Modified adeno-associated virus-binding protein

Abstract

The object of this disclosure is to provide an AAV-binding protein that is alkali-resistant and has binding activity to serotype 5. [Means of Solution] The above problem is solved by substituting amino acid residues at specific positions in the extracellular domains 1 (PKD1) and 2 (PKD2) of KIAA0319L (UniProt No. Q8IZA0) with other specific amino acid residues.

Description

Technical Field

[0001] The present disclosure relates to a protein having binding affinity for a modified adeno-associated virus (AAV).

Background Art

[0002] 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 in a ratio of approximately VP1:VP2:VP3 = 1:1:10 to form an icosahedral shape with a diameter of 20 nm to 30 nm.

[0003] Natural AAV lacks autonomous growth ability, and replication depends on helper viruses such as adenovirus and herpesvirus. When the helper virus is present, the AAV genome is replicated within the host cell, complete AAV particles containing the AAV genome are formed, and the AAV particles are released from the host cell. 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).

[0004] 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 low concern about 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.

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

[0006] One method for recovering and purifying AAV vectors from AAV-producing cells is affinity chromatography based on AAV binding, 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 contaminating substances.

[0007] For example, Patent Document 1 discloses a polypeptide comprising extracellular domain 1 (PKD1) and domain 2 (PKD2) of KIAA0319L (UniProt No. Q8IZA0), wherein stability against heat, acid, or alkali is improved by substituting amino acid residues at specific positions within these domains with other amino acid residues. It also discloses that the AAV vector can be purified to high purity by immobilizing the polypeptide on an insoluble carrier. Furthermore, Patent Document 2 discloses an AAV-binding protein with further improved binding activity to AAV. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] WO2021 / 106882 issue [Patent Document 2] WO2023 / 140197 issue [Overview of the project] [Problems that the invention aims to solve]

[0009] It was found that AAV-binding proteins with significantly improved alkali resistance compared to naturally occurring adeno-associated virus (AAV)-binding proteins exhibited significantly reduced binding activity to serotype 5.

[0010] The object of this disclosure is to provide an AAV-binding protein that has alkali resistance and sufficient binding activity to serotype 5. In one embodiment, the object of this disclosure may also be to provide a specific AAV-binding protein that has improved alkali resistance compared to a conventional AAV-binding protein, and an AAV-binding protein that has equivalent alkali resistance and improved binding activity to serotype 5 compared to the aforementioned specific AAV-binding protein. [Means for solving the problem]

[0011] To solve the above problems, the present inventors conducted diligent research and found an AAV-binding protein that is alkali-resistant and has sufficient binding activity to serotype 5 by substituting specific amino acid residues among the amino acid residues constituting the adeno-associated virus (AAV)-binding protein with other amino acid residues.

[0012] In other words, this disclosure encompasses the following [1] to

[11] aspects: [1] AAV-binding protein selected from any of the following (i) to (iii): (i) AAV-binding proteins containing the amino acid sequence from the 25th serine (S) to the 213th aspartic acid (D) among the amino acid sequences shown in SEQ ID NOs. 4, SEQ ID NOs. 13 to 30, and SEQ ID NOs. 55; (ii) An AAV-binding protein having the amino acid sequence shown in SEQ ID NOs. 4, SEQ ID NOs. 13 to 30, and SEQ ID NOs. 55, from the 25th serine (S) to the 213th aspartic acid (D), 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 (1) to (29) shown below, provided that the amino acid substitutions (2) to (9), (11), and (13) to (29) shown below are maintained, and having binding activity for at least serotype 5; (1) The valine (V) at position 317 in SEQ ID NO: 1 (position 30 in SEQ ID NO: 2) is replaced with aspartic acid (D). (2) The asparagine (N) at position 324 in sequence number 1 (position 37 in sequence number 2) is replaced with histidine (H). (3) The valine (V) at position 326 in sequence number 1 (position 39 in sequence number 2) is replaced with alanine (A). (4) The asparagine (N) at position 329 in SEQ ID NO: 1 (position 42 in SEQ ID NO: 2) is replaced with lysine (K). (5) The 330th alanine (A) in sequence number 1 (the 43rd in sequence number 2) is replaced with valine (V). (6) The glutamine (Q) at position 334 in sequence number 1 (position 47 in sequence number 2) is replaced with leucine (L). (7) The glutamic acid (E) at position 335 in SEQ ID NO: 1 (position 48 in SEQ ID NO: 2) is replaced with valine (V). (8) The 341st threonine (T) in sequence number 1 (54th in sequence number 2) is replaced with alanine (A). (9) The tyrosine (Y) at position 342 in sequence number 1 (position 55 in sequence number 2) is replaced with serine (S). (10) Lysine (K) at position 362 in SEQ ID NO: 1 (position 75 in SEQ ID NO: 2) is replaced with glutamic acid (E). (11) The isoleucine (I) at position 366 in SEQ ID NO: 1 (position 79 in SEQ ID NO: 2) is replaced with phenylalanine (F). (12) Lysine (K) at position 371 in SEQ ID NO: 1 (position 84 in SEQ ID NO: 2) is replaced with asparagine (N). (13) Phenylalanine (F) at position 379 in SEQ ID NO: 1 (position 92 in SEQ ID NO: 2) is replaced with tyrosine (Y). (14) Lysine (K) at position 380 in SEQ ID NO: 1 (position 93 in SEQ ID NO: 2) is replaced with arginine (R). (15) The valine (V) at position 381 in sequence number 1 (position 94 in sequence number 2) is replaced with alanine (A). (16) The isoleucine (I) at position 382 in sequence number 1 (position 95 in sequence number 2) is replaced with valine (V). (17) The glycine (G) at position 390 in sequence number 1 (position 103 in sequence number 2) is replaced with serine (S). (18) The valine (V) at position 394 in sequence number 1 (position 107 in sequence number 2) is replaced with alanine (A). (19) Lysine (K) at position 399 in SEQ ID NO: 1 (position 112 in SEQ ID NO: 2) is replaced with glutamic acid (E). (20) The glutamic acid (E) at position 401 in SEQ ID NO: 1 (position 114 in SEQ ID NO: 2) is replaced with glycine (G). (21) The threonine (T) at position 426 in sequence number 1 (position 139 in sequence number 2) is replaced with alanine (A). (22) Lysine (K) at position 455 in SEQ ID NO: 1 (position 168 in SEQ ID NO: 2) is replaced with arginine (R). (23) The alanine (A) at position 461 in sequence number 1 (position 174 in sequence number 2) is replaced with proline (P). (24) Lysine (K) at position 467 of SEQ ID NO. 1 (position 180 in SEQ ID NO. 2) is replaced with asparagine (N). (25) The serine (S) at position 476 of sequence number 1 (position 189 in sequence number 2) is replaced with arginine (R). (26) The serine (S) at position 482 in sequence number 1 (position 195 in sequence number 2) is replaced with threonine (T). (27) The asparagine (N) at position 487 in SEQ ID NO: 1 (position 200 in SEQ ID NO: 2) is replaced with aspartic acid (D). (28) The asparagine (N) at position 492 in SEQ ID NO: 1 (position 205 in SEQ ID NO: 2) is replaced with aspartic acid (D). (29) The 497th lysine (K) of SEQ ID NO: 1 (the 210th in SEQ ID NO: 2) is substituted with glutamic acid (E). (iii) An amino acid sequence having 70% or more identity to the amino acid sequence shown from the 25th serine (S) to the 213th aspartic acid (D) among the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NOs: 13 to 30, and SEQ ID NO: 55, and having the above amino acid substitutions (2) to (9), (11), (13) to (29) maintained, provided that the amino acid sequence shown in SEQ ID NO: 3 is excluded, and having at least binding activity against serotype 5, an AAV-binding protein. [2] The AAV-binding protein according to [1], selected from any one of the following (iv) to (vi): (iv) An AAV-binding protein containing the amino acid sequence shown from the 25th serine (S) to the 213th aspartic acid (D) among the amino acid sequences shown in SEQ ID NOs: 13, 15, 19, 21, and 23 to 27; (v) An amino acid sequence shown from the 25th serine (S) to the 213th aspartic acid (D) among the amino acid sequences shown in SEQ ID NOs: 13, 15, 19, 21, and 23 to 27, further containing any one or more of substitutions, deletions, insertions, and additions of one or several amino acid residues at one or several positions, provided that the above amino acid substitutions (2), (4) to (9), (14), (16) to (17), (19) to (29) are maintained, and having at least binding activity against serotype 5, an AAV-binding protein; (vi) An amino acid sequence having 70% or more identity to the amino acid sequence shown from the 25th serine (S) to the 213th aspartic acid (D) among the amino acid sequences shown in SEQ ID NOs: 13, 15, 19, 21, and 23 to 27, and having the above amino acid substitutions (4), (9), (17), (19) to (29) maintained, provided that the amino acid sequence shown in SEQ ID NO: 3 is excluded, and having at least binding activity against serotype 5, an AAV-binding protein. [3] An AAV-binding protein according to [1], selected from any one of the following (vii) to (ix): (vii) An AAV-binding protein comprising an amino acid sequence shown from serine (S) at position 25 to aspartic acid (D) at position 213 among the amino acid sequences shown in SEQ ID NO: 13 and SEQ ID NO: 15; (viii) An amino acid sequence shown from serine (S) at position 25 to aspartic acid (D) at position 213 among the amino acid sequences shown in SEQ ID NO: 13 and SEQ ID NO: 15, further comprising any one or more of substitution, deletion, insertion and addition of one or several amino acid residues at one or several positions, provided that the above amino acid substitutions (4), (9), (17), (19) to (29) are maintained and having at least binding activity to serotype 5, an AAV-binding protein; (ix) An amino acid sequence having 70% or more identity to the amino acid sequence shown from serine (S) at position 25 to aspartic acid (D) at position 213 among the amino acid sequences shown in SEQ ID NO: 13 and SEQ ID NO: 15, having an amino acid sequence in which the above amino acid substitutions (4), (9), (17), (19) to (29) are maintained, provided that the amino acid sequence shown in SEQ ID NO: 3 is excluded and having at least binding activity to serotype 5, an AAV-binding protein. [4] A polynucleotide encoding the AAV-binding protein according to any one of [1] to [3]. [5] An expression vector containing the polynucleotide according to [4]. [6] A transformant obtained by transforming Escherichia coli with the expression vector according to [5]. [7] A method for producing an AAV-binding protein, comprising a step of expressing the AAV-binding protein by culturing the transformant according to [6] and a step of 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 according to [8]. A method for purifying or analyzing AAV and / or VLP, comprising the steps of adding a solution containing AAV and / or VLP to the column described in

[10] [9] to adsorb the AAV and / or VLP onto the adsorbent, and eluting the AAV and / or VLP adsorbed onto the adsorbent using an eluent. A method for purifying or analyzing serotype 5, comprising the steps of adding a solution containing serotype 5 to the column described in

[11] [9] to adsorb the serotype 5 onto the adsorbent, and eluting the serotype 5 adsorbed onto the adsorbent using an eluent. [Effects of the Invention]

[0013] 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 replaced with other amino acid residues.

[0014] The AAV-binding protein disclosed herein exhibits significantly improved alkali resistance compared to conventional AAV-binding proteins and retains sufficient binding activity to serotype 5. Therefore, the AAV adsorbent using the AAV-binding protein disclosed herein as a ligand is suitable for alkaline washing and can be repeatedly used for AAV purification, making it useful for the industrial production of AAV. Furthermore, this AAV adsorbent can be used for the purification or analysis of multiple serotypes. [Brief explanation of the drawing]

[0015] [Figure 1] This compares the Biacore results for AVR29c and AVRM9. [Figure 2] These are chromatograms obtained when AAV5-EGFP is purified. (a) and (e) show chromatograms obtained using an AVR29c column, (b) an AVRM9 column, (c) an AVRM12 column, (d) an AVRM14b column, and (f) an AVRM20 column. [Figure 3] These are chromatograms obtained when AAV5-EGFP is purified. (a) shows the chromatogram obtained using an AVR29c column, (b) shows the chromatogram obtained using an AVRM12 column, and (c) shows the chromatogram obtained using an AVRM14b column. [Figure 4] This graph shows the amount of AAV5-EGFP applied on the x-axis and the percentage of AAV vector that leaked out during flow-through immediately after application on the y-axis. It compares the results using AVRM12, AVRM14b, and AVR29c columns. [Figure 5] These are chromatograms obtained when VLP8 or VLP5 is purified. The chromatograms obtained using an AVRM12 analytical column or an AVRM14b analytical column are shown, respectively. [Modes for carrying out the invention]

[0016] The details of this disclosure are described below.

[0017] In this specification and the attached claims, the singular nouns "a," "an," and "the" include plural nouns unless the context clearly indicates otherwise. Furthermore, numerical ranges indicated by "~" include the numbers before and after the "~" as the minimum and maximum values, respectively. Also, unless explicitly stated otherwise, the units of the numbers before and after the "~" are the same. In numerical ranges described in stages within this specification, the upper or lower limit of one stage of the range may be replaced by the upper or lower limit of another stage. Furthermore, in numerical ranges described within this specification, the upper or lower limit of that range may be replaced by the values ​​shown in the examples. Additionally, individually described upper and lower limits can be combined in any way.

[0018] The adeno-associated virus (AAV)-binding protein of this disclosure is a protein that contains at least the amino acid residues from the 312th serine (Ser) to the 500th aspartic acid (Asp) in the extracellular domain 1 (PKD1) and domain 2 (PKD2) of the amino acid sequence of KIAA0319L (UniProt No. Q8IZA0) described in Sequence ID No. 1, wherein a specific amino acid substitution has occurred in the amino acid residues from the 312th to the 500th. 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.

[0019] The protein in which the aforementioned specific amino acid substitution occurs is, specifically, an AAV-binding protein selected from any of (i) to (iii). (i) AAV-binding proteins containing the amino acid sequence from the 25th serine (S) to the 213th aspartic acid (D) among the amino acid sequences shown in SEQ ID NOs. 4, SEQ ID NOs. 13 to 30, and SEQ ID NOs. 55; (ii) An AAV-binding protein having the amino acid sequence shown in SEQ ID NOs. 4, SEQ ID NOs. 13 to 30, and SEQ ID NOs. 55, from the 25th serine (S) to the 213th aspartic acid (D), 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 (1) to (29) shown below, provided that the amino acid substitutions (2) to (9), (11), and (13) to (29) shown below are maintained, and having binding activity for at least serotype 5; (1) The valine (V) at position 317 in SEQ ID NO: 1 (position 30 in SEQ ID NO: 2) is replaced with aspartic acid (D). (2) The asparagine (N) at position 324 in sequence number 1 (position 37 in sequence number 2) is replaced with histidine (H). (3) The valine (V) at position 326 in sequence number 1 (position 39 in sequence number 2) is replaced with alanine (A). (4) The asparagine (N) at position 329 in SEQ ID NO: 1 (position 42 in SEQ ID NO: 2) is replaced with lysine (K). (5) The 330th alanine (A) in sequence number 1 (the 43rd in sequence number 2) is replaced with valine (V). (6) The glutamine (Q) at position 334 in sequence number 1 (position 47 in sequence number 2) is replaced with leucine (L). (7) The glutamic acid (E) at position 335 in SEQ ID NO: 1 (position 48 in SEQ ID NO: 2) is replaced with valine (V). (8) The 341st threonine (T) in sequence number 1 (54th in sequence number 2) is replaced with alanine (A). (9) The tyrosine (Y) at position 342 in sequence number 1 (position 55 in sequence number 2) is replaced with serine (S). (10) Lysine (K) at position 362 in SEQ ID NO: 1 (position 75 in SEQ ID NO: 2) is replaced with glutamic acid (E). (11) The isoleucine (I) at position 366 in SEQ ID NO: 1 (position 79 in SEQ ID NO: 2) is replaced with phenylalanine (F). (12) Lysine (K) at position 371 in SEQ ID NO: 1 (position 84 in SEQ ID NO: 2) is replaced with asparagine (N). (13) Phenylalanine (F) at position 379 in SEQ ID NO: 1 (position 92 in SEQ ID NO: 2) is replaced with tyrosine (Y). (14) Lysine (K) at position 380 in SEQ ID NO: 1 (position 93 in SEQ ID NO: 2) is replaced with arginine (R). (15) The valine (V) at position 381 in sequence number 1 (position 94 in sequence number 2) is replaced with alanine (A). (16) The isoleucine (I) at position 382 in sequence number 1 (position 95 in sequence number 2) is replaced with valine (V). (17) The glycine (G) at position 390 in sequence number 1 (position 103 in sequence number 2) is replaced with serine (S). (18) The valine (V) at position 394 in sequence number 1 (position 107 in sequence number 2) is replaced with alanine (A). (19) Lysine (K) at position 399 in SEQ ID NO: 1 (position 112 in SEQ ID NO: 2) is replaced with glutamic acid (E). (20) The glutamic acid (E) at position 401 in SEQ ID NO: 1 (position 114 in SEQ ID NO: 2) is replaced with glycine (G). (21) The threonine (T) at position 426 in sequence number 1 (position 139 in sequence number 2) is replaced with alanine (A). (22) Lysine (K) at position 455 in SEQ ID NO: 1 (position 168 in SEQ ID NO: 2) is replaced with arginine (R). (23) The alanine (A) at position 461 in sequence number 1 (position 174 in sequence number 2) is replaced with proline (P). (24) Lysine (K) at position 467 of SEQ ID NO. 1 (position 180 in SEQ ID NO. 2) is replaced with asparagine (N). (25) The serine (S) at position 476 of sequence number 1 (position 189 in sequence number 2) is replaced with arginine (R). (26) The serine (S) at position 482 in sequence number 1 (position 195 in sequence number 2) is replaced with threonine (T). (27) The asparagine (N) at position 487 in SEQ ID NO: 1 (position 200 in SEQ ID NO: 2) is replaced with aspartic acid (D). (28) The asparagine (N) at position 492 in SEQ ID NO: 1 (position 205 in SEQ ID NO: 2) is replaced with aspartic acid (D). (29) Lysine (K) at position 497 of SEQ ID NO: 1 (position 210 in SEQ ID NO: 2) is replaced with glutamic acid (E). (iii) An AAV-binding protein having an amino acid sequence that is 70% or more identical to the amino acid sequence shown from the 25th serine (S) to the 213th aspartic acid (D) among the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NOs: 13 to 30, and SEQ ID NO: 55, and in which the above amino acid substitutions (2) to (9), (11), (13) to (29) are maintained, excluding the amino acid sequence shown in SEQ ID NO: 3, and having binding activity for at least serotype 5.

[0020] Furthermore, as an AAV-binding protein having high alkali resistance and sufficient binding activity to serotype 5, a more preferred embodiment may be an AAV-binding protein selected from any of the following (iv) to (vi). (iv) AAV-binding proteins containing the amino acid sequence from the 25th serine (S) to the 213th aspartic acid (D) among the amino acid sequences shown in SEQ ID NOs. 13, 15, 19, 21, and 23-27; (v) An AAV-binding protein having an amino acid sequence from the 25th serine (S) to the 213th aspartic acid (D) of the amino acid sequences shown in SEQ ID NOs. 13, 15, 19, 21, and 23-27, further comprising one or more substitutions, deletions, insertions, and additions of one or more amino acid residues at one or more positions, provided that the above amino acid substitutions (2), (4)-(9), (14), (16)-(17), (19)-(29) are maintained, and having binding activity for at least serotype 5; (vi) An AAV-binding protein having an amino acid sequence that is 70% or more identical to the amino acid sequence shown from the 25th serine (S) to the 213th aspartic acid (D) among the amino acid sequences shown in SEQ ID NOs. 13, SEQ ID NOs. 15, SEQ ID NOs. 19, SEQ ID NOs. 21, and SEQ ID NOs. 23 to 27, and having an amino acid sequence in which the above-mentioned amino acid substitutions (4), (9), (17), (19) to (29) are maintained, excluding the amino acid sequence shown in SEQ ID NO. 3, and having binding activity to at least serotype 5.

[0021] Furthermore, as an AAV-binding protein having high alkali resistance and sufficient binding activity to serotype 5, a more preferred embodiment may be an AAV-binding protein selected from any of the following (vii) to (ix). (vii) AAV-binding proteins containing the amino acid sequence from the 25th serine (S) to the 213th aspartic acid (D) in the amino acid sequences shown in SEQ ID NOs. 13 and 15; (viii) An AAV-binding protein having an amino acid sequence from the 25th serine (S) to the 213th aspartic acid (D) of the amino acid sequences shown in SEQ ID NOs. 13 and SEQ ID NOs. 15, further comprising one or more substitutions, deletions, insertions, and additions of one or more amino acid residues at one or more positions, provided that the above amino acid substitutions (4), (9), (17), (19) to (29) are maintained, and having binding activity for at least serotype 5; (ix) An AAV-binding protein having an amino acid sequence that is 70% or more identical to the amino acid sequence shown in SEQ ID NO: 13 and SEQ ID NO: 15, from the 25th serine (S) to the 213th aspartic acid (D), and in which the above amino acid substitutions (4), (9), (17), (19) to (29) are maintained, excluding the amino acid sequence shown in SEQ ID NO: 3, and having binding activity to at least serotype 5.

[0022] In (ii), (v), and (viii) above, "one or several" means one of the following, although it also depends on the position of amino acid substitutions and the type of amino acid residues in the three-dimensional structure of the AAV-binding protein. For example, it means one to 50, one to 30, 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.

[0023] Examples of substitutions, deletions, insertions, or additions described in (ii), (v), and (viii) above include the amino acid residue substitutions disclosed in WO2021 / 106882. For example, substitutions of at least Val317Asp, Tyr342Ser, Lys362Glu, Lys371Asn, Val381Ala, Ile382Val, Gly390Ser, Lys399Glu, Ser476Arg, and Asn487Asp are particularly preferred for the AAV-binding protein of this disclosure because they particularly improve stability to heat, acid, and alkali.

[0024] In addition to the amino acid substitutions at specific positions mentioned above, the "substitution of one or more amino acid residues" in (ii), (v), and (viii) above may also include conservative substitutions, which occur between amino acids with similar physical and / or chemical properties. In the case of conservative substitutions, it is generally known to those skilled in the art that the function of the protein is maintained between the substituted and unsubstituted parts. Examples of conservative substitutions include substitutions between glycine and alanine, serine and proline, or glutamic acid and alanine (Protein Structure and Function, Medical Science International, 9, 2005). Another example of such amino acid substitutions is substitution to monomerize the AAV-binding protein of this disclosure. Specifically, this includes amino acid substitutions in which a cysteine ​​residue, which readily forms higher-order structures, is replaced with a serine or methionine residue.

[0025] Furthermore, the "substitution, deletion, insertion, or addition of one or more amino acid residues" in (ii), (v), and (viii) above also include naturally occurring mutations (mutants or variants) based on differences in the origin of AAV-binding proteins or differences in species.

[0026] The amino acid sequence identity in (iii), (vi), and (ix) above only needs to be 70% or more, but it may have a higher degree of identity (e.g., 80% or more, 85% or more, 90% or more, or 95% or more). "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). The identity of amino acid sequences can be determined using alignment programs such as BLAST (Basic Local Alignment Search Tool) or FASTA.

[0027] The phrase "having binding activity to at least serotype 5" as described in (ii), (iii), (v), (vi), (viii), and (ix) above may also mean having activity to bind to at least AAV5 and / or VLP5. Furthermore, the phrase "having binding activity to at least serotype 5" may also mean that as long as it has binding activity to serotype 5, it may or may not have binding activity to other serotypes.

[0028] 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. Furthermore, 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.

[0029] 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 activity or stability of the AAV-binding protein. When attaching the oligopeptide to the AAV-binding protein, a 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, when attaching the oligopeptide to the AAV-binding protein, a chemically synthesized oligopeptide may be chemically bonded to the N-terminus or C-terminus of the AAV-binding protein before attachment.

[0030] 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).

[0031] A method for producing the AAV-binding protein of this disclosure may include, for example, the steps of expressing the AAV-binding protein by culturing a transformant obtained by transforming Escherichia coli with an expression vector containing a polynucleotide coding the AAV-binding protein of this disclosure, and recovering the expressed AAV-binding protein from the resulting culture.

[0032] As an example of a method for producing a polynucleotide that codes for 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 an AAV-binding protein into a nucleotide sequence and artificially synthesizing a polynucleotide containing the 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.

[0033] 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 (Arg), AGA / AGG / CGG / CGA, for isoleucine (Ile), ATA, for leucine (Leu), CTA, for glycine (Gly), GGA, and for proline (Pro), CCC are used infrequently (they are so-called rare codons), so conversion should be performed while avoiding 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).

[0034] When introducing mutations into the polynucleotides of this disclosure, the ella-prone PCR method may be used. The reaction conditions in the ella-prone PCR method are not particularly limited as long as they are conditions that can introduce the desired mutations into the polynucleotides coding for the AAV-binding protein. For example, mutations can be introduced into the polynucleotides 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 ella-prone PCR method, other methods for introducing mutations into polynucleotides include methods in which mutations are introduced into polynucleotides containing the entire or partial sequence of the AAV-binding protein by contacting and acting on the polynucleotide with a mutagenic drug or by irradiating it with ultraviolet light. In this method, any mutagenic agent commonly used by those skilled in the art may be used as a mutagenic agent, such as hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine, nitrite, sulfite, or hydrazine.

[0035] When transforming a host E. coli using the polynucleotides of this disclosure, the polynucleotides themselves may be used, but it is more preferable to use an expression vector (for example, a bacterophage, cosmid, or plasmid commonly used for the transformation of prokaryotic or eukaryotic cells) into which the polynucleotides 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.

[0036] 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 polynucleotide of this disclosure into the expression vector, it is preferable to insert it in a state where it is 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.

[0037] Transformation of the 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 carried out 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. The transformants obtained by the above-described method can be screened using an appropriate method to obtain transformants capable of expressing the AAV-binding protein of this disclosure (hereinafter also simply referred to as "transformants of this disclosure").

[0038] To prepare the expression vector of the present disclosure from the transformant of the present disclosure, the culture obtained by culturing the transformant may be prepared using an alkaline extraction method or a commercially available extraction kit such as the QIAprep Spin Miniprep kit (Qiagen).

[0039] The AAV-binding protein can be produced by culturing the transformant and recovering the AAV-binding protein from the resulting culture. In this specification, the term "culture" includes not only the cultured cells of the transformant of this disclosure but also the culture medium used for cultivation.

[0040] The transformants used in the protein production method of this disclosure should be cultured in a medium suitable for culturing the target host (Escherichia coli). An example of a preferred medium is LB (Luria-Bertani) medium supplemented with the necessary nutrients. In order to selectively grow the transformants of this disclosure 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.

[0041] 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 dithiothrateol. 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.

[0042] IPTG (Isopropyl-β-D-thiogalactopyranoside) can be used as an example of an inducer. The turbidity of the culture medium (absorbance at 600 nm) is measured, and when it is approximately between 0.5 and 1.0, an appropriate amount of IPTG is added, and the culture is continued to induce the expression of AAV-binding proteins. The concentration of IPTG added can be appropriately selected from the range of 0.005 mmol / L to 1.0 mmol / L, but a range of 0.01 mmol / L to 0.5 mmol / L is preferred. Various conditions for IPTG induction can be carried out under conditions that are well known in the art.

[0043] 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 mode of the AAV-binding protein in the transformant of this disclosure. 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 in 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.

[0044] To purify the AAV-binding protein, methods known in the art can be used, one example being separation and purification using liquid chromatography. Liquid chromatography includes ion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, affinity chromatography, etc. By combining these chromatography methods in the purification process, the AAV-binding protein can be prepared in high purity.

[0045] The AAV-binding proteins of this disclosure are alkali-resistant and have sufficient binding activity to serotype 5. In this specification, "alkali-resistant" may mean that the AAV-binding proteins have improved alkali resistance compared to conventional AAV-binding proteins and are equivalent in alkali resistance to a particular AAV-binding protein. In this specification, a particular AAV-binding protein with improved alkali resistance compared to conventional AAV-binding proteins may be a protein containing the amino acid sequence described in SEQ ID NO: 3. In this specification, "equivalent alkali resistance" may mean that the binding activity to AAV (hereinafter also referred to as residual activity) of the AAV-binding proteins of this disclosure after treatment with an alkali such as NaOH is equivalent to the residual activity of a protein containing the amino acid sequence described in SEQ ID NO: 3 after alkali treatment. In this specification, "equivalent" or "equivalent" may mean that the residual activity of the AAV-binding proteins of this disclosure after alkali treatment is 70% or more of the residual activity of a protein containing the amino acid sequence described in SEQ ID NO: 3 after alkali treatment. Furthermore, "equivalent" and "of the same degree" more preferably mean that the residual activity of the AAV-binding protein of this disclosure after alkali treatment is 75% or more, 80% or more, 90% or more, or 95% of the residual activity of the protein containing the amino acid sequence described in SEQ ID NO: 3 after alkali treatment. The alkali resistance of the AAV-binding protein can be tested as shown in the examples.

[0046] A method for measuring the binding activity of the obtained AAV-binding protein of this disclosure to serum type 5 includes, for example, measuring the binding activity of the AAV-binding protein to AAV5 using surface plasmon resonance.

[0047] The AAV-binding proteins of this disclosure can be used, for example, for the purification or analysis of multiple types of AAVs and VLPs (Virus-like Particles). These AAV-binding proteins can be used, for example, immobilized on an insoluble carrier. Specifically, the purification or analysis of AAVs and VLPs 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 AAVs and / or VLPs is not limited to the purification of AAVs and / or VLPs from solutions containing contaminants, but also includes the purification of AAVs and / or VLPs based on their structure, properties, or activity. There are no limitations on the material or shape of the insoluble carrier; for example, the insoluble carrier disclosed in WO2021 / 106882 can be cited. Furthermore, the insoluble carrier may be porous or non-porous.

[0048] The AAV-binding protein of this disclosure can be immobilized on an insoluble carrier, for example, via covalent bonding. Specifically, the AAV-binding protein can be immobilized on an insoluble carrier by covalent bonding between the AAV-binding protein and the insoluble carrier via active groups present on the insoluble carrier. The immobilization of the AAV-binding protein on the insoluble carrier can be carried out, for example, in accordance with the disclosure in WO2021 / 106882.

[0049] The AAV adsorbent of this disclosure can be used, for example, by packing it into a column for the purification or analysis of AAV and / or VLPs. For example, the purification or analysis of AAV and / or VLPs can be performed by adding a solution containing AAV and / or VLPs to a column packed with the AAV adsorbent of this disclosure (hereinafter also simply referred to as "the column of this disclosure"), allowing the AAV and / or VLPs to be adsorbed onto the adsorbent, and then eluting the AAV and / or VLPs adsorbed onto the adsorbent. That is, the method for the purification or analysis of AAV and / or VLPs of this disclosure may include, for example, the steps of adding a solution containing AAV and / or VLPs to the column of this disclosure, allowing the AAV and / or VLPs to be adsorbed onto the adsorbent, and eluting the AAV and / or VLPs adsorbed onto the adsorbent. The purification of AAV and / or VLPs using the column may be performed, for example, in accordance with the disclosure in WO2021 / 106882.

[0050] Furthermore, the AAV adsorbent of this disclosure can be used, for example, by packing it into a column for the purification or analysis of serotype 5. For example, the purification or analysis of serotype 5 can be performed by adding a solution containing serotype 5 to a column packed with the AAV adsorbent of this disclosure, allowing the serotype 5 to be adsorbed onto the adsorbent, and then eluting the serotype 5 adsorbed onto the adsorbent. That is, the method for the purification or analysis of serotype 5 of this disclosure may include, for example, the steps of adding a solution containing serotype 5 to a column of this disclosure, allowing the serotype 5 to be adsorbed onto the adsorbent, and eluting the serotype 5 adsorbed onto the adsorbent. The purification of serotype 5 using the column may be performed, for example, in accordance with the disclosure in WO2021 / 106882.

[0051] By purifying AAV, VLP, or serotype 5 (also referred to as AAV, etc.) using the AAV adsorbent of this disclosure, purified AAV, etc. can be obtained, for example. That is, in one embodiment, the method for purifying AAV, etc. may be a method for producing AAV, etc., and more specifically, it may be a method for producing purified AAV, etc. AAV, etc. can be obtained, for example, as an eluted fraction containing AAV, etc. That is, the fraction containing the eluted AAV, etc. can be separated. Separation of the AAV, etc. fraction can be carried out, for example, by a conventional method. Methods for separating the AAV, etc. 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, etc.

[0052] When purifying or analyzing AAVs using the AAV adsorbent of this disclosure, alkaline washing can prevent contamination with remaining AAVs or other impurities. That is, the washing method for the AAV adsorbent may be, for example, an alkaline washing method performed after the purification of AAVs, or, because it is acid-resistant, it may be a method of washing with a low pH solution followed by alkaline washing. The alkaline solution used for washing may be 0.1 mol / L NaOH or 0.5 mol / L NaOH.

[0053] The serotypes to be analyzed and purified are not particularly limited; they may be naturally occurring serotypes or artificially created serotypes. Examples of naturally occurring serotypes 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-specificity or infectivity) from these serotypes.

[0054] For example, serotype 1 may mean AAV1 and / or VLP1. Also, for example, serotype 2 may mean AAV2 and / or VLP2. Also, for example, serotype 3 may mean AAV3 and / or VLP3. Also, for example, serotype 4 may mean AAV4 and / or VLP4. Also, for example, serotype 5 may mean AAV5 and / or VLP5. Also, for example, serotype 6 may mean AAV6 and / or VLP6. Also, for example, serotype 7 may mean AAV7 and / or VLP7. Also, for example, serotype 8 may mean AAV8 and / or VLP8. Also, for example, serotype 9 may mean AAV9 and / or VLP9. Also, for example, serotype 10 may mean AAV10 and / or VLP10. Also, for example, serotype 11 may mean AAV11 and / or VLP11. For example, serotype 12 may mean AAV12 and / or VLP12. For example, serotype 13 may mean AAV13 and / or VLP13. [Examples]

[0055] Example 1: Fabrication of AAV vector (1) Escherichia coli strain JM109 was transformed using the plasmids pRC5 Vector (Takara Bio Inc.) and pRC8 Vector (Takara Bio Inc.), which contain polynucleotides encoding the capsids of AAV serotype 5 (AAV5) and AAV serotype 8 (AAV8), as well as the pHelper Vector (Takara Bio Inc.) and pAAV-EGFP, which encodes EGFP (Enhanced Green Fluorescent Protein) (SEQ ID NO: 56). 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. (2) After recovering the bacterial cells by centrifugation of the culture medium from (1), large quantities of pRC5 Vector, pRC8 Vector, pHelper Vector, and pAAV-EGFP were prepared from the recovered bacterial cells using Plasmid Mega Kit (Qiagen). (3) HEK293F cells were cultured in a 500 mL Erlenmeyer flask (Corning) containing 100 mL of Viral Production Medium (Thermo Fisher Scientific). (4) AAV-MAX Enhancer (manufactured by Thermo Fisher Scientific) was added to the culture medium and shaken at 37°C. (5) A mixture of AAV-MAX Transfection Reagent (manufactured by Thermo Fisher Scientific) and AAV-MAX Transfection Booster (manufactured by Thermo Fisher Scientific) was prepared. (6) The pRC5 Vector (or pRC8 Vector) and pHelper Vector prepared in (2), or these plasmids in addition to pAAV-EGFP, were diluted using Viral Plex Comlexation (Thermo Fisher Scientific). (7) The solutions prepared in (5) and (6) were mixed, and the genes were introduced into the HEK293F cells that had been shaken in (4), and the cells were cultured statically for 3 days under conditions of 8% carbon dioxide and 37°C. (5) The cells cultured in (4) were harvested and extracted with AAV-MAX Lysis Buffer (Thermo Fisher Scientific), MgCl2 final concentration 2 mmol / L, and benzonase final concentration 1 U / mL. By purifying the extract, VLP5 and VLP8 (virus-like particles), or AAV5-EGFP and AAV8-EGFP, were obtained. VLP5 is the outer shell protein particle of AAV serotype 5, and VLP8 is the outer shell protein particle of AAV serotype 8. Example 2: Preparation of AVRM9 (Part 1) The AAV-binding protein AVR29c, consisting of the amino acid sequence described from serine (Ser) at position 25 to aspartic acid (Asp) at position 213 in SEQ ID NO: 3, has 20 amino acid substitutions on PKD1, which is involved in binding to serotype 5. We investigated whether restoring the 20 amino acids on PKD1 to the wild-type sequence while retaining the 9 amino acids on PKD2 that contribute to improved alkali tolerance would improve the binding activity to serotype 5 (SEQ ID NO: 4, named AVRM9). Of the polypeptide consisting of the amino acid sequence described in Sequence ID No. 4, the first methionine (Met) to the 22nd alanine (Ala) is the PelB signal peptide, the 25th serine (Ser) to the 213th aspartic acid (Asp) is the AAV-binding protein AVRM9, and the 214th to 219th histidine (His) is the tag sequence. AVRM9 is a polypeptide in which the following nine amino acid substitutions have occurred in amino acid residues from the 312th to the 500th amino acid residues of Sequence ID No. 1, corresponding to the extracellular domain 1 (PKD1) and domain 2 (PKD2) of KIAA0319L (UniProt No. Q8IZA0). (i) The threonine at position 426 in sequence number 1 (position 139 in sequence number 2) is replaced with alanine. (ii) Lysine at position 455 in SEQ ID NO: 1 (position 168 in SEQ ID NO: 2) is replaced with arginine. (iii) The 461st alanine in SEQ ID NO: 1 (174th in SEQ ID NO: 2) is replaced with proline. (iv) Lysine at position 467 of SEQ ID NO: 1 (position 180 in SEQ ID NO: 2) is replaced with asparagine. (v) The 476th serine in SEQ ID NO: 1 (189th in SEQ ID NO: 2) is replaced with arginine. (vi) The 482nd serine in SEQ ID NO: 1 (195th in SEQ ID NO: 2) is replaced with threonine. (vii) The asparagine at position 487 in SEQ ID NO: 1 (position 200 in SEQ ID NO: 2) is replaced with aspartic acid. (viii) The 492nd asparagine molecule in SEQ ID NO: 1 (the 205th in SEQ ID NO: 2) is replaced with aspartic acid. (ix) Lysine at position 497 in SEQ ID NO: 1 (position 210 in SEQ ID NO: 2) is replaced with glutamic acid. AVRM9 was synthesized by replacing the PKD1-coding sequence of AVR29c with the PKD1-coding sequence of wild-type AVR. Specifically, sequence numbers 5 (Forward) and 6 (Reverse) were designed as PCR primers to amplify the non-PKD1 portion of the AVR29c sequence, and sequence numbers 7 (Forward) and 8 (Reverse) were designed as PCR primers to amplify the PKD1 portion of the wild-type AVR sequence. (1) In-verse PCR was performed using a vector pET-AVR29c capable of expressing a polypeptide containing AVR29c (SEQ ID NO: 3) as a template, with oligonucleotides consisting of the sequences described in SEQ ID NO: 5 (5'-CCGCGCAAGAATCGTCCTCC-3') and SEQ ID NO: 6 (5'-GGAGCAGCAGACCAGCAGCAG-3') as PCR primers. In-verse PCR was performed by preparing a reaction solution with the composition shown in Table 1, heat-treating the reaction solution at 98°C for 30 seconds, performing 30 cycles of a reaction consisting of a first step of 30 seconds at 98°C, a second step of 15 seconds at 55°C, and a third step of 60 seconds at 72°C, and finally heat-treating at 72°C for 5 minutes. The in-verse PCR amplified the polynucleotides of the AAV-binding protein excluding the PKD1 domain.

[0056] [Table 1]

[0057] (2) PCR was performed using a vector pET-AVRwt capable of expressing a polypeptide containing AVRwt (SEQ ID NO: 2) as a template, with oligonucleotides consisting of the sequences described in SEQ ID NO: 7 (5'-CTGGTCTGCTGCTCCTCGCTG-3') and SEQ ID NO: 8 (5'-ACGATTCTTGCGCGGCTCCGGTTTCACGGTAACG-3') as PCR primers. PCR was performed by preparing a reaction solution with the composition shown in Table 1, heat-treating the reaction solution at 98°C for 30 seconds, performing 30 cycles of a reaction consisting of a first step of 30 seconds at 98°C, a second step of 15 seconds at 55°C, and a third step of 10 seconds at 72°C, and finally heat-treating at 72°C for 5 minutes. The PCR amplified the polynucleotide of the PKD1 domain portion of the AAV-binding protein. (3) Using the DNA fragments amplified in (1) and (2), a reaction solution with the composition shown in Table 2 was prepared, and the infusion reaction was carried out by heat treatment at 50°C for 15 minutes.

[0058] [Table 2]

[0059] (4) After the infusion reaction was complete, Escherichia coli BL21(DE3) was transformed with the reaction solution and cultured in LB plate medium containing 50 μg / mL kanamycin (at 37°C for 18 hours). (5)(4) Polynucleotides were extracted from the colonies obtained in (4), and the nucleotide sequence of the polynucleotide region coding 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: 9 (5'-TAATACgACTCACTATAggg-3') or SEQ ID NO: 10 (5'-ATgCTAgTTATTgCTCAgCgg-3') were used as sequencing primers. Example 3: Preparation of AVRM9 (Part 2) (1) As transformants capable of expressing AAV-binding proteins, Escherichia coli strain BL21 (DE3) was transformed with a plasmid containing a polynucleotide encoding either AVRM9 (SEQ ID NO: 4) or AVR29c (SEQ ID NO: 3) obtained in Example 2. The transformed cells were then inoculated into 3 mL of 2×YT liquid medium containing 50 μg / mL of kanamycin. The inoculated medium was pre-cultured overnight at 37°C under aerobic shaking conditions. (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. (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. (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. (5) Bugbuster 10× (Novagen) was added to 1 / 10 of the total volume, and Bensonase nuclease, Purity > 90% (Merck) was added to 25 U / mL, in 20 mM Tris-HCl buffer (pH 7.4) containing 20 mM imidazole (hereinafter also referred to as "equilibrium solution A"). The bacterial cells collected in (4) were suspended in this mixture at a concentration of 5 mL / 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. (6) The supernatant obtained in (2) was applied to an open column packed with 1.5 mL of Ni-NTA Agarose (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), which had been pre-equilibriumized 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. (7)(3) Each eluate obtained in (3) was desalted using PD MiniTrap G-25 (Cytiva Corporation) to prepare an AAV-binding protein solution. Example 4 Evaluation of the binding activity between AVRM9 and AAV5 by surface plasmon resonance method (1) Using a Biacore T-200 (manufactured by Cytiva), the AAV5-EGFP obtained in Example 1 was immobilized on a Series S Sensor chip CM5 (manufactured by Cytiva) by amine coupling. (2) The concentrations of the AAV-binding proteins AVRM9 (SEQ ID NO: 4) and AVR29c (SEQ ID NO: 3) obtained in Example 3 were adjusted to 5, 2.5, 1.25, 0.625, and 0.313 μmol / L using 20 mmol / L Tris-HCl buffer (pH 7.4). (3) Using the sample obtained in (1) as a sensor chip and the AAV-binding protein prepared in (2) as an analyte, the binding response was measured by surface plasmon resonance using a Biacore T-200.

[0060] The results are shown in Figure 1 and Table 3. The value at which the binding response reached its maximum, indicated by the arrow in Figure 1, was named the maximum binding response, and the binding activity was evaluated by comparing the maximum binding responses. From Figure 1 and Table 3, AVRM9 showed a significantly larger maximum binding response to AAV5 compared to AVR29c. From this, it can be seen that AVRM9's binding activity to AAV5 was greatly improved compared to AVR29c.

[0061] [Table 3]

[0062] Example 5: Preparation of AVRM9 amino acid substitution aggregate (Part 1) We decided to sequentially add mutations to AVRM9 that do not affect its binding activity to AAV5. First, we added the mutation found in PKD1 of the mutant AVR5e (SEQ ID NO: 11) obtained in Patent Document 2. Furthermore, for the 342nd mutation, we adopted the modification obtained from the study of AVR10s (SEQ ID NO: 12). Specifically, we selected Y342S, G390S, and K399E, and investigated whether accumulating these amino acid substitutions in AVRM9 (SEQ ID NO: 4) affected its binding activity to serotype 5. Specifically, we designed and produced the AAV-binding protein shown in (a) below. (a) AAV-binding protein (SEQ ID NO: 13, named AVRM12) obtained by introducing the amino acid substitutions Y342S, G390S, and K399E into AVRM9. The method for producing the AAV-binding protein described in (a) above will be explained below.

[0063] The target sequence was obtained from a total synthesis by FASMAC. A PCR primer set was designed to transfer the target sequence into an expression vector. Specifically, Sequence IDs 5 (Forward) and 6 (Reverse) were designed as PCR primers for amplifying the synthetic sequence, and sequence IDs 7 (Forward) and 8 (Reverse) were designed as PCR primers for amplifying the expression vector.

[0064] This protein was prepared by selecting Y342S, G390S, and K399E from the amino acid substitutions disclosed in Patent Document 3, and introducing these amino acid substitutions into AVRM9 (SEQ ID NO: 4). (a-1) The same procedure as in Example 2(2) was performed, except that pUCFk-AVRM12, which codes for a polypeptide (SEQ ID NO: 13) containing the PKD1 domain into which the three mutations described above were introduced, was used as a template. (a-2) The same procedure as in Examples 2(3) to (5) was followed, except that the DNA fragment amplified in Example 2(1) and (a-1) was used.

[0065] The amino acid sequence of AVRM12 with the signal sequence and polyhistidine tag attached is shown in Sequence ID No. 13. In Sequence ID No. 13, 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 AVRM12 (corresponding to the region from positions 25 to 213 in Sequence ID No. 13), and the 214th to 219th histidine (H) is the tag sequence. Furthermore, in sequence number 13, serine from Y342S is located at position 55, serine from G390S at position 103, glutamic acid from K399E at position 112, 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. Example 6: Preparation of AVRM9 amino acid substitution aggregate (Part 2) (1) As a transformant capable of expressing AAV-binding protein, the transformant obtained by transforming Escherichia coli strain BL21(DE3) with a plasmid containing a polynucleotide encoding one of the AVRM9 amino acid substitution aggregates obtained in Example 5, AVRM12 (SEQ ID NO: 13), AVR29c (SEQ ID NO: 3), and AVRM9 (SEQ ID NO: 4) was used, and the same procedure as in Example 3(1) to (7) was followed. Example 7 Evaluation of the binding activity between AVRM9 amino acid substitution aggregates and AAV5 by surface plasmon resonance method (1) The concentrations of the AVRM9 amino acid substitution aggregates AVRM12 (SEQ ID NO: 13), AVR29c (SEQ ID NO: 3), and AVRM9 (SEQ ID NO: 4) obtained in Example 6 were adjusted to 5, 2.5, 1.25, 0.625, and 0.313 μmol / L, respectively. (2) Using the sensor chip obtained in Example 4(1) and the AAV-binding protein prepared in (1) as the analyte, the binding response was measured by surface plasmon resonance using a Biacore T-200.

[0066] The results are shown in Table 4. Binding activity was evaluated by comparing the maximum binding response against AVRM9 as the baseline. Specifically, the maximum binding response value of each sample was divided by the maximum binding response value of AVRM9 and multiplied by 100 (named the activity maintenance rate (%)) for comparison.

[0067] Table 4 shows that AVRM12 maintained the same activity level as AVRM9, indicating that Y342S, G390S, and K399E introduced into AVRM12 are stability-enhancing mutations that do not adversely affect binding to AAV5.

[0068] [Table 4]

[0069] Example 8: Preparation of AVRM12 amino acid substitution aggregate (Part 1) From the remaining 17 amino acid substitutions in AVR29c that are involved in improving the stability of AAV-binding proteins, N324H, V326A, N329K, A330V, Q334L, E335V, T341A, I366F, F379Y, K380R, V381A, I382V, V394A, and E401G were selected. These amino acid substitutions were then aggregated in AVRM12 (SEQ ID NO: 13) to select mutations that do not affect the binding activity to serotype 5. Specifically, four types of AAV-binding proteins shown in (a) to (d) below were designed and produced. (a) AAV-binding protein (SEQ ID NO: 14, named AVRM14a) obtained by introducing the amino acid substitutions V326A and K380R into AVRM12. (b) AAV-binding protein (SEQ ID NO: 15, named AVRM14b) obtained by introducing the amino acid substitutions N329K and E401G into AVRM12. (c) AAV-binding protein (SEQ ID NO: 16, named AVRM14c) obtained by introducing amino acid substitutions I366F and V394A into AVRM12. (d) AAV-binding protein (SEQ ID NO: 17, named AVRM15a) obtained by introducing the amino acid substitutions A330V, V381A, and I382V into AVRM12. (e) AAV-binding protein (SEQ ID NO: 18, named AVRM17) obtained by introducing the amino acid substitutions N324H, Q334L, E335V, T341A, and F379Y into AVRM12. The following describes the methods for producing the four types of AAV-binding proteins shown in (a) to (d) above.

[0070] The target sequence was obtained from a total synthesis by FASMAC. A PCR primer set was designed to transfer the target sequence into an expression vector. Specifically, Sequence IDs 5 (Forward) and 6 (Reverse) were designed as PCR primers for amplifying the synthetic sequence, and sequence IDs 7 (Forward) and 8 (Reverse) were designed as PCR primers for amplifying the expression vector.

[0071] (a) AVRM14a This protein was created by selecting V326A and K380R and introducing these amino acid substitutions into AVRM12 (SEQ ID NO: 13). (a-1) The same procedure as in Example 2(2) was performed, except that pUCFk-AVRM14a, which codes for a polypeptide (SEQ ID NO: 14) containing the PKD1 domain of AVRM12 into which the two mutations described above were introduced, was used as a template. (a-2) The same procedure as in Examples 2(3) to (5) was followed, except that the DNA fragment amplified in Example 2(1) and (a-1) was used.

[0072] The amino acid sequence of AVRM14a with the signal sequence and polyhistidine tag attached is shown in Sequence ID No. 14. In Sequence ID No. 14, 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 AVRM14a (corresponding to the region from positions 25 to 213 in Sequence ID No. 14), and the sequence from positions 214 to 219 histidine (H) is the tag sequence. Furthermore, in sequence number 14, alanine (V326A) is located at position 39, serine (Y342S) at position 55, arginine (K380R) at position 93, serine (G390S) at position 103, glutamic acid (K399E) at position 112, alanine (T426A) at position 139, arginine (K455R) at position 168, 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, aspartic acid (N492D) at position 205, and glutamic acid (K497E) at position 210.

[0073] (b)AVRM14b This protein was created by selecting N329K and E401G and introducing these amino acid substitutions into AVRM12 (SEQ ID NO: 13). (b-1) The same procedure as in Example 2(2) was performed, except that pUCFk-AVRM14b, which codes for a polypeptide (SEQ ID NO: 15) containing the PKD1 domain of AVRM12 into which the two mutations described above were introduced, was used as a template. (b-2) The same procedure as in Examples 2(3) to (5) was followed, except that the DNA fragments amplified in Example 2(1) and (b-1) were used.

[0074] The amino acid sequence of AVRM14b with the signal sequence and polyhistidine tag attached is shown in Sequence ID No. 15. In Sequence ID No. 15, 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 AVRM14b (corresponding to the region from position 25 to 213 in Sequence ID No. 15), and the histidine (H) at positions 214 to 219 is the tag sequence. Furthermore, in Sequence ID No. 15, lysine (N329K) is located at position 42, serine (Y342S) at position 55, serine (G390S) at position 103, 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 (S476R) at position 189, threonine (S482T) at position 195, aspartic acid (N487D) at position 200, aspartic acid (N492D) at position 205, and glutamic acid (K497E) at position 210.

[0075] (c)AVRM14c This protein was created by selecting the mutations I366F and V394A, which were first introduced into PKD1 in AVR25a and AVR29c, and introducing these amino acid substitutions into AVRM12 (SEQ ID NO: 13). (c-1) The same procedure as in Example 2(2) was performed, except that pUCFk-AVRM14c, which codes for a polypeptide (SEQ ID NO: 16) containing the PKD1 domain of AVRM12 into which the two mutations described above were introduced, was used as a template. (c-2) The same procedure as in Examples 2(3) to (5) was followed, except that the DNA fragments amplified in Example 2(1) and (c-1) were used.

[0076] The amino acid sequence of AVRM14c with the signal sequence and polyhistidine tag attached is shown in Sequence ID No. 16. In Sequence ID No. 16, 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 AVRM14c (corresponding to the region from positions 25 to 213 in Sequence ID No. 16), and the 214th to 219th histidine (H) is the tag sequence. Furthermore, in Sequence ID No. 16, serine (Y342S) is located at position 55, phenylalanine (I366F) at position 79, 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 (S476R) at position 189, threonine (S482T) at position 195, aspartic acid (N487D) at position 200, aspartic acid (N492D) at position 205, and glutamic acid (K497E) at position 210. (d)AVRM15a This protein was created by selecting A330V, V381A, and I382V and introducing the corresponding amino acid substitutions into AVRM12 (SEQ ID NO: 13). (d-1) The same procedure as in Example 2(2) was performed, except that pUCFk-AVRM15a, which codes for a polypeptide (SEQ ID NO: 17) containing the PKD1 domain of AVRM12 into which the three above mutations were introduced, was used as a template. (d-2) The same procedure as in Examples 2(3) to (5) was followed, except that the DNA fragments amplified in Example 2(1) and (d-1) were used.

[0077] The amino acid sequence of AVRM15a with the signal sequence and polyhistidine tag attached is shown in Sequence ID No. 17. In Sequence ID No. 17, 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 AVRM15a (corresponding to the region from position 25 to 213 in Sequence ID No. 17), and the histidine (H) at positions 214 to 219 is the tag sequence. Furthermore, in sequence number 17, valine from A330V is located at position 43, serine from Y342S at position 55, alanine from V381A at position 94, valine from I382V at position 95, serine from G390S at position 103, glutamic acid from K399E at position 112, 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. (e)AVRM17 This protein was created by selecting N324H, Q334L, E335V, T341A, and F379Y and introducing these amino acid substitutions into AVRM12 (SEQ ID NO: 13). (e-2) The same procedure as in Example 2(2) was performed, except that pUCFk-AVRM17, which encodes a polypeptide containing the PKD1 domain of AVRM12 into which the above five mutations were introduced (SEQ ID NO: 18), was used as a template. (e-2) The same procedure as in Examples 2(3) to (5) was followed, except that the DNA fragment amplified in Example 2(2) and (e-1) was used.

[0078] The amino acid sequence of AVRM17 with the signal sequence and polyhistidine tag attached is shown in Sequence ID No. 18. In Sequence ID No. 18, 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 AVRM17 (corresponding to the region from positions 25 to 213 in Sequence ID No. 18), and the 214th to 219th histidine (H) is the tag sequence. Furthermore, in Sequence ID No. 18, histidine (N324H) is at position 37, leucine (Q334L) is at position 47, valine (E335V) is at position 48, alanine (T341A) is at position 54, serine (Y342S) is at position 55, tyrosine (F379Y) is at position 92, serine (G390S) is at position 103, glutamic acid (K399E) is at position 112, and alanine (T426A) is at position 139. Arginine from K455R is located 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. Example 9: Preparation of AVRM12 amino acid substitution aggregate (Part 2) As transformants capable of expressing AAV-binding proteins, transformants obtained by transforming E. coli strain BL21(DE3) with plasmids containing polynucleotides encoding any of the four AAV-binding proteins (AVRM12 amino acid substitution aggregates) obtained in Example 8, specifically AVR14a (SEQ ID NO: 14), AVR14b (SEQ ID NO: 15), AVR14c (SEQ ID NO: 16), AVRM15a (SEQ ID NO: 17), AVRM17 (SEQ ID NO: 18), and AVR29c (SEQ ID NO: 3) and AVRM12 (SEQ ID NO: 13) were used. Otherwise, the same procedure as in Examples 3(1) to (7) was followed. Example 10 Evaluation of the binding activity between AVRM12 mutant aggregates and AAV5 by surface plasmon resonance. (1) The concentrations of the five types of AAV-binding proteins obtained in Example 8 (AVRM12 amino acid substitution aggregates, specifically AVR14a (SEQ ID NO: 14), AVR14b (SEQ ID NO: 15), AVR14b (SEQ ID NO: 16), AVRM15a (SEQ ID NO: 17)), AVRM17 (SEQ ID NO: 18), and AVR29c (SEQ ID NO: 3) and AVRM12 (SEQ ID NO: 13) were prepared to 5, 2.5, 1.25, 0.625, and 0.313 μmol / L. (2) Using the sample obtained in Example 4(1) as a sensor chip and the AAV-binding protein prepared in (2) as an analyte, the binding response was measured by surface plasmon resonance using a Biacore T-200. The binding activity was evaluated by comparing the maximum binding response with AVRM12 as the reference. Specifically, the maximum binding response value of each sample was divided by the maximum binding response value of AVRM12 and multiplied by 100 (named as the activity maintenance rate (%)) for comparison.

[0079] The results are shown in Table 5. From Table 5, it can be seen that AVR14a, AVR14b, AVR14b, AVRM15a, and AVRM17 all showed improved activity retention rates compared to AVR29c. In particular, AVRM14b had an activity retention rate equivalent to AVRM12, meaning that AVRM14b showed a significantly improved activity retention rate compared to AVR29c. In other words, it was found that N329K and E401G introduced into AVRM14b are stability-enhancing mutations that do not adversely affect binding to AAV5.

[0080] [Table 5]

[0081] Example 11: Preparation of AVRM14b amino acid substitutions (Part 1) Of the 14 mutations examined in Example 8, N324H, V326A, A330V, Q334L, E335V, T341A, I366F, F379Y, K380R, V381A, I382V, and V394A were introduced as monomutations into AVRM14b (SEQ ID NO: 15) to select mutations that do not affect binding activity to serotype 5. Specifically, 12 types of AAV-binding proteins shown in (a) to (i) below were designed and produced. (a) AAV-binding protein (SEQ ID NO: 19, named AVRM15b) obtained by introducing the N324H amino acid substitution into AVRM14b. (b) AAV-binding protein (SEQ ID NO: 20, named AVRM15c) obtained by introducing the amino acid substitution V326A into AVRM14b. (c) AAV-binding protein (SEQ ID NO: 21, named AVRM15d) obtained by introducing the E335V amino acid substitution into AVRM14b. (d) AAV-binding protein (SEQ ID NO: 22, named AVRM15e) obtained by introducing the I366F amino acid substitution into AVRM14b. (e) AAV-binding protein (SEQ ID NO: 23, named AVRM15f) obtained by introducing the K380R amino acid substitution into AVRM14b. (f) AAV-binding protein (SEQ ID NO: 24, named AVRM15g) obtained by introducing the I382V amino acid substitution into AVRM14b. (g) An AAV-binding protein (SEQ ID NO: 25, named AVRM15h) obtained by introducing the amino acid substitution A330V into AVRM14b. (h) An AAV-binding protein (SEQ ID NO: 26, named AVRM15i) obtained by introducing the Q334L amino acid substitution into AVRM14b. (i) An AAV-binding protein (SEQ ID NO: 27, named AVRM15j) obtained by introducing the T341A amino acid substitution into AVRM14b. (j) An AAV-binding protein (SEQ ID NO: 28, named AVRM15k) obtained by introducing the F379Y amino acid substitution into AVRM14b. (k) An AAV-binding protein (SEQ ID NO: 29, named AVRM15l) obtained by introducing the V381A amino acid substitution into AVRM14b. (l) An AAV-binding protein (SEQ ID NO: 30, named AVRM15m) obtained by introducing the amino acid substitution V394A into AVRM14b. The following describes the methods for producing the nine types of AAV-binding proteins shown in (a) to (l) above.

[0082] We designed a PCR primer set to create a mutation aggregate. Specifically, SEQ ID NOs. 31 (Forward) and 32 (Reverse) were used as PCR primers to introduce the N324H amino acid substitution. For introducing amino acid substitutions into V326A, SEQ ID NO: 33 (Forward) and SEQ ID NO: 34 (Reverse) were used as PCR primers. For introducing amino acid substitutions into E335V, SEQ ID NO: 35 (Forward) and SEQ ID NO: 36 (Reverse) were used as PCR primers. SEQ ID NOs. 37 (Forward) and SEQ ID NOs. 38 (Reverse) were used as PCR primers to introduce the amino acid substitution of I366F. For introducing amino acid substitutions in K380R, SEQ ID NOs. 39 (Forward) and 40 (Reverse) were used as PCR primers. SEQ ID NOs. 41 (Forward) and 42 (Reverse) were used as PCR primers to introduce amino acid substitutions into I382V. For introducing amino acid substitutions into A330V, SEQ ID NOs. 43 (Forward) and SEQ ID NOs. 44 (Reverse) were used as PCR primers. SEQ ID NOs. 45 (Forward) and 46 (Reverse) were used as PCR primers to introduce the amino acid substitution of Q334L. They each designed it. For introducing amino acid substitutions in T341A, SEQ ID NOs. 47 (Forward) and SEQ ID NOs. 48 (Reverse) were used as PCR primers. They each designed it. SEQ ID NOs. 49 (Forward) and SEQ ID NOs. 50 (Reverse) were used as PCR primers to introduce the amino acid substitution of F379Y. They each designed it. For introducing amino acid substitutions into V381A, SEQ ID NOs. 51 (Forward) and SEQ ID NOs. 52 (Reverse) were used as PCR primers. They each designed it. For introducing amino acid substitutions into V394A, SEQ ID NO: 53 (Forward) and SEQ ID NO: 54 (Reverse) were used as PCR primers. They each designed it. (a) AVRM15b This protein was created by selecting N324H and introducing the corresponding amino acid substitution into AVRM14b (SEQ ID NO: 15). (a-1) Using oligonucleotides consisting of the sequences described in SEQ ID NO: 31 (5'-CGAAGCATGAAGTACAACTGAAAGCGTATGTGC-3') and SEQ ID NO: 32 (5'-ACTTCATGCTTCGGCAGGGTAATCTGAAC-3') as PCR primers, a reaction solution with the composition shown in Table 1 was prepared. The reaction solution was then heat-treated at 98°C for 5 minutes. A reaction cycle consisting of a first step of 10 seconds at 98°C, a second step of 5 seconds at 55°C, and a third step of 60 seconds at 72°C was repeated for 30 cycles, and finally, PCR was performed by heat-treating at 72°C for 5 minutes. (a-2) The PCR product obtained in (a-1) was treated with DpnI (New England Biolabs) to digest the template chain, and then 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 plasmid (expression vector-)pET-AVRM15b. (a-3) The nucleotide sequence of pET-AVRM15b was analyzed and confirmed using the same method as in Example 2(5).

[0083] The amino acid sequence of AVRM15b with the signal sequence and polyhistidine tag attached is shown in Sequence ID No. 19. In Sequence ID No. 19, 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 AVRM15b (corresponding to the region from position 25 to 213 in Sequence ID No. 19), and the histidine (H) at positions 214 to 219 is the tag sequence. Furthermore, in sequence number 19, histidine (N324H) is located at position 37, lysine (N329K) at position 42, serine (Y342S) at position 55, serine (G390S) at position 103, 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 (S476R) at position 189, threonine (S482T) at position 195, aspartic acid (N487D) at position 200, aspartic acid (N492D) at position 205, and glutamic acid (K497E) at position 210. (b) AVRM15c This protein was created by selecting V326A and introducing the corresponding amino acid substitution into AVRM14b (SEQ ID NO: 15). (b-1) The same procedure as in (a-1) was performed, except that oligonucleotides consisting of the sequences described in SEQ ID NO: 33 (5'-ACGAAGCGCAACTGAAAGCGTATGTGCTG-3') and SEQ ID NO: 34 (5'-AGTTGCGCTTCGTTCTTCGGCAGGG-3') were used as PCR primers. (b-2) By performing the same procedure as in (a-2) using the PCR product obtained in (b-1), Plasmid (expression vector-)pET-AVRM15c was obtained. (b-3) The nucleotide sequence of pET-AVRM15c was analyzed and confirmed using the same method as in Example 2(5).

[0084] The amino acid sequence of AVRM15c with the signal sequence and polyhistidine tag attached is shown in Sequence ID No. 20. In Sequence ID No. 20, 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 AVRM15c (corresponding to the region from positions 25 to 213 in Sequence ID No. 20), and the 214th to 219th histidine (H) is the tag sequence. Furthermore, in Sequence ID No. 20, alanine (V326A) is located at position 39, lysine (N329K) at position 42, serine (Y342S) at position 55, serine (G390S) at position 103, 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 (S476R) at position 189, threonine (S482T) at position 195, aspartic acid (N487D) at position 200, aspartic acid (N492D) at position 205, and glutamic acid (K497E) at position 210. (c)AVRM15d This protein was created by selecting E335V and introducing the corresponding amino acid substitution into AVRM14b (SEQ ID NO: 15). (c-1) The same procedure as in (a-1) was performed, except that oligonucleotides consisting of the sequences described in SEQ ID NO: 35 (5'-TGCAGGTGCCACCGAAAGGGGAAACC-3') and SEQ ID NO: 36 (5'-CGCAGTTTCTGAAACTGTCCAAACTGAC-3') were used as PCR primers. (c-2) Plasmid (expression vector-)pET-AVRM15d was obtained by performing the same procedure as in (a-2) using the PCR product obtained in (c-1). (c-3) The nucleotide sequence of pET-AVRM15d was analyzed and confirmed using the same method as in Example 2(5).

[0085] The amino acid sequence of AVRM15d with the signal sequence and polyhistidine tag is shown in Sequence ID No. 21. In Sequence ID No. 21, 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 AVRM15d (corresponding to the region from positions 25 to 213 in Sequence ID No. 211), and the sequence from positions 214 to 219 histidine (H) is the tag sequence. Furthermore, in Sequence ID No. 21, lysine (N329K) is located at position 42, valine (E335V) at position 48, serine (Y342S) at position 55, serine (G390S) at position 103, 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 (S476R) at position 189, threonine (S482T) at position 195, aspartic acid (N487D) at position 200, aspartic acid (N492D) at position 205, and glutamic acid (K497E) at position 210. (d) AVRM15e This protein was created by selecting I366F and introducing the corresponding amino acid substitution into AVRM14b (SEQ ID NO: 15). (d-1) The same procedure as in (a-1) was performed, except that oligonucleotides consisting of the sequences described in SEQ ID NO: 37 (5'-CGCAGTTTCTGAAACTGTCCAAACTGAC-3') and SEQ ID NO: 38 (5'-TTCAGAAACTGCGAATGTTTTCCTTCC-3') were used as PCR primers. Plasmid (expression vector-)pET-AVRM15e was obtained by performing the same procedure as in (a-2) using the PCR product obtained in (d-1). (d-3) The nucleotide sequence of pET-AVRM15e was analyzed and confirmed using the same method as in Example 2(5).

[0086] The amino acid sequence of AVRM15e with the signal sequence and polyhistidine tag attached is shown in Sequence ID No. 22. In Sequence ID No. 22, 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 AVRM15e (corresponding to the region from positions 25 to 213 in Sequence ID No. 22), and the sequence from positions 214 to 219 histidine (H) is the tag sequence. Furthermore, in Sequence ID No. 22, lysine (N329K) is located at position 42, serine (Y342S) at position 55, phenylalanine (I366F) at position 79, serine (G390S) at position 103, 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 (S476R) at position 189, threonine (S482T) at position 195, aspartic acid (N487D) at position 200, aspartic acid (N492D) at position 205, and glutamic acid (K497E) at position 210. (e)AVRM15f This protein was created by selecting K380R and introducing the corresponding amino acid substitution into AVRM14b (SEQ ID NO: 15). (e-1) The same procedure as in (a-1) was performed, except that oligonucleotides consisting of the sequences described in SEQ ID NO: 39 (5'-AATTCCGCGTTATCGTTGAAGGTCAGAATGCAC-3') and SEQ ID NO: 40 (5'-ATAACGCGGAATTCATACAGACCCGGCG-3') were used as PCR primers. Plasmid (expression vector-)pET-AVRM15f was obtained by performing the same procedure as in (a-2) using the PCR product obtained in (e-1). (e-3) The nucleotide sequence of pET-AVRM15f was analyzed and confirmed using the same method as in Example 2(5).

[0087] The amino acid sequence of AVRM15f with the signal sequence and polyhistidine tag attached is shown in Sequence ID No. 23. In Sequence ID No. 23, 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 AVRM15f (corresponding to the region from positions 25 to 213 in Sequence ID No. 23), and the 214th to 219th histidine (H) is the tag sequence. Furthermore, in sequence number 23, lysine (N329K) is located at position 42, serine (Y342S) at position 55, arginine (K380R) at position 93, serine (G390S) at position 103, 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 (S476R) at position 189, threonine (S482T) at position 195, aspartic acid (N487D) at position 200, aspartic acid (N492D) at position 205, and glutamic acid (K497E) at position 210. (f) AVRM 15g This protein was created by selecting I382V and introducing the corresponding amino acid substitution into AVRM14b (SEQ ID NO: 15). (f-1) The same procedure as in (a-1) was performed, except that oligonucleotides consisting of the sequences described in SEQ ID NO: 41 (5'-AGGTTGTGGTTGAAGGTCAGAATGCACATAGC-3') and SEQ ID NO: 42 (5'-TCAACCACAACCTTGAATTCATACAGACCCG-3') were used as PCR primers. Plasmid (expression vector-)pET-AVRM15g was obtained by performing the same procedure as in (a-2) using the PCR product obtained in (f-1). (f-3) The nucleotide sequence of pET-AVRM15g was analyzed and confirmed using the same method as in Example 2(5).

[0088] The amino acid sequence of AVRM15g with the signal sequence and polyhistidine tag attached is shown in Sequence ID No. 24. In Sequence ID No. 24, 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 AVRM15g (corresponding to the region from position 25 to 213 in Sequence ID No. 24), and the histidine (H) at positions 214 to 219 is the tag sequence. Furthermore, in sequence number 24, lysine (N329K) is located at position 42, serine (Y342S) at position 55, 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, arginine (K455R) at position 168, 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, aspartic acid (N492D) at position 205, and glutamic acid (K497E) at position 210. (g)AVRM15h This protein was created by selecting A330V and introducing the corresponding amino acid substitution into AVRM14b (SEQ ID NO: 15). (g-1) The same procedure as in (a-1) was performed, except that oligonucleotides consisting of the sequences described in SEQ ID NO: 43 (5'-TGAAAGTGTATGTGCTGCAGGAACC-3') and SEQ ID NO: 44 (5'-ACATACACTTTCAGTTGTACTTCGTTCTTCGG-3') were used as PCR primers. Plasmid (expression vector-)pET-AVRM15h was obtained by performing the same procedure as in (a-2) using the PCR product obtained in (g-1). The nucleotide sequence of (g-3)pET-AVRM15h was analyzed and confirmed using the same method as in Example 2(5).

[0089] The amino acid sequence of AVRM15h with the signal sequence and polyhistidine tag attached is shown in Sequence ID No. 25. In Sequence ID No. 25, 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 AVRM15h (corresponding to the region from position 25 to 213 in Sequence ID No. 25), and the 214th to 219th histidine (H) is the tag sequence. Furthermore, in sequence number 25, lysine (N329K) is located at position 42, valine (A330V) at position 43, serine (Y342S) at position 55, serine (G390S) at position 103, 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 (S476R) at position 189, threonine (S482T) at position 195, aspartic acid (N487D) at position 200, aspartic acid (N492D) at position 205, and glutamic acid (K497E) at position 210. (h)AVRM15i This protein was created by selecting Q334L and introducing the corresponding amino acid substitution into AVRM14b (SEQ ID NO: 15). (h-1) The same procedure as in (a-1) was performed, except that oligonucleotides consisting of the sequences described in SEQ ID NO: 45 (5'-TGCTGCTGGAACCACCGAAAGGG-3') and SEQ ID NO: 46 (5'-GGTTCCAGCAGCACATACGCTTTCAG-3') were used as PCR primers. (h-2) By performing the same procedure as in (a-2) using the PCR product obtained in (h-1), Plasmid (expression vector-)pET-AVRM15i was obtained. The nucleotide sequence of (h-3)pET-AVRM15i was analyzed and confirmed using the same method as in Example 2(5).

[0090] The amino acid sequence of AVRM15i with the signal sequence and polyhistidine tag attached is shown in Sequence ID No. 26. In Sequence ID No. 26, 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 AVRM15i (corresponding to the region from positions 25 to 213 in Sequence ID No. 26), and the sequence from positions 214 to 219 histidine (H) is the tag sequence. Furthermore, in Sequence ID No. 26, lysine (N329K) is located at position 42, leucine (Q334L) at position 47, serine (Y342S) at position 55, serine (G390S) at position 103, 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 (S476R) at position 189, threonine (S482T) at position 195, aspartic acid (N487D) at position 200, aspartic acid (N492D) at position 205, and glutamic acid (K497E) at position 210. (i)AVRM15j This protein was created by selecting T341A and introducing the corresponding amino acid substitution into AVRM14b (SEQ ID NO: 15). (i-1) The same procedure as in (a-1) was performed, except that oligonucleotides consisting of the sequences described in SEQ ID NO: 47 (5'-GGGAAGCGAGCACGTATGACTGGCAGC-3') and SEQ ID NO: 48 (5'-GTGCTCGCTTCCCCTTTCGGTGGTTCCTG-3') were used as PCR primers. (i-2) Plasmid (expression vector-)pET-AVRM15j was obtained by performing the same procedure as in (a-2) using the PCR product obtained in (i-1). (i-3) The nucleotide sequence of pET-AVRM15j was analyzed and confirmed using the same method as in Example 2(5).

[0091] The amino acid sequence of AVRM15j with the signal sequence and polyhistidine tag attached is shown in Sequence ID No. 27. In Sequence ID No. 27, 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 AVRM15j (corresponding to the region from positions 25 to 213 in Sequence ID No. 27), and the sequence from positions 214 to 219 histidine (H) is the tag sequence. Furthermore, in sequence number 27, lysine (N329K) is located at position 42, alanine (T341A) at position 54, serine (Y342S) at position 55, serine (G390S) at position 103, 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 (S476R) at position 189, threonine (S482T) at position 195, aspartic acid (N487D) at position 200, aspartic acid (N492D) at position 205, and glutamic acid (K497E) at position 210. (j)AVRM15k This protein was created by selecting F379Y and introducing the corresponding amino acid substitution into AVRM14b (SEQ ID NO: 15). (j-1) The same procedure as in (a-1) was performed, except that oligonucleotides consisting of the sequences described in SEQ ID NO: 49 (5'-ATGAATATAAGGTTATCGTTGAAGGTCAGAATG-3') and SEQ ID NO: 50 (5'-ACCTTATATTCATACAGACCCGGCGTC-3') were used as PCR primers. Plasmid (expression vector-)pET-AVRM15k was obtained by performing the same procedure as in (a-2) using the PCR product obtained in (j-1). The nucleotide sequence of (j-3)pET-AVRM15k was analyzed and confirmed using the same method as in Example 2(5).

[0092] The amino acid sequence of AVRM15k with the signal sequence and polyhistidine tag attached is shown in Sequence ID No. 28. In Sequence ID No. 28, 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 AVRM15k (corresponding to the region from positions 25 to 213 in Sequence ID No. 28), and the 214th to 219th histidine (H) is the tag sequence. Furthermore, in sequence number 28, lysine (N329K) is located at position 42, serine (Y342S) at position 55, tyrosine (F379Y) at position 92, serine (G390S) at position 103, 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 (S476R) at position 189, threonine (S482T) at position 195, aspartic acid (N487D) at position 200, aspartic acid (N492D) at position 205, and glutamic acid (K497E) at position 210. (k)AVRM15l This protein was created by selecting V381A and introducing the corresponding amino acid substitution into AVRM14b (SEQ ID NO: 15). (k-1) The same procedure as in (a-1) was performed, except that oligonucleotides consisting of the sequences described in SEQ ID NO: 51 (5'-TCAAGGCGATCGTTGAAGGTCAGAATGCAC-3') and SEQ ID NO: 52 (5'-ACGATCGCCTTGAATTCATACAGACCCGGC-3') were used as PCR primers. Plasmid (expression vector-)pET-AVRM15l was obtained by performing the same procedure as in (a-2) using the PCR product obtained in (k-2)(k-1). The nucleotide sequence of (k-3)pET-AVRM15l was analyzed and confirmed using the same method as in Example 2(5).

[0093] The amino acid sequence of AVRM15l with the signal sequence and polyhistidine tag attached is shown in Sequence ID No. 29. In Sequence ID No. 29, 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 AVRM15l (corresponding to the region from positions 25 to 213 in Sequence ID No. 29), and the 214th to 219th histidine (H) is the tag sequence. Furthermore, in sequence number 29, lysine (N329K) is located at position 42, serine (Y342S) at position 55, alanine (V381A) at position 94, serine (G390S) at position 103, 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 (S476R) at position 189, threonine (S482T) at position 195, aspartic acid (N487D) at position 200, aspartic acid (N492D) at position 205, and glutamic acid (K497E) at position 210. (l)AVRM15m This protein was created by selecting V394A and introducing the corresponding amino acid substitution into AVRM14b (SEQ ID NO: 15). (l-1) The same procedure as in (a-1) was performed, except that oligonucleotides consisting of the sequences described in SEQ ID NO: 53 (5'-GCTATGCGAACGTTACCGTGGAACCGGGC-3') and SEQ ID NO: 54 (5'-ACGTTCGCATAGCCTTCGCTATGTGCATTC-3') were used as PCR primers. Plasmid (expression vector-)pET-AVRM15m was obtained by performing the same procedure as in (a-2), except that the PCR product obtained in (l-1) was used. The nucleotide sequence of (l-3)pET-AVRM15m was analyzed and confirmed using the same method as in Example 2(5).

[0094] The amino acid sequence of AVRM15m with the signal sequence and polyhistidine tag attached is shown in Sequence ID No. 30. In Sequence ID No. 30, 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 AVRM15m (corresponding to the region from positions 25 to 213 in Sequence ID No. 30), and the 214th to 219th histidine (H) is the tag sequence. Furthermore, in sequence number 30, lysine (N329K) is located at position 42, serine (Y342S) at position 55, 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 (S476R) at position 189, threonine (S482T) at position 195, aspartic acid (N487D) at position 200, aspartic acid (N492D) at position 205, and glutamic acid (K497E) at position 210. Example 12: Preparation of AVRM14b amino acid substitutions (Part 2) As transformants capable of expressing AAV-binding proteins, the transformants obtained by transforming E. coli strain BL21(DE3) with plasmids containing nine types of AAV-binding proteins obtained in Example 9 (AVRM14b amino acid substitution aggregates, specifically AVRM15b (SEQ ID NO: 19), AVRM15c (SEQ ID NO: 20), AVRM15d (SEQ ID NO: 21), AVRM15e (SEQ ID NO: 22), AVRM15f (SEQ ID NO: 23), AVRM15g (SEQ ID NO: 24), AVRM15h (SEQ ID NO: 25), AVRM15i (SEQ ID NO: 26), AVRM15j (SEQ ID NO: 27), AVRM15k (SEQ ID NO: 28), AVRM15l (SEQ ID NO: 29), and AVRM15m (SEQ ID NO: 30)), as well as one of AVRM12 (SEQ ID NO: 13), AVRM14b (SEQ ID NO: 15), and AVR29c (SEQ ID NO: 3), were used. Otherwise, the same procedure as in Examples 3(1) to (7) was followed. Example 13: Evaluation of the binding activity of AVRM14b amino acid substitutions and AAV5 by surface plasmon resonance (Part 1) (1) The concentrations of the five types of AAV-binding proteins obtained in Example 12 (AVRM12 amino acid substitution aggregates, specifically AVRM15b (SEQ ID NO: 19), AVRM15c (SEQ ID NO: 20), AVRM15d (SEQ ID NO: 21), AVRM15e (SEQ ID NO: 22), AVRM15f (SEQ ID NO: 23), AVRM15g (SEQ ID NO: 24), AVRM15h (SEQ ID NO: 25), AVRM15i (SEQ ID NO: 26), AVRM15j (SEQ ID NO: 27), AVRM15k (SEQ ID NO: 28), AVRM15l (SEQ ID NO: 29), and AVRM15m (SEQ ID NO: 30), as well as AVRM12 (SEQ ID NO: 13), AVRM14b (SEQ ID NO: 15), and AVR29c (SEQ ID NO: 3) were prepared to 5, 2.5, 1.25, 0.625, and 0.313 μmol / L. (2) Using the sample obtained in Example 4(1) as a sensor chip and the AAV-binding protein prepared in (2) as an analyte, the binding response was measured by surface plasmon resonance using a Biacore T-200. The binding activity was evaluated by comparing the maximum binding response with AVRM12 as the reference. Specifically, the maximum binding response value of each sample was divided by the maximum binding response value of AVRM12 and multiplied by 100 (named as the activity maintenance rate (%)) for comparison.

[0095] The results are shown in Tables 6 and 7. From Tables 6 and 7, it can be seen that AVRM15b, AVRM15c, AVRM15d, AVRM15e, AVRM15f, AVRM15g, AVRM15h, AVRM15i, AVRM15j, AVRM15k, AVRM15l, and AVRM15m all showed improved activity retention rates compared to AVR29c.

[0096] In particular, AVRM15b, AVRM15d, AVRM15f, AVRM15g, AVRM15h, AVRM15i, and AVRM15j showed activity retention rates of over 90%, which were equivalent to those of AVRM12. In other words, it was found that AVRM15b, AVRM15d, AVRM15f, AVRM15g, AVRM15i, and AVRM15j showed significantly improved activity retention rates compared to AVR29c. This indicates that N324H introduced into AVRM15b, Q334L introduced into AVRM15d, E335V introduced into AVRM15f, T341A introduced into AVRM15g, A330V introduced into AVRM15h, K380R introduced into AVRM15i, and I382V introduced into AVRM15j are stability-enhancing mutations that do not adversely affect binding to AAV5.

[0097] [Table 6]

[0098] [Table 7]

[0099] Example 14: Preparation of AVRM14b amino acid substitutions (Part 3) In Example 13, we selected N324H, Q334L, E335V, T341A, K380R, and I382V, which are involved in improving the stability of AAV-binding proteins and do not adversely affect the binding activity to serotype 5. By accumulating these amino acid substitutions on AVRM14b (SEQ ID NO: 15), we aimed to improve alkaline stability while maintaining the binding activity to serotype 5. Specifically, we designed and fabricated one type of AAV-binding protein as shown in (a) below. (a) AAV-binding protein (SEQ ID NO: 55, named AVRM20) obtained by introducing the amino acid substitutions N324H, Q334L, E335V, T341A, K380R, and I382V into AVRM14b. The following describes a method for producing one type of AAV-binding protein as shown in (a) above.

[0100] The target sequence was obtained from a total synthesis by FASMAC. A PCR primer set was designed to transfer the target sequence into an expression vector. Specifically, Sequence IDs 5 (Forward) and 6 (Reverse) were designed as PCR primers for amplifying the synthetic sequence, and sequence IDs 7 (Forward) and 8 (Reverse) were designed as PCR primers for amplifying the expression vector.

[0101] (a) AVRM20 This protein was created by introducing the amino acid substitutions involved in stability, N324H, Q334L, E335V, T341A, K380R, and I382V, which were identified in Example 13, into AVRM14b (SEQ ID NO: 15). (a-1) The same procedure as in Example 2(2) was performed, except that pUCFk-AVRM20, which codes for a polypeptide (SEQ ID NO: 55) containing the PKD1 domain of AVRM14b into which N324H, Q334L, E335V, T341A, K380R, and I382V were introduced, was used as a template. (a-2) The same procedure as in Examples 2(3) to (5) was followed, except that the DNA fragment amplified in Example 2(1) and (a-1) was used.

[0102] The amino acid sequence of AVRM20 with the signal sequence and polyhistidine tag attached is shown in Sequence ID No. 55. In Sequence ID No. 55, 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 AVRM20 (corresponding to the region from position 25 to 213 in Sequence ID No. 55), and the histidine (H) at positions 214 to 219 is the tag sequence. Furthermore, in Sequence ID No. 55, histidine (N324H) is ranked 37th, lysine (N329K) 42nd, leucine (Q334L) 47th, valine (E335V) 48th, alanine (T341A) 54th, serine (Y342S) 55th, arginine (K380R) 93rd, valine (I382V) 95th, serine (G390S) 103rd, glutamic acid (K399E) 112th, and glycymethyl N is located at position 114, alanine (T426A) at position 139, arginine (K455R) at position 168, 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, aspartic acid (N492D) at position 205, and glutamic acid (K497E) at position 210. Example 15: Preparation of AVRM14b amino acid substitutions (Part 4) The same procedure as in Examples 3(1) to (7) was followed, except that the transformants used were obtained by transforming E. coli strain BL21(DE3) with a plasmid containing the AAV-binding protein obtained in Example 14 (AVRM14b amino acid substitution aggregate, specifically AVRM20 (SEQ ID NO: 53)) and a polynucleotide encoding one of AVR29c (SEQ ID NO: 3), AVRM12 (SEQ ID NO: 13), or AVRM14b (SEQ ID NO: 15), as transformants capable of expressing AAV-binding proteins. Example 16: Evaluation of the binding activity of AVRM14b amino acid substitution aggregates and AAV5 by surface plasmon resonance (Part 2) (1) The concentrations of the five types of AAV-binding proteins obtained in Example 15 (AVRM12 amino acid substitution aggregate, AVRM20 (SEQ ID NO: 55), AVR29c (SEQ ID NO: 3), AVRM12 (SEQ ID NO: 13), and AVRM14b (SEQ ID NO: 15) were adjusted to 5, 2.5, 1.25, 0.625, and 0.313 μmol / L. (2) Using the sensor chip obtained in Example 4 and the AAV-binding protein prepared in (2) as the analyte, the binding response was measured by surface plasmon resonance using a Biacore T-200. The binding activity was evaluated by comparing the maximum binding response with AVRM12 as the reference. Specifically, the maximum binding response value of each sample was divided by the maximum binding response value of AVRM12 and multiplied by 100 (named as the activity maintenance rate (%)) for comparison. If the activity maintenance rate was 95% or higher, the introduced mutation was considered not to adversely affect binding to AAV5.

[0103] The results are shown in Table 8. From Table 8, it can be seen that AVRM20 has an improved activity retention rate compared to AVR29c.

[0104] [Table 8]

[0105] Example 17: Preparation of AAV-binding protein-immobilized gel A gel was prepared by introducing maleimide groups to the hydroxyl groups on the surface of a hydrophilic vinyl polymer for separation agents (Toyopal, manufactured by Tosoh Corporation) through chemical modification. To 1 g of the prepared gel, 6 mg of the proteins AVRwt, AVRm29c, AVRM9, AVRM12, AVRM14b, and AVRM20 prepared in Examples 3, 6, 9, 12, and 15, and TCEP (Tris(2-CarboxyEthyl)Phosphine) at a final concentration of 0.1 mmol / L as a reducing agent were added, and the mixture was reacted by shaking for 3 hours under conditions of pH 7.4 and 25°C. This prepared gels immobilized with AVRwt, AVRm29c, AVRM9, AVRM12, AVRM14b, and AVRM20 (named AVRwt-immobilized gel, AVRM9-immobilized gel, AVRM12-immobilized gel, AVRM14b-immobilized gel, and AVRM20-immobilized gel). Example 18: Evaluation of alkali resistance of AAV-binding protein-immobilized gel (1) 0.2 mL each of the AVRwt immobilized gel, AVRm29c immobilized gel, AVRM9 immobilized gel, AVRM12 immobilized gel, and AVRM14b immobilized gel prepared in Example 17 were packed into an empty column (φ5 mm × 20 mm, manufactured by Cytiva) to prepare an AAV adsorbent column (hereinafter also referred to as the AVRwt column, AVRm29c column, AVRM9 column, AVRM12 column, AVRM14b column, and AVRM20 column). (2) The AAV8-EGFP obtained in Example 1 was applied to the flow-through fraction in an amount sufficient to confirm that AAV8-EGFP was leaking out. The final amount of AAV8-EGFP applied per column was 6.0 × 10⁶. 13 The result was cp. After application, gradient elution was performed with 0.1M acetate buffer (pH 2.0) containing 0.5M sodium chloride and 0.01% Tween20, and 0.1M Tris-HCl buffer (pH 7.4) containing 0.5M sodium chloride and 0.01% Tween20 (hereinafter also referred to as "equilibrium solution B"). (3) Each AVR column was alkaline washed by equilibrating it for 2 minutes with a 0.1 M sodium hydroxide aqueous solution containing 1 mM calcium chloride (hereinafter also referred to as "alkaline washing solution") and then letting it stand for 13 minutes. (4) Each AVR column was equilibrated with equilibration solution B for 2 minutes and then allowed to stand for 13 minutes. (5)(3)(4) was performed any number of times, and then (2) was performed again. (6) The residual activity after alkaline washing was calculated by dividing the elution peak area obtained in (5) by the elution peak area obtained in (2).

[0106] The results are shown in Table 9. AVRwt had 0% residual activity after 10 alkaline washes, indicating low alkali resistance. On the other hand, AVRM9, AVRM12, AVRM14b, AVRM20, and AVR29c showed significantly improved alkali resistance compared to AVRwt. In particular, AVR29c, AVRM12, AVRM14b, and AVRM20 all retained approximately 90% residual activity even after 100 alkaline washes, demonstrating higher alkali resistance.

[0107] AVRM14a, AVRM14c, M15a, M15b, M15c, M15d, M15e, M15f, M15g, M15h, M15i, M15j, M15k, M15l, M15m, and M17, which have been studied so far, share more than 95% amino acid sequence identity with either AVR29c, AVRM12, AVRM14b, or AVRM20. Therefore, this result suggests that AVRM14a, AVRM14c, M15a, M15b, M15c, M15d, M15e, M15f, M15g, M15h, M15i, M15j, M15k, M15l, M15m, and M17 also possess high alkali resistance.

[0108] [Table 9]

[0109] Example 19: Measurement of static binding capacity of AAV5 on AAV-binding protein-immobilized gel (1) Approximately 10 μL each of the AVRm29c immobilized gel, AVRM9 immobilized gel, AVRM12 immobilized gel, AVRM14b immobilized gel, and AVRM20 immobilized gel prepared in Example 17 was packed into a Cosmos Spin Filter-G (manufactured by Nacalai Tesque), and two mini columns (named AVRm29c immobilized gel mini column, AVRM9 immobilized gel mini column, AVRM12 immobilized gel mini column, AVRM14b immobilized gel mini column, and AVRM20 immobilized gel mini column, respectively) were prepared. (2) Each mini-column was equilibrated with 150 mM sodium chloride and 50 mM acetate buffer (pH 6.0) (hereinafter also referred to as "Solution A"), and then the cell lysate containing AAV5-EGFP obtained in Example 1 was applied to the flow-through fraction in an amount sufficient to confirm that AAV5-EGFP was leaking out. The final amount of AAV5-EGFP applied per column was 1.0 × 10⁶ 14 It became cp. (3) To elute and recover the AAV bound to the gel, the column was washed twice with 200 μL of solution A, and each filtrate was collected along with the flow-through fraction from (2). (Hereafter also referred to as "FT") (Hereafter also referred to as "Wash 1") Then, 200 μL of 50 mM acetate buffer (pH 2.0) containing 500 mM sodium chloride (hereinafter also referred to as "solution B") was added to each mini-column, and after shaking for 5 minutes, the eluate was collected by centrifugation. (4) Add 200 μL of solution B to the mini column again and shake for 1 minute in the same manner, then centrifuge and collect 400 μL of the eluate together with the previous eluate. Furthermore, add 400 μL of 50 mM acetate buffer (pH 1.5) (hereinafter also referred to as "solution C") containing 200 μL of 500 mM sodium chloride, centrifuge, and collect the eluate. (5) The eluates collected in (3) and (4) were neutralized by adding 80 μL of 1 M Tris-HCl buffer (pH 8.5) containing 20 mM magnesium chloride to each (hereinafter referred to as "Elute 1" and "Elute 2"). (6) The number of AAV5-EGFP capsids in Elute 1 and Elute 2 obtained in (5) was quantified using TSKgel G6000PWxL (size exclusion chromatography column, manufactured by Toso Corporation). The static binding capacity (cp / mL-gel) was calculated by dividing the total number of capsids in each of Elute 1 and Elute 2 by the amount of AVR-immobilized gel used in the experiment.

[0110] The results are shown in Table 10. Compared to AVR29c, the static binding capacity of AVRM9, AVRM12, AVRM14b, and AVRM20 was more than 10 times higher. This indicates that, under static conditions, AVRM9, AVRM12, AVRM14b, and AVRM20 have improved binding activity to AAV5 compared to AVR29c.

[0111] [Table 10]

[0112] Example 20: Purification of AAV5 using an AAV-binding protein immobilized gel (Part 1) (1) 0.5 mL each of the AVRm29c immobilized gel, AVRM9 immobilized gel, AVRM12 immobilized gel, AVRM14b immobilized gel, and AVRM20 immobilized gel prepared in Example 17 were packed into an empty column (φ5 mm × 20 mm, manufactured by Cytiva) to prepare an AAV adsorbent column (hereinafter also referred to as the AVRm29c column, AVRM9 column, AVRM12 column, AVRM14b column, and AVRM20 column). (2) The column prepared in (1) was equilibrated with 20 mM Tris-HCl buffer (pH 7.4) containing 0.5 M sodium chloride, 10 mM calcium chloride, and 0.01% Tween 20 (hereinafter also referred to as "equilibrium solution C"). (3) The cell disruption solution containing AAV5-EGFP obtained in Example 1 was applied to the column, washed with equilibration solution E, and then eluted with 0.1 M acetate buffer (pH 2.0) containing 0.5 M sodium chloride and 0.01% Tween 20 (hereinafter also referred to as "elution solution A") to obtain the eluted fraction.

[0113] The chromatograms obtained using each column are shown in Figure 2. In Figure 2, the amount of cell lysate containing AAV5-EGFP applied differs between (e)AVR29c (same conditions as M20) to (f)AVRM20 and (a)AVR29c to (d)AVRM14b. (e)AVR29c (same conditions as M20) to (f)AVRM20 had 6 times more AAV capsids applied than (a)AVR29c to (d)AVRM14b. Also, (e)AVR29c (same conditions as M20) to (f)AVRM20 had 3.3 times more cell lysate applied than (a)AVR29c to (d)AVRM14b.

[0114] In Figure 2, the peak indicated by the black arrow corresponds to the peak of AAV5-EGFP in the elution fraction. Comparing the peak areas of the peaks indicated by the black arrows for (e)AVR29c (same conditions as M20) and (f)AVRM20, the peak area of ​​(f)AVRM20 was 3.7 times that of (e)AVR29c (same conditions as M20).

[0115] When using the AVR29c column, the small peak corresponding to AAV5-EGFP in the eluted fraction indicates that the AAV5-EGFP contained in the solution did not bind to the AVR29c column and was not purified. On the other hand, when using the AVRM9, AVRM12, AVRM14b, or AVRM20 columns, the peak corresponding to AAV5-EGFP in the eluted fraction was large, and most of the impurities contained in the AAV5-EGFP solution passed through the column.

[0116] This indicates that AVRM9, AVRM12, AVRM14b, and AVRM20 columns can purify the AAV5 vector contained in cell lysates. Example 21: Purification of AAV5 using an AAV-binding protein immobilized gel (Part 2) (1) 0.2 mL each of the AVRm29c immobilized gel, AVRM12 immobilized gel, and AVRM14b immobilized gel prepared in Example 17 were packed into an empty column (φ5 mm × 20 mm, manufactured by Cytiva) to prepare an AAV adsorbent column (hereinafter also referred to as the AVRm29c column, AVRM12 column, and AVRmM14b column). (2) 6 mL of the AAV5-EGFP solution obtained in Example 1 was placed in a dialysis tube and dialyzed with 1 L of 1 × PBS (pH 7.4) at 4°C for 18 hours. (3) The column prepared in (1) was equilibrated with 20 mM Tris-HCl buffer (pH 7.4) containing 0.5 M sodium chloride, 10 mM calcium chloride, and 0.01% Tween 20 (hereinafter also referred to as "equilibrium solution C"). (4) The AAV5-EGFP solution obtained in (2) was applied to the column, washed with equilibration solution E, and then eluted with 0.1 M acetate buffer (pH 1.5) containing 0.5 M sodium chloride, 0.01% Pluronic F-68, and 0.01% Tween 20 (also referred to as "elution solution B") to obtain the eluted fraction. (5) The number of AAV5-EGFP capsids contained in the AAV5-EGFP solution applied in (4), the flow-through fraction obtained in (4), and the elution fraction was measured using TSKgel G6000PW XL Quantitative analysis was performed using SEC (Size Exclusion Chromatography) with a size exclusion chromatography column (manufactured by Tosoh Corporation).

[0117] The obtained chromatogram is shown in Figure 3, and the percentage of AAV vectors that leaked into the flow-through solution immediately after application is shown in Figure 4. This percentage was calculated by dividing the AAV5-EGFP concentration of the pass-through fraction by the AAV5-EGFP concentration of the application solution.

[0118] In Figure 3, the peak indicated by the black arrow corresponds to the peak of AAV5-EGFP in the elution fraction. When using the AVR29c column, the peak corresponding to AAV5-EGFP in the elution fraction is small, indicating that the AAV vector contained in the solution was not able to bind to the AVR29c column and was not purified.

[0119] On the other hand, when using AVRM12 or AVRM14b columns, the peak corresponding to AAV5-EGFP in the eluted fraction is large, and most of the impurities in the AAV5-EGFP solution pass through the column. From this, it can be seen that AVRM12 and AVRM14b columns can purify the AAV5 vector contained in 1×PBS. Example 22 Analysis of AAV using mutants (1) A gel was prepared by introducing maleimide groups to the hydroxyl groups on the surface of a hydrophilic vinyl polymer for separation (Toyopal, manufactured by Tosoh Corporation) through chemical modification. 2 mg of AVRM12 and AVRM14b proteins prepared in Examples 9 and 12, and 0.1 mM TCEP (Tris(2-CarboxyEthyl)Phosphine) as a reducing agent were added to 1 g of the prepared gel and reacted by shaking for 3 hours at pH 7.4 and 25°C. This produced gels immobilized with AVRM12 and AVRM14b. 1.25 mL of each gel was packed into a stainless steel empty column (φ4.6 mm × 75 mm, manufactured by Tosoh Corporation) to prepare an AAV analysis column (hereinafter referred to as the AVRM12 analysis column and the AVRM14b analysis column). (2) Connect the prepared analytical column to an HPLC M40A (Shimadzu Corporation), equilibrate it with solution A having the composition shown in Table 11, and then use the VLP5 or VLP8 solution prepared in Example 1 (both with a concentration of 1.0 × 10⁻¹⁶). 14 A solution (cp / mL) was applied at a flow rate of 0.5 mL / min at a rate of 0.01 mL. Then, over 45 minutes, solution B, consisting of the composition shown in Table 11, was eluted from 0% to 100% using a gradient to obtain a chromatogram.

[0120] The results are shown in Figure 5. Both the AVRM12 and AVRM14b analytical columns were found to have sufficient binding activity not only for VLP5 but also for VLP8. Furthermore, the chromatograms obtained using both the AVRM12 and AVRM14b analytical columns showed that the retention times of the VLP5 and VLP8 peaks were different. In other words, it was confirmed that both the AVRM12 and AVRM14b analytical columns can recognize structural differences in serotypes and are suitable for analysis. Reference Example 1: Preparation of AAV-binding protein variants (1) AVR21 (1-1) A plasmid (expression vector) capable of expressing AVR21, an AAV-binding protein having the amino acid sequence described in Sequence ID No. 57, was prepared by the method described in WO2023 / 140197. (1-2) E. coli BL21 strain (DE3) was transformed with the AVR21 expression vector to prepare transformants. The transformants were then cultured. (1-3) After the culture was completed, the bacterial cells were collected by centrifugation and ultrasonically disrupted using an ultrasonic generator (Insonator 201M, manufactured by Kubota Shoji Co., Ltd.). (1-4) The supernatant after sonication was collected. The collected supernatant was applied to an open column packed with 1.5 mL of Ni Sepharose 6 Fast Flow (Cytiva), and eluted with 20 mM Tris-HCl buffer (pH 7.4) containing 0.5 M imidazole and 150 mM sodium chloride. The eluates obtained in (1-5) and (1-4) were dialyzed with 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride to prepare the AVR21 solution. (2) AVR25a (2-1) A plasmid (expression vector) capable of expressing an AAV-binding protein (named AVR25a) having the amino acid sequence described in Sequence ID No. 58 was prepared by introducing the amino acid substitutions N329K, E401G, T426A, and A461P into AVR21. (2-2) Except for transforming E. coli with the AVR25a expression vector, the same procedure as in (1-2) to (1-5) was performed to prepare the AVR25a solution. (3) AVR25c (3-1) A plasmid (expression vector) capable of expressing an AAV-binding protein (named AVR25c) having the amino acid sequence described in Sequence ID No. 59 was prepared by introducing the K467N amino acid substitution to AVR25a. (3-2) Except for transforming E. coli with the AVR25c expression vector, the same procedure as in (1-2) to (1-5) was performed to prepare the AVR25c solution. (4) AVR29c (4-1) A plasmid (expression vector) capable of expressing AVR29c having the amino acid sequence described in Sequence ID No. 3 was prepared. (4-2) Except for transforming E. coli with the AVR29c expression vector, the same procedure as in (1-2) to (1-5) was performed to prepare the AVR29c solution. Reference Example 2: Preparation of VLP2 (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. (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). (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. (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. 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. Reference Example 3: Evaluation of alkali tolerance of AAV-binding protein mutants (Part 1) (1) The binding activity of AVR21 or AVR25a prepared in Reference Example 1 to VLP2 was measured by the ELISA method shown below. (1-1) The VLP2 prepared in Reference Example 2 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. (1-2) 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 prepared AAV-binding protein was reacted with VLP2 (at 30°C for 1 hour). (1-3) 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. (1-4) 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). (2) To ensure that the measured values ​​for AVR21 and AVR25a were equivalent, the AVR21 solution and AVR25a were each diluted with pure water. (3) The AAV-binding protein solutions diluted in (2) 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 letting it stand at a constant temperature for a constant time (treatment temperature: 30°C, treatment time: 0, 30, 60, 90 minutes), while the other fraction was not subjected to the alkaline treatment. (4) The fractions after processing in (3) 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 around 6, and then the binding activity to VLP2 was measured using the ELISA method described in (1-1) to (1-4).

[0121] 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.

[0122] The results are shown in Table 12. It was found that AVR25a has significantly improved alkali resistance compared to AVR21. Note that "Start" in Tables 12 to 14 indicates that no alkali treatment was performed.

[0123] [Table 11]

[0124] Reference Example 4: Evaluation of alkali tolerance of AAV-binding protein mutants (Part 2) The same procedure as in Reference Example 3 was followed for AVR25a or AVR25c prepared in Reference Example 1, except that alkali resistance evaluation was performed.

[0125] The results are shown in Table 13. It was found that AVR25c has significantly improved alkali resistance compared to AVR25a.

[0126] [Table 12]

[0127] Reference Example 5: Evaluation of alkali tolerance of AAV-binding protein mutants (Part 3) (1) The concentrations of the AVR25c solution and AVR29c solution prepared in Reference Example 1 were measured using NanoDrop OneC (Thermo Fisher Scientific), and then each solution was diluted with 20 mM Tris-HCl buffer (pH 7.4) to a protein concentration of 5 μg / mL. (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, 45 minutes).

[0128] (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 Reference Examples (1-1) to (1-4).

[0129] (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.

[0130] The results are shown in Table 14. It was found that AVR29c has significantly improved alkali resistance compared to AVR25c. In other words, it was found that AVR29c has significantly improved alkali resistance compared to AVR21 disclosed in WO2023 / 140197.

[0131] Table 13

[0132] Table 14

Claims

1. AAV-binding protein selected from any of the following (i) through (iii): (i) AAV-binding proteins containing the amino acid sequence from the 25th serine (S) to the 213th aspartic acid (D) among the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NOs: 13 to 30, and SEQ ID NO: 55; (ii) An AAV-binding protein having an amino acid sequence from the 25th serine (S) to the 213th aspartic acid (D) among the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 13 to SEQ ID NO: 30, and SEQ ID NO: 55, 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 (1) to (29) shown below, provided that the amino acid substitutions (2) to (9), (11), (13) to (29) shown below are maintained, and having binding activity for at least serotype 5; (1) The valine (V) at position 317 in SEQ ID NO: 1 (position 30 in SEQ ID NO: 2) is replaced with aspartic acid (D). (2) The asparagine (N) at position 324 in SEQ ID NO: 1 (position 37 in SEQ ID NO: 2) is replaced with histidine (H). (3) The valine (V) at position 326 in SEQ ID NO: 1 (position 39 in SEQ ID NO: 2) is replaced with alanine (A). (4) The asparagine (N) at position 329 in SEQ ID NO: 1 (position 42 in SEQ ID NO: 2) is replaced with lysine (K). (5) The alanine (A) at position 330 in SEQ ID NO: 1 (position 43 in SEQ ID NO: 2) is replaced with valine (V). (6) The glutamine (Q) at position 334 in SEQ ID NO: 1 (position 47 in SEQ ID NO: 2) is replaced with leucine (L). (7) The glutamic acid (E) at position 335 in SEQ ID NO: 1 (position 48 in SEQ ID NO: 2) is replaced with valine (V). (8) The 341st threonine (T) in SEQ ID NO: 1 (54th in SEQ ID NO: 2) is replaced with alanine (A). (9) The tyrosine (Y) at position 342 in SEQ ID NO: 1 (position 55 in SEQ ID NO: 2) is replaced with serine (S). (10) Lysine (K) at position 362 in SEQ ID NO: 1 (position 75 in SEQ ID NO: 2) is replaced with glutamic acid (E). (11) The isoleucine (I) at position 366 in SEQ ID NO: 1 (position 79 in SEQ ID NO: 2) is replaced with phenylalanine (F). (12) The lysine (K) at position 371 in SEQ ID NO: 1 (position 84 in SEQ ID NO: 2) is replaced with asparagine (N). (13) Phenylalanine (F) at position 379 in SEQ ID NO: 1 (position 92 in SEQ ID NO: 2) is replaced with tyrosine (Y). (14) Lysine (K) at position 380 in SEQ ID NO: 1 (position 93 in SEQ ID NO: 2) is replaced with arginine (R). (15) The valine (V) at position 381 in SEQ ID NO: 1 (position 94 in SEQ ID NO: 2) is replaced with alanine (A). (16) The isoleucine (I) at position 382 in SEQ ID NO: 1 (position 95 in SEQ ID NO: 2) is replaced with valine (V). (17) The glycine (G) at position 390 in SEQ ID NO: 1 (position 103 in SEQ ID NO: 2) is replaced with serine (S). (18) The valine (V) at position 394 in SEQ ID NO: 1 (position 107 in SEQ ID NO: 2) is replaced with alanine (A). (19) The lysine (K) at position 399 in SEQ ID NO: 1 (position 112 in SEQ ID NO: 2) is replaced with glutamic acid (E). (20) The glutamic acid (E) at position 401 in SEQ ID NO: 1 (position 114 in SEQ ID NO: 2) is replaced with glycine (G). (21) The threonine (T) at position 426 in sequence number 1 (position 139 in sequence number 2) is replaced with alanine (A). (22) The lysine (K) at position 455 in SEQ ID NO: 1 (position 168 in SEQ ID NO: 2) is replaced with arginine (R). (23) The alanine (A) at position 461 in SEQ ID NO: 1 (position 174 in SEQ ID NO: 2) is replaced with proline (P). (24) The lysine (K) at position 467 in SEQ ID NO: 1 (position 180 in SEQ ID NO: 2) is replaced with asparagine (N). (25) The serine (S) at position 476 in SEQ ID NO: 1 (position 189 in SEQ ID NO: 2) is replaced with arginine (R). (26) The serine (S) at position 482 in SEQ ID NO: 1 (position 195 in SEQ ID NO: 2) is replaced with threonine (T). (27) The asparagine (N) at position 487 in SEQ ID NO: 1 (position 200 in SEQ ID NO: 2) is replaced with aspartic acid (D). (28) The asparagine (N) at position 492 in SEQ ID NO: 1 (position 205 in SEQ ID NO: 2) is replaced with aspartic acid (D). (29) The lysine (K) at position 497 in SEQ ID NO: 1 (position 210 in SEQ ID NO: 2) is replaced with glutamic acid (E). (iii) An AAV-binding protein having an amino acid sequence that is 70% or more identical to the amino acid sequence shown from the 25th serine (S) to the 213th aspartic acid (D) among the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NOs: 13 to 30, and SEQ ID NO: 55, and in which the above amino acid substitutions (2) to (9), (11), (13) to (29) are maintained, except for the amino acid sequence shown in SEQ ID NO: 3, and having binding activity for at least serotype 5.

2. AAV-binding protein according to claim 1, selected from any of (iv) to (vi) below: (iv) AAV-binding protein containing the amino acid sequence from the 25th serine (S) to the 213th aspartic acid (D) among the amino acid sequences shown in SEQ ID NOs. 13, 15, 19, 21, and 23-27; (v) An AAV-binding protein having an amino acid sequence from the 25th serine (S) to the 213th aspartic acid (D) among the amino acid sequences shown in SEQ ID NOs. 13, 15, 19, 21, and 23 to 27, further comprising one or more substitutions, deletions, insertions, and additions of one or more amino acid residues at one or more positions, provided that the above amino acid substitutions (2), (4) to (9), (14), (16) to (17), (19) to (29) are maintained, and having binding activity for at least serotype 5; (vi) An AAV-binding protein having an amino acid sequence that is 70% or more identical to the amino acid sequence shown from the 25th serine (S) to the 213th aspartic acid (D) among the amino acid sequences shown in SEQ ID NOs. 13, SEQ ID NOs. 15, SEQ ID NOs. 19, SEQ ID NOs. 21, and SEQ ID NOs. 23 to 27, and having an amino acid sequence in which the above amino acid substitutions (4), (9), (17), (19) to (29) are maintained, excluding the amino acid sequence shown in SEQ ID NO. 3, and having binding activity to at least serotype 5.

3. AAV-binding protein according to claim 1, selected from any of (vii) to (ix) below: (vii) AAV-binding protein containing the amino acid sequence from the 25th serine (S) to the 213th aspartic acid (D) among the amino acid sequences shown in SEQ ID NOs. 13 and 15; (viiii) An AAV-binding protein having an amino acid sequence from the 25th serine (S) to the 213th aspartic acid (D) of the amino acid sequences shown in SEQ ID NOs. 13 and SEQ ID NOs. 15, further comprising one or more substitutions, deletions, insertions, and additions of one or more amino acid residues at one or more positions, provided that the above amino acid substitutions (4), (9), (17), (19) to (29) are maintained, and having binding activity for at least serotype 5; (ix) An AAV-binding protein having an amino acid sequence that is 70% or more identical to the amino acid sequence shown from the 25th serine (S) to the 213th aspartic acid (D) among the amino acid sequences shown in SEQ ID NO: 13 and SEQ ID NO: 15, wherein the above amino acid substitutions (4), (9), (17), (19) to (29) are maintained, except for the amino acid sequence shown in SEQ ID NO: 3, and which has binding activity to at least serotype 5.

4. A polynucleotide encoding an AAV-binding protein according to any one of claims 1 to 3.

5. An expression vector comprising the polynucleotide described in claim 4.

6. A transformant obtained by transforming Escherichia coli with the expression vector described in claim 5.

7. A method for producing an AAV-binding protein, comprising the steps of: culturing the transformant described in claim 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 claims 1 to 3 immobilized on the carrier.

9. A column comprising the AAV adsorbent described in claim 8.

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

11. A method for purifying or analyzing serotype 5, comprising the steps of: adding a solution containing serotype 5 to the column described in claim 9 to adsorb the serotype 5 onto the adsorbent; and eluting the serotype 5 adsorbed onto the adsorbent using an eluent.

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