for recovering and purifying AAV vectors from AAV-producing cells involves affinity chromatography based on the binding affinity to AAV, using an adsorbent containing an insoluble carrier and an AAV-binding protein immobilized on the carrier. This method allows for the recovery and purification of AAV vectors from solutions containing AAV vectors with contaminants. Specifically, Patent Document 1 describes a method for achieving high-purity purification of AAV vectors by using a polypeptide containing extracellular domain 1 (PKD1) and domain 2 (PKD2) of KIAA0319L (UniProt No. Q8IZA0), but with improved stability against heat, acid, and alkali by substituting specific amino acid residues in these domains with other amino acid residues, as an AAV-binding protein (hereinafter also simply referred to as "ligand protein") immobilized on an insoluble carrier.

[0008] On the other hand, when the above-mentioned adsorbent is used for purification purposes, after AAV purification, the remaining AAV and contaminants are usually washed with an alkaline solution of sodium hydroxide at a high concentration (e.g., 0.1 M to 0.5 M). Therefore, the creation of ligand proteins that can withstand this alkaline washing has been desired.

[0009] The manufactured AAV vectors include those containing genes (Full AAV vectors) and those not containing genes (Empty AAV vectors). Of these, Empty AAV vectors may reduce the efficacy of the drug as a therapeutic agent or induce side effects due to overdose (Non-Patent Literature 2). Therefore, in order to use them as therapeutic AAV vector preparations, it is necessary to purify the Full AAV vectors from the manufactured AAV vectors to a high degree of purity.

[0010] A method for purifying AAV vectors is known, which involves affinity chromatography using an AAV adsorbent containing an insoluble carrier and an AAV-binding protein immobilized on the carrier (Patent Documents 1 and 2, and Non-Patent Document 2). However, even with this affinity chromatography method, it has been difficult to remove empty AAV vectors.

[0011] One known method for obtaining a full AAV vector involves ultracentrifugation of a solution containing the manufactured AAV vector to obtain a fraction rich in the full AAV vector. However, ultracentrifugation requires complex steps such as preparing a density gradient solution, ultracentrifugation, and fraction acquisition, and generally takes several days to obtain a full AAV vector. Furthermore, because it is difficult to distinguish the fraction rich in the full AAV vector, there is a risk of acquiring layers before or after the fraction during fraction recovery, and there is a high risk of contamination with impurities.

[0012] Furthermore, a method has been reported for separating full AAV vectors and empty AAV vectors using Tosoh TSKgel Q-STAT as an anion exchange gel and choline chloride as the eluent (Non-Patent Literature 3). [Prior art documents] [Patent Documents]

[0013] [Patent Document 1] WO2021 / 106882 issue [Patent Document 2] Special Publication No. 2018-507707 [Non-patent literature]

[0014] [Non-Patent Document 1] Gerard A et al.,Pharm Res,36,29(2019) [Non-Patent Document 2] Kai G et al.,Mol Ther Methods Clin Dev,1:9(2014) [Non-Patent Document 3] Sam Kurth et al.,Anal Biochem, 686,115421(2024) [Overview of the project] [Problems that the invention aims to solve]

[0015] As mentioned above, the adeno-associated virus (AAV) binding protein disclosed in WO2021 / 106882 exhibits improved stability against heat, acid, and alkali compared to the native type (AAV binding protein without amino acid substitutions). However, there was a need for a ligand protein that could withstand alkaline washing better than the AAV binding protein disclosed in the aforementioned publication.

[0016] Therefore, in one embodiment, the object of this disclosure is to provide an AAV-binding protein with improved alkali resistance than the AAV-binding protein disclosed in the aforementioned publication.

[0017] As mentioned above, AAV vectors that do not contain genes (empty AAV vectors) may reduce the efficacy of the drug as a therapeutic agent or induce side effects due to overdose. Therefore, in order to use them as therapeutic AAV vector preparations, it was necessary to purify the gene-containing AAV vectors (full AAV vectors) from the manufactured AAV vectors to a high degree of purity.

[0018] Therefore, in one aspect, an object of the present disclosure is to provide a method for purifying a full AAV vector with high purity and simplicity. In an example of such an aspect, an object of the present disclosure is to provide a method for purifying a full AAV vector with high purity and simplicity from a solution containing an AAV vector, which is obtained by culturing AAV-producing cells, for example. **Means for Solving the Problems**

[0019] As a result of intensive studies to solve the above problems, the present inventors have found that by substituting specific residues among the amino acid residues constituting an adeno-associated virus (AAV)-binding protein with other amino acid residues, the alkali resistance is remarkably improved.

[0020] The present inventors have also found that a full AAV vector can be purified to high purity by subjecting a solution containing an AAV vector to affinity chromatography using an AAV-binding protein as a ligand and then further subjecting it to anion exchange chromatography.

[0021] That is, the invention of the present application includes the following aspects [1] to

[15] . [1] An AAV-binding protein selected from any of the following (i) to (iii): (i) An AAV-binding protein that contains at least the amino acid residues from the 25th serine to the 213th asparagine in the amino acid sequence set forth in SEQ ID NO: 2, provided that at least one amino acid substitution among the following (1) to (74) occurs in the amino acid residues from the 25th to the 213th and has AAV-binding activity; (1) The 139th threonine in SEQ ID NO: 2 is substituted with alanine (2) The 32nd isoleucine in SEQ ID NO: 2 is substituted with asparagine (3) The 34th leucine in SEQ ID NO: 2 is substituted with glutamine (4) The 41st leucine in SEQ ID NO: 2 is substituted with proline (5) The 42nd asparagine in Sequence ID No. 2 is replaced with lysine or serine. (6) The 45th valine in sequence number 2 is replaced with alanine. (7) The 48th valine in sequence number 2 is replaced with alanine. (8) The 51st lysine in sequence number 2 is replaced with arginine. (9) The 53rd glutamic acid in sequence number 2 is replaced with glycine. (10) The threonine at position 56 of sequence number 2 is replaced with alanine or serine. (11) The tyrosine at position 57 of sequence number 2 is replaced with phenylalanine. (12) The 58th aspartic acid in Sequence ID No. 2 is replaced with glycine. (13) The glutamine at position 60 of sequence number 2 is replaced with lysine. (14) The 63rd threonine in sequence number 2 is replaced with alanine. (15) The 73rd glutamic acid in sequence number 2 is replaced with aspartic acid. (16) The glutamine at position 78 of sequence number 2 is replaced with arginine. (17) The 79th isoleucine in sequence number 2 is replaced with phenylalanine. (18) The leucine at position 80 of sequence number 2 is replaced with proline. (19) Lysine at position 81 of sequence number 2 is replaced with arginine. (20) Leucine at position 82 of sequence number 2 is replaced with serine. (21) The 84th asparagine in Sequence ID No. 2 is replaced with aspartic acid or serine. (22) Leucine at position 89 of sequence number 2 is replaced with proline. (23) The tyrosine at position 90 of SEQ ID NO: 2 is replaced with phenylalanine or asparagine. (24) The glutamic acid at position 91 of sequence number 2 is replaced with glycine. (25) The tyrosine at position 92 of sequence number 2 is replaced with serine. (26) The 94th alanine in sequence number 2 is replaced with threonine. (27) The valine at position 95 of sequence number 2 is replaced with isoleucine. (28) The 97th glutamic acid in sequence number 2 is replaced with aspartic acid. (29) The 102nd histidine in sequence number 2 is replaced with arginine. (30) The valine at position 107 of sequence number 2 is replaced with alanine or isoleucine. (31) The valine at position 109 of sequence number 2 is replaced with glutamic acid. (32) The 110th threonine in sequence number 2 is replaced with alanine or serine. (33) The glutamic acid at position 114 of Sequence ID No. 2 is replaced with glycine. (34) The 115th proline in sequence number 2 is replaced with serine. (35) The valine at position 125 of sequence number 2 is replaced with alanine. (36) The aspartic acid at position 148 of SEQ ID NO: 2 is replaced with alanine. (37) The glutamic acid at position 166 of SEQ ID NO. 2 is replaced with aspartic acid. (38) The glutamic acid at position 167 of SEQ ID NO. 2 is replaced with aspartic acid. (39) The 174th alanine in sequence number 2 is replaced with proline. (40) Serine at position 179 of sequence number 2 is replaced with threonine. (41) The glutamine at position 180 of sequence number 2 is replaced with asparagine. (42) The asparagine at position 185 of sequence number 2 is replaced with lysine. (43) Lysine at position 210 of Sequence ID No. 2 is replaced with glutamic acid. (44) The 213th aspartic acid in SEQ ID NO. 2 is replaced with asparagine. (45) The 27th glycine in SEQ ID NO: 2 is replaced with aspartic acid. (46) The 30th aspartic acid in SEQ ID NO. 2 is replaced with glycine. (47) The glutamine at position 31 of sequence number 2 is replaced with arginine. (48) The 35th proline in sequence number 2 is replaced with alanine. (49) The 38th glutamic acid in sequence number 2 is replaced with valine. (50) The glutamine at position 60 of sequence number 2 is replaced with arginine. (51) The 75th glutamic acid in sequence number 2 is replaced with aspartic acid. (52) The histidine at position 76 of sequence number 2 is replaced with proline. (53) The tyrosine at position 92 of sequence number 2 is replaced with asparagine. (54) The glycine at position 105 of sequence number 2 is replaced with glutamic acid. (55) The valine at position 111 of sequence number 2 is replaced with alanine. (56) The glutamic acid at position 112 of sequence number 2 is replaced with lysine. (57) Arginine at position 119 of sequence number 2 is replaced with histidine. (58) The glutamine at position 128 of sequence number 2 is replaced with arginine. (59) Phenylalanine at position 129 of Sequence ID No. 2 is substituted with leucine. (60) Leucine at position 134 of sequence number 2 is replaced with proline. (61) Serine at position 146 of sequence number 2 is replaced with glycine. (62) The threonine at position 147 of sequence number 2 is replaced with serine. (63) The aspartic acid at position 150 of SEQ ID NO. 2 is replaced with valine. (64) The glutamic acid at position 158 of sequence number 2 is replaced with lysine. (65) Lysine at position 168 of sequence number 2 is replaced with arginine. (66) Serine at position 170 of sequence number 2 is replaced with arginine. (67) The 187th threonine in sequence number 2 is replaced with arginine. (68) The 191st threonine in sequence number 2 is replaced with isoleucine. (69) The 198th alanine in sequence number 2 is replaced with valine. (70) The 199th threonine in sequence number 2 is replaced with alanine. (71) Leucine at position 206 of sequence number 2 is replaced with methionine. (72) The asparagine at position 209 of sequence number 2 is replaced with glutamic acid. (73) The valine at position 212 of sequence number 2 is replaced with isoleucine. (74) The aspartic acid at position 213 of Sequence ID No. 2 is replaced with glycine. (ii) An AAV-binding protein having AAV-binding activity, comprising at least the amino acid residues from the 25th serine to the 213th aspartic acid of the amino acid sequence described in Sequence ID No. 2, wherein at least one of the amino acid substitutions from (1) to (74) above occurs in the amino acid residues from the 25th to the 213th, and further comprising one or more substitutions, deletions, insertions, and additions of one or more amino acid residues at one or more positions in addition to the amino acid substitutions shown in (1) to (74); (iii) An AAV-binding protein having AAV-binding activity, which includes an amino acid sequence in which at least one of the amino acid substitutions from (1) to (74) occurs in the amino acid sequence from the 25th serine to the 213th aspartic acid of the amino acid sequence described in Sequence ID No. 2, and which contains an amino acid sequence in which at least one of the amino acid substitutions remains. [2] AAV-binding protein described in [1], selected from any of the following (iv) to (vi): (iv) An AAV-binding protein having AAV-binding activity, comprising at least the amino acid residues from the 25th serine to the 213th aspartic acid of the amino acid sequence described in Sequence ID No. 2, wherein at least the amino acid substitutions described in (1) below occur in the 25th to 213th amino acid residues; (1) The threonine at position 139 of sequence number 2 is replaced with alanine. (v) An AAV-binding protein having AAV-binding activity, comprising at least the amino acid residues from the 25th serine to the 213th aspartic acid of the amino acid sequence described in Sequence ID No. 2, wherein at least the amino acid substitutions described in (1) above occur at the 25th to the 213th amino acid residues, and further comprising one or more additional substitutions, deletions, insertions, and additions of one or more amino acid residues at one or more positions in addition to the amino acid substitutions described in (1) above; (vi) An AAV-binding protein having AAV-binding activity, wherein the amino acid sequence from the 25th serine to the 213th aspartic acid of the amino acid sequence described in Sequence ID No. 2 has 70% or more identity with the entire amino acid sequence in which at least one of the above amino acid substitutions occurs, and includes an amino acid sequence in which at least one of the above amino acid substitutions remains. [3] An AAV-binding protein according to [1] or [2], comprising at least the amino acid residues from the 25th serine to the 213th aspartic acid of the amino acid sequence described in Sequence ID No. 2, wherein at least one of the amino acid substitutions shown in (A) to (P) below occurs in the 25th to 213th amino acid residues, and having AAV-binding activity; (A) The 42nd asparagine in SEQ ID NO: 2 is replaced with lysine, the 114th glutamic acid in SEQ ID NO: 2 is replaced with glycine, and the 139th threonine in SEQ ID NO: 2 is replaced with alanine. (B) Lysine at position 51 of SEQ ID NO: 2 is replaced with arginine, threonine at position 139 of SEQ ID NO: 2 is replaced with alanine, and alanine at position 174 of SEQ ID NO: 2 is replaced with proline. (C) The 42nd asparagine in SEQ ID NO: 2 is replaced with lysine, the 114th glutamic acid in SEQ ID NO: 2 is replaced with glycine, the 139th threonine in SEQ ID NO: 2 is replaced with alanine, and the 174th alanine in SEQ ID NO: 2 is replaced with proline. (D) Lysine at position 51 of SEQ ID NO: 2 is replaced with arginine, glutamic acid at position 114 of SEQ ID NO: 2 is replaced with glycine, threonine at position 139 of SEQ ID NO: 2 is replaced with alanine, and alanine at position 174 of SEQ ID NO: 2 is replaced with proline. (E) The 42nd asparagine in SEQ ID NO: 2 is replaced with lysine, the 51st lysine in SEQ ID NO: 2 is replaced with arginine, the 114th glutamic acid in SEQ ID NO: 2 is replaced with glycine, the 139th threonine in SEQ ID NO: 2 is replaced with alanine, and the 174th alanine in SEQ ID NO: 2 is replaced with proline. (F) The asparagine at position 42 of SEQ ID NO: 2 is replaced with lysine, the glutamic acid at position 114 of SEQ ID NO: 2 is replaced with glycine, the threonine at position 139 of SEQ ID NO: 2 is replaced with alanine, the alanine at position 174 of SEQ ID NO: 2 is replaced with proline, and the glutamine at position 180 of SEQ ID NO: 2 is replaced with asparagine. (G) The asparagine at position 42 of SEQ ID NO: 2 is replaced with lysine, the isoleucine at position 79 of SEQ ID NO: 2 is replaced with phenylalanine, the glutamic acid at position 114 of SEQ ID NO: 2 is replaced with glycine, the threonine at position 139 of SEQ ID NO: 2 is replaced with alanine, the alanine at position 174 of SEQ ID NO: 2 is replaced with proline, the glutamine at position 180 of SEQ ID NO: 2 is replaced with asparagine, and the threonine at position 187 of SEQ ID NO: 2 is replaced with arginine. (H) The asparagine at position 42 of SEQ ID NO: 2 is replaced with lysine, the valine at position 107 of SEQ ID NO: 2 is replaced with alanine, the glutamic acid at position 114 of SEQ ID NO: 2 is replaced with glycine, the threonine at position 139 of SEQ ID NO: 2 is replaced with alanine, the alanine at position 174 of SEQ ID NO: 2 is replaced with proline, the glutamine at position 180 of SEQ ID NO: 2 is replaced with asparagine, and the threonine at position 187 of SEQ ID NO: 2 is replaced with arginine. (I) The asparagine at position 42 of SEQ ID NO: 2 is replaced with lysine, the glutamic acid at position 114 of SEQ ID NO: 2 is replaced with glycine, the threonine at position 139 of SEQ ID NO: 2 is replaced with alanine, the lysine at position 168 of SEQ ID NO: 2 is replaced with arginine, the alanine at position 174 of SEQ ID NO: 2 is replaced with proline, the glutamine at position 180 of SEQ ID NO: 2 is replaced with asparagine, and the threonine at position 187 of SEQ ID NO: 2 is replaced with arginine. (J) The asparagine at position 42 of SEQ ID NO: 2 is replaced with lysine, the isoleucine at position 79 of SEQ ID NO: 2 is replaced with phenylalanine, the valine at position 107 of SEQ ID NO: 2 is replaced with alanine, the glutamic acid at position 114 of SEQ ID NO: 2 is replaced with glycine, the threonine at position 139 of SEQ ID NO: 2 is replaced with alanine, the lysine at position 168 of SEQ ID NO: 2 is replaced with arginine, the alanine at position 174 of SEQ ID NO: 2 is replaced with proline, and the glutamine at position 180 of SEQ ID NO: 2 is replaced with asparagine. (K) The asparagine at position 42 of SEQ ID NO: 2 is replaced with lysine, the isoleucine at position 79 of SEQ ID NO: 2 is replaced with phenylalanine, the valine at position 107 of SEQ ID NO: 2 is replaced with alanine, the glutamic acid at position 114 of SEQ ID NO: 2 is replaced with glycine, the threonine at position 139 of SEQ ID NO: 2 is replaced with alanine, the lysine at position 168 of SEQ ID NO: 2 is replaced with arginine, the alanine at position 174 of SEQ ID NO: 2 is replaced with proline, the glutamine at position 180 of SEQ ID NO: 2 is replaced with asparagine, and the threonine at position 187 of SEQ ID NO: 2 is replaced with arginine. (L) The glycine at position 27 of SEQ ID NO: 2 is replaced with aspartic acid, the asparagine at position 42 of SEQ ID NO: 2 is replaced with lysine, the isoleucine at position 79 of SEQ ID NO: 2 is replaced with phenylalanine, the valine at position 107 of SEQ ID NO: 2 is replaced with alanine, the glutamic acid at position 114 of SEQ ID NO: 2 is replaced with glycine, the threonine at position 139 of SEQ ID NO: 2 is replaced with alanine, the lysine at position 168 of SEQ ID NO: 2 is replaced with arginine, the alanine at position 174 of SEQ ID NO: 2 is replaced with proline, and the glutamine at position 180 of SEQ ID NO: 2 is replaced with asparagine. (M) The asparagine at position 42 of SEQ ID NO: 2 is replaced with lysine, the isoleucine at position 79 of SEQ ID NO: 2 is replaced with phenylalanine, the valine at position 107 of SEQ ID NO: 2 is replaced with alanine, the glutamic acid at position 114 of SEQ ID NO: 2 is replaced with glycine, the threonine at position 139 of SEQ ID NO: 2 is replaced with alanine, the lysine at position 168 of SEQ ID NO: 2 is replaced with arginine, the alanine at position 174 of SEQ ID NO: 2 is replaced with proline, the glutamine at position 180 of SEQ ID NO: 2 is replaced with asparagine, and the lysine at position 210 of SEQ ID NO: 2 is replaced with glutamic acid. (N) The asparagine at position 42 of SEQ ID NO: 2 is replaced with lysine, the isoleucine at position 79 of SEQ ID NO: 2 is replaced with phenylalanine, the valine at position 107 of SEQ ID NO: 2 is replaced with alanine, the glutamic acid at position 114 of SEQ ID NO: 2 is replaced with glycine, the threonine at position 139 of SEQ ID NO: 2 is replaced with alanine, the lysine at position 168 of SEQ ID NO: 2 is replaced with arginine, the alanine at position 174 of SEQ ID NO: 2 is replaced with proline, the glutamine at position 180 of SEQ ID NO: 2 is replaced with asparagine, and the valine at position 212 of SEQ ID NO: 2 is replaced with isoleucine. (O) The glycine at position 27 of SEQ ID NO: 2 is replaced with aspartic acid, the asparagine at position 42 of SEQ ID NO: 2 is replaced with lysine, the isoleucine at position 79 of SEQ ID NO: 2 is replaced with phenylalanine, the valine at position 107 of SEQ ID NO: 2 is replaced with alanine, the glutamic acid at position 114 of SEQ ID NO: 2 is replaced with glycine, the threonine at position 139 of SEQ ID NO: 2 is replaced with alanine, the lysine at position 168 of SEQ ID NO: 2 is replaced with arginine, the alanine at position 174 of SEQ ID NO: 2 is replaced with proline, the glutamine at position 180 of SEQ ID NO: 2 is replaced with asparagine, the lysine at position 210 of SEQ ID NO: 2 is replaced with glutamic acid, and the valine at position 212 of SEQ ID NO: 2 is replaced with isoleucine. [4] A polynucleotide encoding an AAV-binding protein as described in any of [1] to [3]. [5] An expression vector containing the polynucleotide described in [4]. [6] [5] A transformant obtained by transforming Escherichia coli with the expression vector described in the description. [7] A method for producing an AAV-binding protein, comprising the steps of culturing the transformant described in [6] to express an AAV-binding protein, and recovering the AAV-binding protein expressed from the obtained culture. [8] An AAV adsorbent comprising an insoluble carrier and an AAV-binding protein according to any one of [1] to [3] immobilized on the carrier. [9] A column containing the AAV adsorbent described in [8].

[10] A method for purifying or analyzing AAV, comprising the steps of adding a solution containing AAV to the column described in [9] to adsorb the AAV onto the adsorbent, and eluting the AAV adsorbed onto the adsorbent using an eluent.

[11] The method for purifying or analyzing AAV according to

[10] , further comprising the step of washing the AAV adsorbent with an alkaline solution after the step of eluting AAV.

[12] A method for purifying AAV contained in a sample, A step of adding a sample containing AAV to an adsorbent containing an insoluble carrier and an AAV-binding protein immobilized on the insoluble carrier, and adsorbing the AAV onto the adsorbent, A step of eluting the AAV adsorbed onto the adsorbent, A step of adding a fraction containing AAV eluted in the elution step to a carrier for anion exchange chromatography and adsorbing the AAV onto the carrier for anion exchange chromatography, The process includes a step of eluting AAV adsorbed onto the carrier for anion exchange chromatography, Purification method.

[13] A method for purifying AAV contained in a sample, A step of adding a sample containing AAV to an adsorbent containing an insoluble carrier and an AAV-binding protein immobilized on the insoluble carrier, and adsorbing the AAV onto the adsorbent, A step of eluting the AAV adsorbed onto the adsorbent, A step of adding a fraction containing AAV eluted in the elution step to a carrier for anion exchange chromatography and adsorbing the AAV onto the carrier for anion exchange chromatography, The process includes eluting AAV adsorbed onto the aforementioned anion exchange chromatography carrier, A purification method wherein the AAV-binding protein is the AAV-binding protein described in any of [1] to [3].

[14] The purification method according to

[12] or

[13] , wherein the step of eluting AAV adsorbed on a carrier for anion exchange chromatography is to elute the AAV adsorbed on the carrier using an eluent with a conductivity of 13.5 mS / cm or less, and then to elute the AAV remaining on the carrier using an eluent with a conductivity of 15.0 mS / cm or more.

[15] The purification method according to

[14] , wherein the eluate contains choline chloride. [Brief explanation of the drawing]

[0022] [Figure 1] This is a chromatogram obtained by applying a solution containing the AAV vector (AAV8-EGFP) to an affinity chromatograph (AVR29c column). [Figure 2] This chromatogram was obtained by applying a fraction containing AAV8-EGFP, purified using an AVR29c column, to anion exchange chromatography (GigaCapQ column). [Figure 3] This is a chromatogram obtained by directly applying a solution containing AAV8-EGFP to a GigaCapQ column. [Figure 4] The chromatograms shown in Figure 3 were obtained by applying size exclusion chromatography (G6000PWXL column) to each peak. [Figure 5] This figure shows the purity of AVR29c and AVR29c(-) confirmed by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). [Figure 6] This figure shows the results of evaluating the binding compatibility of AVR29c or AVR29c(-) to VLP2. [Figure 7] This figure shows the results of evaluating the elution properties of AAV8 adsorbed onto an AVR29c column or an AVR29c(-) column. [Figure 8]These are chromatograms obtained by applying a fraction containing AAV8-EGFP purified using an AVR29c column to an anion exchange chromatography column (SkillPak GigaCapQ column, 1.0 mL). The chromatograms are obtained by adjusting the mixing ratio for eluting impurities of solution AEX-A and solution AEX-C, which is 20 mmol / L Tris-HCl buffer (pH 9.0) containing 300 mmol / L choline chloride, to (a) 49.0%, (b) 48.0%, (c) 47.0%, and (d) 46.5%. [Figure 9] These are chromatograms obtained by applying a fraction containing AAV8-EGFP, purified using an AVR29c column, to a SkillPak GigaCapQ column (volume 5.0 mL). (a) is a chromatogram obtained by affinity chromatography (AF) purification using an AVR29c column. (b) is a chromatogram obtained by anion exchange chromatography (AEX) purification using a SkillPak GigaCapQ column. [Figure 10] This figure compares the positive rates when cells were infected using the AAV8-EGFP solution obtained in Example 24(1) (peak indicated by the black arrow in Figure 9(a)), and the eluted fractions obtained in Examples 24(3) (peak indicated by the white arrow in Figure 9(b)) and (4) (peak indicated by the black arrow in Figure 9(b)). The infection dose shown on the X axis is represented by the number of particles per cell in (a), and by the number of vector genomes (VGs) per cell in (b). [Modes for carrying out the invention]

[0023] The details of this disclosure are described below.

[0024] In this specification, an AAV-binding protein is defined as a protein containing at least the amino acid residues from the 312th serine (S) to the 500th aspartic acid (D) in the amino acid sequence of KIAA0319L (UniProt No. Q8IZA0), described in Sequence ID No. 1, which is a native AAV-binding protein, and which corresponds to the extracellular domain 1 (PKD1) and domain 2 (PKD2), provided that the protein has undergone specific amino acid substitutions at the 312th to 500th amino acid residues. Therefore, the AAV-binding protein of this disclosure may also contain all or part of the other extracellular domains (domain 3 (PKD3), domain 4 (PKD4), and domain 5 (PKD5)) located on the C-terminal side of the protein. Furthermore, the AAV-binding protein of this disclosure may also contain all or part of signal sequences and cysteine-rich regions, such as the MANSC (Motif At N terminus with Seven Cysteines) domain located on the N-terminal side of PKD1. Furthermore, the AAV-binding protein of this disclosure may include all or part of the transmembrane region located at the N-terminal and / or C-terminal end of the extracellular region, as well as the intracellular region.

[0025] The amino acid substitution at the aforementioned specific position specifically includes at least the amino acid residues from the 312th serine to the 500th aspartic acid residue in the amino acid sequence described in Sequence ID No. 1, provided that in the amino acid residues from the 312th to the 500th, It contains at least the amino acid substitutions V317D (this notation indicates that the valine at position 317 of SEQ ID NO: 1 is substituted with aspartic acid, and so on), N324H, V326A, A330V, Q334L, E335V, T341A, Y342S, K362E, K371N, F379Y, K380R, V381A, I382V, G390S, K399E, K467Q, S476R, S482T, N487D, and N492D, and also I319N, L321Q , L328P, N329K, N329S, V332A, E(V)335A (This notation indicates that the 335th glutamic acid in SEQ ID NO: 1 was initially substituted with valine in SEQ ID NO: 2, and then further substituted with alanine; the same applies below), K338R, E340G, T343A, T343S, Y344F, D345G, Q347K, T350A, E360D, Q365R, I366F, L367P, K368R, L369S, K(N)371D, K(N)371S, L376P, Y377F, Y377N, E378G, F(Y)379S, V(A)381T, I(V)382I, E384D, H389R, V394A, V394I, V396E, T397A, T397S, E401G, P402 S, V412A, T426A, D435A, E453D, E454D, A461P, S466T, K(Q)467N, N472K, K497E, D500N, G314D, V(D)317G, Q318R, P322A, E325V, It is preferable that at least one amino acid substitution of any of the following occurs: Q347R, E362D, H363P, F(Y)379N, G392E, V398A, K(E)399K, R406H, Q415R, F416L, L421P, S433G, T434S, D437V, E445K, K455R, S457R, T474R, T478I, A485V, T486A, L493M, N496D, V499I, and D500G, as this improves stability to alkali. Among these, an AAV-binding protein with at least one amino acid substitution of T426A is a more preferred embodiment.

[0026] Furthermore, an AAV-binding protein consisting of amino acid residues from the 312th serine to the 500th aspartic acid residue in the amino acid sequence described in Sequence ID No. 1, provided that the amino acid substitutions V317D, N324H, V326A, A330V, Q334L, E335V, T341A, Y342S, K362E, K371N, F379Y, K380R, V381A, I382V, G390S, K399E, K467Q, S476R, S482T, N487D, and N492D occur in the amino acid sequence from the 25th serine to the 213th aspartic acid residue in the amino acid sequence described in Sequence ID No. 2 are equivalent to an AAV-binding protein consisting of amino acid residues from the 25th serine to the 213th aspartic acid residue in the amino acid sequence described in Sequence ID No. 2. Also, the position of the amino acid residue in Sequence ID No. 2 corresponds to the position 287 amino acid residues from the position in Sequence ID No. 1. Specifically, the amino acid substitution in V317D corresponds to the substitution of the amino acid residue located at position 30 in SEQ ID NO: 2, and the amino acid substitution in T426A corresponds to the substitution of the amino acid residue located at position 139 in SEQ ID NO: 2.

[0027] In the AAV-binding proteins of this disclosure, there are no particular restrictions on the number of substituted amino acids. As an example, the AAV-binding proteins shown in any of (A) to (P) below are given. These AAV-binding proteins are more preferable in that they have improved stability against alkali.

[0028] (A) An AAV-binding protein comprising the amino acid residues from the 25th serine to the 213th aspartic acid of the amino acid sequence described in Sequence ID No. 2, wherein at least the amino acid substitutions N329K, E401G and T426A occur in the amino acid residues from the 25th to the 213th.

[0029] (B) An AAV-binding protein comprising the amino acid residues from the 25th serine to the 213th aspartic acid of the amino acid sequence described in Sequence ID No. 2, wherein at least the amino acid substitutions K338R, T426A, and A461P occur in the amino acid residues from the 25th to the 213th.

[0030] (C) An AAV-binding protein comprising the amino acid residues from the 25th serine to the 213th aspartic acid of the amino acid sequence described in Sequence ID No. 2, wherein at least the amino acid substitutions N329K, E401G, T426A, and A461P occur in the amino acid residues from the 25th to the 213th.

[0031] (D) An AAV-binding protein comprising the amino acid residues from the 25th serine to the 213th aspartic acid of the amino acid sequence described in Sequence ID No. 2, wherein at least the amino acid substitutions K338R, E401G, T426A, and A461P occur in the amino acid residues from the 25th to the 213th.

[0032] (E) An AAV-binding protein comprising the amino acid residues from the 25th serine to the 213th aspartic acid of the amino acid sequence described in Sequence ID No. 2, wherein at least the amino acid substitutions N329K, K338R, E401G, T426A, and A461P occur in the amino acid residues from the 25th to the 213th.

[0033] (F) An AAV-binding protein comprising the amino acid residues from the 25th serine to the 213th aspartic acid of the amino acid sequence described in Sequence ID No. 2, wherein at least the amino acid substitutions N329K, E401G, T426A, A461P and K(Q)467N occur in the amino acid residues from the 25th to the 213th.

[0034] (G) An AAV-binding protein comprising the amino acid residues from the 25th serine to the 213th aspartic acid of the amino acid sequence described in Sequence ID No. 2, wherein at least the amino acid substitutions N329K, I366F, E401G, T426A, A461P, K(Q)467N and T474R occur in the amino acid residues from the 25th to the 213th.

[0035] (H) An AAV-binding protein comprising the amino acid residues from the 25th serine to the 213th aspartic acid of the amino acid sequence described in Sequence ID No. 2, wherein at least the amino acid substitutions N329K, V394A, E401G, T426A, A461P, K(Q)467N and T474R occur in the amino acid residues from the 25th to the 213th.

[0036] (I) An AAV-binding protein comprising the amino acid residues from the 25th serine to the 213th aspartic acid of the amino acid sequence described in Sequence ID No. 2, wherein at least the amino acid substitutions N329K, E401G, T426A, K455R, A461P, K(Q)467N and T474R occur in the amino acid residues from the 25th to the 213th.

[0037] (J) An AAV-binding protein comprising the amino acid residues from the 25th serine to the 213th aspartic acid of the amino acid sequence described in Sequence ID No. 2, wherein at least the amino acid substitutions N329K, I366F, V394A, E401G, T426A, K455R, A461P and K(Q)467N occur in the amino acid residues from the 25th to the 213th.

[0038] (K) An AAV-binding protein comprising the amino acid residues from the 25th serine to the 213th aspartic acid in the amino acid sequence described in Sequence ID No. 2, wherein at least the amino acid substitutions N329K, I366F, V394A, E401G, T426A, K455R, A461P, K(Q)467N, and T474R occur in the amino acid residues from the 25th to the 213th.

[0039] (L) An AAV-binding protein comprising the amino acid residues from the 25th serine to the 213th aspartic acid of the amino acid sequence described in Sequence ID No. 2, wherein at least the amino acid substitutions G314D, N329K, I366F, V394A, E401G, T426A, K455R, A461P and K(Q)467N occur in the amino acid residues from the 25th to the 213th.

[0040] (M) An AAV-binding protein comprising the amino acid residues from the 25th serine to the 213th aspartic acid of the amino acid sequence described in Sequence ID No. 2, wherein at least the amino acid substitutions N329K, I366F, V394A, E401G, T426A, K455R, A461P, K(Q)467N, and K497E occur in the amino acid residues from the 25th to the 213th.

[0041] (N) An AAV-binding protein comprising the amino acid residues from the 25th serine to the 213th aspartic acid of the amino acid sequence described in Sequence ID No. 2, wherein at least the amino acid substitutions N329K, I366F, V394A, E401G, T426A, K455R, A461P, K(Q)467N and V499I occur in the amino acid residues from the 25th to the 213th.

[0042] (O) An AAV-binding protein comprising the amino acid residues from the 25th serine to the 213th aspartic acid of the amino acid sequence described in Sequence ID No. 2, wherein at least the amino acid substitutions G314D, N329K, I366F, V394A, E401G, T426A, K455R, A461P, K(Q)467N, K497E, and V499I occur in the amino acid residues from the 25th to the 213th.

[0043] In (ii) and (v) above, "one or several" means one of the following, although it varies depending on the position of amino acid substitutions and the type of amino acid residue in the three-dimensional structure of the AAV-binding protein. For example, it means one to 50, one to 40, one to 30, one to 25, one to 20, one to 18, one to 15, one to 10, one to 9, one to 8, one to 7, one to 6, one to 5, one to 4, one to 3, one to 2, or one.

[0044] Examples of substitutions, deletions, insertions, or additions described in (ii) and (v) above include the amino acid residue substitutions disclosed in WO2021 / 106882 and WO2023 / 140197.

[0045] In addition to the amino acid substitutions at specific positions mentioned above, the "substitution of one or more amino acid residues" in (ii) and (v) above may also include conservative substitutions, which occur between amino acids with similar physical and / or chemical properties. It is generally known to those skilled in the art that conservative substitutions maintain the function of the protein between the substituted and unsubstituted portions. Examples of conservative substitutions include substitutions between glycine and alanine, serine and threonine, or glutamic acid and aspartic acid (Protein Structure and Function, Medical Science International, 9, 2005). Another example of the above amino acid substitutions is substitution to monomerize AAV-binding proteins. Specifically, this includes amino acid substitutions where a cysteine ​​residue, which readily forms higher-order structures, is replaced with a serine or methionine residue.

[0046] Furthermore, the "substitution, deletion, insertion, or addition of one or more amino acid residues" in (ii) and (v) above also includes naturally occurring mutations (mutants or variants) based on differences in the origin of AAV-binding proteins or differences in species.

[0047] The amino acid sequence identity in (iii) and (vi) above only needs to be 70% or more, and may have a higher degree of identity (e.g., 80% or more, 85% or more, 90% or more, or 95% or more). "Amino acid sequence identity" means identity of the entire amino acid sequence. "Identity" between amino acid sequences means the ratio of amino acid residues of the same type in those amino acid sequences (Experimental Medicine, 31(3), Yodosha). Amino acid sequence identity can be determined using alignment programs such as BLAST (Basic Local Alignment Search Tool) or FASTA.

[0048] This disclosure provides a method for purifying AAV contained in a sample using an adsorbent (hereinafter also referred to as "AAV adsorbent") comprising an insoluble carrier and an AAV-binding protein immobilized on the carrier. The AAV-binding protein is not particularly limited as long as it is a polypeptide capable of binding to AAV, and examples include laminin receptors such as integrins, anti-AAV antibodies, and AAV receptors (AAVRs).

[0049] A preferred embodiment when the AAV-binding protein is an anti-AAV antibody is a polypeptide containing at least a heavy chain antibody variable region (VHH) capable of binding to AAV. A preferred embodiment when the AAV-binding protein is AAVR is the following: from <iii>A polypeptide selected from one of the following is an example. A polypeptide comprising at least the amino acid residues from the 312th serine to the 500th aspartic acid of the amino acid sequence described in SEQ ID NO: 1. <ii>A polypeptide comprising at least the amino acid residues from the 312th serine to the 500th aspartic acid of the amino acid sequence described in Sequence ID No. 1, wherein one or more substitutions, deletions, insertions, and additions of one or more amino acid residues occur at one or more positions within the amino acid residues from the 312th to the 500th, and which has AAV binding activity. <iii>It contains at least the amino acid residues from the 312th serine to the 500th aspartic acid of the amino acid sequence described in Sequence ID No. 1, provided that it has 70% or more identity with the entire amino acid sequence consisting of the amino acid residues from the 312th to the 500th. A polypeptide that also has AAV binding activity.

[0050] The amino acid sequence described in Sequence ID No. 1 is the amino acid sequence of KIAA0319L (official database: UniProt, accession number: Q8IZA0), which is one form of AAVR. The amino acid residues from the 312th serine (S) to the 500th aspartic acid (D) in the amino acid sequence described in Sequence ID No. 1 correspond to the extracellular domain 1 (PKD1) and domain 2 (PKD2) of KIAA0319L.

[0051] The aforementioned from <iii>The polypeptides shown in any of the above only need to include at least the regions corresponding to PKD1 and PKD2 of KIAA0319L as described above. For example, they may include all or part of the regions corresponding to other extracellular domains on the C-terminal side of PKD2 (domain 3 (PKD3), domain 4 (PKD4), and domain 5 (PKD5)), or all or part of the regions corresponding to signal sequences such as the MANSC (Motif At N terminus with Seven Cysteines) domain on the N-terminal side of PKD1, or all or part of the transmembrane and intracellular regions on the N-terminal and / or C-terminal sides of the extracellular region.

[0052] The aforementioned <ii>Examples include the AAV-binding proteins disclosed in WO2021 / 106882 and WO2023 / 140197, and AAV-binding proteins having at least one of the amino acid substitutions shown in (I) through (LXIX) below; (I) The glycine at position 314 of SEQ ID NO: 1 is replaced with aspartic acid. (II) The valine at position 317 of sequence number 1 is replaced with glycine. (III) The glutamine at position 318 of sequence number 1 is replaced with arginine. (IV) The isoleucine at position 319 of Sequence ID No. 1 is replaced with asparagine. (V) Leucine at position 321 of sequence number 1 is replaced with glutamine. (VI) The 322nd proline in sequence number 1 is replaced with alanine. (VII) The glutamic acid at position 325 of sequence number 1 is replaced with valine. (VIII) Leucine at position 328 of sequence number 1 is replaced with proline. (IX) The asparagine at position 329 of Sequence ID No. 1 is replaced with lysine or serine. (X) The valine at position 332 of sequence number 1 is replaced with alanine. (XI) The glutamic acid at position 335 of sequence number 1 is replaced with alanine. (XII) Lysine at position 338 of sequence number 1 is replaced with arginine. (XIII) The 340th glutamic acid in sequence number 1 is replaced with glycine. (XIV) The 343rd threonine in sequence number 1 is replaced with alanine or serine. (XV) The tyrosine at position 344 of sequence number 1 is replaced with phenylalanine. (XVI) The aspartic acid at position 345 of Sequence ID No. 1 is replaced with glycine. (XVII) The glutamine at position 347 of sequence number 1 is replaced with lysine or arginine. (XVIII) The 350th threonine in sequence number 1 is replaced with alanine. (XIX) The 360th glutamic acid in sequence number 1 is replaced with aspartic acid. (XX) Lysine at position 362 of Sequence ID No. 1 is replaced with aspartic acid. (XXI) The histidine at position 363 of sequence number 1 is replaced with proline. (XXII) The glutamine at position 365 of sequence number 1 is replaced with arginine. (XXIII) The 366th isoleucine in sequence number 1 is replaced with phenylalanine. (XXIV) Leucine at position 367 of sequence number 1 is replaced with proline. (XXV) Lysine at position 368 of sequence number 1 is replaced with arginine. (XXVI) Leucine at position 369 of sequence number 1 is replaced with serine. (XXVII) Lysine at position 371 of Sequence ID No. 1 is replaced with aspartic acid or serine. (XXVIII) Leucine at position 376 of sequence number 1 is replaced with proline. (XXIX) The tyrosine at position 377 of SEQ ID NO: 1 is replaced with phenylalanine or asparagine. (XXX) The glutamic acid at position 378 of sequence number 1 is replaced with glycine. (XXXI) Phenylalanine at position 379 of Sequence ID No. 1 is replaced with serine or asparagine. (XXXII) The valine at position 381 of sequence number 1 is replaced with threonine. (XXXIII) The 384th glutamic acid in sequence number 1 is replaced with aspartic acid. (XXXIV) The histidine at position 389 in sequence number 1 is replaced with arginine. (XXXV) The glycine at position 392 of sequence number 1 is replaced with glutamic acid. (XXXVI) The valine at position 394 of sequence number 1 is replaced with alanine or isoleucine. (XXXVII) The valine at position 396 of sequence number 1 is replaced with glutamic acid. (XXXVIII) The 397th threonine in sequence number 1 is replaced with alanine or serine. (XXXIV) The valine at position 398 of sequence number 1 is replaced with alanine. (XL) The glutamic acid at position 401 of sequence number 1 is replaced with glycine. (XLI) The 402nd proline in sequence number 1 is replaced with serine. (XLII) Arginine at position 406 of sequence number 1 is replaced with histidine. (XLIII) The 412th valine in sequence number 1 is replaced with alanine. (XLIV) The glutamine at position 415 of sequence number 1 is replaced with arginine. (XLV) Phenylalanine at position 416 of Sequence ID No. 1 is substituted with leucine. (XLVI) Leucine at position 421 of sequence number 1 is replaced with proline. (XLVII) The threonine at position 426 of sequence number 1 is replaced with alanine. (XLVIII) Serine at position 433 of sequence number 1 is replaced with glycine. (XLIX) The threonine at position 434 of sequence number 1 is replaced with serine. (L) The aspartic acid at position 435 of SEQ ID NO: 1 is replaced with alanine. (LI) The aspartic acid at position 437 of SEQ ID NO: 1 is replaced with valine. (LII) The glutamic acid at position 445 of sequence number 1 is replaced with lysine. (LIII) The 453rd glutamic acid in sequence number 1 is replaced with aspartic acid. (LIV) The glutamic acid at position 454 of sequence number 1 is replaced with aspartic acid. (LV) Lysine at position 455 of sequence number 1 is replaced with arginine. (LVI) Serine at position 457 of sequence number 1 is replaced with arginine. (LVII) Alanine at position 461 of sequence number 1 is replaced with proline. (LVIII) Serine at position 466 of sequence number 1 is replaced with threonine. (LIX) Lysine at position 467 of sequence number 1 is replaced with asparagine. (LX) The asparagine at position 472 of sequence number 1 is substituted with lysine. (LXI) The threonine at position 474 of sequence number 1 is replaced with arginine. (LXII) The threonine at position 478 of sequence number 1 is replaced with isoleucine. (LXIII) Alanine at position 485 of sequence number 1 is replaced with valine. (LXIV) The threonine at position 486 of sequence number 1 is replaced with alanine. (LXV) Leucine at position 493 of sequence number 1 is replaced with methionine. (LXVI) The 496th asparagine molecule in sequence number 1 is replaced with glutamic acid. (LXVII) Lysine at position 497 of sequence number 1 is replaced with glutamic acid. (LXVIII) The valine at position 499 of sequence number 1 is replaced with isoleucine. (LXIX) The aspartic acid at position 500 of SEQ ID NO: 1 is replaced with glycine or asparagine. Also, the above <ii>Examples of substitutions, deletions, insertions, or additions described herein include the amino acid residue substitutions disclosed in WO2021 / 106882 and WO2023 / 140197, as well as the amino acid substitutions shown in (I) through (LXIX) above.

[0053] The aforementioned <ii>In this context, "one or several" means one or more amino acid residues, although this can vary depending on the position and type of amino acid substitution in the three-dimensional structure of AAVR. For example, it can mean one to 50, one to 40, one to 30, one to 25, one to 20, one to 15, one to 10, one to 9, one to 8, one to 7, one to 6, one to 5, one to 4, one to 3, one to 2, or one. The substitution of "one or several" amino acid residues may occur at positions other than those disclosed in WO2021 / 106882 and WO2023 / 140197, as well as those shown in (I) to (LXIX), as long as AAV binding activity is maintained.

[0054] Note the above <ii>In the context of "substitution of one or more amino acid residues," the term "substitution of one or more amino acid residues" may include not only amino acid substitutions at specific positions as described above, but also conservative substitutions that occur between amino acids with similar physical and / or chemical properties. It is generally known to those skilled in the art that conservative substitutions maintain the function of the protein between the substituted and unsubstituted portions. Examples of conservative substitutions include substitutions between glycine and alanine, serine and threonine, or glutamic acid and aspartic acid (Protein Structure and Function, Medical Science International, 9, 2005). <ii>The term "substitution, deletion, insertion, or addition of one or more amino acid residues" in this context includes naturally occurring mutations (mutants or variants) based on differences in the origin of AAVR or differences in species.

[0055] The aforementioned <iii>The amino acid sequence identity in a given sequence only needs to be 70% or higher, and may have a higher degree of identity (e.g., 80% or higher, 85% or higher, 90% or higher, or 95% or higher). "Identity" between amino acid sequences refers to the ratio of amino acid residues of the same type in those sequences (Experimental Medicine, 31(3), Yodosha). The identity of amino acid sequences can be determined using alignment programs such as BLAST (Basic Local Alignment Search Tool) or FASTA.

[0056] In one embodiment, the AAV-binding protein of this disclosure may have an oligopeptide added to its N-terminus or C-terminus that is useful for separating it from a solution in the presence of contaminants. Examples of such oligopeptides include polyhistidine, polylysine, polyarginine, polyglutamic acid, and polyaspartic acid. Alternatively, an oligopeptide containing cysteine, which is useful for immobilizing the AAV-binding protein on a solid phase such as a support for chromatography, may be added to the N-terminus or C-terminus of the AAV-binding protein.

[0057] The length of the oligopeptide to be attached to the N-terminus 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. When attaching the oligopeptide to the AAV-binding protein of this disclosure, the polynucleotide encoding the oligopeptide may be prepared and then genetically engineered to attach it to the N-terminus or C-terminus of the AAV-binding protein using a method well known to those skilled in the art. Alternatively, the oligopeptide may be chemically synthesized and chemically attached to the N-terminus or C-terminus of the AAV-binding protein.

[0058] Furthermore, a signal peptide may be added to the N-terminus of the AAV-binding protein of this disclosure to promote efficient expression in Escherichia coli used as a host. Examples of such signal peptides include those that induce protein secretion into the periplasm, such as PelB, OmpA, DsbA, DsbC, MalE, and TorT (Japanese Patent Publication No. 2011-097898). Alternatively, the addition of a signal peptide to the N-terminus may be omitted. In this case, it is preferable in that the homogeneity of the protein is improved during protein preparation.

[0059] As an example of a method for producing the polynucleotide encoding the AAV-binding protein of this disclosure (hereinafter also referred to as "the polynucleotide of this disclosure"), (I) A method for converting the amino acid sequence of the AAV-binding protein of this disclosure into a nucleotide sequence and artificially synthesizing a polynucleotide containing said nucleotide sequence, (II) An example of a method is to prepare polynucleotides containing the whole or partial sequence of the AAV-binding protein directly or artificially, or from the cDNA of the AAV-binding protein using a DNA amplification method such as PCR, and then ligate the prepared polynucleotides in an appropriate manner.

[0060] In the method described in (I) above, when converting from an amino acid sequence to a nucleotide sequence, it is preferable to consider the frequency of codon use in the host E. coli being transformed. Specifically, for arginine (R), AGA / AGG / CGG / CGA, for isoleucine (I), ATA, for leucine (L), CTA, for glycine (G), GGA, and for proline (P), CCC are used infrequently (they are so-called rare codons), so the conversion should avoid these codons. Codon usage frequency analysis can also be performed using public databases (for example, the Codon Usage Database on the Kazusa DNA Research Institute website).

[0061] When introducing mutations into the polynucleotides of this disclosure, the error-prone PCR method can be used. The reaction conditions in the error-prone PCR method are not particularly limited as long as they are conditions that can introduce the desired mutations into the polynucleotide encoding the AAV-binding protein. For example, mutations can be introduced into the polynucleotide by making the concentrations of the four types of deoxynucleotides (dATP / dTTP / dCTP / dGTP), which are substrates, heterogeneous, and adding MnCl2 at a concentration of 0.01 mM to 10 mM (preferably 0.1 mM to 1 mM) to the PCR reaction mixture and performing PCR. In addition to the error-prone PCR method, other methods for introducing mutations include introducing mutations into polynucleotides containing the entire or partial sequence of the AAV-binding protein by contacting and acting on them with a mutagenic agent or by irradiating them with ultraviolet light. As the agent used as the mutagenic agent in this method, any mutagenic agent commonly used by those skilled in the art may be used, such as hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine, nitrite, sulfite, hydrazine, etc.

[0062] When transforming a host E. coli using the polynucleotides disclosed herein, the polynucleotides themselves may be used, but it is more preferable to use an expression vector (for example, a bacteriophage, cosmid, or plasmid commonly used for the transformation of prokaryotic or eukaryotic cells) into which the polynucleotides disclosed herein have been inserted at an appropriate position. The expression vector is not particularly limited as long as it can stably exist and replicate within the host (E. coli) to be transformed, and examples include pET plasmid vectors, pUC plasmid vectors, pTrc plasmid vectors, and pCDF plasmid vectors.

[0063] Furthermore, the aforementioned appropriate location may mean a location that does not disrupt the replication function of the expression vector, the desired antibiotic marker, or the region involved in transduction. When inserting the polynucleotides of this disclosure into the expression vector, it is preferable to insert them in a state where they are linked to a functional polynucleotide such as a promoter necessary for expression. Examples of such promoters include the trp promoter, tac promoter, trc promoter, lac promoter, T7 promoter, recA promoter, and lpp promoter.

[0064] Transforming host Escherichia coli using the expression vector containing the polynucleotides of this disclosure, prepared by the method described above (hereinafter also simply referred to as "the expression vector of this disclosure") can be done by methods commonly used by those skilled in the art. Specifically, transformation can be performed by methods described in known literature such as Molecular Cloning (Cold Spring Harbor Laboratory), 256, 1992. Transformants obtained by the above-described method can be screened using appropriate methods to obtain transformants capable of expressing the AAV-binding protein of this disclosure (hereinafter also simply referred to as "transformants of this disclosure").

[0065] To prepare the expression vector of this disclosure from the transformant of this disclosure, the culture obtained by culturing the transformant of this disclosure may be prepared using alkaline extraction or a commercially available extraction kit such as the QIAprep Spin Miniprep kit (Qiagen).

[0066] The AAV-binding protein can be produced by culturing the transformant of this disclosure and recovering the AAV-binding protein from the resulting culture. In this specification, the culture may include not only the cultured cells of the transformant of this disclosure themselves, but also the culture medium used for culturing.

[0067] The transformants used in the protein production method can be cultured in a medium suitable for culturing the target host (Escherichia coli). A preferred medium is LB (Luria-Bertani) medium supplemented with the necessary nutrients. In order to selectively grow the transformants depending on whether or not the vector of this disclosure has been introduced, it is preferable to add a drug corresponding to the drug resistance gene contained in the vector to the culture medium. For example, if the vector contains a kanamycin resistance gene, kanamycin should be added to the culture medium.

[0068] In addition to carbon, nitrogen, and inorganic salt sources, the culture medium may also contain appropriate nutrients, and optionally, one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine, thioglycolate, and dithiothreitol. The culture temperature is generally 10°C to 40°C, preferably 20°C to 37°C, and more preferably around 25°C, but should be selected according to the characteristics of the protein to be expressed. The pH of the culture medium is pH 6.8 to 7.4, preferably around pH 7.0. Furthermore, if the vector of this disclosure contains an inducible promoter, induction is preferably performed under conditions that allow for good expression of the AAV-binding protein of this disclosure.

[0069] IPTG (Isopropyl-β-D-thiogalactopyranoside) can be used as an example of an inducer. By measuring the turbidity of the culture medium (absorbance at 600 nm) and adding an appropriate amount of IPTG when it is approximately between 0.5 and 1.0, and then continuing the culture, the expression of AAV-binding proteins can be induced. The concentration of IPTG added can be appropriately selected from the range of 0.005 mM to 1.0 mM, but the range of 0.01 mM to 0.5 mM is preferred. Various conditions for IPTG induction can be carried out under conditions that are well known in the art.

[0070] To recover the AAV-binding protein of this disclosure from a culture obtained by culturing the transformant of this disclosure, the AAV-binding protein can be recovered by isolating and purifying it from the culture using a method suitable for the expression morphology of the AAV-binding protein in the transformant. For example, if the protein is expressed in the culture supernatant, the bacterial cells can be separated by centrifugation, and the AAV-binding protein can be purified from the resulting culture supernatant. Alternatively, if the protein is expressed intracellularly (including the periplasm), the bacterial cells can be collected by centrifugation, then the cells can be disrupted by adding an enzyme treatment agent or surfactant to extract the AAV-binding protein, and then it can be purified.

[0071] To purify the AAV-binding protein of this disclosure, methods known in the art may be used, one example being separation / purification using liquid chromatography. Examples of liquid chromatography include ion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, and affinity chromatography. By combining these chromatography methods in the purification process, the AAV-binding protein of this disclosure can be prepared in high purity.

[0072] As a method for measuring the binding activity of the obtained AAV-binding protein of this disclosure to AAV, for example, the binding activity to AAV can be measured using the Enzyme-Linked ImmunoSorbent Assay (ELISA) method. The AAV used for measuring the binding activity may be VLP (virus-like particles). Furthermore, any serotype of AAV vector and VLP can be used as long as they show binding activity to the AAV-binding protein of this disclosure.

[0073] The AAV-binding proteins of this disclosure can be used, for example, for the purification or analysis of AAVs. When used for this purpose, there is no particular limitation on the AAVs to which they bind; they may be naturally occurring AAVs or artificially created AAVs. Examples of naturally occurring AAVs 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 created AAVs include AAVrh8, AAVrh10, and chimeric AAVs that possess two or more characteristics (cell-targeting or infectivity) from these serotypes.

[0074] The AAV-binding proteins of this disclosure can be used, for example, by immobilizing them on an insoluble carrier. Specifically, the purification or analysis of AAV can be carried out using an AAV adsorbent comprising, for example, an insoluble carrier and the AAV-binding proteins of this disclosure immobilized on the insoluble carrier. In this specification, an AAV adsorbent comprising an insoluble carrier and the AAV-binding proteins of this disclosure immobilized on the insoluble carrier is also referred to as the AAV adsorbent of this disclosure. Note that the purification of AAV is not limited to the purification of AAV from a solution containing impurities, but also includes the purification of AAV based on its structure, properties, or activity.

[0075] The insoluble carrier, which is a component of the AAV adsorbent of this disclosure, is not particularly limited as long as it is insoluble in the sample containing AAV or in the solution used for purification (eluent, equilibration solution, washing solution, etc.). Examples of insoluble carriers include carriers made from polysaccharides such as agarose, alginate (alginate salt), carrageenan, chitin, cellulose, dextrin, dextran, and starch; carriers made from synthetic polymers such as polyvinyl alcohol, polymethacrylate, poly(2-hydroxyethyl methacrylate), and polyurethane; and carriers made from ceramics such as silica. Among these, carriers made from polysaccharides and carriers made from synthetic polymers are preferred as insoluble carriers. Examples of the preferred carriers include polymethacrylate gels with introduced hydroxyl groups such as Toyopal (manufactured by Tosoh Corporation), agarose gels such as Sepharose (manufactured by Cytiva Corporation), and cellulose gels such as Cellfine (manufactured by JNC Corporation). The shape of the insoluble carrier is not particularly limited and may be granular, monolithic, film-like, or fibrous, and may be porous or non-porous. A shape that can be packed into a column is preferred.

[0076] When manufacturing an AAV adsorbent, the immobilization of the AAV-binding protein onto the insoluble carrier can be done, for example, by covalent bonding. Specifically, the AAV adsorbent can be manufactured by covalently bonding the AAV-binding protein to the insoluble carrier via an active group present on the insoluble carrier. Examples of such active groups include N-hydroxysuccinimide (NHS) activated ester group, epoxy group, carboxyl group, maleimide group, haloacetyl group, tresyl group, formyl group, and haloacetamide group. As the insoluble carrier having an active group, for example, a commercially available insoluble carrier having an active group may be used as is, or an insoluble carrier into which an active group has been introduced may be used. Examples of commercially available carriers containing active groups include TOYOPEARL AF-Epoxy-650M, TOYOPEARL AF-Tresyl-650M (both manufactured by Tosoh Corporation), HiTrap NHS-activated HP Columns, NHS-activated Sepharose 4 Fast Flow, Epoxy-activated Sepharose 6B (all manufactured by Cytiva Corporation), and SulfoLink Coupling Resin (manufactured by Thermo Fisher Scientific).

[0077] One example of a method for introducing active groups to a support surface is to react one of two or more active sites of a compound with hydroxyl groups, epoxy groups, carboxyl groups, amino groups, etc., present on the support surface.

[0078] Examples of compounds that introduce epoxy groups to hydroxyl or amino groups present on the support surface include epichlorohydrin, ethanediol diglycidyl ether, butanediol diglycidyl ether, and hexanediol diglycidyl ether.

[0079] Examples of compounds that introduce carboxyl groups to epoxy groups present on the support surface include 2-mercaptoacetic acid, 3-mercaptopropionic acid, 4-mercaptobutyric acid, 6-mercaptobutyric acid, glycine, 3-aminopropionic acid, 4-aminobutyric acid, and 6-aminohexanoic acid.

[0080] Compounds that introduce maleimide groups to hydroxyl groups, epoxy groups, carboxyl groups, and amino groups present on the carrier surface include N-(ε-maleimidocaproic acid)hydrazide, N-(ε-maleimidopropionic acid)hydrazide, 4-(4-N-maleimidophenyl)acetic acid hydrazide, 2-aminomaleimide, 3-aminomaleimide, 4-aminomaleimide, 6-aminomaleimide, 1-(4-aminophenyl)maleimide, 1-(3-aminophenyl)maleimide, 4-(maleimide)phenylisocyanate, 2-maleimidoacetic acid, and 3-maleimidopropionic acid. Examples include pionic 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, and (m-maleimidobenzoyl)N-hydroxysuccinimide ester.

[0081] Examples of compounds that introduce haloacetyl groups to hydroxyl or amino groups present on the carrier surface include chloroacetic acid, bromoacetic acid, iodoacetic acid, chloroacetic acid chloride, bromoacetic acid chloride, bromoacetic acid bromide, chloroacetic acid anhydride, bromoacetic acid anhydride, iodoacetic acid anhydride, 2-(iodoacetamide)acetic acid-N-hydroxysuccinimide, 3-(bromoacetamide)propionic acid-N-hydroxysuccinimide, and 4-(iodoacetyl)aminobenzoic acid-N-hydroxysuccinimide.

[0082] Another method for introducing active groups to the support surface involves reacting hydroxyl groups or amino groups present on the support surface with an ω-alkenyl alkane glycidyl ether, and then activating it by halogenating the ω-alkenyl moiety with a halogenating agent. Examples of ω-alkenyl alkane glycidyl ethers include allyl glycidyl ether, 3-butenyl glycidyl ether, and 4-pentenyl glycidyl ether. Examples of halogenating agents include N-chlorosuccinimide, N-bromosuccinimide, and N-iodosuccinimide.

[0083] Another method for introducing active groups to the support surface involves introducing active groups to carboxyl groups present on the support surface using a condensing agent and an additive. Examples of condensing agents include 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), dicyclohexylcarbodiamide, and carbonyldiimidazole. Examples of additives include NHS, 4-nitrophenol, and 1-hydroxybenztriazole.

[0084] Immobilization of AAV-binding proteins onto insoluble carriers can be carried out, for example, in a buffer solution. Examples of buffer solutions include acetate buffer, phosphate buffer, MES (2-MorpholinoEthaneSulfonic acid) buffer, HEPES (4-(2-HydroxyEthyl)-1-PiperazineEthaneSulfonic acid) buffer, Tris (Tris(hydroxymethyl)aminomethane) buffer, and borate buffer. The reaction temperature during immobilization can be appropriately set according to various conditions, such as the reactivity of the active group and the stability of the AAV-binding protein. The reaction temperature during immobilization may be, for example, 4°C to 50°C, and preferably 10°C to 35°C.

[0085] The AAV adsorbent of this disclosure can be used, for example, by packing it into a column for the purification or analysis of AAV. Specifically, for example, a solution containing AAV can be added to a column packed with the AAV adsorbent of this disclosure (hereinafter also simply referred to as "the column of this disclosure") to adsorb the AAV onto the adsorbent, and the AAV adsorbed onto the adsorbent can be eluted to purify or analyze the AAV. That is, in one embodiment, this disclosure provides a method for purifying or analyzing AAV, which includes the steps of adding a solution containing AAV to the column of this disclosure to adsorb the AAV onto the adsorbent, and eluting the AAV adsorbed onto the adsorbent. Purification of AAV using the column of this disclosure can be carried out, for example, in accordance with the disclosure in WO2021 / 106882.

[0086] Purified AAV can be obtained by purifying AAV using the AAV adsorbent of this disclosure. That is, in one embodiment, the method for purifying AAV may be a method for producing AAV, and more specifically, a method for producing purified AAV. AAV can be obtained, for example, as an eluted fraction containing AAV. That is, the fraction containing the eluted AAV can be separated. Separation of the AAV fraction can be carried out, for example, by conventional methods. Methods for separating the AAV fraction include changing the collection container at regular intervals or at regular volume intervals, changing the collection container according to the shape of the chromatogram of the eluate, or separating the fraction using an automated fraction collector such as an autosampler. Furthermore, AAV can also be recovered from the fraction containing AAV. Recovery of AAV from the fraction containing AAV can be carried out, for example, by known methods used for protein purification.

[0087] By alkaline washing the AAV adsorbent of this disclosure, it is possible to prevent contamination with, for example, any remaining AAV or impurities. In other words, in one embodiment, the washing method for the AAV adsorbent may be an alkaline washing method performed after purifying the AAV. Furthermore, since the AAV-binding protein of this disclosure is also acid-resistant, the method may also involve washing with a low pH solution followed by alkaline washing. An example of an alkaline solution used for washing is an aqueous sodium hydroxide solution of 0.1 M to 0.5 M.

[0088] In one embodiment, the AAV purification method of this disclosure may mean purifying a gene-containing AAV vector (Full AAV vector).

[0089] In one embodiment, the AAV purification method of this disclosure is: The process involves adding a sample containing AAV to an AAV adsorbent and adsorbing the AAV onto the adsorbent (hereinafter also referred to as the "first adsorption process"), The process involves eluting the AAV adsorbed onto the adsorbent (hereinafter also referred to as the "first elution process"), The process involves adding a fraction containing AAV eluted in the first elution step to a carrier for anion exchange chromatography and adsorbing the AAV onto the carrier (hereinafter also referred to as the "second adsorption step"), The process involves eluting the AAV adsorbed onto the anion exchange chromatography carrier (hereinafter also referred to as the "second elution step"), This includes the following. Furthermore, a configuration in which the AAV adsorbent and the anion exchange chromatography support are packed into a column (hereinafter also referred to as the "AAV adsorbent column" and the "anion exchange column," respectively) is preferable because it simplifies these processes. The following describes in detail the configurations using the AAV adsorbent column and the anion exchange column as examples.

[0090] A sample containing AAV can be added to an AAV adsorbent column using, for example, a liquid delivery means such as a pump. In this specification, adding a liquid to a column is also referred to as "delivering a liquid to the column." Before adding a sample containing AAV to the AAV adsorbent column, the solvent may be replaced using a suitable buffer solution. Alternatively, before adding a sample containing AAV to the AAV adsorbent column (i.e., before the first adsorption step), the AAV adsorbent column may be equilibrated using a suitable buffer solution (equilibrium solution). This equilibration is expected to allow for higher purity purification of AAV, for example. Any neutral buffer solution with buffering capacity in the neutral range (in this specification, pH 5.0 to 9.0, preferably pH 5.5 to 8.0) can be used for solvent replacement or equilibration. Specifically, examples include phosphate buffer, acetate buffer, succinate buffer, citrate buffer, Tris buffer, HEPES buffer, and MES buffer. Such buffers may also contain, for example, 0.1 mmol / L to 50 mmol / L of a chelating agent. The buffer used for solvent substitution and the equilibration solution may or may not be the same.

[0091] Furthermore, if non-AAV components, such as contaminants, remain on the AAV adsorbent column after passing a sample containing AAV through it, such components may be removed (washed) from the AAV adsorbent column before eluting the AAV adsorbed on the AAV (i.e., before the elution step). Non-AAV components can be removed from the AAV adsorbent column, for example, by using an appropriate buffer solution as a washing solution. For such washing solutions, the descriptions for buffer solutions used for solvent replacement and equilibration can be applied mutatis mutandis.

[0092] In one embodiment, in the first elution step, the AAV adsorbed onto the AAV adsorbent in the first adsorption step can be eluted using, for example, a buffer with a pH lower than that of the equilibration solution or washing solution, a neutral buffer containing 800 mmol / L or more of chloride ions or anions with a Hofmeister series smaller than those ions, or a buffer containing a chelating agent (such as ethylenediaminetetraacetic acid) adjusted to the pH used in the second adsorption step. By passing the aforementioned elution solution through the AAV adsorbent column, the interaction between AAV and AAV-binding proteins (ligands of the AAV adsorbent) within the column is weakened, so that the AAV adsorbed onto the AAV adsorbent is eluted, and a fraction containing AAV is obtained.

[0093] The fraction containing AAV obtained in the first elution step may be diluted, for example, by adjusting the pH to around 8.0 to 9.5 using Tris, or by diluting it with the buffer used in the second adsorption step.

[0094] Anion exchange columns can be manufactured, for example, by immersing an anion exchange chromatography carrier in a 1 mol / L sodium chloride aqueous solution and then packing it into the column. The buffer component used in the second adsorption step and the second elution step can be any buffer component having a buffering capacity around pH 8.0 to 9.5, and Tris is preferred.

[0095] In one embodiment, examples of preferred salts to be included in the eluate in the second elution step include sodium chloride, sodium acetate, ammonium chloride, ammonium acetate, tetramethylammonium chloride, magnesium chloride, calcium chloride, ammonium sulfate, tetraethylammonium chloride, choline chloride, acetylcholine chloride, carnitine hydrochloride, and trimethylglycine, with tetraethylammonium chloride and choline chloride being more preferred. In particular, including choline chloride in the eluate is preferred because it allows for the high-purity purification of infectious full AAV. Full AAV may refer to AAV containing genes.

[0096] In the second elution step, for example, the salt concentration is increased linearly to an appropriate concentration, the first peak is eluted, and then the salt concentration is increased stepwise to elute the second peak. For example, when using AAV8, a fraction with a high proportion of Empty AAV is obtained in the first peak, and a fraction with a high proportion of Full AAV is obtained in the second peak.

[0097] In one embodiment, it is preferable to set the conductivity of the eluate when eluting the first peak to 13.5 mS / cm or less and the conductivity of the eluate when eluting the second peak to 15.0 mS / cm or more, as this improves the proportion of Full AAV included in the second peak. It is even more preferable to set the conductivity of the eluate when eluting the first peak to 5.0 mS / cm or more and 13.5 mS / cm or less and the conductivity of the eluate when eluting the second peak to 15.0 mS / cm or more and 50.0 mS / cm or less. It is even more preferable to set the conductivity of the eluate when eluting the first peak to 12.0 mS / cm or more and 13.5 mS / cm or less and the conductivity of the eluate when eluting the second peak to 20.0 mS / cm or more and 40.0 mS / cm or less. The conductivity can be measured using a conductivity monitor built into a liquid chromatography system such as AKTA go (manufactured by Cytiva).

[0098] In this specification, the percentage of AAV vectors containing genes is also referred to as the Full rate. The Full rate is best measured using a mass photometry method such as the Refeyn Two (manufactured by Refeyn). [Examples]

[0099] The present disclosure will be described in more detail below with reference to examples and reference examples, but the present disclosure is not limited to these examples. Reference examples do not constitute part of the present disclosure.

[0100] Example 1: Preparation of AAV vectors (Part 1) (1) Escherichia coli strain JM109 was transformed using the plasmid pRC2-mi342 Vector (Takara Bio Inc.) and pHelper Vector (Takara Bio Inc.), which contain a polynucleotide encoding the capsid of AAV serotype 2 (AAV2). The resulting transformants were cultured overnight at 37°C in a 5L baffled flask containing 1L of 2×YT medium (1.6% (w / v) Tryptone, 1% (w / v) Yeast Extract, 0.5% (w / v) Sodium Chloride) with 100 μg / mL of carbenicillin.

[0101] (2) After recovering the bacterial cells from the culture solution of (1) by centrifugation, large quantities of pRC2-mi342 Vector and pHelper Vector were prepared from the recovered bacterial cells using Plasmid Mega Kit (Qiagen).

[0102] (3) HEK293T cells were cultured in five T-225 flasks (Thermo Fisher Scientific) containing 40 mL of D-MEM medium (Fujifilm Wako Pure Chemical Industries, Ltd.) with 10% (v / v) bovine serum.

[0103] (4) Using TransIT-ViruGEN Transfection Reagent (manufactured by Takara Bio Inc.), the pRC2-mi342 Vector and pHelper Vector prepared in (2) were introduced into HEK293T cells cultured in (3), and the cells were cultured statically for 3 days under conditions of 5% carbon dioxide and 37°C.

[0104] (5) The cells cultured in (4) were harvested and extracted and purified using the AAVpro Purification Kit (Takara Bio Inc.) to obtain VLP2 (virus-like particles, outer shell protein particles of AAV serotype 2). Approximately 1 mL of purified VLP2 solution was prepared from five T-225 flasks.

[0105] Example 2: Creation and screening of a mutant library of AAV-binding proteins (Part 1) Using the vector pET-AVR21, which is capable of expressing a polypeptide containing the AAV-binding protein AVR21 (SEQ ID NO: 2), as a template, random mutations were introduced into the polynucleotide portion encoding the protein using error-prone PCR. Of the polypeptide consisting of the amino acid sequence described in SEQ ID NO: 2, the methionine (M) at position 1 to the alanine (A) at position 22 is the PelB signal peptide (the N-terminal 22 amino acid residues of UniProt No. P0C1C1), the serine (S) at position 25 to the aspartic acid (D) at position 213 is the AAV-binding protein AVR21, and the histidine (H) at positions 214 to 219 is the tag sequence. Furthermore, AVR21 is a polypeptide in which 21 amino acid substitutions have occurred in amino acid residues from position 312 to 500 of Sequence ID No. 1, corresponding to the extracellular domain 1 (PKD1) and domain 2 (PKD2) of the native AAV-binding protein KIAA0319L (UniProt No. Q8IZA0) (WO2023 / 140197). The valine (V) at position 317 in SEQ ID NO: 1 (position 30 in SEQ ID NO: 2) is replaced with aspartic acid (D). The asparagine (N) at position 324 in sequence number 1 (position 37 in sequence number 2) is replaced with histidine (H). The valine (V) at position 326 in sequence number 1 (position 39 in sequence number 2) is replaced with alanine (A). The alanine (A) at position 330 in sequence number 1 (position 43 in sequence number 2) is replaced with valine (V). The glutamine (Q) at position 334 in sequence number 1 (position 47 in sequence number 2) is replaced with leucine (L). The glutamic acid (E) at position 335 in SEQ ID NO: 1 (position 48 in SEQ ID NO: 2) is replaced with valine (V). The 341st threonine (T) in sequence number 1 (54th in sequence number 2) is replaced with alanine (A). The tyrosine (Y) at position 342 in sequence number 1 (position 55 in sequence number 2) is replaced with serine (S). The lysine (K) at position 362 in SEQ ID NO: 1 (position 75 in SEQ ID NO: 2) is replaced with glutamic acid (E). The lysine (K) at position 371 in sequence number 1 (position 84 in sequence number 2) is replaced with asparagine (N). Phenylalanine (F) at position 379 in SEQ ID NO: 1 (position 92 in SEQ ID NO: 2) is replaced with tyrosine (Y). The lysine (K) at position 380 in SEQ ID NO: 1 (position 93 in SEQ ID NO: 2) is replaced with arginine (R). The valine (V) at position 381 in sequence number 1 (position 94 in sequence number 2) is replaced with alanine (A). The isoleucine (I) at position 382 in sequence number 1 (position 95 in sequence number 2) is replaced with valine (V). The glycine (G) at position 390 in sequence number 1 (position 103 in sequence number 2) is replaced with serine (S). The lysine (K) at position 399 in sequence number 1 (position 112 in sequence number 2) is replaced with glutamic acid (E). Lysine (K) at position 467 in sequence number 1 (position 180 in sequence number 2) is replaced with glutamine (Q). The serine (S) at position 476 in sequence number 1 (position 189 in sequence number 2) is replaced with arginine (R). The serine (S) at position 482 in sequence number 1 (position 195 in sequence number 2) is replaced with threonine (T). The asparagine (N) at position 487 in SEQ ID NO: 1 (position 200 in SEQ ID NO: 2) is replaced with aspartic acid (D). The asparagine (N) at position 492 in SEQ ID NO: 1 (position 205 in SEQ ID NO: 2) is substituted with aspartic acid (D).

[0106] (1) Error-prone PCR was performed using pET-AVR21, a vector capable of expressing a polypeptide containing AVR21 (SEQ ID NO: 2), as a template. Error-prone PCR was performed by preparing a reaction solution with the composition shown in Table 1, heat-treating the reaction solution at 98°C for 2 minutes, performing 30 cycles of a reaction consisting of a first step at 98°C for 30 seconds, a second step at 55°C for 20 seconds, and a third step at 72°C for 90 seconds, and finally heat-treating at 72°C for 5 minutes. The error-prone PCR successfully introduced mutations into the polynucleotide encoding the AAV-binding protein, with an average mutation rate of 1.4 amino acid mutations per molecule.

[0107] [Table 1]

[0108] (2) After purifying the PCR product obtained in (1), it was digested with restriction enzymes NcoI and XhoI and ligated into the expression vector pET26b (Merck millipore), which had been previously digested with the same restriction enzymes.

[0109] (3) After the ligation reaction was complete, Escherichia coli BL21(DE3) was transformed with the reaction solution and cultured in LB (Luria-Bertani) plate medium containing 50 μg / mL kanamycin (at 37°C for 18 hours). The colonies formed on the plate were then used as a random mutant library.

[0110] (4) The random mutant library (transformers) prepared in (3) was inoculated into 200 μL of 2YT liquid medium containing 50 μg / mL kanamycin, and incubated overnight with shaking at 37°C in a 96-well deep-well plate.

[0111] (5) The culture solution from (4) was centrifuged, and the resulting culture supernatant was diluted 2-fold with ultrapure water. 60 μL of the diluted culture solution was mixed with 60 μL of 0.5 M sodium hydroxide aqueous solution, and the mixture was subjected to alkaline treatment at 30°C for 30 minutes.

[0112] The binding activity between the AAV-binding protein and VLP2 when the treatment described in (6)(5) was performed, and the binding activity between the AAV-binding protein and VLP2 when the treatment described in (5) was not performed, were evaluated using the ELISA (Enzyme-Linked Immuno Sorbent Assay) method shown below.

[0113] (6-1) The VLP2 prepared in Example 1 was diluted 200-fold with 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride, and 100 μL / well was added to a 96-well microplate (Thermo Fisher Scientific) and immobilized (at 4°C for 18 hours). After immobilization, the plates were blocked with 2% (w / v) skim milk (Becton Dickinson) and 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride.

[0114] (6-2) After washing the wells with a washing buffer (20 mM Tris hydrochloride buffer (pH 7.4) containing 0.05% (w / v) Tween 20 (Sigma-Aldrich) and 150 mM sodium chloride), the culture supernatant prepared in (3) was added and the AAV-binding protein was reacted with VLP2 (at 30°C for 1 hour).

[0115] (6-3) After the reaction was complete, the samples were washed with the washing buffer and 100 μL / well of Anti-6His antibody (Bethyl Laboratories), diluted to 100 ng / mL, was added.

[0116] (6-4) The mixture was reacted at 30°C for 1 hour, washed with the aforementioned washing buffer, and then 50 μL / well of TMB Peroxidase Substrate (KPL) was added. The 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).

[0117] The remaining activity was calculated by dividing the binding activity between the AAV-binding protein and VLP2 after alkaline treatment by the binding activity between the AAV-binding protein and VLP2 without alkaline treatment.

[0118] (7)(6) A random mutant library of approximately 1800 strains was evaluated using the method described in (6), and transformants expressing an AAV-binding protein with improved residual activity compared to the parent molecule AVR21 were selected from among them. The selected transformants were cultured, and expression vectors were prepared using the QIAprep Spin Miniprep kit (Qiagen).

[0119] (8) The sequence of the polynucleotide region encoding the AAV-binding protein inserted into the obtained expression vector was analyzed using a fully automated DNA sequencer, Genetic Analyzer 3500 (Thermo Fisher Scientific), to identify amino acid substitution sites. For this analysis, oligonucleotides consisting of the sequences described in SEQ ID NO: 3 (5'-TAATACgACTCACTATAggg-3') or SEQ ID NO: 4 (5'-ATgCTAgTTATTgCTCAgCgg-3') were used as sequencing primers.

[0120] Table 2 summarizes the amino acid substitution positions relative to AVR21 and the residual activity [%] after alkali treatment of the AAV-binding protein expressed by the transformants selected in (7) above.

[0121] In the amino acid sequence described in SEQ ID NO: 2, from the 25th serine (S) to the 213th aspartic acid (D), the following amino acids are present: I319N (this notation indicates that the isoleucine at position 319 in SEQ ID NO: 1 (position 32 in SEQ ID NO: 2) is replaced with asparagine, and so on), L321Q, L328P, N329K, N329S, V332A, E(V)335A (this notation indicates that the glutamic acid at position 335 in SEQ ID NO: 1 (position 48 in SEQ ID NO: 2) is initially replaced with valine in SEQ ID NO: 2, and then further replaced with alanine, and so on), K338R, E340G, T343A, T343S, Y344F, D345G, Q347K , T350A, E360D, Q365R, I366F, L367P, K368R, L369S, K(N)371D, K(N)371S, L376P, Y377F, Y377N, E378G, F(Y)379S, V(A)381T, I(V)382I, E384D, H389R, V394A, V394I, V396E, T397A AAV-binding proteins that have at least one amino acid substitution among T397S, E401G, P402S, V412A, T426A, D435A, E453D, E454D, A461P, S466T, N472K, K497E, and D500N exhibit improved stability to alkalis compared to AVR21.

[0122] [Table 2]

[0123] Reference example 1 Using the vector pET-AVR8g, which is capable of expressing a polypeptide containing the AAV-binding protein AVR8g (SEQ ID NO: 22), random mutations were introduced into the polynucleotide portion encoding the protein using error-prone PCR. Of the polypeptide consisting of the amino acid sequence described in SEQ ID NO: 22, the methionine (M) at position 1 to the alanine (A) at position 22 is the PelB signal peptide, the serine (S) at position 25 to the aspartic acid (D) at position 213 is the AAV-binding protein AVR8g, and the histidine (H) at positions 214 to 219 is the tag sequence. Furthermore, AVR8g is a polypeptide in which amino acid substitutions V317D, Y342C, K362E, K371N, G390S, K399E, S476R, and N487D have occurred in amino acid residues from position 312 to 500 of Sequence ID No. 1, corresponding to the extracellular domain 1 (PKD1) and domain 2 (PKD2) of KIAA0319L (UniProt No. Q8IZA0) (WO2021 / 106882).

[0124] (1) Error-prone PCR was performed in the same manner as in Example 2(1), except that a plasmid pET-AVR8g containing the polynucleotide encoding AVR8g (sequence number 29) (sequence number 30) was used as a template and the concentration of the template was set to 10 ng / μL. Mutations were introduced into the polynucleotide encoding AVR8g by the error-prone PCR, and the average mutagenesis rate was 2.3 amino acid mutations per molecule.

[0125] (2) After purifying the PCR product obtained in (1), a random mutant library was prepared using the method described in Examples 2(2) and (3).

[0126] (3) The random mutant library (transformed organisms) prepared in (2) was inoculated into 200 μL of 2YT liquid medium containing 50 μg / mL kanamycin, and incubated overnight with shaking in a 96-well deep-well plate at 37°C.

[0127] (4) 10 μL of the culture medium prepared in (3) was subpoenaed into 500 μL of 2YT liquid medium containing 0.1 mM IPTG (IsoPropyl-β-D-ThioGalactopyranoside) and 50 μg / mL kanamycin, and then incubated overnight with shaking at 25°C in a 96-well deep-well plate.

[0128] (5) The culture supernatant obtained by centrifugation of the culture medium from (4) was diluted 16-fold with ultrapure water. 60 μL of this diluted culture supernatant was mixed with 60 μL of 0.1 M sodium glycine hydroxide buffer (pH 10.0) and subjected to heat and alkali treatment at 51.3°C for 15 minutes.

[0129] (6) The binding activity of AAV-binding protein to VLP2 when the treatment in (5) is performed. The sex and the binding activity between the AAV-binding protein and VLP2 when the treatment described in (5) was not performed were measured by the ELISA method described in Example 2(6).

[0130] The residual activity was calculated by dividing the binding activity between the AAV-binding protein and VLP2 after heat treatment by the binding activity between the AAV-binding protein and VLP2 without heat treatment.

[0131] A random mutant library of approximately 1800 strains was evaluated using the method described in (7)(6), and transformants expressing an AAV-binding protein with improved residual activity compared to the parent molecule AVR8g were selected from among them. The selected transformants were cultured, and expression vectors were prepared using the QIAprep Spin Miniprep kit (Qiagen).

[0132] (6) The sequence of the polynucleotide region encoding the AAV-binding protein inserted into the obtained expression vector was analyzed using the method described in Example 2(8) to identify amino acid substitution sites.

[0133] Table 3 summarizes the amino acid substitution positions relative to AVR8g and the residual activity [%] after heat and alkali treatment of the AAV-binding protein expressed by the transformants selected in (7)(6).

[0134] [Table 3]

[0135] Reference example 2 We investigated whether introducing any one of the amino acid substitutions (Y331H, F379S, K467N, and A491T) that were identified in Reference Example 1 as being involved in improving the thermal and alkaline stability of AAV-binding proteins into AVR8g (SEQ ID NO: 22) would improve thermal and alkaline stability. Specifically, we examined the thermal and alkaline stability of the four types of AAV-binding proteins shown in (a) to (d) below. (a) A protein obtained by introducing the Y331H amino acid substitution into AVR8g (SEQ ID NO: 24, named AVR9a) (b) A protein obtained by introducing the F379S amino acid substitution into AVR8g (SEQ ID NO: 25, named AVR9b) (c) A protein obtained by introducing the K467N amino acid substitution into AVR8g (SEQ ID NO: 26, named AVR9c) (d) A protein obtained by introducing the amino acid substitution A491T into AVR8g (SEQ ID NO: 27, named AVR9d) (1) Transformants capable of expressing any of AVR9a (SEQ ID NO: 24), AVR9b (SEQ ID NO: 25), AVR9c (SEQ ID NO: 26), and AVR9d (SEQ ID NO: 27) (host: E. coli BL21 strain (DE3)) were inoculated into 3 mL of 2×YT liquid medium containing 50 μg / mL of kanamycin, and pre-cultured by aerobic shaking at 37°C overnight.

[0136] (2) 2 mL of the pre-culture solution from (1) was inoculated into 200 mL of 2×YT liquid medium containing 50 μg / mL of kanamycin in a 500 mL baffled flask, and cultured aerobically with shaking at 37°C.

[0137] (3) Two hours after the start of culture, the samples were cooled on ice, and IPTG was added to each sample to a final concentration of 0.1 mM. The samples were then cultured aerobically with shaking at 25°C for 20 hours.

[0138] (4) After the culturing was complete, the culture medium was centrifuged at 4°C and 8000 rpm for 20 minutes to collect each bacterial cell.

[0139] (5) The bacterial cells recovered in (4) were suspended in 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride and 20 mM imidazole (hereinafter also referred to as "equilibrium solution A") at a concentration of 5 mL / 1 g (bacterial cells). The bacterial cells were then lysed using an ultrasonic generator (Insonator 201M [manufactured by Kubota Shoji Co., Ltd.]) at 4°C for approximately 10 minutes at an output of approximately 150 W. The lysate was centrifuged twice at 8000 rpm for 20 minutes at 4°C, and the supernatant was collected.

[0140] (6) The supernatant obtained in (5) was applied to an open column packed with 1.5 mL of Ni Sepharose 6 Fast Flow (Cytiva), which had been pre-equilibrated with equilibration solution A. After washing with equilibration solution A, the column was eluted with 20 mM Tris-HCl buffer (pH 7.4) containing 0.5 M imidazole and 150 mM sodium chloride.

[0141] AAV-binding proteins were prepared by dialyzing each eluate obtained in (7) and (6) with 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride.

[0142] The binding activity of the AAV-binding protein prepared in (8)(7) to the VLP2 prepared in Example 1 was measured using the ELISA method described in Example 2(6). Based on the absorbance at 450 nm, which was the measurement result, the AAV-binding protein obtained in (7) was diluted with pure water so that the measurement value would be similar.

[0143] (9) 60 μL of diluted AAV-binding protein solution was mixed with 60 μL of 0.1 M sodium glycine hydroxide buffer (pH 10.0), and the mixture was subjected to heat and alkali treatment at 55.7°C, 60.1°C, 66.1°C, or 70°C for 15 minutes.

[0144] The binding activity between the AAV-binding protein and VLP2 after treatment (10)(9) and the binding activity between the AAV-binding protein and VLP2 without treatment (9) were measured using the ELISA method described in Example 2(6). The residual activity was calculated by dividing the binding activity between the AAV-binding protein and VLP2 after treatment (9) by the binding activity between the AAV-binding protein and VLP2 without heat treatment (9).

[0145] The results are shown in Table 4. In the amino acid sequence of AVR8g (SEQ ID NO: 29), AAV-binding proteins in which at least one of the following amino acid substitutions occurred between the 25th serine (S) residue (S) and the 213th aspartic acid (D) residues were Y331H, F379S, K467N, and A491T showed improved stability to heat and alkali compared to AVR8g.

[0146] [Table 4]

[0147] Example 3: Preparation of AVR21 amino acid substitution aggregates From the amino acid substitutions involved in improving the alkali stability of AAV-binding proteins, as revealed in Example 2, N329K, K338R, E401G, T426A, and A461P were selected. From the amino acid substitutions involved in improving the thermal and alkali stability of AAV-binding proteins, as revealed in Reference Examples 1 and 2, K467N was selected. These amino acid substitutions were then added to AVR21 (SEQ ID NO: 2) to further improve alkali stability. Specifically, five types of AAV-binding proteins shown in (a) to (e) below were designed and fabricated. (a) AAV-binding protein (SEQ ID NO: 5, named AVR24a) obtained by introducing amino acid substitutions N329K, E401G, and T426A into AVR21. (b) AAV-binding protein (SEQ ID NO: 6, named AVR24b) obtained by introducing amino acid substitutions K338R, T426A, and A461P into AVR21. (c) AAV-binding protein (SEQ ID NO: 7, named AVR25a) obtained by introducing amino acid substitutions N329K, E401G, T426A, and A461P into AVR21. (d) AAV-binding protein (SEQ ID NO: 8, named AVR25b) obtained by introducing amino acid substitutions K338R, E401G, T426A, and A461P into AVR21. (e) AAV-binding protein (SEQ ID NO: 9, named AVR26) obtained by introducing the amino acid substitutions N329K, K338R, E401G, T426A, and A461P into AVR21. The following describes the methods for producing the six types of AAV-binding proteins shown in (a) to (e) above.

[0148] We designed a PCR primer set for creating mutation aggregates. Specifically, SEQ ID NOs. 10 (Forward) and 11 (Reverse) were used as PCR primers to introduce the N329K amino acid substitution. For introducing amino acid substitutions in K338R, SEQ ID NOs. 12 (Forward) and 13 (Reverse) were used as PCR primers. For introducing amino acid substitutions in E401G, SEQ ID NOs. 14 (Forward) and 15 (Reverse) were used as PCR primers. For introducing amino acid substitutions in T426A, SEQ ID NOs. 16 (Forward) and 17 (Reverse) were used as PCR primers. For introducing amino acid substitutions in A461P, SEQ ID NOs. 18 (Forward) and 19 (Reverse) were used as PCR primers. They each designed it.

[0149] (a) AVR24a This protein was prepared by selecting N329K, E401G, and T426A from the amino acid substitutions involved in alkaline stability identified in Example 2, and introducing these amino acid substitutions into AVR21 (SEQ ID NO: 2).

[0150] (a-1) Using the vector pET-AVR21, which can express a polypeptide containing AVR21 (SEQ ID NO: 2), as a template, and using oligonucleotides consisting of the sequences described in SEQ ID NO: 10 (5'-AGCTGAAAGTCTACGTACTGCTGGT-3') and SEQ ID NO: 11 (5'-TAGACTTTCAGCTGGGCTTCATGTT-3') as PCR primers, a reaction solution with the composition shown in Table 5 was prepared. The reaction solution was then heat-treated at 98°C for 5 minutes, and the reaction was carried out for 30 cycles, with the first step being at 98°C for 10 seconds, the second step at 55°C for 5 seconds, and the third step at 72°C for 6 minutes, and finally the reaction was heat-treated at 72°C for 5 minutes to perform PCR.

[0151] [Table 5]

[0152] (a-2) Using the PCR product obtained in (a-1) as a template, and oligonucleotides consisting of the sequences described in SEQ ID NO: 14 (5'-AGCCTGGCCCGCGCAAGAATCGTCC-3') and SEQ ID NO: 15 (5'-CGCGGGCCAGGCTCAACCGTTACGT-3') as PCR primers, a reaction solution with the composition shown in Table 5 was prepared, and the reaction solution was heat-treated at 98°C for 5 minutes. The reaction was performed for 30 cycles, with the first step being at 98°C for 10 seconds, the second step at 55°C for 5 seconds, and the third step at 72°C for 6 minutes, and finally the reaction was heat-treated at 72°C for 5 minutes to perform PCR.

[0153] Using the PCR product obtained in (a-3)(a-2) as a template, and oligonucleotides consisting of the sequences described in SEQ ID NO: 16 (5'-CAAGTGCGGTGATCGACGGATCGCA-3') and SEQ ID NO: 17 (5'-ATCACCGCACTTGTAGTCGGGAGTG-3') as PCR primers, a reaction solution with the composition shown in Table 5 was prepared. The reaction solution was then heat-treated at 98°C for 5 minutes, and the reaction was performed for 30 cycles, with the first step being at 98°C for 10 seconds, the second step at 55°C for 5 seconds, and the third step at 72°C for 6 minutes, followed by heat treatment at 72°C for 5 minutes to perform PCR.

[0154] (a-4)(a-3) was used to transform E. coli BL21 strain (DE3). The resulting transformants were cultured in LB medium supplemented with 50 μg / mL kanamycin, and the plasmid was extracted from the cells (transformants) recovered by centrifugation to obtain a plasmid (expression vector) pET-AVR24a containing a polynucleotide encoding AVR24a, in which the native AAV-binding protein had 24 amino acid substitutions.

[0155] (a-5) The nucleotide sequence of pET-AVR24a was analyzed and confirmed using the same method as in Example 2(8).

[0156] The amino acid sequence of AVR24a with the signal sequence and polyhistidine tag attached is shown in Sequence ID No. 5. In Sequence ID No. 5, the methionine (M) at position 1 to the alanine (A) at position 22 is the PelB signal peptide, the serine (S) at position 25 to the aspartic acid (D) at position 213 is the AAV-binding protein AVR24a (corresponding to the region from position 25 to 213 in Sequence ID No. 2), and the histidine (H) at positions 214 to 219 is the tag sequence. Furthermore, in Sequence ID No. 5, aspartic acid (V317D) is at position 30, histidine (N324H) is at position 37, alanine (V326A) is at position 39, lysine (N329K) is at position 42, valine (A330V) is at position 43, leucine (Q334L) is at position 47, valine (E335V) is at position 48, alanine (T341A) is at position 54, serine (Y342S) is at position 55, glutamic acid (K362E) is at position 75, asparagine (K371N) is at position 84, tyrosine (F379Y) is at position 92, and K380R Arginine is located at position 93, alanine (V381A) at position 94, valine (I382V) at position 95, serine (G390S) at position 103, glutamic acid (K399E) at position 112, glycine (E401G) at position 114, alanine (T426A) at position 139, glutamine (K467Q) at position 180, arginine (S476R) at position 189, threonine (S482T) at position 195, aspartic acid (N487D) at position 200, and aspartic acid (N492D) at position 205.

[0157] (b) AVR24b This protein was prepared by selecting K338R, T426A, and A461P from among the amino acid substitutions involved in alkali stability identified in Example 2, and introducing these amino acid substitutions into AVR21 (SEQ ID NO: 2).

[0158] (b-1) Using the vector pET-AVR21, which can express a polypeptide containing AVR21 (SEQ ID NO: 2), as a template, and using oligonucleotides consisting of the sequences described in SEQ ID NO: 12 (5'-CACCGCGCGGGGAAGCGTCAACGTA-3') and SEQ ID NO: 13 (5'-TCCCCGCGCGGTGGAACCAGCAGTA-3') as PCR primers, a reaction solution with the composition shown in Table 5 was prepared. The reaction solution was then heat-treated at 98°C for 5 minutes, and the reaction was performed for 30 cycles, with the first step being at 98°C for 10 seconds, the second step at 55°C for 5 seconds, and the third step at 72°C for 6 minutes, and finally heat-treated at 72°C for 5 minutes to perform PCR.

[0159] (b-2) Using the PCR product obtained in (b-1) as a template, and oligonucleotides consisting of the sequences described in SEQ ID NO: 16 (5'-CAAGTGCGGTGATCGACGGATCGCA-3') and SEQ ID NO: 17 (5'-ATCACCGCACTTGTAGTCGGGAGTG-3') as PCR primers, a reaction solution with the composition shown in Table 5 was prepared, and the reaction solution was heat-treated at 98°C for 5 minutes. The reaction was performed for 30 cycles, with the first step being at 98°C for 10 seconds, the second step at 55°C for 5 seconds, and the third step at 72°C for 6 minutes, and finally the reaction was heat-treated at 72°C for 5 minutes to perform PCR.

[0160] Using the PCR product obtained in (b-3)(b-2) as a template, and oligonucleotides consisting of the sequences described in SEQ ID NO: 18 (5'-ATACCCCGATTCTTAAGCTCTCGCA-3') and SEQ ID NO: 19 (5'-AGAATCGGGGTATCCTCGCTGATCT-3') as PCR primers, a reaction solution with the composition shown in Table 5 was prepared. The reaction solution was then heat-treated at 98°C for 5 minutes, and the reaction was performed for 30 cycles, with the first step being at 98°C for 10 seconds, the second step at 55°C for 5 seconds, and the third step at 72°C for 6 minutes, followed by heat treatment at 72°C for 5 minutes to perform PCR.

[0161] (b-4)(b-3) was used to transform E. coli BL21 strain (DE3). The resulting transformants were cultured in LB medium supplemented with 50 μg / mL kanamycin, and the plasmid was extracted from the cells (transformants) recovered by centrifugation to obtain the plasmid (expression vector) pET-AVR24b, which contains a polynucleotide encoding AVR24b with 24 amino acid substitutions from the native AAV-binding protein.

[0162] (b-5) The nucleotide sequence of pET-AVR24b was analyzed and confirmed using the same method as in Example 2(8).

[0163] The amino acid sequence of AVR24b with the signal sequence and polyhistidine tag is shown in Sequence ID No. 6. In Sequence ID No. 6, the sequence from the 1st methionine (M) to the 22nd alanine (A) is the PelB signal peptide, the sequence from the 25th serine (S) to the 213th aspartic acid (D) is the AAV-binding protein AVR24b (corresponding to the region from the 25th to the 213th position in Sequence ID No. 2) is the histidine (H) from the 214th to the 219th position is the tag sequence. Furthermore, in Sequence ID No. 6, aspartic acid (V317D) is ranked 30th, histidine (N324H) 37th, alanine (V326A) 39th, valine (A330V) 43rd, leucine (Q334L) 47th, valine (E335V) 48th, arginine (K338R) 51st, alanine (T341A) 54th, serine (Y342S) 55th, glutamic acid (K362E) 75th, asparagine (K371N) 84th, tyrosine (F379Y) 92nd, and K380R Arginine in V381A is located at position 93, alanine in V381A at position 94, valine in I382V at position 95, serine in G390S at position 103, glutamic acid in K399E at position 112, alanine in T426A at position 139, proline in A461P at position 174, glutamine in K467Q at position 180, arginine in S476R at position 189, threonine in S482T at position 195, aspartic acid in N487D at position 200, and aspartic acid in N492D at position 205.

[0164] (c)AVR25a This protein was prepared by selecting N329K, E401G, T426A, and A461P from among the amino acid substitutions involved in alkaline stability identified in Example 2, and introducing these amino acid substitutions into AVR21 (SEQ ID NO: 2).

[0165] (c-1) Using the vector pET-AVR21, which can express a polypeptide containing AVR21 (SEQ ID NO: 2), as a template, and using oligonucleotides consisting of the sequences described in SEQ ID NO: 10 (5'-AGCTGAAAGTCTACGTACTGCTGGT-3') and SEQ ID NO: 11 (5'-TAGACTTTCAGCTGGGCTTCATGTT-3') as PCR primers, a reaction solution with the composition shown in Table 5 was prepared. The reaction solution was then heat-treated at 98°C for 5 minutes, and the reaction was performed for 30 cycles, with the first step being at 98°C for 10 seconds, the second step at 55°C for 5 seconds, and the third step at 72°C for 6 minutes, and finally heat-treated at 72°C for 5 minutes to perform PCR.

[0166] (c-2) Using the PCR product obtained in (c-1) as a template, and oligonucleotides consisting of the sequences described in SEQ ID NO: 14 (5'-AGCCTGGCCCGCGCAAGAATCGTCC-3') and SEQ ID NO: 15 (5'-CGCGGGCCAGGCTCAACCGTTACGT-3') as PCR primers, a reaction solution with the composition shown in Table 5 was prepared. The reaction solution was then heat-treated at 98°C for 5 minutes, and the reaction was performed for 30 cycles, with the first step being at 98°C for 10 seconds, the second step at 55°C for 5 seconds, and the third step at 72°C for 6 minutes, and finally the reaction was heat-treated at 72°C for 5 minutes to perform PCR.

[0167] Using the PCR product obtained in (c-3)(c-2) as a template, and oligonucleotides consisting of the sequences described in SEQ ID NO: 16 (5'-CAAGTGCGGTGATCGACGGATCGCA-3') and SEQ ID NO: 17 (5'-ATCACCGCACTTGTAGTCGGGAGTG-3') as PCR primers, a reaction solution with the composition shown in Table 5 was prepared. The reaction solution was then heat-treated at 98°C for 5 minutes, and the reaction was performed for 30 cycles, with the first step being at 98°C for 10 seconds, the second step at 55°C for 5 seconds, and the third step at 72°C for 6 minutes, followed by heat treatment at 72°C for 5 minutes to perform PCR.

[0168] Using the PCR product obtained in (c-4)(c-3) as a template, and oligonucleotides consisting of the sequences described in SEQ ID NO: 18 (5'-ATACCCCGATTCTTAAGCTCTCGCA-3') and SEQ ID NO: 19 (5'-AGAATCGGGGTATCCTCGCTGATCT-3') as PCR primers, a reaction solution with the composition shown in Table 5 was prepared. The reaction solution was then heat-treated at 98°C for 5 minutes, and the reaction was performed for 30 cycles, with the first step being at 98°C for 10 seconds, the second step at 55°C for 5 seconds, and the third step at 72°C for 6 minutes, followed by heat treatment at 72°C for 5 minutes to perform PCR.

[0169] (c-5)(c-4) was used to transform E. coli BL21 strain (DE3). The resulting transformants were cultured in LB medium supplemented with 50 μg / mL kanamycin, and the plasmid was extracted from the cells (transformants) recovered by centrifugation to obtain a plasmid (expression vector) pET-AVR25a containing a polynucleotide encoding AVR25a, in which 25 amino acids were substituted from the native AAV-binding protein.

[0170] (c-6) The nucleotide sequence of pET-AVR25a was analyzed and confirmed using the same method as in Example 2(8).

[0171] The amino acid sequence of AVR25a with the signal sequence and polyhistidine tag attached is shown in Sequence ID No. 7. In Sequence ID No. 7, the sequence from the 1st methionine (M) to the 22nd alanine (A) is the PelB signal peptide, the sequence from the 25th serine (S) to the 213th aspartic acid (D) is the AAV-binding protein AVR25a (corresponding to the region from the 25th to the 213th position in Sequence ID No. 2) is the histidine (H) from the 214th to the 219th position is the tag sequence. Furthermore, in Sequence ID No. 7, aspartic acid (V317D) is at position 30, histidine (N324H) is at position 37, alanine (V326A) is at position 39, lysine (N329K) is at position 42, valine (A330V) is at position 43, leucine (Q334L) is at position 47, valine (E335V) is at position 48, alanine (T341A) is at position 54, serine (Y342S) is at position 55, glutamic acid (K362E) is at position 75, asparagine (K371N) is at position 84, tyrosine (F379Y) is at position 92, and arginine (K380R) is at position 93. In the eye, alanine (V381A) is located at position 94, valine (I382V) at position 95, serine (G390S) at position 103, glutamic acid (K399E) at position 112, glycine (E401G) at position 114, alanine (T426A) at position 139, proline (A461P) at position 174, glutamine (K467Q) at position 180, arginine (S476R) at position 189, threonine (S482T) at position 195, aspartic acid (N487D) at position 200, and aspartic acid (N492D) at position 205.

[0172] (d) AVR25b This protein was prepared by selecting K338R, E401G, T426A, and A461P from among the amino acid substitutions involved in alkali stability identified in Example 2, and introducing these amino acid substitutions into AVR21 (SEQ ID NO: 2).

[0173] (d-1) Using the vector pET-AVR21, which can express a polypeptide containing AVR21 (SEQ ID NO: 2), as a template, and using oligonucleotides consisting of the sequences described in SEQ ID NO: 12 (5'-CACCGCGCGGGGAAGCGTCAACGTA-3') and SEQ ID NO: 13 (5'-TCCCCGCGCGGTGGAACCAGCAGTA-3') as PCR primers, a reaction solution with the composition shown in Table 5 was prepared. The reaction solution was then heat-treated at 98°C for 5 minutes, and the reaction was performed for 30 cycles, with the first step being at 98°C for 10 seconds, the second step at 55°C for 5 seconds, and the third step at 72°C for 6 minutes, and finally heat-treated at 72°C for 5 minutes to perform PCR.

[0174] (d-2) Using the PCR product obtained in (d-1) as a template, and oligonucleotides consisting of the sequences described in SEQ ID NO: 14 (5'-AGCCTGGCCCGCGCAAGAATCGTCC-3') and SEQ ID NO: 15 (5'-CGCGGGCCAGGCTCAACCGTTACGT-3') as PCR primers, a reaction solution with the composition shown in Table 5 was prepared. The reaction solution was then heat-treated at 98°C for 5 minutes, and the reaction was performed for 30 cycles, with the first step being at 98°C for 10 seconds, the second step at 55°C for 5 seconds, and the third step at 72°C for 6 minutes, followed by heat treatment at 72°C for 5 minutes to perform PCR.

[0175] Using the PCR product obtained in (d-3)(d-2) as a template, and oligonucleotides consisting of the sequences described in SEQ ID NO: 16 (5'-CAAGTGCGGTGATCGACGGATCGCA-3') and SEQ ID NO: 17 (5'-ATCACCGCACTTGTAGTCGGGAGTG-3') as PCR primers, a reaction solution with the composition shown in Table 5 was prepared. The reaction solution was then heat-treated at 98°C for 5 minutes, and the reaction was performed for 30 cycles, with the first step being at 98°C for 10 seconds, the second step at 55°C for 5 seconds, and the third step at 72°C for 6 minutes, followed by heat treatment at 72°C for 5 minutes to perform PCR.

[0176] Using the PCR product obtained in (d-4)(d-3) as a template, and oligonucleotides consisting of the sequences described in SEQ ID NO: 18 (5'-ATACCCCGATTCTTAAGCTCTCGCA-3') and SEQ ID NO: 19 (5'-AGAATCGGGGTATCCTCGCTGATCT-3') as PCR primers, a reaction solution with the composition shown in Table 5 was prepared. The reaction solution was then heat-treated at 98°C for 5 minutes, and the reaction was performed for 30 cycles, with the first step being at 98°C for 10 seconds, the second step at 55°C for 5 seconds, and the third step at 72°C for 6 minutes, followed by heat treatment at 72°C for 5 minutes to perform PCR.

[0177] E. coli BL21 strain (DE3) was transformed using the PCR products obtained in (d-5) and (d-4). The resulting transformants were cultured in LB medium supplemented with 50 μg / mL kanamycin, and the plasmid was extracted from the cells (transformants) recovered by centrifugation to obtain the plasmid (expression vector) pET-AVR25b, which contains a polynucleotide encoding AVR25b with 25 amino acid substitutions from the native AAV-binding protein.

[0178] (d-6) The nucleotide sequence of pET-AVR25b was analyzed and confirmed using the same method as in Example 2(8).

[0179] The amino acid sequence of AVR25b with the signal sequence and polyhistidine tag is shown in Sequence ID No. 8. In Sequence ID No. 8, the first methionine (M) to the 22nd alanine (A) is the PelB signal peptide, the 25th serine (S) to the 213th aspartic acid (D) is the AAV-binding protein AVR25b (corresponding to the region from positions 25 to 213 in Sequence ID No. 2), and the 214th to 219th histidine (H) is the tag sequence. Furthermore, in Sequence ID No. 8, aspartic acid (V317D) is ranked 30th, histidine (N324H) 37th, alanine (V326A) 39th, valine (A330V) 43rd, leucine (Q334L) 47th, valine (E335V) 48th, arginine (K338R) 51st, alanine (T341A) 54th, serine (Y342S) 55th, glutamic acid (K362E) 75th, asparagine (K371N) 84th, tyrosine (F379Y) 92nd, and arginine (K380R) 93rd As for the second position, alanine from V381A is located at 94th, valine from I382V at 95th, serine from G390S at 103rd, glutamic acid from K399E at 112th, glycine from E401G at 114th, alanine from T426A at 139th, proline from A461P at 174th, glutamine from K467Q at 180th, arginine from S476R at 189th, threonine from S482T at 195th, aspartic acid from N487D at 200th, and aspartic acid from N492D at 205th.

[0180] (e) AVR26 This protein was prepared by selecting N329K, K338R, E401G, T426A, and A461P from among the amino acid substitutions involved in alkaline stability identified in Example 2, and introducing these amino acid substitutions into AVR21 (SEQ ID NO: 2).

[0181] (e-1) PCR was performed using pET-AVR21, a vector capable of expressing a polypeptide containing AVR21 (SEQ ID NO: 2), as a template, and oligonucleotides consisting of the sequences described in SEQ ID NO: 10 (5'-AGCTGAAAGTCTACGTACTGCTGGT-3') and SEQ ID NO: 11 (5'-TAGACTTTCAGCTGGGCTTCATGTT-3') as PCR primers. After preparing a reaction solution with the composition shown in Table 5, the reaction solution was heat-treated at 98°C for 5 minutes, and the reaction was carried out for 30 cycles, with the first step being 10 seconds at 98°C, the second step being 5 seconds at 55°C, and the third step being 6 minutes at 72°C. Finally, PCR was performed by heat-treating at 72°C for 5 minutes.

[0182] Using the PCR product obtained in (e-2)(e-1) as a template, and oligonucleotides consisting of the sequences described in SEQ ID NO: 12 (5'-CACCGCGCGGGGAAGCGTCAACGTA-3') and SEQ ID NO: 13 (5'-TCCCCGCGCGGTGGAACCAGCAGTA-3') as PCR primers, a reaction solution with the composition shown in Table 5 was prepared. The reaction solution was then heat-treated at 98°C for 5 minutes, and the reaction was performed for 30 cycles, with the first step being at 98°C for 10 seconds, the second step at 55°C for 5 seconds, and the third step at 72°C for 6 minutes, followed by heat treatment at 72°C for 5 minutes to perform PCR.

[0183] Using the PCR product obtained in (e-3)(e-2) as a template, and oligonucleotides consisting of the sequences described in SEQ ID NO: 14 (5'-AGCCTGGCCCGCGCAAGAATCGTCC-3') and SEQ ID NO: 15 (5'-CGCGGGCCAGGCTCAACCGTTACGT-3') as PCR primers, a reaction solution with the composition shown in Table 5 was prepared. The reaction solution was then heat-treated at 98°C for 5 minutes, and the reaction was performed for 30 cycles, with the first step being at 98°C for 10 seconds, the second step at 55°C for 5 seconds, and the third step at 72°C for 6 minutes, followed by heat treatment at 72°C for 5 minutes to perform PCR.

[0184] Using the PCR product obtained in (e-4)(e-3) as a template, oligonucleotides consisting of the sequences described in SEQ ID NO: 16 (5’-CAAGTGCGGTGATCGACGGATCGCA-3’) and SEQ ID NO: 17 (5’-ATCACCGCACTTGTAGTCGGGAGTG-3’) as PCR primers, after preparing a reaction solution with the composition shown in Table 5, the reaction solution was heat-treated at 98°C for 5 minutes, and a reaction with a first step of 10 seconds at 98°C, a second step of 5 seconds at 55°C, and a third step of 6 minutes at 72°C per cycle was performed for 30 cycles, and finally, PCR was performed by heat-treating at 72°C for 5 minutes.

[0185] Using the PCR product obtained in (e-5)(e-4) as a template, oligonucleotides consisting of the sequences described in SEQ ID NO: 18 (5’-ATACCCCGATTCTTAAGCTCTCGCA-3’) and SEQ ID NO: 19 (5’-AGAATCGGGGTATCCTCGCTGATCT-3’) as PCR primers, after preparing a reaction solution with the composition shown in Table 5, the reaction solution was heat-treated at 98°C for 5 minutes, and a reaction with a first step of 10 seconds at 98°C, a second step of 5 seconds at 55°C, and a third step of 6 minutes at 72°C per cycle was performed for 30 cycles, and finally, PCR was performed by heat-treating at 72°C for 5 minutes.

[0186] E. coli BL21 strain (DE3) was transformed using the PCR product obtained in (e-6)(e-5). After culturing the obtained transformant in LB medium supplemented with 50 μg / mL kanamycin and collecting the cells (transformants) by centrifugation, a plasmid (expression vector) pET-AVR26 containing a polynucleotide encoding AVR26 with 26 amino acid substitutions in the native AAV-binding protein was obtained by extracting the plasmid from the cells.

[0187] The nucleotide sequence of pET-AVR26 was analyzed and confirmed in the same manner as in Example 2(8).

[0188] The amino acid sequence of AVR26 with the signal sequence and polyhistidine tag attached is shown in Sequence ID No. 9. In Sequence ID No. 9, the methionine (M) at position 1 to the alanine (A) at position 22 is the PelB signal peptide, the serine (S) at position 25 to the aspartic acid (D) at position 213 is the AAV-binding protein AVR26 (corresponding to the region from position 25 to 213 in Sequence ID No. 2), and the histidine (H) at positions 214 to 219 is the tag sequence. Furthermore, in Sequence ID No. 9, aspartic acid (V317D) is ranked 30th, histidine (N324H) 37th, alanine (V326A) 39th, lysine (N329K) 42nd, valine (A330V) 43rd, leucine (Q334L) 47th, valine (E335V) 48th, arginine (K338R) 51st, alanine (T341A) 54th, serine (Y342S) 55th, glutamic acid (K362E) 75th, asparagine (K371N) 84th, tyrosine (F379Y) 92nd, and K380R Arginine is located at position 93, alanine (V381A) at position 94, valine (I382V) at position 95, serine (G390S) at position 103, glutamic acid (K399E) at position 112, glycine (E401G) at position 114, alanine (T426A) at position 139, proline (A461P) at position 174, glutamine (K467Q) at position 180, arginine (S476R) at position 189, threonine (S482T) at position 195, aspartic acid (N487D) at position 200, and aspartic acid (N492D) at position 205.

[0189] Example 4: Evaluation of the alkaline stability of AVR21 amino acid substitution aggregates (1) As transformants capable of expressing AAV-binding proteins, Escherichia coli strain BL21 (DE3) was transformed with plasmids containing one of the six AAV-binding proteins obtained in Example 3 (AVR21 amino acid substitution aggregates, specifically AVR24a [SEQ ID NO: 5], AVR24b [SEQ ID NO: 6], AVR25a [SEQ ID NO: 7], AVR25b [SEQ ID NO: 8], and AVR26 [SEQ ID NO: 9], as well as AVR21 (SEQ ID NO: 2)). The transformed organisms were then inoculated into 3 mL of 2×YT liquid medium containing 50 μg / mL of kanamycin and cultured overnight at 37°C under aerobic shaking conditions for pre-culture.

[0190] (2) 2 mL of the pre-culture solution from (1) was inoculated into 200 mL of 2×YT liquid medium containing 50 μg / mL of kanamycin in a 1 L baffled flask, and cultured aerobically with shaking at 37°C.

[0191] (3) Two hours after the start of culture, the samples were cooled on ice, and IPTG was added to each sample to a final concentration of 0.1 mM. The samples were then cultured aerobically with shaking at 25°C for 20 hours.

[0192] (4) After the culturing was complete, each cultured bacterial cell (transformed organism) was collected by centrifuging the culture solution at 4°C and 8000 rpm for 20 minutes.

[0193] (5) The bacterial cells recovered in (4) were suspended in 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride and 20 mM imidazole (hereinafter also referred to as "equilibrium solution A") at a concentration of 5 mL / 1 g (bacterial cells). The bacterial cells were then lysed using an ultrasonic generator (Insonator 201M (manufactured by Kubota Shoji Co., Ltd.)) at 8°C for approximately 10 minutes at an output of approximately 150 W. The lysates were centrifuged twice at 8000 rpm for 20 minutes at 4°C, and the supernatants were collected.

[0194] (6) The supernatant obtained in (5) was applied to an open column packed with 1.5 mL of Ni Sepharose 6 Fast Flow (Cytiva), which had been pre-equilibrated with equilibration solution A. After washing with equilibration solution A, the column was eluted with 20 mM Tris-HCl buffer (pH 7.4) containing 0.5 M imidazole and 150 mM sodium chloride.

[0195] AAV-binding proteins were prepared by dialyzing each eluate obtained in (7) and (6) with 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride.

[0196] (8) The binding activity of the AAV-binding protein prepared in (7) and the VLP2 prepared in Example 1 was measured using the ELISA method described in Example 2(6). Based on the absorbance at 450 nm, which was the measurement result, the AAV-binding protein obtained in (7) was diluted with pure water so that the measurement value was similar.

[0197] (9) The AAV-binding protein solutions diluted in (8) were divided into two fractions. One fraction was subjected to alkaline treatment by mixing an equal amount of 0.5 M sodium hydroxide aqueous solution and allowing it to stand at a constant temperature for a constant time (treatment temperature: 30°C, treatment time: 0, 30, 60 minutes), while the other fraction was not subjected to the alkaline treatment (corresponding to the "start" of the alkaline treatment).

[0198] The fractions after processing in (10) and (9) were mixed with 0.5 M MES (2-(N-Morpholino)EthaneSulfonic acid) buffer (pH 6.0) in an 8:2 ratio to adjust the pH to approximately 6, and then the binding activity to VLP2 was measured using the ELISA method described in Example 2(6).

[0199] The residual activity was calculated by dividing the absorbance at 450 nm after the alkaline treatment described in (11)(10) by the absorbance at 450 nm when the treatment time was 0 hours.

[0200] The results are shown in Table 6. The AVR21 amino acid substitution aggregates obtained in Example 3 all exhibited higher residual activity and improved stability against alkali compared to AVR21 (SEQ ID NO: 2).

[0201] [Table 6]

[0202] Example 5: Preparation of AAV vectors (Part 2) The AAV vectors used as AAVs in the following examples were prepared by the following method.

[0203] (1) A nucleotide sequence (SEQ ID NO: 21) was designed by adding the restriction enzyme EcoRI recognition sequence (GAATTC) to the 5' end of a polynucleotide encoding EGFP (Enhanced Green Fluorescent Protein) consisting of the amino acid sequence described in SEQ ID NO: 20, and a stop codon (TAG) and a BamHI recognition sequence (GGATTC) to the 3' end.

[0204] (2) A polynucleotide consisting of the sequence described in Sequence ID No. 21 was totally synthesized and cloned into a plasmid (commissioned to FASMAC, named pUC-EGFP). Escherichia coli strain JM109 was transformed with pUC-EGFP, and the resulting transformants were cultured. pUC-EGFP was extracted from the culture medium using the QIAprep Spin Miniprep kit (Qiagen).

[0205] (3) The pUC-EGFP obtained in (2) was digested with restriction enzymes EcoRI and BamHI, and then ligated into the expression vector pAAV-CMV (manufactured by Takara Bio Inc.), which had been previously digested with restriction enzymes EcoRI and BamHI. The ligation product was then used to transform E. coli strain JM109.

[0206] (4) The transformant obtained in (3) was cultured overnight with shaking at 37 °C 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 completion of the culture, the cells were collected by centrifugation, and a large amount of pAAV-EGFP was prepared from the collected cells using a Plasmid Mega Kit (manufactured by Qiagen).

[0207] (5) Escherichia coli JM109 strain was transformed using a plasmid containing a polynucleotide encoding the capsid of serotype 8 (AAV8) (hereinafter also referred to as "pRC8 Vector") and pHelper Vector (manufactured by Takara Bio Inc.). By performing the same operations as in (4) using the obtained transformant, a large amount of pRCX Vector and pHelper were prepared.

[0208] (6) HEK293T cells were cultured in 10 T-225 flasks (manufactured by Thermo Fisher Scientific) each containing 45 mL of D-MEM medium (manufactured by Fujifilm Wako Pure Chemical Corporation) containing 10% (v / v) fetal bovine serum. Gene introduction was performed by adding a complex of pAAV-EGFP prepared in (4), pRC8 Vector and pHelper prepared in (5), and polyethyleneimine (manufactured by Polysciences), and the cells were statically cultured for 3 days under the conditions of 5% (v / v) carbon dioxide and 37 °C. After the culture, the detached cells were collected by centrifugation and cryopreserved at -80 °C for each set of cells obtained from 5 T-225 flasks.

[0209] (7) The frozen cells obtained in (6) were thawed and suspended in 10 mL of 20 mM Tris-HCl buffer (pH 7.4) containing 0.5 M sodium chloride, 4 mM magnesium chloride, and 0.01% (w / v) Tween 20 (Sigma-Aldrich). Benzonase (Merck Millipore) was added at 1 / 2000 volume, and the mixture was allowed to stand at 37°C for 1 hour. The mixture was then centrifuged at 13000 × g at 4°C for 10 minutes to obtain the supernatant. Ammonium sulfate was added to the supernatant to a 15% saturation level, and the mixture was centrifuged again under the same conditions. The supernatant was passed through a 0.45 μm pore size filter to remove suspended solids.

[0210] (8) The supernatant from which suspended solids were removed was applied to a 7 mL POROS AAVX column (Thermo Fisher Scientific) that had been pre-equilibrated with 20 mM Tris-HCl buffer (pH 8.0) containing 0.5 M sodium chloride (hereinafter also referred to as "equilibrium solution B").

[0211] (9) After washing with equilibration solution B, the cells were eluted with 0.1 M acetate buffer (pH 2.5) containing 0.5 M sodium chloride. The resulting eluate was neutralized by adding 1 / 4 volume of 1 M Tris-HCl buffer (pH 8.5) containing 20 mM magnesium chloride to obtain the AAV8-EGFP solution, which is the AAV vector.

[0212] The AAV8-EGFP concentration in the solution obtained in (10)(9) was quantified by qPCR using the AAVpro Titration Kit (Takara Bio Inc.). The purity of the AAV vector contained in the solution was confirmed by subjecting the solution to SDS-PAGE and staining it with silver using the Pierce Silver Stain Kit (Thermo Fisher Scientific Inc.).

[0213] Example 6: Amino acid substitution introduction into AAV-binding protein AVR25a From the AVR21 amino acid substitution aggregates whose alkaline stability was evaluated in Example 4, AVR25a (SEQ ID NO: 7) was selected, and an AVR21 amino acid substitution aggregate (named AVR25c) was prepared by introducing the K467N amino acid substitution, which was found to improve thermal and alkaline stability in Reference Examples 1 and 2, into AVR25a. SEQ ID NOs: 29 (Forward) and 30 (Reverse) were designed as PCR primers for introducing the K467N amino acid substitution, respectively.

[0214] (1) PCR was performed in the same manner as in Example 3(a-1), except that the vector pET-AVR25a, which is capable of expressing a polypeptide containing AVR25a (SEQ ID NO: 7), was used as a template, and oligonucleotides consisting of the sequences described in SEQ ID NO: 29 (5'-GAGAGCTTAAGAATGGGGGTATCCTC-3') and SEQ ID NO: 30 (5'-TTCTTAAGCTCTCGAATCTGGTACC-3') were used as PCR primers.

[0215] (2) From the PCR product obtained in (1), a plasmid (expression vector) pET-AVR25c containing a polynucleotide encoding AVR25c, in which 25 amino acids were substituted from the native AAV-binding protein, was obtained by the same method as in Example 3(a-4).

[0216] (3) The nucleotide sequence of pET-AVR25c was analyzed and confirmed using the same method as in Example 2(8).

[0217] The amino acid sequence of AVR25c with the signal sequence and polyhistidine tag is shown in Sequence ID No. 28. In Sequence ID No. 28, the sequence from the 1st methionine (M) to the 22nd alanine (A) is the PelB signal peptide, the sequence from the 25th serine (S) to the 213th aspartic acid (D) is the AAV-binding protein AVR25c (corresponding to the region from positions 25 to 213 in Sequence ID No. 2) is the histidine (H) from positions 214 to 219 is the tag sequence. Furthermore, in Sequence ID No. 28, aspartic acid (V317D) is ranked 30th, histidine (N324H) 37th, alanine (V326A) 39th, lysine (N329K) 42nd, valine (A330V) 43rd, leucine (Q334L) 47th, valine (E335V) 48th, alanine (T341A) 54th, serine (Y342S) 55th, glutamic acid (K362E) 75th, asparagine (K371N) 84th, tyrosine (F379Y) 92nd, and arginine (K380R) 93rd. In the eye, alanine (V381A) is located at position 94, valine (I382V) at position 95, serine (G390S) at position 103, glutamic acid (K399E) at position 112, glycine (E401G) at position 114, alanine (T426A) at position 139, proline (A461P) at position 174, asparagine (K467N) at position 180, arginine (S476R) at position 189, threonine (S482T) at position 195, aspartic acid (N487D) at position 200, and aspartic acid (N492D) at position 205.

[0218] Example 7: Evaluation of the alkaline stability of the AAV-binding protein AVR25c E. coli strain BL21(DE3) was transformed with a plasmid containing the polynucleotide encoding AVR25c (SEQ ID NO: 28) obtained in Example 6, or with a plasmid encoding the AAV-binding protein AVR25a (SEQ ID NO: 7) obtained in Example 3(c). The transformed cells were cultured in the same manner as in Examples 4(1) to (3), the cultured cells were collected using the method described in Example 4(4), the AAV-binding protein was prepared using the methods described in Examples 4(5) to (8), and the alkaline stability was evaluated using the methods described in Examples 4(9) to (11).

[0219] The results are shown in Table 7. It can be seen that AVR25c (SEQ ID NO: 28), which has the K467N amino acid substitution introduced, has higher residual activity and improved stability against alkali compared to AVR25a (SEQ ID NO: 7), which does not have the amino acid substitution.

[0220] [Table 7]

[0221] Example 8: Creation and screening of a mutant library of AAV-binding proteins (Part 3) Using the pET-AVR25c vector, which is capable of expressing the polypeptide (SEQ ID NO: 28) containing the AAV-binding protein AVR25c prepared in Example 6, random mutations were introduced into the polynucleotide portion encoding the protein using error-prone PCR.

[0222] (1) Error-prone PCR was performed in the same manner as described in Example 2(1), except that a vector pET-AVR25c capable of expressing a polypeptide containing AVR25c (SEQ ID NO: 28) was used as a template. The error-prone PCR successfully introduced mutations into the polynucleotide encoding the AAV-binding protein, with an average mutation rate of 1.8 amino acid mutations per molecule.

[0223] (2) A random mutant library was prepared from the PCR products obtained in (1) using the method described in Examples 2(2) and (3).

[0224] (3) The random mutant library (transformers) prepared in (2) was inoculated into 200 μL of 2×YT liquid medium containing 50 μg / mL kanamycin, and incubated overnight with shaking at 37°C in a 96-well deep-well plate.

[0225] (4) The culture solution from (3) was centrifuged, and the resulting culture supernatant was diluted 2-fold with ultrapure water. 50 μL of the diluted culture supernatant was mixed with 50 μL of 1 M sodium hydroxide aqueous solution, and then subjected to alkaline treatment at 30°C for 15 minutes.

[0226] The binding activity between the AAV-binding protein and VLP2 after treatment (5)(4) and the binding activity between the AAV-binding protein and VLP2 without treatment (4) were evaluated using the ELISA method described in Example 2(6). The residual activity was calculated by dividing the binding activity between the AAV-binding protein and VLP2 after alkaline treatment by the binding activity between the AAV-binding protein and VLP2 without alkaline treatment.

[0227] (6) We evaluated a random mutant library of approximately 1800 strains using the method described in (5), and selected transformants from which we expressed an AAV-binding protein with improved residual activity compared to the parent molecule AVR25c.

[0228] The transformants selected in (7)(6) were cultured, and an expression vector was prepared using the QIAprep Spin Miniprep kit (Qiagen). The sequence of the polynucleotide region encoding the AAV-binding protein inserted into the vector was analyzed using the method described in Example 2(8) (commissioned to FASMAC) to identify the amino acid substitution sites.

[0229] Table 8 summarizes the amino acid substitution positions relative to AVR25c and the residual activity [%] after alkali treatment of the AAV-binding protein expressed by the transformants selected in (7) above.

[0230] Among the amino acid sequences described in Sequence ID No. 2, the amino acid residues from the 25th serine (S) to the 500th aspartic acid (D) are G314D, V(D)317G, Q318R, P322A, E325V, Q347R, T350A, E362D, H363P, I366F, E378G, F(Y)379N, G392E, V394A, V398A, E399K, R406H, Q415R, F4 AAV-binding proteins that have at least one amino acid substitution among 16L, L421P, S433G, T434S, D437V, E445K, K455R, S457R, T474R, T478I, A485V, T486A, L493M, N496D, K497E, V499I, and D500G exhibit improved stability to alkalis compared to AVR25c (SEQ ID NO: 28).

[0231] [Table 8]

[0232] Example 9: Preparation of AVR25c amino acid substitution aggregate (Part 1) From the amino acid substitutions involved in improving the alkali stability of AAV-binding proteins, as revealed in Example 8, G314D, I366F, V394A, K455R, T474R, K467E, and V499I were selected, and these amino acid substitutions were accumulated in AVR25c (SEQ ID NO: 28) to further improve alkali stability. Specifically, nine types of AAV-binding proteins shown in (a) to (i) below were designed and fabricated. (a) AAV-binding protein (SEQ ID NO: 31, named AVR27b) obtained by introducing the amino acid substitutions I366F and T474R into AVR25c. (b) AAV-binding protein (SEQ ID NO: 32, named AVR27c) obtained by introducing the amino acid substitutions V394A and T474R into AVR25c. (c) AAV-binding protein (SEQ ID NO: 33, named AVR27d) obtained by introducing amino acid substitutions K455R and T474R into AVR25c. (d) AAV-binding protein (SEQ ID NO: 34, named AVR28) obtained by introducing the amino acid substitutions I366F, V394A, and K455R into AVR25c. (e) AAV-binding protein (SEQ ID NO: 35, named AVR29a) obtained by introducing amino acid substitutions I366F, V394A, K455R, and T474R into AVR25c. (f) AAV-binding protein (SEQ ID NO: 36, named AVR29b) obtained by introducing amino acid substitutions G314D, I366F, V394A, and K455R into AVR25c. (g) AAV-binding protein (SEQ ID NO: 37, named AVR29c) obtained by introducing amino acid substitutions I366F, V394A, K455R, and K497E into AVR25c. (h) AAV-binding protein (SEQ ID NO: 38, named AVR29d) obtained by introducing amino acid substitutions I366F, V394A, K455R, and V499I into AVR25c. (i) AAV-binding protein (SEQ ID NO: 39, named AVR31) obtained by introducing amino acid substitutions G314D, I366F, V394A, K455R, K497E, and V499I into AVR25c. The following describes the methods for producing the nine types of AAV-binding proteins shown in (a) to (i) above.

[0233] We designed a PCR primer set for creating mutation aggregates. Specifically, For introducing amino acid substitutions in G314D, SEQ ID NOs. 40 (Forward) and 41 (Reverse) were used as PCR primers. For introducing the amino acid substitution of I366F, SEQ ID NOs. 42 (Forward) and 43 (Reverse) were used as PCR primers. For introducing amino acid substitutions in V394A, SEQ ID NOs. 44 (Forward) and 45 (Reverse) were used as PCR primers. For introducing amino acid substitutions in K455R, SEQ ID NOs. 46 (Forward) and 47 (Reverse) were used as PCR primers. For introducing amino acid substitutions in T474R, SEQ ID NOs. 48 (Forward) and 49 (Reverse) were used as PCR primers. For introducing amino acid substitutions in K497E, SEQ ID NOs. 50 (Forward) and 51 (Reverse) were used as PCR primers. For introducing amino acid substitutions in V499I, SEQ ID NOs. 52 (Forward) and 53 (Reverse) were used as PCR primers. For introducing amino acid substitutions in K497E and V499I, SEQ ID NOs. 54 (Forward) and 55 (Reverse) were used as PCR primers. They each designed it.

[0234] (a) AVR27b This protein was prepared by selecting I366F and T474R from among the amino acid substitutions involved in alkali stability identified in Example 9, and introducing I366F into the AAV-binding protein obtained in Example 8, which was obtained by introducing the T474R amino acid substitution into AVR25c (see Table 8, hereinafter also referred to as "AVR25c_T474R").

[0235] (a-1) Using a vector capable of expressing AVR25c_T474R as a template, and oligonucleotides consisting of the sequences described in SEQ ID NO: 42 (5'-GTCAGTTTCTGAAATTATCCAACTT-3') and SEQ ID NO: 43 (5'-TTCAGAAACTGACTGTGTTCGCCTT-3') as PCR primers, a reaction solution with the composition shown in Table 9 was prepared. The reaction solution was then heat-treated at 98°C for 5 minutes, and the reaction was carried out for 30 cycles, with the first step being at 98°C for 10 seconds, the second step at 55°C for 5 seconds, and the third step at 72°C for 6 minutes, and finally the reaction was heat-treated at 72°C for 5 minutes to perform PCR.

[0236] [Table 9]

[0237] (a-2) After digesting the template chain by treating the PCR product obtained in (a-1) with DpnI (New England Biolabs), the PCR product was used to transform E. coli BL21 strain (DE3). The resulting transformants were cultured in LB medium supplemented with 50 μg / mL kanamycin, and the plasmid was extracted from the cells (transformants) recovered by centrifugation to obtain the plasmid (expression vector) pET-AVR27b, which contains a polynucleotide encoding AVR27b with 27 amino acid substitutions from the native AAV-binding protein.

[0238] (a-3) The nucleotide sequence of pET-AVR27b was analyzed and confirmed using the same method as in Example 2(8).

[0239] The amino acid sequence of AVR27b with the signal sequence and polyhistidine tag attached is shown in Sequence ID No. 31. In Sequence ID No. 31, the sequence from the 1st methionine (M) to the 22nd alanine (A) is the PelB signal peptide, the sequence from the 25th serine (S) to the 213th aspartic acid (D) is the AAV-binding protein AVR27b (corresponding to the region from the 25th to the 213th position in Sequence ID No. 2) is the histidine (H) from the 214th to the 219th position is the tag sequence. Furthermore, in Sequence ID No. 31, aspartic acid (V317D) is ranked 30th, histidine (N324H) 37th, alanine (V326A) 39th, lysine (N329K) 42nd, valine (A330V) 43rd, leucine (Q334L) 47th, valine (E335V) 48th, alanine (T341A) 54th, serine (Y342S) 55th, glutamic acid (K362E) 75th, phenylalanine (I366F) 79th, asparagine (K371N) 84th, tyrosine (F379Y) 92nd, and arginine (K380R) 93rd. As for the second position, alanine from V381A is located at 94th, valine from I382V at 95th, serine from G390S at 103rd, glutamic acid from K399E at 112th, glycine from E401G at 114th, alanine from T426A at 139th, proline from A461P at 174th, asparagine from K467N at 180th, arginine from T474R at 187th, arginine from S476R at 189th, threonine from S482T at 195th, aspartic acid from N487D at 200th, and aspartic acid from N492D at 205th.

[0240] (b) AVR27c This protein was prepared by selecting V394A and T474R from among the amino acid substitutions involved in alkali stability identified in Example 8, and introducing V394A into AVR25c_T474R.

[0241] (b-1) PCR was performed in the same manner as in (a-1), except that a vector capable of expressing AVR25c_T474R was used as a template, and oligonucleotides consisting of the sequences described in SEQ ID NO: 44 (5'-GGTATGCGAACGTAACGGTTGAGCC-3') and SEQ ID NO: 45 (5'-ACGTTCGCATACCCCTCGGAATGCG-3') were used as PCR primers.

[0242] (b-2) From the PCR product obtained in (b-1), a plasmid (expression vector) pET-AVR27c containing a polynucleotide encoding AVR27c, in which the native AAV-binding protein has 27 amino acid substitutions, was obtained using the same method as in (a-2).

[0243] (b-3) The nucleotide sequence of pET-AVR27c was analyzed and confirmed using the same method as in Example 2(8).

[0244] The amino acid sequence of AVR27c with the signal sequence and polyhistidine tag attached is shown in Sequence ID No. 32. In Sequence ID No. 32, the methionine (M) at position 1 to the alanine (A) at position 22 is the PelB signal peptide, the serine (S) at position 25 to the aspartic acid (D) at position 213 is the AAV-binding protein AVR27c (corresponding to the region from position 25 to 213 in Sequence ID No. 2), and the histidine (H) at positions 214 to 219 is the tag sequence. Furthermore, in Sequence ID No. 32, aspartic acid (V317D) is ranked 30th, histidine (N324H) 37th, alanine (V326A) 39th, lysine (N329K) 42nd, valine (A330V) 43rd, leucine (Q334L) 47th, valine (E335V) 48th, alanine (T341A) 54th, serine (Y342S) 55th, glutamic acid (K362E) 75th, asparagine (K371N) 84th, tyrosine (F379Y) 92nd, arginine (K380R) 93rd, and alanine (V381A) 94th. In this study, valine from I382V is located at position 95, serine from G390S at position 103, alanine from V394A at position 107, glutamic acid from K399E at position 112, glycine from E401G at position 114, alanine from T426A at position 139, proline from A461P at position 174, asparagine from K467N at position 180, arginine from T474R at position 187, arginine from S476R at position 189, threonine from S482T at position 195, aspartic acid from N487D at position 200, and aspartic acid from N492D at position 205.

[0245] (c)AVR27d This protein was prepared by selecting K455R and T474R from the amino acid substitutions involved in alkali stability identified in Example 8, and introducing K455R into a vector capable of expressing AVR25c_T474R using that as a template.

[0246] (c-1) PCR was performed in the same manner as in (a-1), except that a vector capable of expressing AVR25c_T474R was used as a template, and oligonucleotides consisting of the sequences described in SEQ ID NO: 46 (5'-AAGAACGCATCAGCGAGGATACCCC-3') and SEQ ID NO: 47 (5'-CTGATGCGTTCTTCACGGAGCGGGC-3') were used as PCR primers.

[0247] (c-2) From the PCR product obtained in (c-1), a plasmid (expression vector) pET-AVR27d containing a polynucleotide encoding AVR27d, in which 27 amino acids were substituted from the native AAV-binding protein, was obtained using the same method as in (a-2).

[0248] (c-3) The nucleotide sequence of pET-AVR27d was analyzed and confirmed using the same method as in Example 2(8).

[0249] The amino acid sequence of AVR27d with the signal sequence and polyhistidine tag is shown in Sequence ID No. 33. In Sequence ID No. 33, the sequence from the 1st methionine (M) to the 22nd alanine (A) is the PelB signal peptide, the sequence from the 25th serine (S) to the 213th aspartic acid (D) is the AAV-binding protein AVR27d (corresponding to the region from positions 25 to 213 in Sequence ID No. 2) is the histidine (H) from positions 214 to 219 is the tag sequence. Furthermore, in Sequence ID No. 33, aspartic acid (V317D) is ranked 30th, histidine (N324H) 37th, alanine (V326A) 39th, lysine (N329K) 42nd, valine (A330V) 43rd, leucine (Q334L) 47th, valine (E335V) 48th, alanine (T341A) 54th, serine (Y342S) 55th, glutamic acid (K362E) 75th, asparagine (K371N) 84th, tyrosine (F379Y) 92nd, arginine (K380R) 93rd, and alanine (V381A) 94th. Valine from I382V is located at position 95, serine from G390S at position 103, glutamic acid from K399E at position 112, glycine from E401G at position 114, alanine from T426A at position 139, arginine from K455R at position 168, proline from A461P at position 174, asparagine from K467N at position 180, arginine from T474R at position 187, arginine from S476R at position 189, threonine from S482T at position 195, aspartic acid from N487D at position 200, and aspartic acid from N492D at position 205.

[0250] (d) AVR28 For this protein, I366F, V394A, and K455R were selected from among the amino acid substitutions involved in alkaline stability identified in Example 8. Since this protein was already obtained in Example 8 (see Table 8), it was reused.

[0251] The amino acid sequence of AVR28 with the signal sequence and polyhistidine tag attached is shown in Sequence ID No. 34. In Sequence ID No. 34, the sequence from the 1st methionine (M) to the 22nd alanine (A) is the PelB signal peptide, the sequence from the 25th serine (S) to the 213th aspartic acid (D) is the AAV-binding protein AVR28a (corresponding to the region from the 25th to the 213th position in Sequence ID No. 2) is the histidine (H) from the 214th to the 219th position is the tag sequence. Furthermore, in Sequence ID No. 34, aspartic acid (V317D) is ranked 30th, histidine (N324H) 37th, alanine (V326A) 39th, lysine (N329K) 42nd, valine (A330V) 43rd, leucine (Q334L) 47th, valine (E335V) 48th, alanine (T341A) 54th, serine (Y342S) 55th, glutamic acid (K362E) 75th, phenylalanine (I366F) 79th, asparagine (K371N) 84th, tyrosine (F379Y) 92nd, arginine (K380R) 93rd, and V381A Alanine is located at position 94, valine at position 95 for I382V, serine at position 103 for G390S, alanine at position 107 for V394A, glutamic acid at position 112 for K399E, glycine at position 114 for E401G, alanine at position 139 for T426A, arginine at position 168 for K455R, proline at position 174 for A461P, asparagine at position 180 for K467N, arginine at position 189 for S476R, threonine at position 195 for S482T, aspartic acid at position 200 for N487D, and aspartic acid at position 205 for N492D.

[0252] (e) AVR29a This protein was prepared by selecting T474R from the amino acid substitutions involved in alkali stability identified in Example 8, and introducing this amino acid substitution into AVR28 (SEQ ID NO: 34).

[0253] (e-1) PCR was performed in the same manner as in (a-1), except that the vector pET-AVR28a, which can express a polypeptide containing AVR28 (SEQ ID NO: 34), was used as a template, and oligonucleotides consisting of the sequences described in SEQ ID NO: 48 (5'-ACTACCGCTTTCGCCTGACCGTGAC-3') and SEQ ID NO: 49 (5'-CGAAAGCGGTAGTTACCTGGTACCA-3') were used as PCR primers.

[0254] (e-2) From the PCR product obtained in (e-1), a plasmid (expression vector) pET-AVR29a containing a polynucleotide encoding AVR29a with 29 amino acid substitutions from the native AAV-binding protein was obtained using the same method as in (a-2).

[0255] (e-3) The nucleotide sequence of pET-AVR29a was analyzed and confirmed using the same method as in Example 2(8).

[0256] The amino acid sequence of AVR29a with the signal sequence and polyhistidine tag attached is shown in Sequence ID No. 35. In Sequence ID No. 35, the first methionine (M) to the 22nd alanine (A) is the PelB signal peptide, the 25th serine (S) to the 213th aspartic acid (D) is the AAV-binding protein AVR29a (corresponding to the region from positions 25 to 213 in Sequence ID No. 2), and the 214th to 219th histidine (H) is the tag sequence. Furthermore, in Sequence ID No. 35, aspartic acid (V317D) is ranked 30th, histidine (N324H) 37th, alanine (V326A) 39th, lysine (N329K) 42nd, valine (A330V) 43rd, leucine (Q334L) 47th, valine (E335V) 48th, alanine (T341A) 54th, serine (Y342S) 55th, glutamic acid (K362E) 75th, phenylalanine (I366F) 79th, asparagine (K371N) 84th, tyrosine (F379Y) 92nd, arginine (K380R) 93rd, and alanine (V381A) 94th. In the eye, valine (I382V) is found at position 95, serine (G390S) at position 103, alanine (V394A) at position 107, glutamic acid (K399E) at position 112, glycine (E401G) at position 114, alanine (T426A) at position 139, arginine (K455R) at position 168, proline (A461P) at position 174, asparagine (K467N) at position 180, arginine (T474R) at position 187, arginine (S476R) at position 189, threonine (S482T) at position 195, aspartic acid (N487D) at position 200, and aspartic acid (N492D) at position 205.

[0257] (f) AVR29b This protein was prepared by selecting G314D from the amino acid substitutions involved in alkali stability identified in Example 9, and introducing this amino acid substitution into AVR28 (SEQ ID NO: 34).

[0258] (f-1) PCR was performed in the same manner as in (e-1), except that oligonucleotides consisting of the sequences described in SEQ ID NO: 40 (5'-CTGCAGATGAAAGCGACCAAATCAC-3') and SEQ ID NO: 41 (5'-CTTTCATCTGCAGAGCCCATGGCCA-3') were used as PCR primers.

[0259] (f-2) From the PCR product obtained in (f-1), a plasmid (expression vector) pET-AVR29b containing a polynucleotide encoding AVR29b with 29 amino acid substitutions from the native AAV-binding protein was obtained using the same method as in (a-2).

[0260] (f-3) The nucleotide sequence of pET-AVR29b was analyzed and confirmed using the same method as in Example 2(8).

[0261] The amino acid sequence of AVR29b with the signal sequence and polyhistidine tag is shown in Sequence ID No. 36. In Sequence ID No. 36, the first methionine (M) to the 22nd alanine (A) is the PelB signal peptide, the 25th serine (S) to the 213th aspartic acid (D) is the AAV-binding protein AVR29b (corresponding to the region from positions 25 to 213 in Sequence ID No. 2), and the 214th to 219th histidine (H) is the tag sequence. Furthermore, in Sequence ID No. 36, aspartic acid (G314D) is ranked 27th, aspartic acid (V317D) is ranked 30th, histidine (N324H) is ranked 37th, alanine (V326A) is ranked 39th, lysine (N329K) is ranked 42nd, valine (A330V) is ranked 43rd, leucine (Q334L) is ranked 47th, valine (E335V) is ranked 48th, alanine (T341A) is ranked 54th, serine (Y342S) is ranked 55th, glutamic acid (K362E) is ranked 75th, phenylalanine (I366F) is ranked 79th, asparagine (K371N) is ranked 84th, tyrosine (F379Y) is ranked 92nd, and arginine (K380R) is ranked 9th. Thirdly, alanine from V381A is located at position 94, valine from I382V at position 95, serine from G390S at position 103, alanine from V394A at position 107, glutamic acid from K399E at position 112, glycine from E401G at position 114, alanine from T426A at position 139, arginine from K455R at position 168, proline from A461P at position 174, asparagine from K467N at position 180, arginine from S476R at position 189, threonine from S482T at position 195, aspartic acid from N487D at position 200, and aspartic acid from N492D at position 205.

[0262] (g) AVR29c This protein was prepared by selecting K497E from the amino acid substitutions involved in alkali stability identified in Example 8, and introducing this amino acid substitution into AVR28 (SEQ ID NO: 34).

[0263] (g-1) PCR was performed in the same manner as in (e-1), except that oligonucleotides consisting of the sequences described in SEQ ID NO: 50 (5'-TTAACGAAGCCGTCGACCATCATCA-3') and SEQ ID NO: 51 (5'-ACGGCTTCGTTAACGGTCAAGTCTG-3') were used as PCR primers.

[0264] (g-2) From the PCR product obtained in (g-1), a plasmid (expression vector) pET-AVR29c containing a polynucleotide encoding AVR29c with 29 amino acid substitutions from the native AAV-binding protein was obtained using the same method as in (a-2).

[0265] The nucleotide sequence of (g-3)pET-AVR29c was analyzed and confirmed using the same method as in Example 2(8).

[0266] The amino acid sequence of AVR29c with the signal sequence and polyhistidine tag is shown in Sequence ID No. 37. In Sequence ID No. 37, the first methionine (M) to the 22nd alanine (A) is the PelB signal peptide, the 25th serine (S) to the 213th aspartic acid (D) is the AAV-binding protein AVR29c (corresponding to the region from positions 25 to 213 in Sequence ID No. 2), and the 214th to 219th histidine (H) is the tag sequence. Furthermore, in Sequence ID No. 37, aspartic acid (V317D) is ranked 30th, histidine (N324H) 37th, alanine (V326A) 39th, lysine (N329K) 42nd, valine (A330V) 43rd, leucine (Q334L) 47th, valine (E335V) 48th, alanine (T341A) 54th, serine (Y342S) 55th, glutamic acid (K362E) 75th, phenylalanine (I366F) 79th, asparagine (K371N) 84th, tyrosine (F379Y) 92nd, arginine (K380R) 93rd, and alanine (V381A) 94th. In this sequence, valine from I382V is located at position 95, serine from G390S at position 103, alanine from V394A at position 107, glutamic acid from K399E at position 112, glycine from E401G at position 114, alanine from T426A at position 139, arginine from K455R at position 168, proline from A461P at position 174, asparagine from K467N at position 180, arginine from S476R at position 189, threonine from S482T at position 195, aspartic acid from N487D at position 200, aspartic acid from N492D at position 205, and glutamic acid from K497E at position 210.

[0267] (h)AVR29d This protein was prepared by selecting V499I from the amino acid substitutions involved in alkali stability identified in Example 8, and introducing this amino acid substitution into AVR28 (SEQ ID NO: 34).

[0268] (h-1) PCR was performed in the same manner as in (e-1), except that oligonucleotides consisting of the sequences described in SEQ ID NO: 52 (5'-AAGCCATTGACCATCATCATCATCA-3') and SEQ ID NO: 53 (5'-TGGTCAATGGCTTTGTTAACGGTCA-3') were used as PCR primers.

[0269] (h-2) From the PCR product obtained in (h-1), a plasmid (expression vector) pET-AVR29d containing a polynucleotide encoding AVR29d, in which 29 amino acids were substituted from the native AAV-binding protein, was obtained using the same method as in (a-2).

[0270] The nucleotide sequence of (h-3)pET-AVR29d was analyzed and confirmed using the same method as in Example 2(8).

[0271] The amino acid sequence of AVR29d with the signal sequence and polyhistidine tag is shown in Sequence ID No. 38. In Sequence ID No. 38, the sequence from the 1st methionine (M) to the 22nd alanine (A) is the PelB signal peptide, the sequence from the 25th serine (S) to the 213th aspartic acid (D) is the AAV-binding protein AVR29d (corresponding to the region from positions 25 to 213 in Sequence ID No. 2) is the histidine (H) from positions 214 to 219 is the tag sequence. Furthermore, in Sequence ID No. 38, aspartic acid (V317D) is ranked 30th, histidine (N324H) 37th, alanine (V326A) 39th, lysine (N329K) 42nd, valine (A330V) 43rd, leucine (Q334L) 47th, valine (E335V) 48th, alanine (T341A) 54th, serine (Y342S) 55th, glutamic acid (K362E) 75th, phenylalanine (I366F) 79th, asparagine (K371N) 84th, tyrosine (F379Y) 92nd, arginine (K380R) 93rd, and alanine (V381A) 94th. In this sequence, valine from I382V is located at position 95, serine from G390S at position 103, alanine from V394A at position 107, glutamic acid from K399E at position 112, glycine from E401G at position 114, alanine from T426A at position 139, arginine from K455R at position 168, proline from A461P at position 174, asparagine from K467N at position 180, arginine from S476R at position 189, threonine from S482T at position 195, aspartic acid from N487D at position 200, aspartic acid from N492D at position 205, and isoleucine from V499I at position 212.

[0272] (i) AVR31 This protein was prepared by selecting K497E and V499I from the amino acid substitutions involved in alkali stability identified in Example 8, and introducing these amino acid substitutions into AVR29b (SEQ ID NO: 36).

[0273] (i-1) PCR was performed in the same manner as in (a-1), except that the vector pET-AVR29b, which can express a polypeptide containing AVR29b (SEQ ID NO: 36), was used as a template, and oligonucleotides consisting of the sequences described in SEQ ID NO: 54 (5'-AACGAAGCCATTGACCATCATCATCATCAT-3') and SEQ ID NO: 55 (5'-GTCAATGGCTTCGTTAACGGTCAAGTCTGC-3') were used as PCR primers.

[0274] (i-2) From the PCR product obtained in (i-1), a plasmid (expression vector) pET-AVR31 containing a polynucleotide encoding AVR31 with 31 amino acid substitutions from the native AAV-binding protein was obtained using the same method as in (a-2).

[0275] (i-3) The nucleotide sequence of pET-AVR31 was analyzed and confirmed using the same method as in Example 2(8).

[0276] The amino acid sequence of AVR31 with the signal sequence and polyhistidine tag attached is shown in Sequence ID No. 39. In Sequence ID No. 39, the sequence from the 1st methionine (M) to the 22nd alanine (A) is the PelB signal peptide, the sequence from the 25th serine (S) to the 213th aspartic acid (D) is the AAV-binding protein AVR31 (corresponding to the region from positions 25 to 213 in Sequence ID No. 2) is the histidine (H) from positions 214 to 219 is the tag sequence. Furthermore, in Sequence ID No. 39, aspartic acid from G314D is ranked 27th, aspartic acid from V317D is ranked 30th, histidine from N324H is ranked 37th, alanine from V326A is ranked 39th, lysine from N329K is ranked 42nd, valine from A330V is ranked 43rd, leucine from Q334L is ranked 47th, valine from E335V is ranked 48th, alanine from T341A is ranked 54th, serine from Y342S is ranked 55th, glutamic acid from K362E is ranked 75th, phenylalanine from I366F is ranked 79th, asparagine from K371N is ranked 84th, tyrosine from F379Y is ranked 92nd, arginine from K380R is ranked 93rd, and alanine from V381A is ranked 94th. In this sequence, valine from I382V is located at position 95, serine from G390S at position 103, alanine from V394A at position 107, glutamic acid from K399E at position 112, glycine from E401G at position 114, alanine from T426A at position 139, arginine from K455R at position 168, proline from A461P at position 174, asparagine from K467N at position 180, arginine from S476R at position 189, threonine from S482T at position 195, aspartic acid from N487D at position 200, aspartic acid from N492D at position 205, glutamic acid from K497E at position 210, and isoleucine from V499I at position 212.

[0277] Example 10: Preparation of AVR25c amino acid substitution aggregate (Part 2) (1) As transformants capable of expressing AAV-binding proteins, transformants obtained by transforming E. coli strain BL21(DE3) with plasmids containing the nine types of AAV-binding proteins obtained in Example 9 (AVR25c amino acid substitution aggregates, specifically AVR27b (SEQ ID NO: 31), AVR27c (SEQ ID NO: 32), AVR27d (SEQ ID NO: 33), AVR28 (SEQ ID NO: 34), AVR29a (SEQ ID NO: 35), AVR29b (SEQ ID NO: 36), AVR29c (SEQ ID NO: 37), AVR29d (SEQ ID NO: 38), and AVR31 (SEQ ID NO: 39)), as well as polynucleotides encoding either AVR21 (SEQ ID NO: 2) or AVR25c (SEQ ID NO: 28), were used. Otherwise, the transformants were cultured and the cells were recovered in the same manner as in Examples 4(1) to (4).

[0278] (2) Bugbuster 10× (Novagen) was added to 1 / 10 of the total volume, and Bensonase nuclease, Purity > 90% (Merck) was added to 25 U / mL, both in 20 mM Tris-HCl buffer (pH 7.4) containing 20 mM imidazole (hereinafter also referred to as "equilibrium solution D"). The bacterial cells collected in (1) were suspended in this mixture at a concentration of 5 mL / 1 g (bacterial cells), and the cells were disrupted by shaking at 15°C for 20 minutes. The lysates were centrifuged at 8000 rpm at 4°C for 20 minutes, and the supernatants were collected.

[0279] (3) The supernatant obtained in (2) was applied to an open column packed with 1.5 mL of Ni-NTAAgarose (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), which had been pre-equilibriumized with equilibration solution D. After washing with equilibration solution A, the column was eluted with 20 mM Tris-HCl buffer (pH 7.4) containing 0.5 M imidazole.

[0280] (4) The eluates obtained in (3) were desalted using PD MiniTrap G-25 (Cytiva Corporation) to prepare AAV-binding protein solutions.

[0281] Example 11: Evaluation of the alkaline stability of AVR25c amino acid substitution aggregate (Part 1) Of the AAV-binding proteins prepared in Example 10, AVR25c (SEQ ID NO: 28), AVR28 (SEQ ID NO: 34), and AVR29a (SEQ ID NO: 35) were used to evaluate their alkaline stability.

[0282] (1) The concentration of the AAV-binding protein solution prepared in Example 10 was measured using NanoDrop OneC (Thermo Fisher Scientific), and then diluted with 20 mM Tris-HCl buffer (pH 7.4) to a protein concentration of 5 μg / mL.

[0283] (2)Each AAV-binding protein solution diluted in (1) was mixed in an equal volume with a 1M sodium hydroxide aqueous solution containing 1 mM calcium chloride, and subjected to alkaline treatment by standing at a constant temperature for a constant time (treatment temperature: 30°C, treatment time: 0, 30, 60 minutes).

[0284] (3) The fraction after processing in (2) was mixed with 0.5 M MES buffer (pH 6.0) in an 8:2 ratio to bring the pH to around 6, and then the binding activity to VLP2 was measured using the ELISA method described in Example 2(6).

[0285] (4) The residual activity was calculated by dividing the absorbance at 450 nm after the alkaline treatment described in (3) by the absorbance at 450 nm when the treatment time was 0 hours.

[0286] The results are shown in Table 10. Both AVR28 (SEQ ID NO: 34) and AVR29a (SEQ ID NO: 35) exhibit higher residual activity and improved stability against alkali compared to AVR25c (SEQ ID NO: 28).

[0287] [Table 10]

[0288] Example 12: Evaluation of the alkaline stability of AVR25c amino acid substitution aggregate (Part 2) The alkaline stability of AAV-binding proteins prepared in Example 10 was evaluated using AVR25c (SEQ ID NO: 28), AVR27b (SEQ ID NO: 31), AVR27c (SEQ ID NO: 32), and AVR27d (SEQ ID NO: 33). The evaluation was carried out in the same manner as in Example 11, except that the treatment time was set to 0, 30 minutes, or 45 minutes.

[0289] The results are shown in Table 11. AVR27b (SEQ ID NO: 31), AVR27c (SEQ ID NO: 32), and AVR27d (SEQ ID NO: 33) all exhibit higher residual activity and improved stability against alkali compared to AVR25c (SEQ ID NO: 28).

[0290] [Table 11]

[0291] Example 13: Evaluation of the alkaline stability of AVR25c amino acid substitution aggregate (Part 3) The alkali stability of AVR25c amino acid substitution aggregates prepared in Example 10 was evaluated using AVR25c (SEQ ID NO: 28), AVR29b (SEQ ID NO: 36), AVR29c (SEQ ID NO: 37), and AVR29d (SEQ ID NO: 38). The evaluation was carried out in the same manner as in Example 11, except that the treatment time was set to 0, 20 minutes, or 45 minutes.

[0292] The results are shown in Table 12. AVR29b (SEQ ID NO: 36), AVR29c (SEQ ID NO: 37), and AVR29d (SEQ ID NO: 38) all exhibit higher residual activity and improved stability against alkali compared to AVR25c (SEQ ID NO: 28).

[0293] [Table 12]

[0294] Example 14: Evaluation of the alkaline stability of AVR25c amino acid substitution aggregate (Part 4) The alkaline stability of AAV-binding proteins prepared in Example 10 was evaluated using AVR28 (SEQ ID NO: 34), AVR29b (SEQ ID NO: 36), AVR29c (SEQ ID NO: 37), and AVR29d (SEQ ID NO: 38). The evaluation was carried out in the same manner as in Example 11, except that the treatment time was set to 0, 20 minutes, or 60 minutes.

[0295] The results are shown in Table 13. AVR29b (SEQ ID NO: 36), AVR29c (SEQ ID NO: 37), and AVR29d (SEQ ID NO: 38) all exhibit higher residual activity and improved stability against alkali compared to AVR28 (SEQ ID NO: 34).

[0296] [Table 13]

[0297] Example 15: Evaluation of the alkaline stability of AVR25c amino acid substitution aggregate (Part 5) Of the AAV-binding proteins prepared in Example 10, AVR25c (SEQ ID NO: 28), AVR28 (SEQ ID NO: 34), AVR29c (SEQ ID NO: 37), and AVR31 (SEQ ID NO: 39) were used, and their alkaline stability was evaluated using the method described in Example 11.

[0298] The results are shown in Table 14. AVR28 (SEQ ID NO: 34), AVR29c (SEQ ID NO: 37), and AVR31 (SEQ ID NO: 39) all exhibit higher residual activity and improved stability against alkali compared to AVR25c (SEQ ID NO: 28).

[0299] [Table 14]

[0300] Example 16: Evaluation of the alkaline stability of AVR25c amino acid substitution aggregate (Part 6) Of the AAV-binding proteins prepared in Example 10, AVR21 (SEQ ID NO: 2), AVR25c (SEQ ID NO: 28), and AVR31 (SEQ ID NO: 39) were used, and their alkaline stability was evaluated using the method described in Example 11.

[0301] The results are shown in Table 15. AVR31 (SEQ ID NO: 39) shows higher residual activity and improved stability against alkalis compared to AVR21 (SEQ ID NO: 2) and AVR25c (SEQ ID NO: 28).

[0302] [Table 15]

[0303] Example 17: Preparation and evaluation of AAV-binding protein-immobilized gel (1) A gel was prepared by introducing iodoacetamide groups to the hydroxyl groups on the surface of a hydrophilic vinyl polymer for separation (Tosoh Corporation: Toyopearl) through chemical modification. 1.0 g of the prepared gel was mixed with 6 mg of AVR29c (SEQ ID NO: 37), an AAV-binding protein prepared in Example 10, and TCEP (Tris(2-CarboxyEthyl)Phosphine) at a final concentration of 0.3 mM as a reducing agent, and reacted by shaking for 3 hours under conditions of pH 7.4 and 25°C. This prepared a gel immobilized with AVR29c, an AAV adsorbent (named AVR29c immobilized gel).

[0304] (2) 2.0 mL of the AVR29c immobilized gel prepared in (1) was packed into an empty column (φ10 mm × 50 mm, manufactured by Cytiva) to prepare an AAV adsorbent column (hereinafter also referred to as the "AVR29c column").

[0305] (3) The AVR29c column prepared in (2) was equilibrated with 20 mM Tris-HCl buffer (pH 8.0) containing 0.15 mM sodium chloride (hereinafter also referred to as "equilibrium solution E"). (4) The AAV8-EGFP solution obtained in Example 5 was applied to the column, washed with equilibration solution E, and then eluted with 0.1 M acetate buffer (pH 2.5) containing 0.15 M sodium chloride (also referred to as "elution solution B") to obtain the AAV8-EGFP solution, which is the AAV vector.

[0306] The resulting chromatogram is shown in Figure 1. In Figure 1, the peak indicated by the black arrow corresponds to the peak of the fraction containing the AAV vector (AAV8-EGFP). Since most of the impurities in the AAV8-EGFP solution pass through the AVR29c column, it can be seen that the AAV vector present in the solution can be purified using the AVR29c column.

[0307] Example 18: Purification using a combination of affinity chromatography and anion exchange chromatography (Part 1) The AAV vector solution, purified by affinity chromatography, was applied to an anion exchange column to attempt further purification of the AAV vector.

[0308] (1) The AAV vector solution obtained in Example 17(4) was diluted fivefold with 20 mM Tris-HCl buffer (pH 9.0) (hereinafter also referred to as solution AEX-A) to prepare a sample to be applied to the anion exchange column.

[0309] (2) A column (hereinafter also simply referred to as "GigaCapQ column") was prepared by packing 0.5 mL of TOYOPEARL GigaCapQ-650S (a highly adsorbent anion exchange chromatography carrier for separation and purification, manufactured by Tosoh Corporation), which had been pre-equilibrated with solution AEX-A, into an empty column (φ5 mm × 50 mm, manufactured by Cytiva Corporation), and the sample prepared in (1) was applied at a rate of 0.1 mL / min.

[0310] (3) After washing the GigaCapQ column by flowing 0.5 mL / min of solution AEX-A over a column volume of 10 CV, a linear gradient was applied until the concentration of solution AEX-A (hereinafter also referred to as solution AEX-B) containing 1 M choline chloride reached 14.5%, and then the column was flowed over a volume of 60 CV while maintaining this state (where AEX-B was at 14.5%) to elute any impurities.

[0311] (4) A step gradient was applied to the solution AEX-B until it reached 35%, and the AAV vector (AAV8-EGFP) was eluted.

[0312] The obtained chromatogram is shown in Figure 2. In Figure 2, the peak indicated by the black arrow corresponds to the peak of the fraction containing the AAV vector (AAV8-EGFP). The impurities remaining in the AAV vector solution obtained in Example 18(4) were removed by an anion exchange column (the peak indicated by the white arrow in Figure 2 corresponds to these impurities). From these results, it can be seen that the purity of the AAV vector can be further improved by purifying the solution containing the AAV vector using affinity chromatography with an AAV-binding protein immobilized gel, and then further purifying it with anion exchange chromatography.

[0313] Example 19: Measurement of the percentage (Full rate) of AAV vectors containing genes. The percentage of AAV vectors containing genes (Full rate) among the AAV vectors obtained in the fraction obtained in Example 17(4) (peak indicated by the black arrow in Figure 1), as well as in the fractions obtained in Example 18(3) (peak indicated by the white arrow in Figure 2) and Example 18(4) (peak indicated by the black arrow in Figure 2), was measured by the method described below.

[0314] (1) Each fraction was diluted four-fold with solution AEX-A and applied to a TSKgel Q-STAT (manufactured by Tosoh Corporation), an anion exchange chromatography column.

[0315] (2) Empty AAV vectors were eluted by applying a linear gradient at a flow rate of 1 mL / min so that the AEX-B solution became 17.0% in 7 minutes.

[0316] (3) After maintaining the AEX-B solution at 17.0% for 3 minutes, the (Full) AAV vector containing the gene was eluted by applying a linear gradient at a flow rate of 1 mL / min so that the AEX-B solution became 45.0% in 10 minutes.

[0317] (4) The peak area based on the fluorescence intensity at 350 nm in response to 280 nm excitation light was quantified for each of the fractions obtained in (2) (containing empty AAV vectors) and (3) (containing full AAV vectors). The proportion of full AAV vectors (full ratio) was calculated by determining the ratio of the peak area of ​​fraction (3) to the sum of the peak areas of fractions (2) and (3).

[0318] The result of calculating the full rate was, The fraction obtained in Example 17(4), i.e., the purified fraction using the AVR29c column, was 12.1%. The fraction obtained in Example 18(3), i.e., the washing fraction on the GigaCapQ column, was 8.5%. The fraction obtained in Example 18(4), i.e., the purified fraction using the GigaCapQ column, was 82%. From these results, it can be seen that purifying a solution containing AAV vectors using affinity chromatography with an AAV adsorbent, followed by further purification using anion exchange chromatography, not only improves the purity of the AAV vectors but also increases the fullness rate (i.e., the proportion of AAV vectors containing genes).

[0319] Reference Example 3: AAV purification using anion exchange chromatography alone. (1) The AAV8-EGFP solution obtained in Example 5 was prepared to pH 9.0 using Tris buffer.

[0320] (2) The AAV8-EGFP solution whose pH was adjusted in (1) was purified using a GigaCapQ column according to the methods described in Examples 18(2) to (4).

[0321] (3) Each obtained fraction (peak) is processed using TSKgel G6000PW XL Analysis was performed using a column packed with size exclusion chromatography gel (manufactured by Tosoh Corporation).

[0322] Figure 3 shows the purification results (chromatograms) using a GigaCapQ column, and the TSKgel G6000PW obtained for each fraction. XL The analysis results are shown in Figure 4. The black arrows in Figure 4 correspond to the peaks in the fraction containing the AAV vector (AAV8-EGFP). Although peaks corresponding to the AAV vector were confirmed in peaks 2 to 4 in Figure 3, it can be seen that a large amount of impurities were also present. From these results, it can be seen that it is difficult to obtain high-purity AAV by purifying a solution containing the AAV vector using only anion exchange chromatography.

[0323] Example 20 Preparation of AAV-binding protein with signal peptide sequence removed (1) PCR was performed in the same manner as in Example 10(a-1), except that a vector capable of expressing AVR29c (SEQ ID NO: 37) was used as a template, and oligonucleotides consisting of the sequences described in SEQ ID NO: 56 (5'-ACATATGTCTGCAGGCGAAAGCGAC-3') and SEQ ID NO: 57 (5'-CCTGCAGACATATGTATATCTCCTTC-3') were used as PCR primers.

[0324] (2) From the PCR product obtained in (1), a plasmid (expression vector) pET-AVR29c(-) was obtained in the same manner as in Example 9(a-2), encoding AVR29c, which has 29 amino acid substitutions from the native AAV-binding protein, and containing a polynucleotide that does not have a signal sequence at its N-terminus.

[0325] (3) The nucleotide sequence of pET-AVR29c(-) was analyzed and confirmed using the same method as in Example 2(8).

[0326] The amino acid sequence of AVR29c(-) with a polyhistidine tag is shown in Sequence ID No. 58. In Sequence ID No. 58, the second serine (S) to the 190th aspartic acid (D) is the AAV-binding protein AVR29c(-) (corresponding to the region from position 25 to 213 in Sequence ID No. 2), and the histidine (H) from position 191 to 196 is the tag sequence. Furthermore, in Sequence ID No. 58, aspartic acid (V317D) is ranked 7th, histidine (N324H) 14th, alanine (V326A) 16th, lysine (N329K) 19th, valine (A330V) 20th, leucine (Q334L) 24th, valine (E335V) 25th, alanine (T341A) 31st, serine (Y342S) 32nd, glutamic acid (K362E) 52nd, phenylalanine (I366F) 56th, asparagine (K371N) 61st, tyrosine (F379Y) 69th, arginine (K380R) 70th, and alanine (V381A) 71st Subsequently, valine from I382V is located at position 72, serine from G390S at position 80, alanine from V394A at position 84, glutamic acid from K399E at position 89, glycine from E401G at position 91, alanine from T426A at position 116, arginine from K455R at position 145, proline from A461P at position 151, asparagine from K467N at position 157, arginine from S476R at position 166, threonine from S482T at position 172, aspartic acid from N487D at position 177, aspartic acid from N492D at position 182, and glutamic acid from K497E at position 187.

[0327] (4)(3) The transformants were obtained by transforming E. coli strain BL21(DE3) with a plasmid containing a polynucleotide encoding the AAV-binding protein AVR29c(-) (SEQ ID NO: 58) obtained in (3), and the transformants were cultured and the bacterial cells were recovered in the same manner as in Examples 4(1) to (4).

[0328] (5) An AVR29c(-) solution was prepared from the bacterial cells recovered in the same manner as in (2) to (4) of Example 10.

[0329] (6) The AVR29c prepared in Example 10 and the AVR29c(-) prepared in (5) were subjected to SDS-PAGE and stained with CBB (Coomassie Brilliant Blue) staining solution (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.).

[0330] The results obtained are shown in Figure 5. In AVR29c(-) (SEQ ID NO: 58, lane 2), the AVR29c+His tag was observed as the main band, whereas in AVR29c (SEQ ID NO: 37, lane 3), a mixture of AVR29c+His tags with residual signal peptide PelB and AVR29c+His tags with cleaved PelB was observed, indicating low homogeneity.

[0331] Example 21: Performance evaluation of AAV-binding protein The binding affinity to AAV was evaluated using AVR29c (SEQ ID NO: 37) prepared in Example 10 and AVR29c(-) prepared in Example 20 (SEQ ID NO: 58).

[0332] (1) The VLP2 prepared in Example 1 was immobilized and blocked in the wells of a 96-well microplate (Thermo Fisher Scientific) using the method described in Example 2 (6-1).

[0333] (2) After washing the wells with the washing buffer described in Example 2 (6-2), a solution containing AVR29c or AVR29c(-) was added to the wells and reacted with VLP2 (at 30°C for 1 hour). (3) After the reaction was complete, the same amount of elution buffer as the solution added in (2) (200 mM acetate buffer containing 0.5 M sodium chloride (pH 3.0 or pH 2.0)) or 20 mM Tris hydrochloride buffer (pH 7.4) was added and the mixture was reacted at 25°C for 10 minutes.

[0334] (4) After the reaction was complete, the solution in the well was removed and washed with the washing buffer described in Example 2 (6-2), and then 100 μL / well of Anti-6His antibody (Bethyl Laboratories) diluted to 100 ng / mL was added.

[0335] (5) The reaction and absorbance measurement were carried out in the same manner as in Example 2 (6-4). The percentage of AAV-binding protein remaining in the well was calculated by dividing the absorbance of the well after adding the elution buffer in (3) by the absorbance of the well after adding the Tris hydrochloride buffer.

[0336] The measurement results are shown in Figure 6. The retention rate of AAV-binding protein in the wells after the addition of elution buffer (acetate buffer) was lower for AVR29c(-)(sequence number 58) than for AVR29c(sequence number 37), indicating that AVR29c(-) dissociated more efficiently from VLP2 immobilized in the wells. As shown in Figure 5 (Example 20), AVR29c(-)(sequence number 58) has higher homogeneity as an AAV-binding protein than AVR29c(sequence number 37). Therefore, it is presumed that this high homogeneity enables the efficient dissociation of AAV upon addition of elution buffer.

[0337] Example 22: Preparation and performance evaluation of AAV-binding protein-immobilized gel (1) Using the same method as in Example 13(1), a gel (named AVR29c(-) immobilized gel) was prepared by immobilizing AVR29c(-) prepared in Example 22 onto a hydrophilic vinyl polymer for separation.

[0338] (2) Add approximately 10 mg each of the AVR29c immobilized gel prepared in Example 13(1) and the AVR29c(-) immobilized gel prepared in (1) to a 0.2 μm Cosmos Spin Filter (Nacalai Tesque), and then add 9.7 × 10⁻⁶ of the AAV8-EGFP solution prepared in Example 5. 12 After adding the solution to form cp, the mixture was stirred at 25°C for 1 hour to adsorb AAV8-EGFP onto the immobilized gel.

[0339] (3) After removing the AAV8-EGFP solution from the immobilized gel by centrifugation, the gel was washed by adding 100 μL of washing solution (20 mM Tris hydrochloride buffer (pH 7.4) containing 0.5 M sodium chloride and 0.5 mM calcium chloride) and stirring at 25°C for 3 minutes.

[0340] (4) After separating and removing the washing solution from (3) from the immobilized gel by centrifugation, 100 μL of eluent A (0.1 M acetate buffer (pH 3.0) containing 0.5 M sodium chloride) was added and stirred at 25°C for 3 minutes. After stirring, eluent A was separated from the immobilized gel by centrifugation to eluate the AAV8 adsorbed on the immobilized gel. This procedure was repeated once more, and the two eluted fractions were combined and designated as "Fraction A".

[0341] (5) 100 μL of eluent B (0.1 M acetic acid (pH 2.5) containing 0.5 M sodium chloride) was added and stirred at 25°C for 3 minutes. After stirring, eluent B was separated from the immobilized gel by centrifugation to eluate the AAV8 remaining in the immobilized gel. This procedure was repeated, and the two eluted fractions were combined and designated as "Fraction B".

[0342] (6) 100 μL of eluent C (0.1 M acetic acid (pH 2.0) containing 0.5 M sodium chloride) was added and stirred at 25°C for 3 minutes. After stirring, eluent C was separated from the immobilized gel by centrifugation to eluate the AAV8 remaining in the immobilized gel. This procedure was repeated, and the two eluted fractions were combined and referred to as "Fraction C".

[0343] (7) A TSKgel G6000PW column for size exclusion chromatography (Tosoh Corporation) was connected to a Nexera ultrahigh-speed liquid chromatograph (Shimadzu Corporation), and equilibrated with eluent A (50 mM sodium acetate buffer (pH 6.0) containing 0.5 M sodium chloride, 0.01% (w / v) Tween 20 (Sigma-Aldrich), and 0.01% (w / v) Pluronic F-68 (Sigma-Aldrich)).

[0344] (8) AAV8-EGFP of known concentration, as well as fraction A obtained in (4), fraction B obtained in (5), and fraction C obtained in (6), were applied to columns equilibrated in (7) and delivered at a flow rate of 1 mL / min to obtain the separated peaks derived from AAV8-EGFP of each sample. The separated peaks were detected by fluorescence intensity (280 nm excitation / 350 nm emission).

[0345] (9) Based on the area of ​​the separation peak obtained with AAV8-EGFP at known concentrations, the AAV8 concentrations in fractions A, B, and C were quantified, respectively.

[0346] The results obtained are shown in Figure 7. In the AVR29c-immobilized gel, the highest AAV8 concentration was observed in fraction C (pH 2.0), while in the AVR29c(-)-immobilized gel, the highest AAV8 concentration was observed in fraction B (pH 2.5). As shown in Figure 5 (Example 20), AVR29c(-) (SEQ ID NO: 58), used as the ligand for the immobilized gel, exhibits higher homogeneity as an AAV-binding protein than AVR29c (SEQ ID NO: 37). Therefore, it is presumed that this higher homogeneity enables AAV elution under milder conditions.

[0347] Example 23: Purification using a combination of affinity chromatography and anion exchange chromatography (Part 2) (1) The AVR29c column prepared in Example 17 was equilibrated with 20 mmol / L Tris-HCl buffer (pH 7.4) containing 500 mmol / L sodium chloride (hereinafter also referred to as "equilibrium solution D").

[0348] (2) The AAV8-EGFP solution obtained in Example 5 was applied to an AVR29c column, washed with equilibration solution D, and then eluted with 20 mmol / L Tris-HCl buffer (pH 7.4) containing 2.0 mol / L magnesium chloride to obtain the AAV8-EGFP solution, which is the AAV vector.

[0349] (3) The AAV vector (AAV8-EGFP) solution obtained in (2) was dialyzed with 100 times the volume of solution AEX-A to prepare a sample to be applied to the anion exchange column.

[0350] (4) The AAV8-EGFP solution prepared in (3) was applied at a rate of 0.1 mL / min to a 1.0 mL SkillPak TOYOPEARL GigaCapQ-650S (a pre-packed column for high-adsorption anion exchange chromatography for separation and purification, manufactured by Tosoh Corporation, hereinafter also simply referred to as "SkillPak GigaCapQ column") which had been pre-equilibrated with solution AEX-A.

[0351] (5) After washing the SkillPak GigaCapQ column by flowing 0.5 mL / min of solution AEX-A through 2CV, the conductivity of the solution was changed by adjusting the mixing ratio of solution AEX-A and 20 mmol / L Tris-HCl buffer (pH 9.0) containing 300 mmol / L choline chloride (hereinafter also referred to as "solution AEX-C"), thereby eluting impurities adsorbed on the column. Specifically, the ratio of solution AEX-C was: (a) 49.0% (conductivity: 13.3 mS / cm (millisiemens per centimeter)), (b) 48.0% (conductivity: 13.0mS / cm), (c) 47.0% (conductivity: 12.8 mS / cm) and (d) The mixing ratio was adjusted to achieve either 46.5% (conductivity: 12.7 mS / cm), and impurities were eluted by flowing the mixture at 30 CV to 60 CV while maintaining this state.

[0352] (6) A step gradient was applied to the solution AEX-C until it reached 100% (conductivity: 24.5 mS / cm) to elute the AAV vector (AAV8-EGFP).

[0353] (7) The proportion of Full AAV contained in the eluted fractions obtained in (5) and (6) was measured by Mass Photometry using Refeyn Two (Refeyn Corporation), respectively.

[0354] The obtained chromatograms are shown in Figure 8. Furthermore, Table 16 shows the results of measuring the fullness of the AAV vectors contained in the eluted fractions obtained in (5) (peak indicated by the white arrow in Figure 8) and (6) (peak indicated by the black arrow in Figure 8). It can be seen that a solution containing AAV vectors with a higher fullness is obtained when the conductivity of the eluent used to elute impurities is 13.5 mS / cm or less, and the conductivity of the eluent used to elute the AAV vectors is 15.0 mS / cm or more.

[0355] [Table 16]

[0356] Example 24: Purification using a combination of affinity chromatography and anion exchange chromatography (Part 3) (1) The AAV8-EGFP solution obtained in Example 5 was purified using an AVR29c column according to the method described in Examples 23(1) to (3) to prepare a sample (AAV8-EGFP solution) to be applied to an anion exchange column.

[0357] (2) The AAV8-EGFP solution prepared in (1) was applied at a rate of 0.1 mL / min to a 5.0 mL SkillPak GigaCapQ column that had been pre-equilibrated with solution AEX-A.

[0358] (3) After washing the SkillPak GigaCapQ column by flowing 0.5 mL / min of solution AEX-A through 2 CV, the mixing ratio was adjusted so that the ratio of solution AEX-C was 46.0% (conductivity: 12.5 mS / cm), and impurities were eluted by flowing 20 CV while maintaining this state.

[0359] (4) A step gradient was applied to the solution AEX-C until it reached 100% (conductivity: 24.5 mS / cm) to elute the AAV vector (AAV8-EGFP).

[0360] (5) The full percentages in the AAV8-EGFP solution obtained in (1), and in the eluted fractions obtained in (3) and (4), were measured by mass photometry using a Refeyn Two (Refeyn Corporation).

[0361] Figure 9(a) shows the chromatogram obtained by affinity chromatography (AF) purification (purification using an AVR29c column), and Figure 9(b) shows the chromatogram obtained by anion exchange chromatography (AEX) purification (purification using a SkillPak GigaCapQ column). Table 17 shows the results of measuring the full percentage of the AAV vector contained in the eluted fractions obtained in (1) (peak indicated by the black arrow in Figure 9(a), AF purified fraction), (3) (peak indicated by the white arrow in Figure 9(b), Fr18), and (4) (peak indicated by the black arrow in Figure 9(b), Fr38). It can be seen that even when the volume of the SkillPak GigaCapQ column is increased from 1.0 mL (Example 23) to 5.0 mL, a solution containing an AAV vector with a high full percentage can be obtained using the purification method of this disclosure.

[0362] [Table 17]

[0363] Example 25 Evaluation of infectivity after purification by anion exchange chromatography The AF purified fraction obtained in Example 24(1), Fr18 obtained in Example 24(3), and Fr38 obtained in Example 24(4) were dialyzed with PBS (Phosphate Buffered Saline) containing 1 mmol / L magnesium chloride, AAV-HT1080 cells (manufactured by Agilent Technologies) are treated with 62,500 particles per cell. 6 It was infected with particles.

[0364] The number of particles per cell is measured using a size-exclusion chromatography column (G6000PW). XL The analysis was performed by drawing a calibration curve using a column.

[0365] After culturing the cells in 5% CO2 at 37°C for 3 days, the cells were harvested. The solution was resuspended several times using a pipette, and the fluorescence intensity derived from EGFP expressed in each cell was measured using Guava easyCyte (Luminex). The percentage of cells infected with the AAV8 mutant vector (infection rate) was calculated by measuring the percentage of cells with enhanced fluorescence intensity compared to cells without the AAV infection solution.

[0366] Figure 10 shows the results of comparing the positive rates three days after infection.

[0367] The infection dose shown on the X axis is represented by the number of particles per cell in Figure 10(a) and by the number of vector genomes (VGs) per cell calculated based on the Full rate measured in Example 10(5) in Figure 10(b). Figure 10(b) does not include the results of infecting cells with particles in Fr18 obtained in Example 24(3) because it is outside the scope.

[0368] The number of vector genomes per cell was measured by qPCR. A QuantStudio3 Real-Time PCR system (Thermo Fisher) was used. For sample preparation, reagents included with the AAVpro Titration Kit (Takara Bio) were used, and DNase I treatment was performed according to the protocol. A portion of the DNase I was then detected by PCR. The PCR reagent used was TaqPath qPCR Master Mix (Thermo Fisher), and primers SEQ ID NOs. 59 (CTCCATCACTAGGGGTTC) and 60 (TTGGGATTCCAGGCATGC) were used. For the probe, a TaqMan probe with SEQ ID NO. 61 (TCCCTTCCCTGTCCTT) modified with FAM was used. A calibration curve was created from the positive standard included with the AAVpro Titration Kit (Takara Bio) to determine the vector concentration in the obtained solution.

[0369] The positive rates corresponded to the order of Fr38 (black circle), AF purified fraction (black triangle), and Fr18 (white circle, not shown in Figure 7(b) as it is outside the range), and to the order of the Full rate measured in Example 24(5). From these results, it can be seen that in the purification method of this disclosure, by adding choline chloride to the eluate of AAV adsorbed on a carrier for anion exchange chromatography, Full AAV can be purified in a highly pure and infectious state. [Industrial applicability]

[0370] In one embodiment, the adeno-associated virus (AAV) binding protein of this disclosure is a protein in which amino acid residues at specific positions in the extracellular domain domain 1 (PKD1) and domain 2 (PKD2) of KIAA0319L (UniProt No. Q8IZA0) are substituted with other amino acid residues. The AAV binding protein of this disclosure can have significantly improved alkali resistance compared to conventional AAV binding proteins. Therefore, an AAV adsorbent using the AAV binding protein as a ligand can be washed with alkali and reused for AAV purification, and is therefore considered useful for the industrial production of AAV.

[0371] In one embodiment, the present disclosure is characterized by purifying AAV contained in a sample by a method comprising the steps of: adding a sample containing AAV to an adsorbent containing an insoluble carrier and an AAV-binding protein immobilized on the carrier, and adsorbing the AAV onto the adsorbent; eluting the AAV adsorbed on the adsorbent using an elution solution; adding a fraction containing the AAV eluted in the elution step to a carrier for anion exchange chromatography, and adsorbing the AAV onto the carrier; and eluting the AAV adsorbed on the carrier using an elution solution. This method can be used to purify AAV vectors containing genes with therapeutic effects (full AAV vectors) from among the AAV vectors contained in the sample in a high-purity and convenient manner.< / iii> < / ii> < / ii> < / ii> < / ii> < / ii> < / iii> < / iii> < / ii> < / iii>

Claims

1. An AAV-binding protein comprising at least the amino acid residues from the 25th serine to the 213th aspartic acid of the amino acid sequence described in Sequence ID No. 2, wherein the amino acid substitutions shown in any of (A) to (O) below occur in the 25th to 213th amino acid residues, and which has AAV-binding activity; (A) The asparagine at position 42 of SEQ ID NO: 2 is replaced with lysine, the glutamic acid at position 114 of SEQ ID NO: 2 is replaced with glycine, and the threonine at position 139 of SEQ ID NO: 2 is replaced with alanine. (B) Lysine at position 51 of SEQ ID NO: 2 is replaced with arginine, threonine at position 139 of SEQ ID NO: 2 is replaced with alanine, and alanine at position 174 of SEQ ID NO: 2 is replaced with proline. (C) The 42nd asparagine molecule in SEQ ID NO: 2 is replaced with lysine, the 114th glutamic acid molecule in SEQ ID NO: 2 is replaced with glycine, the 139th threonine molecule in SEQ ID NO: 2 is replaced with alanine, and the 174th alanine molecule in SEQ ID NO: 2 is replaced with proline. (D) Lysine at position 51 of SEQ ID NO: 2 is replaced with arginine, glutamic acid at position 114 of SEQ ID NO: 2 is replaced with glycine, threonine at position 139 of SEQ ID NO: 2 is replaced with alanine, and alanine at position 174 of SEQ ID NO: 2 is replaced with proline. (E) The asparagine at position 42 of SEQ ID NO: 2 is replaced with lysine, the lysine at position 51 of SEQ ID NO: 2 is replaced with arginine, the glutamic acid at position 114 of SEQ ID NO: 2 is replaced with glycine, the threonine at position 139 of SEQ ID NO: 2 is replaced with alanine, and the alanine at position 174 of SEQ ID NO: 2 is replaced with proline. (F) The asparagine at position 42 of SEQ ID NO: 2 is replaced with lysine, the glutamic acid at position 114 of SEQ ID NO: 2 is replaced with glycine, the threonine at position 139 of SEQ ID NO: 2 is replaced with alanine, the alanine at position 174 of SEQ ID NO: 2 is replaced with proline, and the glutamine at position 180 of SEQ ID NO: 2 is replaced with asparagine. (G) The asparagine at position 42 of SEQ ID NO: 2 is replaced with lysine, the isoleucine at position 79 of SEQ ID NO: 2 is replaced with phenylalanine, the glutamic acid at position 114 of SEQ ID NO: 2 is replaced with glycine, the threonine at position 139 of SEQ ID NO: 2 is replaced with alanine, the alanine at position 174 of SEQ ID NO: 2 is replaced with proline, the glutamine at position 180 of SEQ ID NO: 2 is replaced with asparagine, and the threonine at position 187 of SEQ ID NO: 2 is replaced with arginine. (H) The asparagine at position 42 of SEQ ID NO: 2 is replaced with lysine, the valine at position 107 of SEQ ID NO: 2 is replaced with alanine, the glutamic acid at position 114 of SEQ ID NO: 2 is replaced with glycine, the threonine at position 139 of SEQ ID NO: 2 is replaced with alanine, the alanine at position 174 of SEQ ID NO: 2 is replaced with proline, the glutamine at position 180 of SEQ ID NO: 2 is replaced with asparagine, and the threonine at position 187 of SEQ ID NO: 2 is replaced with arginine. (I) The asparagine at position 42 of SEQ ID NO: 2 is replaced with lysine, the glutamic acid at position 114 of SEQ ID NO: 2 is replaced with glycine, the threonine at position 139 of SEQ ID NO: 2 is replaced with alanine, the lysine at position 168 of SEQ ID NO: 2 is replaced with arginine, the alanine at position 174 of SEQ ID NO: 2 is replaced with proline, the glutamine at position 180 of SEQ ID NO: 2 is replaced with asparagine, and the threonine at position 187 of SEQ ID NO: 2 is replaced with arginine. (J) The asparagine at position 42 of SEQ ID NO: 2 is replaced with lysine, the isoleucine at position 79 of SEQ ID NO: 2 is replaced with phenylalanine, the valine at position 107 of SEQ ID NO: 2 is replaced with alanine, the glutamic acid at position 114 of SEQ ID NO: 2 is replaced with glycine, the threonine at position 139 of SEQ ID NO: 2 is replaced with alanine, the lysine at position 168 of SEQ ID NO: 2 is replaced with arginine, the alanine at position 174 of SEQ ID NO: 2 is replaced with proline, and the glutamine at position 180 of SEQ ID NO: 2 is replaced with asparagine. (K) The asparagine at position 42 of SEQ ID NO: 2 is replaced with lysine, the isoleucine at position 79 of SEQ ID NO: 2 is replaced with phenylalanine, the valine at position 107 of SEQ ID NO: 2 is replaced with alanine, the glutamic acid at position 114 of SEQ ID NO: 2 is replaced with glycine, the threonine at position 139 of SEQ ID NO: 2 is replaced with alanine, the lysine at position 168 of SEQ ID NO: 2 is replaced with arginine, the alanine at position 174 of SEQ ID NO: 2 is replaced with proline, the glutamine at position 180 of SEQ ID NO: 2 is replaced with asparagine, and the threonine at position 187 of SEQ ID NO: 2 is replaced with arginine. (L) The glycine at position 27 of SEQ ID NO: 2 is replaced with aspartic acid, the asparagine at position 42 of SEQ ID NO: 2 is replaced with lysine, the isoleucine at position 79 of SEQ ID NO: 2 is replaced with phenylalanine, the valine at position 107 of SEQ ID NO: 2 is replaced with alanine, the glutamic acid at position 114 of SEQ ID NO: 2 is replaced with glycine, the threonine at position 139 of SEQ ID NO: 2 is replaced with alanine, the lysine at position 168 of SEQ ID NO: 2 is replaced with arginine, the alanine at position 174 of SEQ ID NO: 2 is replaced with proline, and the glutamine at position 180 of SEQ ID NO: 2 is replaced with asparagine. (M) The asparagine at position 42 of SEQ ID NO: 2 is replaced with lysine, the isoleucine at position 79 of SEQ ID NO: 2 is replaced with phenylalanine, the valine at position 107 of SEQ ID NO: 2 is replaced with alanine, the glutamic acid at position 114 of SEQ ID NO: 2 is replaced with glycine, the threonine at position 139 of SEQ ID NO: 2 is replaced with alanine, the lysine at position 168 of SEQ ID NO: 2 is replaced with arginine, the alanine at position 174 of SEQ ID NO: 2 is replaced with proline, the glutamine at position 180 of SEQ ID NO: 2 is replaced with asparagine, and the lysine at position 210 of SEQ ID NO: 2 is replaced with glutamic acid. (N) The asparagine at position 42 of SEQ ID NO: 2 is replaced with lysine, the isoleucine at position 79 of SEQ ID NO: 2 is replaced with phenylalanine, the valine at position 107 of SEQ ID NO: 2 is replaced with alanine, the glutamic acid at position 114 of SEQ ID NO: 2 is replaced with glycine, the threonine at position 139 of SEQ ID NO: 2 is replaced with alanine, the lysine at position 168 of SEQ ID NO: 2 is replaced with arginine, the alanine at position 174 of SEQ ID NO: 2 is replaced with proline, the glutamine at position 180 of SEQ ID NO: 2 is replaced with asparagine, and the valine at position 212 of SEQ ID NO: 2 is replaced with isoleucine. (O) The glycine at position 27 of SEQ ID NO: 2 is replaced with aspartic acid, the asparagine at position 42 of SEQ ID NO: 2 is replaced with lysine, the isoleucine at position 79 of SEQ ID NO: 2 is replaced with phenylalanine, the valine at position 107 of SEQ ID NO: 2 is replaced with alanine, the glutamic acid at position 114 of SEQ ID NO: 2 is replaced with glycine, the threonine at position 139 of SEQ ID NO: 2 is replaced with alanine, the lysine at position 168 of SEQ ID NO: 2 is replaced with arginine, the alanine at position 174 of SEQ ID NO: 2 is replaced with proline, the glutamine at position 180 of SEQ ID NO: 2 is replaced with asparagine, the lysine at position 210 of SEQ ID NO: 2 is replaced with glutamic acid, and the valine at position 212 of SEQ ID NO: 2 is replaced with isoleucine.

2. A polynucleotide encoding an AAV-binding protein as described in claim 1.

3. An expression vector comprising the polynucleotide described in claim 2.

4. A transformant obtained by transforming Escherichia coli with the expression vector described in claim 3.

5. A method for producing an AAV-binding protein, comprising the steps of: culturing the transformant described in claim 4 to express an AAV-binding protein; and recovering the AAV-binding protein expressed from the obtained culture.

6. An AAV adsorbent comprising an insoluble carrier and an AAV-binding protein according to claim 1 immobilized on the carrier.

7. A column comprising the AAV adsorbent described in claim 6.

8. A method for purifying or analyzing AAV, comprising the steps of: adding a solution containing AAV to the column described in claim 7 to adsorb the AAV onto the adsorbent; and eluting the AAV adsorbed onto the adsorbent using an eluent.

9. The method for purifying or analyzing AAV according to claim 8, further comprising the step of washing the AAV adsorbent with an alkaline solution after the step of eluting AAV.

10. A method for purifying AAV contained in a sample, A step of adding a sample containing AAV to an adsorbent containing an insoluble carrier and an AAV-binding protein immobilized on the insoluble carrier, and adsorbing the AAV onto the adsorbent, A step of eluting the AAV adsorbed on the adsorbent, A step of adding a fraction containing AAV eluted in the elution step to a carrier for anion exchange chromatography and adsorbing the AAV onto the carrier for anion exchange chromatography, The process includes eluting AAV adsorbed onto the carrier for anion exchange chromatography, A purification method wherein the AAV-binding protein is the AAV-binding protein described in claim 1.

11. The purification method according to claim 10, wherein the step of eluting AAV adsorbed on a carrier for anion exchange chromatography is to elute the AAV adsorbed on the carrier using an eluent with a conductivity of 13.5 mS / cm or less, and then to elute the AAV remaining on the carrier using an eluent with a conductivity of 15.0 mS / cm or more.

12. The purification method according to claim 11, wherein the eluate contains choline chloride.

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