Improved adeno-associated virus binding protein, method for producing said protein, and adeno-associated virus adsorbent using said protein

Abstract

The present invention provides an improved adeno-associated virus (AAV)-binding protein having enhanced stability, in particular, heat stability, acid stability and alkali stability, of an AAV-binding protein, a method for producing the improved AAV-binding protein, and an AAV adsorber using the same.

Description

[Technical field]

[0001] The present invention relates to a protein that has binding affinity to an adeno-associated virus (AAV). More specifically, the present invention relates to an improved AAV-binding protein that has higher stability against heat, acid, and alkali than the wild-type AAV, a method for producing the protein, and an AAV adsorbent using the protein. [Background technology]

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

[0003] In nature, AAV lacks the ability to replicate autonomously, and its replication depends on helper viruses such as adenoviruses and herpes viruses. When the helper virus is present, the AAV genome replicates in the host cell, forming complete AAV particles containing the AAV genome, and the AAV particles are released from the host cell. On the other hand, when the helper virus is not present, the AAV genome is maintained in an episome or integrated into the host chromosome (latent state).

[0004] AAV is capable of infecting the cells of a wide range of species, including humans, and can also infect non-dividing cells that have completed differentiation, such as blood cells, muscles, and nerve cells. It is not pathogenic to humans, so there is little risk of side effects. In addition, the viral particles are physicochemically stable. For these reasons, AAV is attracting attention as a potential vector for gene transfer to treat congenital genetic diseases.

[0005] A recombinant AAV vector (hereinafter, simply referred to as AAV vector) is usually produced by introducing a nucleic acid encoding an element essential for AAV particle formation into a cell to produce a cell capable of producing AAV (hereinafter, also referred to as AAV-producing cell), and culturing the cell to express the element essential for AAV particle formation. In general, AAV is produced by introducing three types of plasmids into a cell: a plasmid (vector plasmid) for supplying a recombinant AAV genome carrying a polynucleotide encoding a protein to be expressed, leaving the ITR (Inverted Terminal Repeat) sequences at both ends; a plasmid (packaging plasmid) for supplying Rep proteins and capsid proteins (coat proteins VP1, VP2, and VP3); and a plasmid (helper plasmid) for supplying only the element essential for AAV particle formation among the elements derived from adenovirus. As other methods, production is also performed using a method using a host cell in which an element essential for AAV particle formation has been introduced in advance, or a method using insect cells (Sf9 cells, etc.).

[0006] In gene transfer using AAV vectors, the AAV vector must enter cells. It is known that the first step of AAV entry into cells is binding to the cell surface and receptors, and this binding involves heparan sulfate on the cell surface, integrins, and receptors such as FGF (fibroblast growth factor) receptors and HGF (hepatocyte growth factor) receptors. In general, it is known that the receptors that bind when entering cells differ depending on the serotype of the AAV vector.

[0007] In addition, a receptor called KIAA0319L (AAVR) has been reported as a universal receptor (Non-Patent Document 1). This AAVR is composed of five extracellular domains, a transmembrane domain, and an intracellular domain, and it has been reported that the domain 2 (PKD2) of the extracellular domain is mainly important for binding to AAV (Non-Patent Document 2). AAVR can bind to AAV vectors, so it can be a ligand for adsorbing AAV. However, since AAVR is a protein derived from humans, there was a problem that it was not very stable for industrial use. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Pillay S.,et.al.,Nature,530,108-112,2016 [Non-Patent Document 2] Zhang. R.,et.al.,Nat.Microbiol.,4,675-682,2019 Summary of the Invention [Problem to be solved by the invention]

[0009] An object of the present invention is to provide an improved AAV binding protein having improved stability, particularly against heat, acid and alkali, a method for producing said protein, and an AAV adsorbent using said protein. [Means for solving the problem]

[0010] As a result of intensive research aimed at solving the above problems, the present inventors identified amino acid residues involved in improving the stability of AAV binding proteins and found that mutants in which these amino acid residues were replaced with other amino acid residues have excellent stability against heat, acid and alkali, thereby completing the present invention.

[0011] That is, the present application includes the following aspects [1] to

[10] : [1] An AAV binding protein selected from any one of the following (i) to (iii): (i) an AAV binding protein comprising at least the amino acid residues from serine at position 312 to aspartic acid at position 500 in the amino acid sequence set forth in SEQ ID NO:1, with the proviso that at least one of the following amino acid substitutions (1) to (137) has occurred in the amino acid residues at positions 312 to 500; (1) Substitution of glycine at position 390 of SEQ ID NO:1 with serine (2) Substitution of isoleucine at position 319 of SEQ ID NO:1 with phenylalanine (3) Substitution of leucine at position 321 of SEQ ID NO:1 with proline (4) Proline at position 322 of SEQ ID NO: 1 is replaced with leucine (5) Substitution of asparagine at position 324 of SEQ ID NO: 1 with aspartic acid or serine (6) Substitution of glutamine at position 327 of SEQ ID NO:1 with arginine (7) Substitution of asparagine at position 329 of SEQ ID NO: 1 with aspartic acid or tyrosine (8) Alanine at position 330 of SEQ ID NO: 1 is replaced with glycine (9) Substitution of tyrosine at position 331 of SEQ ID NO:1 with cysteine (10) Substitution of valine at position 332 of SEQ ID NO:1 with glutamine (11) Leucine at position 333 of SEQ ID NO: 1 is replaced with valine or proline (12) Substitution of glutamine at position 334 of SEQ ID NO:1 with arginine (13) Proline at position 337 of SEQ ID NO: 1 is replaced with leucine (14) Substitution of lysine at position 338 of SEQ ID NO:1 with arginine or glutamic acid (15) Substitution of glutamic acid at position 340 of SEQ ID NO:1 with glycine (16) Substitution of threonine at position 341 of SEQ ID NO:1 with alanine (17) Tyrosine at position 342 of SEQ ID NO: 1 is substituted with cysteine, histidine, or asparagine (18) Substitution of threonine at position 343 of SEQ ID NO:1 with methionine (19) Tyrosine at position 344 of SEQ ID NO:1 is substituted with histidine (20) Substitution of aspartic acid at position 345 of SEQ ID NO:1 with asparagine (21) Tryptophan at position 346 of SEQ ID NO:1 is substituted with leucine or arginine (22) Substitution of glutamine at position 347 of SEQ ID NO:1 with leucine or proline (23) Substitution of leucine at position 348 of SEQ ID NO:1 with proline (24) Substitution of isoleucine at position 349 of SEQ ID NO:1 with threonine (25) Substitution of threonine at position 350 of SEQ ID NO:1 with methionine (26) Substitution of histidine at position 351 of SEQ ID NO:1 with leucine (27) Proline at position 352 of SEQ ID NO:1 is replaced with leucine (28) Substitution of arginine at position 353 of SEQ ID NO:1 with cysteine (29) Aspartic acid at position 354 of SEQ ID NO:1 is replaced with glycine (30) Tyrosine at position 355 of SEQ ID NO: 1 is substituted with histidine, asparagine, or cysteine (31) Substitution of serine at position 356 of SEQ ID NO:1 with cysteine (32) Substitution of glycine at position 357 of SEQ ID NO:1 with cysteine (33) Histidine at position 363 of SEQ ID NO:1 is substituted with arginine or leucine (34) Serine at position 364 of SEQ ID NO:1 is replaced with proline (35) Substitution of glutamine at position 365 of SEQ ID NO:1 with arginine (36) Substitution of isoleucine at position 366 of SEQ ID NO:1 with threonine (37) Substitution of leucine at position 367 of SEQ ID NO:1 with proline (38) Substitution of lysine at position 368 of SEQ ID NO:1 with arginine (39) Substitution of leucine at position 369 of SEQ ID NO:1 with glutamine or proline (40) Substitution of serine at position 370 of SEQ ID NO:1 with alanine (41) Lysine at position 371 of SEQ ID NO:1 is replaced with glutamic acid (42) Substitution of threonine at position 373 of SEQ ID NO:1 with alanine (43) Substitution of leucine at position 376 of SEQ ID NO:1 with proline (44) Tyrosine at position 377 of SEQ ID NO:1 is replaced with cysteine (45) Substitution of phenylalanine at position 379 of SEQ ID NO:1 with serine (46) Valine at position 381 of SEQ ID NO:1 is substituted with alanine (47) Glutamic acid at position 384 of SEQ ID NO:1 is replaced with glycine (48) Substitution of asparagine at position 387 of SEQ ID NO:1 with serine (49) Histidine at position 389 of SEQ ID NO:1 is substituted with glutamine, leucine, or arginine (50) Substitution of glutamic acid at position 391 of SEQ ID NO:1 with lysine (51) Tyrosine at position 393 of SEQ ID NO:1 is replaced with cysteine (52) Valine at position 396 of SEQ ID NO:1 is substituted with alanine (53) Substitution of lysine at position 399 of SEQ ID NO:1 with glutamic acid or arginine (54) Substitution of arginine at position 406 of SEQ ID NO:1 with serine (55) Valine at position 412 of SEQ ID NO:1 is substituted with alanine (56) Substitution of glutamine at position 415 of SEQ ID NO:1 with leucine (57) Substitution of phenylalanine at position 416 of SEQ ID NO:1 with serine (58) Substitution of glutamine at position 441 of SEQ ID NO:1 with leucine (59) Tyrosine at position 442 of SEQ ID NO:1 is replaced with phenylalanine (60) Substitution of lysine at position 448 of SEQ ID NO:1 with arginine (61) Substitution of glutamic acid at position 453 of SEQ ID NO:1 with glycine (62) Substitution of lysine at position 455 of SEQ ID NO:1 with arginine (63) Substitution of glutamic acid at position 458 of SEQ ID NO:1 with glycine (64) Substitution of alanine at position 461 of SEQ ID NO:1 with serine (65) Serine at position 476 of SEQ ID NO:1 is substituted with arginine (66) Substitution of leucine at position 477 of SEQ ID NO:1 with proline (67) Asparagine at position 487 of SEQ ID NO:1 is substituted with aspartic acid (68) Asparagine at position 492 of SEQ ID NO:1 is substituted with aspartic acid (69) Substitution of isoleucine at position 319 of SEQ ID NO:1 with asparagine or serine (70) Substitution of threonine at position 320 of SEQ ID NO:1 with isoleucine (71) Lysine at position 323 of SEQ ID NO:1 is replaced with glutamic acid (72) Substitution of leucine at position 328 of SEQ ID NO:1 with glutamine or proline (73) Tyrosine at position 331 of SEQ ID NO:1 is replaced with histidine (74) Valine at position 332 of SEQ ID NO:1 is substituted with alanine or glutamic acid (75) Proline at position 336 of SEQ ID NO:1 is replaced with glutamine (76) Proline at position 337 of SEQ ID NO:1 is replaced with glutamine (77) Substitution of lysine at position 338 of SEQ ID NO:1 with asparagine (78) Tyrosine at position 342 of SEQ ID NO:1 is replaced with arginine (79) Substitution of threonine at position 350 of SEQ ID NO:1 with serine (80) Isoleucine at position 366 of SEQ ID NO:1 is replaced with phenylalanine (81) Valine at position 383 of SEQ ID NO:1 is substituted with alanine (82) Substitution of glutamine at position 386 of SEQ ID NO:1 with arginine (83) Substitution of histidine at position 389 of SEQ ID NO:1 with aspartic acid (84) Glycine at position 392 of SEQ ID NO:1 is replaced with cysteine (85) Valine at position 394 of SEQ ID NO:1 is substituted with alanine (86) Isoleucine at position 409 of SEQ ID NO:1 is replaced with valine (87) Serine at position 476 of SEQ ID NO:1 is replaced with glycine (88) Substitution of threonine at position 490 of SEQ ID NO:1 with serine (89) Serine at position 312 of SEQ ID NO:1 is replaced with proline (90) Substitution of alanine at position 313 of SEQ ID NO:1 with serine (91) Valine at position 317 of SEQ ID NO:1 is replaced with aspartic acid (92) Substitution of glutamine at position 318 of SEQ ID NO:1 with proline (93) Substitution of threonine at position 320 of SEQ ID NO:1 with alanine (94) Lysine at position 323 of SEQ ID NO:1 is substituted with arginine (95) Valine at position 326 of SEQ ID NO:1 is substituted with alanine or glutamic acid (96) Substitution of glutamine at position 327 of SEQ ID NO:1 with histidine or leucine (97) Asparagine at position 329 of SEQ ID NO:1 is substituted with histidine or isoleucine (98) Valine at position 332 of SEQ ID NO:1 is substituted with alanine (99) Glutamic acid at position 335 of SEQ ID NO:1 is substituted with glycine or valine (100) Glutamic acid at position 340 of SEQ ID NO:1 is replaced with Valine (101) Substitution of threonine at position 341 of SEQ ID NO:1 with proline (102) Substitution of threonine at position 343 of SEQ ID NO:1 with serine (103) Tyrosine at position 344 of SEQ ID NO:1 is replaced with phenylalanine (104) Tryptophan at position 346 of SEQ ID NO:1 is replaced with cysteine (105) Substitution of methionine at position 359 of SEQ ID NO:1 with leucine (106) Substitution of glutamic acid at position 360 of SEQ ID NO:1 with lysine or valine (107) Glycine at position 361 of SEQ ID NO:1 is replaced with cysteine (108) Lysine at position 362 of SEQ ID NO:1 is substituted with glutamic acid, asparagine, or glycine (109) Serine at position 364 of SEQ ID NO:1 is replaced with Leucine (110) Substitution of lysine at position 371 of SEQ ID NO:1 with asparagine or aspartic acid (111) Leucine at position 372 of SEQ ID NO:1 is replaced with proline or glutamine (112) Proline at position 374 of SEQ ID NO:1 is replaced with leucine (113) Glutamic acid at position 378 of SEQ ID NO:1 is substituted with glycine or valine (114) Substitution of phenylalanine at position 379 of SEQ ID NO:1 with tyrosine or cysteine (115) Lysine at position 380 of SEQ ID NO:1 is replaced with glutamic acid (116) Valine at position 381 of SEQ ID NO:1 is replaced with aspartic acid (117) Isoleucine at position 382 of SEQ ID NO:1 is replaced with valine (118) Glutamic acid at position 384 of SEQ ID NO:1 is replaced with Valine (119) Valine at position 394 of SEQ ID NO:1 is substituted with aspartic acid or isoleucine (120) Asparagine at position 395 of SEQ ID NO:1 is substituted with serine (121) Substitution of threonine at position 397 of SEQ ID NO:1 with serine (122) Glutamic acid at position 401 of SEQ ID NO:1 is replaced with Valine (123) Substitution of arginine at position 403 of SEQ ID NO:1 with histidine (124) Arginine at position 406 of SEQ ID NO:1 is substituted with histidine (125) Substitution of glutamine at position 415 of SEQ ID NO:1 with arginine (126) Substitution of threonine at position 426 of SEQ ID NO:1 with alanine (127) Substitution of glutamine at position 432 of SEQ ID NO:1 with leucine or arginine (128) Substitution of glutamine at position 441 of SEQ ID NO:1 with arginine (129) Histidine at position 443 of SEQ ID NO:1 is substituted with leucine (130) Lysine at position 448 of SEQ ID NO:1 is replaced with glutamic acid (131) Isoleucine at position 456 of SEQ ID NO:1 is replaced with valine (132) Substitution of aspartic acid at position 483 of SEQ ID NO:1 with asparagine (133) Serine at position 488 of SEQ ID NO:1 is replaced with Leucine (134) The valine at position 499 of SEQ ID NO: 1 is substituted with glutamic acid or isoleucine. (ii) An AAV binding protein comprising at least the amino acid residues from serine at position 312 to aspartic acid at position 500 in the amino acid sequence set forth in SEQ ID NO:1, wherein at least one of the amino acid substitutions selected from (1) to (134) has occurred in the amino acid residues at positions 312 to 500, and further comprising one or more substitutions, deletions, insertions, and additions of one or more amino acid residues at one or more positions in addition to the amino acid substitutions selected from (1) to (134), and having AAV-binding activity. (iii) An AAV binding protein comprising an amino acid sequence from the serine at position 312 to the aspartic acid at position 500 of the amino acid sequence set forth in SEQ ID NO: 1, the amino acid sequence having 70% or more homology to the entire amino acid sequence in which at least one of the amino acid substitutions (1) to (134) has occurred, the amino acid sequence in which at least one of the amino acid substitutions remains, and the AAV binding protein has AAV-binding activity.

[0012] [2] The AAV binding protein according to [1], selected from any one of the following (iv) to (vi): (iv) an AAV binding protein that contains at least the amino acid residues from serine at position 312 to aspartic acid at position 500 of the amino acid sequence set forth in SEQ ID NO:1, provided that at least the following amino acid substitution (1) has occurred in the amino acid residues at positions 312 to 500: (1) Substitution of glycine at position 390 of SEQ ID NO:1 with serine (v) An AAV binding protein comprising at least the amino acid residues from serine at position 312 to aspartic acid at position 500 in the amino acid sequence set forth in SEQ ID NO:1, wherein at least the amino acid substitutions described in (1) above have occurred in the amino acid residues at positions 312 to 500, and further comprising one or more substitutions, deletions, insertions, and additions of one or more amino acid residues at one or more positions in addition to the amino acid substitutions described in (1) above, and having AAV-binding activity. (vi) An AAV binding protein comprising an amino acid sequence that has 70% or more homology to the entire amino acid sequence from the serine at position 312 to the aspartic acid at position 500 of the amino acid sequence set forth in SEQ ID NO:1 in which at least the amino acid substitution of (1) has occurred, and in which the amino acid substitution of (1) remains, and which has AAV-binding activity.

[0013] [3] The AAV binding protein according to [2], in which at least any one of the amino acid substitutions shown in (A) to (K) below has occurred; (A) Glycine at position 390 of SEQ ID NO: 1 is replaced with serine, and tyrosine at position 342 of SEQ ID NO: 1 is replaced with cysteine (B) Glycine at position 390 of SEQ ID NO: 1 is replaced with serine, tyrosine at position 342 of SEQ ID NO: 1 is replaced with cysteine, and serine at position 476 of SEQ ID NO: 1 is replaced with arginine (C) Glycine at position 390 of SEQ ID NO: 1 is replaced with serine, isoleucine at position 319 of SEQ ID NO: 1 is replaced with phenylalanine, tyrosine at position 342 of SEQ ID NO: 1 is replaced with cysteine, and serine at position 476 of SEQ ID NO: 1 is replaced with arginine (D) Glycine at position 390 of SEQ ID NO: 1 is replaced with serine, tyrosine at position 342 of SEQ ID NO: 1 is replaced with cysteine, serine at position 476 of SEQ ID NO: 1 is replaced with arginine, and asparagine at position 487 of SEQ ID NO: 1 is replaced with aspartic acid (E) Glycine at position 390 of SEQ ID NO:1 is replaced with serine, isoleucine at position 319 of SEQ ID NO:1 is replaced with phenylalanine, lysine at position 323 of SEQ ID NO:1 is replaced with glutamic acid, tyrosine at position 342 of SEQ ID NO:1 is replaced with cysteine, and serine at position 476 of SEQ ID NO:1 is replaced with arginine (F) Glycine at position 390 of SEQ ID NO:1 is replaced with serine, tyrosine at position 342 of SEQ ID NO:1 is replaced with cysteine, lysine at position 399 of SEQ ID NO:1 is replaced with glutamic acid, serine at position 476 of SEQ ID NO:1 is replaced with arginine, and asparagine at position 487 of SEQ ID NO:1 is replaced with aspartic acid (G) Glycine at position 390 of SEQ ID NO:1 is replaced with serine, asparagine at position 324 of SEQ ID NO:1 is replaced with aspartic acid, tyrosine at position 342 of SEQ ID NO:1 is replaced with cysteine, lysine at position 362 of SEQ ID NO:1 is replaced with glutamic acid, lysine at position 399 of SEQ ID NO:1 is replaced with glutamic acid, serine at position 476 of SEQ ID NO:1 is replaced with arginine, and asparagine at position 487 of SEQ ID NO:1 is replaced with aspartic acid. (H) Glycine at position 390 of SEQ ID NO:1 is replaced with serine, valine at position 317 of SEQ ID NO:1 is replaced with aspartic acid, tyrosine at position 342 of SEQ ID NO:1 is replaced with cysteine, lysine at position 371 of SEQ ID NO:1 is replaced with asparagine, lysine at position 399 of SEQ ID NO:1 is replaced with glutamic acid, serine at position 476 of SEQ ID NO:1 is replaced with arginine, and asparagine at position 487 of SEQ ID NO:1 is replaced with aspartic acid. (I) Glycine at position 390 of SEQ ID NO:1 is replaced with serine, valine at position 317 of SEQ ID NO:1 is replaced with aspartic acid, asparagine at position 324 of SEQ ID NO:1 is replaced with aspartic acid, tyrosine at position 342 of SEQ ID NO:1 is replaced with cysteine, lysine at position 362 of SEQ ID NO:1 is replaced with glutamic acid, lysine at position 399 of SEQ ID NO:1 is replaced with glutamic acid, serine at position 476 of SEQ ID NO:1 is replaced with arginine, and asparagine at position 487 of SEQ ID NO:1 is replaced with aspartic acid. (J) Glycine at position 390 of SEQ ID NO:1 is replaced with serine, valine at position 317 of SEQ ID NO:1 is replaced with aspartic acid, tyrosine at position 342 of SEQ ID NO:1 is replaced with cysteine, lysine at position 362 of SEQ ID NO:1 is replaced with glutamic acid, lysine at position 371 of SEQ ID NO:1 is replaced with asparagine, lysine at position 399 of SEQ ID NO:1 is replaced with glutamic acid, serine at position 476 of SEQ ID NO:1 is replaced with arginine, and asparagine at position 487 of SEQ ID NO:1 is replaced with aspartic acid. (K) Glycine at position 390 of SEQ ID NO:1 is replaced with serine, valine at position 317 of SEQ ID NO:1 is replaced with aspartic acid, tyrosine at position 342 of SEQ ID NO:1 is replaced with serine, lysine at position 362 of SEQ ID NO:1 is replaced with glutamic acid, lysine at position 371 of SEQ ID NO:1 is replaced with asparagine, lysine at position 381 of SEQ ID NO:1 is replaced with alanine, isoleucine at position 382 of SEQ ID NO:1 is replaced with valine, glycine at position 390 of SEQ ID NO:1 is replaced with serine, lysine at position 399 of SEQ ID NO:1 is replaced with glutamic acid, serine at position 476 of SEQ ID NO:1 is replaced with arginine, and asparagine at position 487 of SEQ ID NO:1 is replaced with aspartic acid.

[0014] [4] The AAV binding protein according to [3], selected from any one of (vii) to (ix) below: (vii) an AAV binding protein comprising at least the amino acid residues from serine at position 25 to aspartic acid at position 213 in the amino acid sequence set forth in any one of SEQ ID NOs: 18, 32, 34, 38, 42, 46, 53, 55, 59, 63, 69, and 76; (viii) an AAV binding protein comprising at least the amino acid residues from serine at position 25 to aspartic acid at position 213 of the amino acid sequence set forth in any one of SEQ ID NOs: 18, 32, 34, 38, 42, 46, 53, 55, 59, 63, 69, and 76, provided that the amino acid residues from position 25 to position 213 further include any one or more substitutions, deletions, insertions, and additions of one or several amino acid residues at one or several positions in addition to the amino acid substitutions contained in the amino acid sequence, and having AAV binding activity; (ix) An AAV binding protein comprising at least the amino acid residues from serine at position 25 to aspartic acid at position 213 of the amino acid sequence set forth in any one of SEQ ID NOs: 18, 32, 34, 38, 42, 46, 53, 55, 59, 63, 69, and 76, wherein the AAV binding protein has 70% or more identity to the amino acid sequence from the 25th to the 213th amino acid residues, and wherein the amino acid substitutions in the amino acid sequence remain, and wherein the AAV binding activity is maintained.

[0015] [5] A polynucleotide encoding an AAV binding protein according to any one of [1] to [4].

[0016] [6] An expression vector comprising the polynucleotide described in [5].

[0017] [7] A transformant obtained by transforming a host with the expression vector described in [6].

[0018] [8] The transformant according to [7], wherein the host is Escherichia coli.

[0019] [9] A method for producing an AAV-binding protein, comprising the steps of expressing the AAV-binding protein by culturing the transformant described in [7] or [8], and recovering the expressed AAV-binding protein from the resulting culture.

[0020]

[10] An AAV adsorbent comprising an insoluble carrier and an AAV binding protein described in any one of [1] to [3] immobilized on the carrier.

[0021]

[11] A column containing the AAV adsorbent described in

[10] .

[12] A method for purifying AAV, comprising the steps of adding a solution containing AAV to the column described in

[11] to adsorb the AAV to the adsorbent, and eluting the AAV adsorbed to the adsorbent using an elution solution.

[0022] The present invention will be described in detail below. However, the present invention is not limited to the following embodiments, and can be embodied in any form without departing from the spirit of the present invention.

[0023] All patent publications, published patent applications, non-patent publications, and the like cited in this specification are hereby incorporated by reference in their entirety for all purposes.

[0024] The AAV-binding protein described herein is a protein that contains at least the amino acid residues from serine (Ser) at position 312 to aspartic acid (Asp) at position 500, which are regions corresponding to extracellular domain 1 (PKD1) and domain 2 (PKD2) in the amino acid sequence of KIAA0319L (official database: UniProt, accession number: Q8IZA0) described in SEQ ID NO: 1, and in which an amino acid substitution has occurred at a specific position in the amino acid residues from position 312 to position 500. Thus, the AAV-binding protein of the present invention may contain all or a part of other extracellular domain domains (domain 3 (PKD3), domain 4 (PKD4) and domain 5 (PKD5)) on the C-terminal side of the protein, may contain all or a part of a signal sequence or a cysteine-rich region such as the MANSC (Motif At N terminus with Seven Cysteines) domain on the N-terminal side of PKD1, or may contain all or a part of a transmembrane region and an intracellular region on the N-terminal and / or C-terminal side of the extracellular region.

[0025] Specifically, the amino acid substitutions at the specific positions are, in the amino acid sequence of SEQ ID NO: 1, Ile319Phe (this notation indicates that the 319th isoleucine in SEQ ID NO: 1 is substituted with phenylalanine, the same applies below), Leu321Pro, Pro322Leu, Asn324Asp, Asn324Ser, Gln327Arg, Asn329Asp, Asn329Tyr, Ala330Gly, Tyr331Cys, Val332Gln, Leu333Val, Leu333Pro, Gln334Arg, Pro337Leu, Lys338Arg, Lys338Glu, Glu340Gly, Thr341Ala, Tyr342Cys, Tyr342His, Tyr342Asn, Th r343Met, Tyr344His, Asp345Asn, Trp346Leu, Trp346Arg, Gln347Leu, Gln347Pro, Leu348Pro, Ile34 9Thr, Thr350Met, His351Leu, Pro352Leu, Arg353Cys, Asp354Gly, Tyr355His, Tyr355Asn, Tyr355Cy s, Ser356Cys, Gly357Cys, His363Arg, His363Leu, Ser364Pro, Gln365Arg, Ile366Thr, Leu367Pro, L ys368Arg, Leu369Gln, Leu369Pro, Ser370Ala, Lys371Glu, Thr373Ala, Leu376Pro, Tyr377Cys, Phe37 9Ser, Val381Ala, Glu384Gly, Asn387Ser, His389Gln, His389Leu, His389Arg, Gly390Ser, Glu391Ly s, Tyr393Cys, Val396Ala, Lys399Glu, Lys399Arg, Arg406Ser, Val412Ala, Gln415Leu, Phe416Ser, G At least one amino acid substitution among In441Leu, Tyr442Phe, Lys448Arg, Glu453Gly, Lys455Arg, Glu458Gly, Ala461Ser, Ser476Arg, Leu477Pro, Asn487Asp and Asn492Asp is preferable since it improves stability against heat and / or acid.

[0026] Among these, Gly390Ser is an amino acid substitution that particularly improves stability against heat and acid, and therefore, an AAV binding protein having at least the amino acid substitution Gly390Ser is a preferred embodiment of the AAV binding protein of the present invention.

[0027] Furthermore, in the amino acid sequence set forth in SEQ ID NO: 1, at least one amino acid substitution among Ile319Asn, Ile319Ser, Thr320Ile, Lys323Glu, Leu328Gln, Leu328Pro, Tyr331His, Val332Ala, Val332Glu, Pro336Gln, Pro337Gln, Lys338Asn, Tyr342Arg, Thr350Ser, Ile366Phe, Val383Ala, Gln386Arg, His389Asp, Gly392Cys, Val394Ala, Ile409Val, Ser476Gly and Thr490Ser is preferably present, as this improves stability against heat and / or acid.

[0028] Furthermore, in the amino acid sequence set forth in SEQ ID NO:1, at least one amino acid substitution among Val317Asp, Gln318Pro, Thr320Ala, Val326Ala, Val326Glu, Leu328Pro, Asn329His, Val332Ala, Lys362Glu, Lys362Asn, Lys371Asn, Leu376Pro, Glu378Gly, Glu378Val, Phe379Tyr, Phe379Ser, Val381Asp, Thr397Ser, Glu401Val, Arg406His and Val499Glu is preferably present, as this improves stability against heat and / or acid.

[0029] Furthermore, in the amino acid sequence of SEQ ID NO:1, Ser312Pro, Ala313Ser, Lys323Arg, Asn324Ser, Gln327His, Gln327Leu, Asn329Ile, Glu335Gly, Glu335Val, Glu340Val, Thr341Pro, Thr343Ser, Tyr344Phe, Trp346Cys, Arg353Cy s, Tyr355Cys, Ser356Cys, Gly357Cys, Met359Leu, Glu360Lys, Glu360Val, Gly361Cys, Lys362Gly, Ser 364Leu, Ile366Thr, Leu369Gln, Lys371Asp, Leu372Pro, Leu372Gln, Thr373Ala, Pro374Leu, Leu376Pro , Glu378Gly, Glu378Val, Phe379Cys, Phe379Ser, Lys380Glu, Val381Ala, Ile382Val, Val383Ala, Glu3 84Val, Gln386Arg, His389Arg, Tyr393Cys, Val394Asp, Val394Ile, Asn395Ser, Val396Ala, Arg403His, At least one of the amino acid substitutions Gln415Arg, Thr426Ala, Gln432Leu, Gln432Arg, Gln441Arg, His443Leu, Lys448Glu, Ile456Val, Asp483Asn, Ser488Leu, and Val499Ile is preferred because it improves stability against heat and / or alkali.

[0030] In the AAV binding protein of the present invention, the number of amino acids to be substituted is not particularly limited. Examples include the AAV binding proteins shown in (A) to (K) below. These AAV binding proteins are more preferred in terms of improved stability against heat and acid.

[0031] (A) An AAV binding protein comprising amino acid residues from serine at position 312 to aspartic acid at position 500 in the amino acid sequence set forth in SEQ ID NO:1, and in which at least the amino acid substitutions Gly390Ser and Tyr342Cys have occurred in the amino acid residues at positions 312 to 500.

[0032] (B) An AAV binding protein comprising amino acid residues from serine at position 312 to aspartic acid at position 500 in the amino acid sequence set forth in SEQ ID NO:1, in which at least the following amino acid substitutions have occurred in the amino acid residues at positions 312 to 500: Gly390Ser, Tyr342Cys, and Ser476Arg.

[0033] (C) An AAV binding protein comprising amino acid residues from serine at position 312 to aspartic acid at position 500 in the amino acid sequence set forth in SEQ ID NO:1, and in which at least the following amino acid substitutions have occurred in the amino acid residues at positions 312 to 500: Gly390Ser, Ile319Phe, Tyr342Cys, and Ser476Arg.

[0034] (D) An AAV binding protein comprising amino acid residues from serine at position 312 to aspartic acid at position 500 in the amino acid sequence set forth in SEQ ID NO:1, in which at least the following amino acid substitutions have occurred in the amino acid residues at positions 312 to 500: Gly390Ser, Tyr342Cys, Ser476Arg, and Asn487Asp.

[0035] (E) An AAV binding protein comprising amino acid residues from serine at position 312 to aspartic acid at position 500 in the amino acid sequence set forth in SEQ ID NO:1, and in which at least the following amino acid substitutions have occurred in the amino acid residues at positions 312 to 500: Gly390Ser, Ile319Phe, Lys323Glu, Tyr342Cys, and Ser476Arg.

[0036] (F) An AAV binding protein comprising amino acid residues from serine at position 312 to aspartic acid at position 500 in the amino acid sequence set forth in SEQ ID NO:1, in which at least the following amino acid substitutions have occurred in the amino acid residues at positions 312 to 500: Gly390Ser, Tyr342Cys, Lys399Glu, Ser476Arg, and Asn487Asp.

[0037] (G) An AAV binding protein comprising amino acid residues from serine at position 312 to aspartic acid at position 500 in the amino acid sequence set forth in SEQ ID NO:1, and in which at least the following amino acid substitutions have occurred in the amino acid residues at positions 312 to 500: Gly390Ser, Asn324Asp, Tyr342Cys, Lys362Glu, Lys399Glu, Ser476Arg, and Asn487Asp.

[0038] (H) An AAV binding protein comprising amino acid residues from serine at position 312 to aspartic acid at position 500 in the amino acid sequence set forth in SEQ ID NO:1, and in which at least the following amino acid substitutions have occurred in the amino acid residues at positions 312 to 500: Gly390Ser, Val317Asp, Tyr342Cys, Lys371Asn, Lys399Glu, Ser476Arg, and Asn487Asp.

[0039] (I) An AAV binding protein comprising amino acid residues from serine at position 312 to aspartic acid at position 500 in the amino acid sequence set forth in SEQ ID NO:1, and in which at least the following amino acid substitutions have occurred in the amino acid residues at positions 312 to 500: Gly390Ser, Val317Asp, Asn324Asp, Tyr342Cys, Lys362Glu, Lys399Glu, Ser476Arg, and Asn487Asp.

[0040] (J) An AAV binding protein comprising amino acid residues from serine at position 312 to aspartic acid at position 500 in the amino acid sequence set forth in SEQ ID NO:1, and in which at least the following amino acid substitutions have occurred in the amino acid residues at positions 312 to 500: Gly390Ser, Val317Asp, Tyr342Cys, Lys362Glu, Lys371Asn, Lys399Glu, Ser476Arg, and Asn487Asp.

[0041] (K) An AAV binding protein comprising amino acid residues from serine at position 312 to aspartic acid at position 500 in the amino acid sequence set forth in SEQ ID NO:1, and in which at least the following amino acid substitutions have occurred in the amino acid residues at positions 312 to 500: Gly390Ser, Val317Asp, Tyr(Cys)342Ser, Lys362Glu, Lys371Asn, Val381Ala, Ile382Val, Lys399Glu, Ser476Arg, and Asn487Asp.

[0042] More specifically, the AAV-binding proteins of the present invention include the following AAV-binding proteins. These AAV-binding proteins are preferred because of their improved stability against heat and acid. An AAV binding protein comprising at least the amino acid sequence from serine at position 25 to aspartic acid at position 213 of the amino acid sequence set forth in SEQ ID NO:18, which is one embodiment of the AAV binding protein shown in (A) above. An AAV binding protein comprising at least the amino acid sequence from serine at position 25 to aspartic acid at position 213 of the amino acid sequence set forth in SEQ ID NO: 32, which is one embodiment of the AAV binding protein shown in (B) above. An AAV binding protein comprising at least the amino acid sequence from serine at position 25 to aspartic acid at position 213 of the amino acid sequence set forth in SEQ ID NO:34, which is one embodiment of the AAV binding protein shown in (C) above. An AAV binding protein comprising at least the amino acid sequence from serine at position 25 to aspartic acid at position 213 of the amino acid sequence set forth in SEQ ID NO:38, which is one embodiment of the AAV binding protein shown in (D) above. An AAV binding protein comprising at least the amino acid sequence from serine at position 25 to aspartic acid at position 213 of the amino acid sequence set forth in SEQ ID NO:42, which is one embodiment of the AAV binding protein shown in (E) above. An AAV binding protein comprising at least the amino acid sequence from serine at position 25 to aspartic acid at position 213 of the amino acid sequence set forth in SEQ ID NO:46, which is one embodiment of the AAV binding protein shown in (F) above. An AAV binding protein comprising at least the amino acid sequence from serine at position 25 to aspartic acid at position 213 of the amino acid sequence set forth in SEQ ID NO:53, which is one embodiment of the AAV binding protein shown in (G) above. An AAV binding protein comprising at least the amino acid sequence from serine at position 25 to aspartic acid at position 213 of the amino acid sequence set forth in SEQ ID NO:55, which is one embodiment of the AAV binding protein shown in (H) above. An AAV binding protein comprising at least the amino acid sequence from serine at position 25 to aspartic acid at position 213 of the amino acid sequence set forth in SEQ ID NO:59, which is one embodiment of the AAV binding protein shown in (I) above. An AAV binding protein comprising at least the amino acid sequence from serine at position 25 to aspartic acid at position 213 of the amino acid sequence set forth in SEQ ID NO:63, which is one embodiment of the AAV binding protein shown in (J) above. An AAV binding protein comprising at least the amino acid sequence from serine at position 25 to aspartic acid at position 231 of the amino acid sequence set forth in SEQ ID NO:69, which is one embodiment of the AAV binding protein shown in (K) above.

[0043] More specifically, examples of the AAV-binding proteins of the present invention include the following AAV-binding proteins. The above-mentioned AAV-binding proteins are preferred in that they have improved stability against heat. An AAV binding protein comprising at least the amino acid sequence from serine at position 25 to aspartic acid at position 213 of the amino acid sequence set forth in SEQ ID NO: 76, which is one embodiment of the AAV binding protein shown in (F) above.

[0044] In the above (ii), (v) and (viii), "one or several" means, for example, 1 to 50, 1 to 30, 1 to 20, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 amino acid residue, depending on the position of the amino acid substitution in the three-dimensional structure of the AAV binding protein and the type of amino acid residue. An example of the amino acid substitution is a conservative substitution in which a substitution occurs between amino acids having similar physical and / or chemical properties. In the case of a conservative substitution, it is generally known to those skilled in the art that the function of a protein is maintained between a substitution and a non-substitution. An example of a conservative substitution is a substitution between glycine and alanine, between serine and proline, or between glutamic acid and alanine (Protein Structure and Function, Medical Science International, 9, 2005). Another example of the amino acid substitution is a substitution for monomerizing the AAV binding protein of the present invention, specifically, an amino acid substitution in which a cysteine ​​residue that easily forms a higher-order structure is replaced with a serine residue or a methionine residue. Furthermore, as used herein, any one or more of the substitutions, deletions, insertions, and additions also include naturally occurring mutations (mutants or variants) based on differences in the origin or species of the AAV binding protein.

[0045] The homology of the amino acid sequences in (iii), (vi) and (ix) may be 70% or more, and may be higher (e.g., 80% or more, 85% or more, 90% or more, 95% or more, or 98% or more). In this specification, "homology" may mean similarity or identity, and may particularly mean identity. "Homology of amino acid sequences" means homology with respect to the entire amino acid sequence. "Identity" between amino acid sequences means the ratio of amino acid residues of the same type in those amino acid sequences (Experimental Medicine, February 2013, Vol. 31, No. 3, Yodosha). "Similarity" between amino acid sequences means the sum of the ratio of amino acid residues of the same type in those amino acid sequences and the ratio of amino acid residues with similar side chain properties (Experimental Medicine, February 2013, Vol. 31, No. 3, Yodosha). Amino acid sequence homology can be determined using alignment programs such as BLAST (Basic Local Alignment Search Tool) and FASTA.

[0046] The AAV-binding protein of the present invention may further have an oligopeptide attached to its N-terminus or C-terminus that is useful for separating the AAV-binding protein of the present invention from a solution in the presence of contaminants. Examples of the oligopeptide include polyhistidine, polylysine, polyarginine, polyglutamic acid, polyaspartic acid, and the like. In addition, an oligopeptide containing cysteine ​​(e.g., an oligopeptide consisting of amino acid residues from cysteine ​​at position 220 to glycine at position 226 in the amino acid sequence set forth in SEQ ID NO: 50) that is useful for immobilizing the AAV-binding protein of the present invention on a solid phase such as a support for chromatography may further be attached to the N-terminus or C-terminus of the AAV-binding protein of the present invention.

[0047] The length of the oligopeptide added to the N- or C-terminus of the AAV-binding protein is not particularly limited as long as it does not impair the AAV-binding ability or stability of the AAV-binding protein of the present invention. When adding the oligopeptide to the AAV-binding protein of the present invention, a polynucleotide encoding the oligopeptide may be prepared and then added to the N- or C-terminus of the AAV-binding protein by genetic engineering using a method well known to those skilled in the art, or a chemically synthesized oligopeptide may be chemically bonded to the N- or C-terminus of the AAV-binding protein of the present invention.

[0048] Furthermore, a signal peptide may be added to the N-terminus of the AAV-binding protein of the present invention to promote efficient expression in a host. When the host is Escherichia coli, examples of the signal peptide include signal peptides that secrete proteins into the periplasm, such as PelB, OmpA, DsbA, DsbC, MalE, and TorT (JP 2011-097898 A).

[0049] As used herein, "improved stability against heat, acid or alkali" may mean improved stability against heat, acid or alkali compared to a wild-type AAV binding protein (a protein containing at least the amino acid residues from serine at position 25 to aspartic acid at position 213 of the amino acid sequence set forth in SEQ ID NO:7), preferably means improved stability against heat and acid or alkali, and more preferably means improved stability against heat, acid and alkali.

[0050] As an example of a method for producing a polynucleotide encoding the AAV binding protein of the present invention (hereinafter referred to as the polynucleotide of the present invention), (I) A method of converting the amino acid sequence of the AAV binding protein of the present invention into a nucleotide sequence and artificially synthesizing a polynucleotide containing the nucleotide sequence; (II) A method in which a polynucleotide containing the entire or partial sequence of an AAV-binding protein is prepared artificially directly or from the cDNA of the AAV-binding protein using a DNA amplification method such as PCR, and the prepared polynucleotides are then ligated by an appropriate method.

[0051] In the method (I) above, when converting an amino acid sequence into a nucleotide sequence, it is preferable to convert the sequence taking into consideration the frequency of codon usage in the host to be transformed. For example, when the host is Escherichia coli, AGA / AGG / CGG / CGA for arginine (Arg), ATA for isoleucine (Ile), CTA for leucine (Leu), GGA for glycine (Gly), and CCC for proline (Pro) are used infrequently (so-called rare codons), so conversion should be performed to avoid these codons. Analysis of the frequency of codon usage can also be performed by using public databases (for example, the Codon Usage Database on the website of the Kazusa DNA Research Institute).

[0052] When introducing a mutation into the polynucleotide of the present invention, an error-prone PCR method can be used. The reaction conditions in the error-prone PCR method are not particularly limited as long as they are conditions that allow the desired mutation to be introduced into the polynucleotide encoding the AAV binding protein. For example, the concentrations of the four types of substrate deoxynucleotides (dATP / dTTP / dCTP / dGTP) are made non-uniform, and MnCl2 is added to the PCR reaction solution at a concentration of 0.01 to 10 mM (preferably 0.1 to 1 mM) to perform PCR, thereby introducing a mutation into the polynucleotide. In addition, as a method for introducing a mutation into a polynucleotide other than the error-prone PCR method, a method of introducing a mutation into a polynucleotide by contacting and acting a mutagenic agent on a polynucleotide containing the entire or partial sequence of an AAV binding protein or irradiating it with ultraviolet light can be mentioned. In this method, as a mutagen used as a mutagen, mutagenic agents commonly used by those skilled in the art, such as hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine, nitrous acid, sulfurous acid, and hydrazine, may be used.

[0053] When transforming a host with the polynucleotide of the present invention, the polynucleotide of the present invention itself may be used, but it is more preferable to use an expression vector (e.g., a bacteriophage, cosmid, or plasmid usually used for transforming prokaryotic or eukaryotic cells) into which the polynucleotide of the present invention has been inserted at an appropriate position. The expression vector is not particularly limited as long as it is stable in the host to be transformed and can be replicated. When Escherichia coli is used as the host, examples of the expression vector include pET plasmid vector, pUC plasmid vector, pTrc plasmid vector, and pCDF plasmid vector.

[0054] The appropriate position means a position that does not disrupt the replication function of the expression vector, the desired antibiotic marker, or the region involved in transduction. When the polynucleotide of the present invention is inserted 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 required for expression. Examples of such promoters when the host is E. coli include the trp promoter, tac promoter, trc promoter, lac promoter, T7 promoter, recA promoter, and lpp promoter.

[0055] A host can be transformed using an expression vector containing the polynucleotide of the present invention (hereinafter referred to as the expression vector of the present invention) prepared by the above method by a method commonly used by those skilled in the art. For example, when a microorganism belonging to the genus Escherichia (E. coli JM109 strain, E. coli BL21 (DE3) strain, E. coli W3110 strain, etc.) is selected as a host, the host can be transformed by a method described in a known document (e.g., Molecular Cloning, Cold Spring Harbor Laboratory, 256, 1992). The transformant obtained by transformation by the above method can be screened by an appropriate method to obtain a transformant capable of expressing the AAV binding protein of the present invention (hereinafter referred to as the transformant of the present invention).

[0056] To prepare the expression vector of the present invention from the transformant of the present invention, it may be prepared from the culture obtained by culturing the transformant of the present invention by alkaline extraction or using a commercially available extraction kit such as QIAprep Spin Miniprep kit (Qiagen).

[0057] The AAV-binding protein of the present invention can be produced by culturing the transformant of the present invention and recovering the AAV-binding protein of the present invention from the resulting culture. In this specification, the culture includes not only the cultured cells of the transformant of the present invention themselves, but also the medium used for the culture.

[0058] The transformant used in the protein production method of the present invention may be cultured in a medium suitable for culturing the target host. When the host is Escherichia coli, a preferred example of the medium is LB (Luria-Bertani) medium supplemented with necessary nutrient sources. In order to selectively grow the transformant of the present invention depending on whether or not the vector of the present invention has been introduced, it is preferable to culture the transformant by adding a drug corresponding to the drug resistance gene contained in the vector to the medium. For example, when the vector contains a kanamycin resistance gene, kanamycin may be added to the medium.

[0059] In addition to carbon, nitrogen and inorganic salt sources, the medium may contain an appropriate nutrient source and, if desired, one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine, thioglycolate and dithiothreitol. When the host is E. coli, the culture temperature is generally 10°C to 40°C, preferably 20°C to 37°C, and more preferably around 25°C, but may be selected depending on the characteristics of the protein to be expressed. When the host is E. coli, the pH of the medium is pH 6.8 to pH 7.4, preferably around pH 7.0. When the vector of the present invention contains an inducible promoter, it is preferable to induce it under conditions that allow good expression of the AAV binding protein of the present invention.

[0060] An example of an inducer is IPTG (isopropyl-β-D-thiogalactopyranoside). When the host is Escherichia coli, the turbidity of the culture solution (absorbance at 600 nm) is measured, and when it reaches about 0.5 to 1.0, an appropriate amount of IPTG is added, followed by continued cultivation, to induce expression of the AAV binding protein. The concentration of IPTG added may be appropriately selected from the range of 0.005 to 1.0 mM, with the range of 0.01 to 0.5 mM being preferred. Various conditions for IPTG induction may be those well known in the art.

[0061] To recover the AAV-binding protein of the present invention from a culture obtained by culturing the transformant of the present invention, the AAV-binding protein of the present invention may be recovered by separating / purifying it from the culture using a method suitable for the expression form of the AAV-binding protein of the present invention in the transformant of the present invention. For example, when the protein is expressed in the culture supernatant, the cells may be separated by centrifugation, and the AAV-binding protein of the present invention may be purified from the resulting culture supernatant. When the protein is expressed intracellularly (including the periplasm), the cells may be collected by centrifugation, disrupted by adding an enzyme treatment agent, a surfactant, or the like, and the AAV-binding protein of the present invention may be extracted and then purified.

[0062] The AAV-binding protein of the present invention can be purified by any method known in the art, for example, separation / purification using liquid chromatography. Liquid chromatography includes ion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, affinity chromatography, etc., and the AAV-binding protein of the present invention can be prepared at a high purity by performing a purification procedure using a combination of these chromatographies.

[0063] The binding activity of the obtained AAV-binding protein of the present invention to AAV can be measured, for example, by using an enzyme-linked immunosorbent assay (hereinafter referred to as ELISA) or a surface plasmon resonance method. The AAV used to measure the binding activity may be an AAV vector or a VLP (virus-like particle). In addition, any serotype (serum type) of AAV vector and VLP may be used as long as they exhibit binding activity to the AAV-binding protein of the present invention.

[0064] The AAV binding protein of the present invention can be used, for example, for the purification or analysis of AAV. The AAV binding protein of the present invention can be used, for example, by immobilizing it on an insoluble carrier. That is, the purification or analysis of AAV can be specifically carried out, for example, using an AAV adsorbent comprising an insoluble carrier and the AAV binding protein of the present invention immobilized on the insoluble carrier. In this specification, the AAV adsorbent comprising an insoluble carrier and the AAV binding protein of the present invention immobilized on the insoluble carrier is also referred to as the AAV adsorbent of the present invention. Note that the purification of AAV is not limited to the purification of AAV from a solution containing impurities, but also includes the purification of AAV based on structure, properties, activity, etc. The insoluble carrier is not particularly limited. Examples of insoluble carriers include carriers made from polysaccharides such as agarose, alginate (alginic acid salt), carrageenan, chitin, cellulose, dextrin, dextran, and starch; carriers made from synthetic polymers such as polyvinyl alcohol, polymethacrylate, poly(2-hydroxyethyl methacrylate), and polyurethane; and carriers made from ceramics such as silica. Among them, carriers made from polysaccharides and carriers made from synthetic polymers are preferred as insoluble carriers. Examples of the preferred carriers include polymethacrylate gels with hydroxyl groups introduced such as Toyopearl (manufactured by Tosoh Corporation), agarose gels such as Sepharose (manufactured by Cytiva Corporation), and cellulose gels such as Cellufine (manufactured by JNC Corporation). The shape of the insoluble carrier is not particularly limited. The insoluble carrier may be in a shape that can be packed in a column, for example. The insoluble carrier may be in a granular, monolithic, membranous, or fibrous form, for example. Additionally, the insoluble carrier may be, for example, porous or non-porous.

[0065] The AAV binding protein of the present invention can be immobilized on an insoluble carrier, for example, via a covalent bond. Specifically, the AAV binding protein of the present invention can be immobilized on an insoluble carrier, for example, by covalently binding the AAV binding protein of the present invention to an insoluble carrier via an active group possessed by the insoluble carrier. That is, the insoluble carrier may have an active group. The insoluble carrier may have an active group on its surface, for example. Examples of the active group include an N-hydroxysuccinimide (NHS) activated ester group, an epoxy group, a carboxy group, a maleimide group, a haloacetyl group, a tresyl group, a formyl group, and a haloacetamide group. As the insoluble carrier having an active group, for example, a commercially available insoluble carrier having an active group may be used as it is, or an active group may be introduced into the insoluble carrier. Examples of commercially available supports having active groups include TOYOPEARL AF-Epoxy-650M, TOYOPEARL AF-Tresyl-650M (both manufactured by Tosoh Corporation), HiTrap NHS-activated HP Columns, NHS-activated Sepharose 4 Fast Flow, Epoxy-activated Sepharose 6B (all manufactured by Cytiva Corporation), and SulfoLink Coupling Resin (manufactured by Thermo Fisher Scientific).

[0066] An example of a method for introducing active groups onto the support surface is a method in which one of a compound having two or more active sites is reacted with a hydroxy group, epoxy group, carboxy group, amino group, etc. present on the support surface.

[0067] Examples of compounds that introduce epoxy groups into hydroxyl groups or amino groups present on the surface of a carrier include epichlorohydrin, ethanediol diglycidyl ether, butanediol diglycidyl ether, and hexanediol diglycidyl ether.

[0068] Examples of compounds that introduce carboxy groups into epoxy groups present on the surface of the carrier include 2-mercaptoacetic acid, 3-mercaptopropionic acid, 4-mercaptobutyric acid, 6-mercaptobutyric acid, glycine, 3-aminopropionic acid, 4-aminobutyric acid, and 6-aminohexanoic acid.

[0069] Compounds that introduce maleimide groups into hydroxyl, epoxy, carboxyl, and amino groups present on the support surface include N-(ε-maleimidocaproic acid) hydrazide, N-(ε-maleimidopropionic acid) hydrazide, 4-(4-N-maleimidophenyl)acetic acid hydrazide, 2-aminomaleimide, 3-aminomaleimide, 4-aminomaleimide, 6-aminomaleimide, 1-(4-aminophenyl)maleimide, 1-(3-aminophenyl)maleimide, 4-(maleimido)phenyl isocyanate, 2-maleimidoacetic acid, 3-maleimidopropionic acid, and Examples include pionic acid, 4-maleimidobutyric acid, 6-maleimidohexanoic acid, N-(α-maleimidoacetoxy)succinimide ester, (m-maleimidobenzoyl)N-hydroxysuccinimide ester, succinimidyl-4-(maleimidomethyl)cyclohexane-1-carbonyl-(6-aminohexanoic acid), succinimidyl-4-(maleimidomethyl)cyclohexane-1-carboxylic acid, (p-maleimidobenzoyl)N-hydroxysuccinimide ester, and (m-maleimidobenzoyl)N-hydroxysuccinimide ester.

[0070] Examples of compounds that introduce haloacetyl groups into hydroxyl groups or amino groups present on the surface of a carrier include chloroacetic acid, bromoacetic acid, iodoacetic acid, chloroacetic acid chloride, bromoacetic acid chloride, bromoacetic acid bromide, chloroacetic acid anhydride, bromoacetic acid anhydride, iodoacetic acid anhydride, 2-(iodoacetamido)acetic acid-N-hydroxysuccinimide ester, 3-(bromoacetamido)propionic acid-N-hydroxysuccinimide ester, and 4-(iodoacetyl)aminobenzoic acid-N-hydroxysuccinimide ester.

[0071] As a method for introducing active groups onto the carrier surface, an example is a method in which an ω-alkenylalkane glycidyl ether is reacted with a hydroxy group or an amino group present on the carrier surface, and then the ω-alkenyl moiety is halogenated with a halogenating agent to activate the group. Examples of the ω-alkenylalkane glycidyl ether include allyl glycidyl ether, 3-butenyl glycidyl ether, and 4-pentenyl glycidyl ether. Examples of the halogenating agent include N-chlorosuccinimide, N-bromosuccinimide, and N-iodosuccinimide.

[0072] Another example of a method for introducing active groups onto the carrier surface is a method for introducing active groups onto carboxy groups present on the carrier surface using a condensation agent and an additive. Examples of condensation agents include 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), dicyclohexylcarbodiamide, and carbonyldiimidazole. Examples of additives include N-hydroxysuccinimide (NHS), 4-nitrophenol, and 1-hydroxybenztriazole.

[0073] The immobilization of the AAV binding protein of the present invention to an insoluble carrier can be carried out, for example, in a buffer solution. Examples of the buffer solution include acetate buffer, phosphate buffer, MES (2-Morpholinoethanesulfonic acid) buffer, HEPES (4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid) buffer, Tris (Tris(hydroxymethyl)aminomethane) buffer, and borate buffer. The reaction temperature during immobilization can be appropriately set depending on various conditions such as the reactivity of the active group and the stability of the AAV binding protein. The reaction temperature during immobilization can be, for example, 5°C to 50°C, and preferably 10°C to 35°C.

[0074] The AAV adsorbent of the present invention can be used, for example, in purifying AAV by filling a column. Specifically, for example, AAV can be purified by adding a solution containing AAV to a column filled with the AAV adsorbent of the present invention to adsorb the AAV to the adsorbent, and eluting the AAV adsorbed to the adsorbent. That is, the present invention provides a method for purifying AAV, which includes, for example, a step of adding a solution containing AAV to a column filled with the AAV adsorbent of the present invention to adsorb the AAV to the adsorbent, and a step of eluting the AAV adsorbed to the adsorbent. The solution containing AAV can be added to the column using a liquid delivery means such as a pump. In this specification, adding a liquid to a column is also referred to as "delivering a liquid to a column". The solution containing AAV may be solvent-substituted with an appropriate buffer solution before being added to the column. In addition, the column may be equilibrated with an appropriate buffer solution before the solution containing AAV is added to the column. It is expected that the equilibration can purify AAV to a higher purity, for example. Examples of buffer solutions used for solvent replacement or equilibration include phosphate buffer, acetate buffer, and MES buffer. For example, inorganic salts such as 10 mM to 100 mM sodium chloride may be added to such buffer solutions. The buffer solution used for solvent replacement and the buffer solution used for equilibration may be the same or different. In addition, if components other than AAV, such as impurities, remain in the column after passing the AAV-containing solution through the column, such components may be removed from the column before eluting the AAV adsorbed to the AAV adsorbent. Components other than AAV can be removed from the column, for example, using an appropriate buffer. For the buffer solution used to remove components other than AAV, the description of the buffer solution used for solvent replacement or equilibration can be applied mutatis mutandis. The AAV adsorbed to the AAV adsorbent can be eluted, for example, by weakening the interaction between AAV and the ligand (the AAV-binding protein of the present invention). Examples of means for weakening the interaction between AAV and the ligand (the AAV-binding protein of the present invention) include lowering the pH with a buffer solution, adding a counter peptide, increasing the temperature, and changing the salt concentration. Specifically, the AAV adsorbed to the AAV adsorbent can be eluted using, for example, a suitable elution solution, such as a buffer solution that is more acidic than the buffer solution used for solvent replacement or equilibration.Examples of such a buffer solution include a citrate buffer solution, a glycine hydrochloride buffer solution, and an acetate buffer solution. The pH of the elution solution can be set within a range that does not impair the function of AAV (such as antigen binding).

[0075] By carrying out the purification of AAV in this manner, for example, purified AAV can be obtained. That is, in one embodiment, the purification method of AAV may be a method for producing AAV, specifically, a method for producing purified AAV. AAV is obtained, for example, as an eluted fraction containing AAV. That is, a fraction containing eluted AAV can be separated. Separation of the AAV fraction can be performed, for example, by a conventional method. Methods for separating the AAV fraction include a method of replacing the collection container at regular intervals or at regular volumes, a method of replacing the collection container according to the shape of the chromatogram of the eluate, and a method of separating the fraction using an automatic fraction collector such as an autosampler. Furthermore, AAV can be recovered from a fraction containing AAV. Recovery of AAV from a fraction containing AAV can be performed, for example, by a known method used for protein purification. Effect of the Invention

[0076] The adeno-associated virus (AAV) binding protein of the present invention is a protein in which an amino acid residue at a specific position in the extracellular domain of the AAV receptor is replaced with another amino acid residue. The AAV binding protein of the present invention has improved stability against heat, acid, and alkali compared to the wild type (AAV binding protein with no amino acid residue replacement). Therefore, the AAV binding protein of the present invention is useful as a ligand for an adsorbent for purifying an AAV vector. In addition, an AAV adsorbent comprising an insoluble carrier and the AAV binding protein of the present invention immobilized on the carrier can purify an AAV vector contained in a solution to a higher purity than an AAV adsorbent comprising an insoluble carrier and a wild type AAV binding protein immobilized on the carrier. [Brief description of the drawings]

[0077] [Figure 1]1 shows the results of ELISA assay of the binding activity of adeno-associated virus (AAV) binding proteins to VLP2 (virus-like particles of AAV2), in which (+) and (-) indicate the results when VLP2 was immobilized on a solid phase and when VLP2 was not immobilized on a solid phase, respectively. [Diagram 2] This is a chromatographic pattern of AAV2 purified from a cell lysate using an AVR5e column. The peak at about 20 minutes elution is the AAV2 elution peak, and the fractions corresponding to this peak (Fr6 to 9, 1 mL each) were collected. [Diagram 3] This figure shows the results of SDS-PAGE confirming the purity of AAV2 contained in the fractions (Fr6 to 9) collected in Figure 2. In the figure, AP is the analysis result of a solution obtained by diluting the sample before purification 100 times, and Fr6 to Fr9 are the analysis results of each fraction obtained in Figure 2. VP1 to VP3 are the positions of the bands corresponding to the three types of outer coat proteins that constitute AAV. [Figure 4] FIG. 1 shows the results of recovering AAV2 contained in a solution using a column packed with a wild-type AAV binding protein (Wild type) immobilized gel or an AVR5eHC immobilized gel. [Diagram 5] This figure shows the results of recovering AAV2 contained in a solution using a column packed with a wild-type AAV binding protein (Wild type) immobilized gel, an AVR3HC immobilized gel, or an AVR8gHC immobilized gel. EXAMPLES

[0078] EXAMPLES In the following, examples are shown to explain the present invention in more detail, but the present invention is not limited to these examples.

[0079] Example 1: Construction of adeno-associated virus (AAV) binding protein expression vector (1) A region from serine (Ser) 312 to aspartic acid (AAS) 500, which corresponds to extracellular domains 1 and 2 (PKD1 and PKD2) in the amino acid sequence of the AAV binding protein set forth in SEQ ID NO: 1 (UniProt accession number: Q8IZA0) The codons of the nucleotide sequence encoding the amino acid residues up to (Asp) were converted from human type to E. coli type. The codon-converted nucleotide sequence is shown in SEQ ID NO:2.

[0080] (2) The gene described in SEQ ID NO: 2 was artificially synthesized and cloned into a plasmid (outsourced to Eurofins Genomics). The prepared plasmid was named pUC-AAVR, and PCR was performed using this as a template and oligonucleotides consisting of the sequences described in SEQ ID NO: 3 (5'-AAT[CCATGG]GCTCTGCAGGCGAGTCGGTTC-3') and SEQ ID NO: 4 (5'-TTA[CTCGAG]TCAATGATGATGATGATGATGGTCTACCGCTTTGTTGACGG-3') as PCR primers (the square brackets in SEQ ID NO: 3 indicate the restriction enzyme NcoI recognition sequence, and the square brackets in SEQ ID NO: 4 indicate the restriction enzyme XhoI recognition sequence). Specifically, a reaction solution with the composition shown in Table 1 was prepared, and the reaction solution was heat-treated at 98°C for 5 minutes, and the reaction was performed by repeating 30 cycles of a first step at 98°C for 10 seconds, a second step at 55°C for 5 seconds, and a third step at 72°C for 90 seconds.

[0081] [Table 1]

[0082] (3) The polynucleotide obtained in (2) was purified, digested with the restriction enzymes NcoI and XhoI, and then ligated into the expression vector pET26b (Merck Millipore) which had been previously digested with the restriction enzymes NcoI and XhoI. The ligation product was used to transform Escherichia coli BL21 strain (DE3).

[0083] (4) The transformant obtained in (3) was cultured in LB (Luria-Bertani) medium containing 50 μg / mL kanamycin, and the vector pET-AAVRD2 expressing the extracellular domain 1 and domain 2 of the AAV binding protein was extracted using a QIAprep Spin Miniprep kit (Qiagen).

[0084] (5) The nucleotide sequence of the polynucleotide encoding the AAV binding protein and its surrounding region in the expression vector pET-AAVRD2 prepared in (4) was analyzed using a fully automated DNA sequencer, Genetic Analyzer 3500 (Thermo Fisher Scientific). In this analysis, an oligonucleotide consisting of the sequence shown in SEQ ID NO: 5 (5'-TAATACGACTCACTATAGGG-3') or SEQ ID NO: 6 (5'-ATGCTAGTTATTGCTCAGCGG-3') was used as a sequencing primer. The amino acid sequence of the polypeptide expressed by the expression vector pET-AAVRD2 is shown in SEQ ID NO: 7, and the sequence of the polynucleotide encoding the peptide is shown in SEQ ID NO: 8. In SEQ ID NO: 7, the region from methionine (Met) at position 1 to alanine (Ala) at position 22 is the PelB signal peptide, the region from serine (Ser) at position 25 to aspartic acid (Asp) at position 213 is the extracellular region of the AAV binding protein (domain 1 and domain 2, the region from positions 312 to 500 of SEQ ID NO: 1), and the histidine (His) at positions 214 to 219 is the tag sequence.

[0085] Example 2 Expression of AAV-binding proteins and evaluation of their binding to AAV (1) The E. coli strain BL21(DE3) transformed with pET-AAVRD2 prepared in Example 1 was inoculated into 3 mL of 2YT liquid medium (peptone 16 g / L, yeast extract 10 g / L, sodium chloride 5 g / L) containing 50 μg / mL of kanamycin, and pre-cultured overnight at 37° C. under aerobic shaking.

[0086] (2) 200 μL of the preculture solution from (1) was inoculated into 20 mL of 2YT liquid medium containing 50 μg / mL kanamycin in a 100 mL baffled flask, and cultured aerobically at 37°C with shaking.

[0087] (3) After 2.0 hours from the start of the culture, the culture was cooled on ice, IPTG (isopropyl-β-D-thiogalactopyranoside) was added to a final concentration of 0.1 mM, and the culture was continued overnight at 25°C under aerobic shaking.

[0088] (4) After the culture was completed, 2 mL of the culture medium was collected and centrifuged to collect the bacteria. The obtained bacteria were subjected to centrifugation using 200 μL of BugBuster Protein extraction kit (Merck Millipore) to prepare a protein extract containing the expressed AAV binding protein. The prepared protein extract was diluted 30-fold with 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride.

[0089] (5) Preparation of VLP2 (AAV2 virus-like particle) (5-1) Escherichia coli JM109 strain was transformed using pRC2-mi342 Vector (Takara Bio Inc.) and pHelper Vector (Takara Bio Inc.). The resulting transformants were cultured overnight at 37°C with shaking in a 5 L baffled flask containing 1 L of 2YT medium supplemented with 100 μg / mL carbenicillin.

[0090] (5-2) The culture medium of (5-1) was centrifuged to collect the bacterial cells. The pRC2-mi342 Vector and pHelper Vector were prepared in large quantities from the obtained bacterial cells using the Plasmid Mega Kit (Qiagen).

[0091] (5-3) HEK293T cells were cultured in five T-225 flasks (manufactured by Thermo Fisher Scientific) containing 40 mL of D-MEM medium (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) containing 10% (v / v) bovine serum. The pRC2-mi342 Vector and pHelper Vector prepared in (5-2) were transfected into the flasks using TransIT-ViruGEN Transfection Reagent (manufactured by Takara Bio Inc.), and the cells were cultured stationary for 3 days under conditions of 5% carbon dioxide and 37°C.

[0092] (5-4) After the culture in (5-3), the cells were collected and extracted and purified using an AAVpro Purification Kit (Takara Bio Inc.) to obtain VLP2. Approximately 1 mL of VLP2 purified solution was prepared from five T-225 flasks.

[0093] (6) The binding affinity between the AAV binding protein in the protein extract prepared in (4) and the VLP2 prepared in (5) was evaluated by ELISA.

[0094] (6-1) The VLP2 prepared in (5) was diluted 200 to 1000 times with 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride, added to a 96-well microplate (Thermo Fisher Scientific) at 100 μL / well, and immobilized (4° C. for 18 hours). After immobilization, the plate was blocked with 2% (w / v) SKIM MILK (Becton Dickinson) and 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride.

[0095] (6-2) After washing with a washing buffer (20 mM Tris-HCl buffer (pH 7.4) containing 0.05% [w / v] Tween 20 (trade name) and 150 mM sodium chloride), a solution containing an AAV-binding protein whose VLP2-binding activity was to be evaluated was added, and the AAV-binding protein was reacted with VLP2 (at 30° C. for 1 hour).

[0096] (6-3) After the reaction was completed, the plate was washed with the washing buffer, and an Anti-6His antibody (manufactured by Bethyl Laboratories) diluted to 100 ng / mL was added at 100 μL / well.

[0097] (6-4) After reacting at 30°C for 1 hour and washing with the washing buffer, TMB Peroxidase Substrate (KPL) was added at 50 μL / well. Color development was stopped by adding 50 μL / well of 1 M phosphoric acid, and the absorbance at 450 nm was measured using a microplate reader (Tecan). The measurement results of the ELISA method are shown in Figure 1. When VLP2 was immobilized on the solid phase (VLP(+)), high absorbance was observed, which indicates that the AAV-binding protein expressed in this example binds to VLP2. Note that since VLP2 is a virus-like particle of AAV2, this result can be said to be synonymous with the binding of the AAV-binding protein to AAV2.

[0098] Example 3: Construction and screening of a mutant library of AAV binding proteins (part 1) In the AAV binding protein expression vector pET-AAVRD2, random mutations were introduced by error-prone PCR into the polynucleotide portion encoding the AAV binding protein.

[0099] (1) Error-prone PCR was performed using pET-AAVRD2 prepared in Example 1 as a template. Error-prone PCR was performed by preparing a reaction solution having the composition shown in Table 2, heat-treating the reaction solution at 98°C for 2 minutes, and carrying out 30 cycles of reaction in which one cycle consisted of a first step at 98°C for 30 seconds, a second step at 55°C for 20 seconds, and a third step at 72°C for 90 seconds, and finally heat-treating the reaction at 72°C for 5 minutes. The error-prone PCR successfully introduced mutations into the polynucleotide encoding the AAV binding protein, and the average mutation introduction rate was 1.8 amino acid mutations per molecule.

[0100] [Table 2]

[0101] (2) The PCR product obtained in (1) was purified and then digested with restriction enzymes NcoI and XhoI, and ligated into an expression vector pET26b (Merck Millipore) that had been previously digested with the same restriction enzymes.

[0102] (3) After the ligation reaction was completed, the reaction mixture was used to transform E. coli BL21(DE3) and the resulting mixture was cultured on an LB plate medium containing 50 μg / mL kanamycin (37°C for 18 hours). The colonies formed on the plate were used as a random mutant library.

[0103] (4) The random mutant library (transformants) prepared in (3) was inoculated into 200 μL of 2YT liquid medium containing 50 μg / mL kanamycin, and cultured overnight at 37°C with shaking in a 96-well deep well plate.

[0104] (5) 10 μL of the culture prepared in (4) was transferred to 500 μL of 2YT liquid medium containing 0.1 mM IPTG and 50 μg / mL kanamycin, and further cultured overnight at 25° C. with shaking using a 96-well deep well plate.

[0105] (6) The culture medium from (5) was centrifuged, and the resulting culture supernatant was diluted 2-fold with 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride. The diluted solution was heat-treated at 43° C. for 15 minutes.

[0106] (7) The binding activity of the AAV binding protein to VLP2 when subjected to the heat treatment in (6) and the binding activity of the AAV binding protein to VLP2 when not subjected to the heat treatment in (6) were measured by the ELISA method described in Example 2(6). The binding activity of the AAV binding protein to VLP2 when subjected to the heat treatment was divided by the binding activity of the AAV binding protein to VLP2 when not subjected to heat treatment to calculate the residual activity.

[0107] (8) Approximately 2700 strains of transformants were evaluated using the method of (7), and transformants expressing AAV binding proteins with improved thermal stability compared to wild-type (no amino acid substitution) AAV binding proteins were selected from among them. The selected transformants were cultured, and expression vectors were prepared using a QIAprep Spin Miniprep kit (Qiagen).

[0108] (9) The sequence of the polynucleotide region encoding the AAV binding protein inserted into the obtained expression vector was analyzed for nucleotide sequence by the same method as described in Example 1(5), and the amino acid mutation sites were identified. Table 3 shows the amino acid substitution positions and the residual activity (%) after heat treatment of the AAV binding protein expressed by the transformant selected in (8) above, relative to the wild-type (no amino acid substitution) AAV binding protein. In the amino acid sequence of SEQ ID NO: 1, from the 312th serine (Ser) to the 500th aspartic acid (Asp), Ile319Phe (this notation indicates that the 319th isoleucine in SEQ ID NO: 1 has been substituted with phenylalanine, the same applies below), Leu321Pro, Pro322Leu, Asn324Asp, Asn324Ser, Gln327Arg, Asn329Asp, A sn329Tyr, Ala330Gly, Tyr331Cys, Val332Gln, Leu333Val, Leu333Pro, Gln334Arg, Pro337Leu, Lys338Arg, Lys338 Glu, Glu340Gly, Thr341Ala, Tyr342Cys, Tyr342His, Tyr342Asn, Thr343Met, Tyr344His, Asp345Asn, Trp346Leu, T rp346Arg, Gln347Leu, Gln347Pro, Leu348Pro, Ile349Thr, Thr350Met, His351Leu, Pro352Leu, A rg353Cys, Asp354Gly, Tyr355His, Tyr355Asn, Tyr355Cys, Ser356Cys, Gly357Cys, His363Arg, H is363Leu, Ser364Pro, Gln365Arg, Ile366Thr, Leu367Pro, Lys368Arg, Leu369Gln, Leu369Pro, S er370Ala, Lys371Glu, Thr373Ala, Leu376Pro, Tyr377Cys, Phe379Ser, Val381Ala, Glu384Gly, As AAV binding proteins having at least one of the following amino acid substitutions are considered to have improved thermal stability compared to wild-type AAV binding proteins: n387Ser, His389Gln, His389Leu, His389Arg, Gly390Ser, Glu391Lys, Tyr393Cys, Val396Ala, Lys399Glu, Lys399Arg, Arg406Ser, Val412Ala, Gln415Leu, Phe416Ser, Gln441Leu, Tyr442Phe, Lys448Arg, Glu453Gly, Lys455Arg, Glu458Gly, Ala461Ser, Ser476Arg, Leu477Pro, Asn487Asp, and Asn492Asp.

[0109] [Table 3]

[0110] Example 4: Evaluation of thermal stability of AAV binding protein mutants (amino acid substitutions) Among the AAV binding proteins with amino acid substitutions (mutated type) shown in Table 3, No. 9 (Pro352Leu substitution), No. 16 (Leu369Gln substitution), No. 29 (Leu333Val and Tyr342Cys substitution), No. 30 (Glu340Gly and Tyr342Cys substitution), No. 32 (Tyr342Cys and Gly390Ser substitution), No. 36 (Tyr355His and Asn387Ser substitution), No. 43 (Leu321P The following substitutions were selected and their thermal stability was evaluated again: No.45 (Asn329Asp, Tyr342His and Tyr355Cys substitutions), No.47 (Asn329Tyr, Tyr377Cys and Arg406Ser substitutions), No.49 (Tyr331Cys, Thr343Met and Tyr355Cys substitutions) and No.53 (Asp354Gly, Leu369Gln and Lys455Arg substitutions).

[0111] The nucleotide sequence and amino acid sequence of No. 9 are shown in SEQ ID NOs: 9 and 10. The nucleotide and amino acid sequences of No. 16 are shown in SEQ ID NOs: 11 and 12. The nucleotide and amino acid sequences of No. 29 are shown in SEQ ID NOs: 13 and 14. The nucleotide and amino acid sequences of No. 30 are shown in SEQ ID NOs: 15 and 16. The nucleotide and amino acid sequences of No. 32 are shown in SEQ ID NOs: 17 and 18. The nucleotide and amino acid sequences of No. 36 are shown in SEQ ID NOs: 19 and 20. The nucleotide and amino acid sequences of No. 43 are shown in SEQ ID NOs: 21 and 22. The nucleotide sequence and amino acid sequence of No. 45 are shown in SEQ ID NOs: 23 and 24. The nucleotide and amino acid sequences of No. 47 are shown in SEQ ID NOs: 25 and 26. The nucleotide and amino acid sequences of No. 49 are shown in SEQ ID NOs: 27 and 28. The nucleotide and amino acid sequences of No. 53 are shown in SEQ ID NOs: 29 and 30, respectively.

[0112] In addition, the substitution Pro352Leu in the amino acid sequence of No. 9 (SEQ ID NO: 10) is located at position 65 in SEQ ID NO: 10, In the amino acid sequence of No. 16 (SEQ ID NO: 12), the Leu369Gln substitution is located at position 82 in SEQ ID NO: 12; In the amino acid sequence of No. 29 (SEQ ID NO: 14), the substitutions Leu333Val and Tyr342Cys are located at positions 46 and 55 in SEQ ID NO: 14, In the amino acid sequence of No. 30 (SEQ ID NO: 16), the substitutions of Glu340Gly and Tyr342Cys are located at positions 53 and 55 in SEQ ID NO: 16, In the amino acid sequence of No. 32 (SEQ ID NO: 18), the substitutions Tyr342Cys and Gly390Ser are located at positions 55 and 103 in SEQ ID NO: 18, In the amino acid sequence of No. 36 (SEQ ID NO: 20), the substitutions Tyr355His and Asn387Ser are located at positions 68 and 100 in SEQ ID NO: 20, In the amino acid sequence of No. 43 (SEQ ID NO: 22), the substitutions Leu321Pro, Tyr342Asn, and Ala461Ser are located at positions 34, 55, and 174, respectively, in SEQ ID NO: 22; In the amino acid sequence of No. 45 (SEQ ID NO: 24), the substitutions Asn329Asp, Tyr342His, and Tyr355Cys are located at positions 42, 55, and 68, respectively, in SEQ ID NO: 24; In the amino acid sequence of No. 47 (SEQ ID NO: 26), the substitutions Asn329Tyr, Tyr377Cys, and Arg406Ser are located at positions 42, 90, and 119, respectively, in SEQ ID NO: 26; In the amino acid sequence of No. 49 (SEQ ID NO: 28), the substitutions of Tyr331Cys, Thr343Met, and Tyr355Cys are located at positions 44, 56, and 68, respectively, in SEQ ID NO: 30; In the amino acid sequence of No. 53 (SEQ ID NO: 30), the substitutions Asp354Gly, Leu369Gln, and Lys455Arg are located at positions 67, 82, and 168, respectively, in SEQ ID NO: 28.

[0113] (1) The transformants expressing the 11 selected mutant AAV binding proteins and the transformant expressing the wild-type AAV binding protein obtained in Example 1(3) were cultured by the methods described in Examples 3(4) and (5), respectively, and the culture solutions were then centrifuged to obtain culture supernatants containing the expressed mutant AAV binding proteins and the wild-type AAV binding protein, respectively.

[0114] (2) The binding activity between the AAV binding protein in the culture supernatant obtained in (1) and VLP2 was measured using the ELISA method described in Example 2(6). Based on the absorbance at 450 nm, which is the measurement result, the culture supernatant obtained in (1) was diluted with 20 mM Tris buffer (pH 7.4) containing 150 mM sodium chloride so that the measured value was similar.

[0115] (3) The diluted protein solution was divided into three portions, two of which were heated at 50.3°C or 66.1°C for 15 minutes using a thermal cycler (Eppendorf), and the remaining portion was not heated.

[0116] (4) The binding activity of the AAV binding activity protein to VLP2 after the heat treatment or without heat treatment in (3) was measured by the ELISA method described in Example 2(6), and the residual activity was calculated by dividing the absorbance at 450 nm after heat treatment by the absorbance at 450 nm without heat treatment. The results are shown in Table 4. All of the mutant AAV binding proteins had higher residual activity than the wild-type AAV binding protein, confirming that the mutant AAV binding proteins had improved thermal stability.

[0117] [Table 4]

[0118] Example 5: Evaluation of acid stability of mutant AAV binding proteins (part 1) (1) Transformants expressing the mutant AAV binding proteins evaluated in Example 4 (i.e., the mutant AAV binding proteins described in Nos. 9, 16, 29, 30, 32, 36, 43, 45, 47, 49, and 53 in Table 3) and the transformants expressing the wild-type AAV binding protein obtained in Example 1(3) were cultured by the methods described in Examples 3(4) and (5), respectively, and the culture solutions were centrifuged to obtain culture supernatants containing the expressed mutant AAV binding proteins or wild-type AAV binding proteins, respectively.

[0119] (2) The binding activity between the AAV binding protein in the culture supernatant obtained in (1) and VLP2 was measured using the ELISA method described in Example 2(6). Based on the absorbance at 450 nm, which is the measurement result, the culture supernatant obtained in (1) was diluted with pure water so that the measured value became similar.

[0120] (3) The diluted protein solution was divided into two, and one was mixed with an equal amount of 0.1 M glycine hydrochloride buffer (pH 2.5). After standing at 30°C for 2 hours and 24 hours, it was mixed with 0.5 M MES buffer (pH 6.0) in a ratio of 3:2 to adjust the pH to about 6, and the binding activity with VLP2 was measured by the ELISA method described in Example 2(6). The other was not subjected to the acid treatment, and the dilution ratio was adjusted to the same as the acid treatment conditions, and then it was mixed with 0.5 M MES buffer (pH 6.0) in a ratio of 3:2 to adjust the pH to about 6, and the binding activity with VLP2 was measured by the ELISA method described in Example 2(6).

[0121] (4) The residual activity was calculated by dividing the absorbance at 450 nm when the acid treatment described in (3) was performed by the absorbance at 450 nm when the acid treatment was not performed. The results are shown in Table 5. In all mutant AAV binding proteins evaluated in Example 4, the residual activity after 2 and 24 hours of treatment at 30°C and pH 2.5 was improved compared to the wild type. In other words, it was confirmed that the acid stability of the mutant AAV binding proteins was improved. Among them, No. 30 (Glu340Gly and Tyr342Cys substitution, SEQ ID NO: 16), No. 32 (Tyr342Cys and Gly390Ser substitution, SEQ ID NO: 18) and No. 45 (Asn329Asp, Tyr342His and Tyr355Cys substitution, SEQ ID NO: 24) had significantly improved acid stability compared to the wild type AAV binding protein.

[0122] [Table 5]

[0123] Example 6: Preparation and screening of a mutant library of AAV binding proteins (part 2) Among the mutant AAV-binding proteins evaluated in Examples 4 and 5, No. 32 (Tyr342Cys and Gly390Ser substitutions; sequence number 18) was selected (designated AVR2), and mutations were randomly introduced into the polynucleotide portion encoding the protein by error-prone PCR.

[0124] (1) A reaction solution having the composition shown in Table 6 was prepared using the plasmid pET-AVR2, into which a polynucleotide (SEQ ID NO: 17) encoding AVR2 (SEQ ID NO: 18) was inserted, as a template, and error-prone PCR was then carried out under the same temperature conditions as in Example 3(1). Mutations were introduced into the polynucleotide encoding AVR2 by the error-prone PCR, with the average mutation introduction rate being 1.5 amino acid mutations per molecule.

[0125] [Table 6]

[0126] (2) The PCR product obtained in (1) was purified and then digested with restriction enzymes NcoI and XhoI, and ligated into an expression vector pET26b (Merck Millipore) that had been previously digested with the same restriction enzymes.

[0127] (3) After the ligation reaction was completed, the reaction mixture was used to transform E. coli BL21(DE3) and the resulting mixture was cultured on an LB plate medium containing 50 μg / mL kanamycin (overnight at 37°C). The colonies formed on the plate were used as a random mutant library.

[0128] (4) The random mutant library (transformants) prepared in (3) was cultured by the method described in Example 3 (4) and (5).

[0129] (5) The culture medium from (4) was centrifuged, and the resulting culture supernatant was diluted 4-fold with ultrapure water. 60 μL of the diluted culture medium was mixed with 60 μL of 0.1 M glycine-HCl buffer (pH 3.0) and subjected to heat and acid treatment at 51.1° C. for 15 minutes.

[0130] (6) The binding activity of the AAV binding protein to VLP2 when treated with (5) and when not treated with (5) were measured by the ELISA method described in Example 2(6). The binding activity of the AAV binding protein to VLP2 when heat treatment was performed was divided by the binding activity of the AAV binding protein to VLP2 when not heat treatment was performed to calculate the residual activity.

[0131] (7) A random mutant library of about 1,800 strains was evaluated using the method of (6), and transformants expressing AAV binding proteins with improved residual activity compared to the parent molecule AVR2 were selected from the library. The selected transformants were cultured, and expression vectors were prepared using a QIAprep Spin Miniprep kit (Qiagen).

[0132] (8) The sequence of the polynucleotide region encoding the AAV binding protein inserted into the obtained expression vector was analyzed for nucleotide sequence by the method described in Example 1(5), and the amino acid mutation sites were identified. Table 7 shows the amino acid substitution positions relative to AVR2 (Tyr342Cys and Gly390Ser substitutions) of the AAV binding proteins expressed by the transformants selected in (7) above, as well as the residual activity (%) after heat and acid treatment.

[0133] In the amino acid sequence of SEQ ID NO:1, the amino acid residues from the 312th serine (Ser) to the 500th aspartic acid (Asp) are Ile319Phe, Ile319Asn, Ile319Ser, Thr320Ile, Leu321Pro, Lys323Glu, Leu328Gln, Leu328Pro, Tyr331His, Val332Ala, Val332Glu, Leu333Pro, Pro336Gln, Pro337Gln, Lys338Asn, and Tyr(Cys)342Arg (this notation indicates that the 342nd tyrosine in SEQ ID NO:1 is once replaced with cysteine ​​and then replaced with arginine). AAV binding proteins having at least one amino acid substitution, among which Glu, Gluginine, Thr350Ser, Gln365Arg, Ile366Phe, Phe379Ser, Val383Ala, Gln386Arg, His389Asp, Gly392Cys, Val394Ala, Val396Ala, Lys399Glu, Ile409Val, Ser476Gly, Ser476Arg, Asn487Asp, Thr490Ser, and Asn492Asp, can be said to have improved heat and acid stability compared to the wild-type AAV binding protein.

[0134] [Table 7]

[0135] Example 7 Accumulation of stabilizing amino acid substitutions (part 1) The amino acid substitutions involved in improving the thermal and acid stability of the AAV binding protein, as identified in Example 6, were accumulated in AVR2 (SEQ ID NO: 18) to further improve stability. Specifically, among the (mutated) AAV binding proteins with amino acid substitutions shown in Table 7, No. 75 (AVR2 with the amino acid substitution Ser476Arg accumulated) was selected (named AVR3), and the protein was accumulated with any of the amino acid substitutions shown in (a) to (d) below. In addition, in the amino acid sequence of AVR3 (SEQ ID NO: 32), the substitution Ser476Arg is located at position 189 in SEQ ID NO: 32. (a) Ile319Phe (designated AVR4a) (b) Asn487Asp (designated AVR4d) (c) Ile319Phe and Lys323Glu (designated AVR5b) (d) Lys399Glu and Asn487Asp (designated AVR5e) The methods for producing each AAV binding protein are described in detail below.

[0136] (a)AVR4a From the amino acid substitutions involved in improving stability to heat and acid, which were revealed in Example 6, Ile319Phe was selected and accumulated in AVR3 (SEQ ID NO: 32) to prepare AVR4a. Specifically, AVR4a was prepared by introducing a mutation that generates Ile319Phe into the polynucleotide (SEQ ID NO: 31) encoding AVR3 (SEQ ID NO: 32).

[0137] (a-1) Using the plasmid pET-AVR3 into which a polynucleotide (SEQ ID NO: 31) encoding AVR3 was inserted as a template, and using oligonucleotides consisting of the sequences described in SEQ ID NO: 4 and SEQ ID NO: 33 (5'-CCATGGGCTCTGCAGGCGAGTCGGTTCAGTTTACCC-3') as PCR primers, a reaction solution with the composition shown in Table 1 was prepared. The reaction solution was heat-treated at 98°C for 5 minutes, and PCR was performed by carrying out 30 cycles of a first step at 98°C for 10 seconds, a second step at 55°C for 5 seconds, and a third step at 72°C for 1 minute, and finally heat-treated at 72°C for 5 minutes.

[0138] (a-2) The amplified PCR product was subjected to agarose gel electrophoresis and purified from the gel using a QIAquick Gel Extraction kit (Qiagen). The purified PCR product was named 4ap.

[0139] (a-3) After purifying 4ap obtained in (a-2), it was digested with the restriction enzymes NcoI and XhoI and ligated into the expression vector pET26b (Merck Millipore) which had been previously digested with the restriction enzymes NcoI and XhoI. The ligation product was used to transform Escherichia coli BL21 strain (DE3).

[0140] (a-4) The transformant obtained in (a-3) was cultured in LB medium supplemented with 50 μg / mL kanamycin. Plasmids were extracted from the collected cells (transformants) to obtain plasmid pET-AVR4a, which contains a polynucleotide encoding AVR4a with four amino acid substitutions relative to the wild-type AAV binding protein.

[0141] (a-5) The nucleotide sequence of pET-AVR4a was analyzed in the same manner as in Example 1(5). The amino acid sequence of AVR4a to which a signal sequence and a polyhistidine tag have been added is shown in SEQ ID NO: 34, and the sequence of a polynucleotide encoding the AVR4a is shown in SEQ ID NO: 35. In SEQ ID NO: 34, the region from the 1st methionine (Met) to the 22nd alanine (Ala) is the PelB signal peptide, the region from the 25th serine (Ser) to the 213th aspartic acid (Asp) is the AAV binding protein AVR4a (corresponding to the region from the 312th to the 500th in domain 1 and domain 2, SEQ ID NO: 1), and the region from the 214th to the 219th histidine (His) is the tag sequence. In SEQ ID NO: 34, the phenylalanine of Ile319Phe is at the 32nd position, the cysteine ​​of Tyr342Cys is at the 55th position, the serine of Gly390Ser is at the 103rd position, and the arginine of Ser476Arg is at the 189th position.

[0142] (b) AVR4d From the amino acid substitutions involved in improving stability to heat and acid, which were revealed in Example 6, Asn487Asp was selected and accumulated in AVR3 (SEQ ID NO: 32) to prepare AVR4d. Specifically, AVR4d was prepared by introducing a mutation that generates Asn487Asp into the polynucleotide (SEQ ID NO: 31) encoding AVR3 (SEQ ID NO: 32).

[0143] (b-1) Using pET-AVR3 as a template and oligonucleotides consisting of the sequences set forth in SEQ ID NO:5 and SEQ ID NO:36 (5'-GATTCTGATGGCGCAACCGACTCCACCACC-3') as PCR primers, a reaction solution having the composition shown in Table 8 was prepared. The reaction solution was heat-treated at 98°C for 5 minutes, and PCR was performed by carrying out 30 cycles of reaction in which one cycle consisted of a first step at 98°C for 10 seconds, a second step at 55°C for 5 seconds, and a third step at 72°C for 1 minute, and finally a heat treatment at 72°C for 5 minutes.

[0144] [Table 8]

[0145] (b-2) The amplified PCR product was purified in the same manner as in (a-2). The purified PCR product was named 4dF.

[0146] (b-3) PCR was carried out in the same manner as in (b-1) and (b-2) except that pET-AVR3 was used as a template and oligonucleotides consisting of the sequences described in SEQ ID NO: 6 and SEQ ID NO: 37 (5'-GGTGGTGGAGTCGGTTGCGCCATCAGAATC-3') were used as PCR primers. The purified PCR product was named 4dR.

[0147] (b-4) After mixing the two PCR products (4dF, 4dR) obtained in (b-2) and (b-3), a reaction solution having the composition shown in Table 9 was prepared. The reaction solution was heat-treated at 98°C for 5 minutes, and then PCR was performed in which 30 cycles of reaction were performed, each cycle consisting of a first step at 98°C for 10 seconds, a second step at 55°C for 5 seconds, and a third step at 72°C for 1 minute, to obtain the PCR product 4dp in which 4dF and 4dR were linked.

[0148] [Table 9]

[0149] (b-5) The PCR product 4dp obtained in (b-4) was treated in the same manner as in (a-3) and (a-4) to obtain plasmid pET-AVR4d containing a polynucleotide encoding AVR4d, which has four amino acid substitutions relative to the wild-type AAV binding protein.

[0150] (b-6) The nucleotide sequence of pET-AVR4d was analyzed in the same manner as in Example 1(5). The amino acid sequence of AVR4d to which a signal sequence and a polyhistidine tag have been added is shown in SEQ ID NO: 38, and the sequence of a polynucleotide encoding the AVR4d is shown in SEQ ID NO: 39. In SEQ ID NO: 38, the PelB signal peptide is from the 1st methionine (Met) to the 22nd alanine (Ala), the AAV binding protein AVR4d is from the 25th serine (Ser) to the 213th aspartic acid (Asp) (corresponding to the region from the 312th to the 500th in SEQ ID NO: 1), and the tag sequence is from the 214th to the 219th histidine (His). In SEQ ID NO: 38, the cysteine ​​of Tyr342Cys is at the 55th position, the serine of Gly390Ser is at the 103rd position, the arginine of Ser476Arg is at the 189th position, and the aspartic acid of Asn487Asp is at the 200th position.

[0151] (c)AVR5b Among the amino acid substitutions involved in improving stability to heat and acid, which were revealed in Example 6, Ile319Phe and Lys323Glu were selected, and AVR5b was prepared by integrating them into AVR3 (SEQ ID NO: 32). Specifically, AVR5b was prepared by introducing mutations into the polynucleotide (SEQ ID NO: 31) encoding AVR3 (SEQ ID NO: 32) to generate Ile319Phe and Lys323Glu.

[0152] (c-1) Using pET-AVR3 as a template and oligonucleotides consisting of the sequences set forth in SEQ ID NO: 40 (5'-TTTT[GGTCTC]AGTTCTCCGGCAGGGTAACTGAACCGAC-3') and SEQ ID NO: 41 (5'-TTTT[GGTCTC]AGAACGAAGTACAACTGAATGCGTATGTG-3') as PCR primers (the brackets in SEQ ID NOs: 40 and 41 indicate the restriction enzyme BsaI recognition sequence), a reaction solution having the composition shown in Table 8 was prepared. The reaction solution was heat-treated at 98°C for 5 minutes, and 30 cycles of reaction were carried out, each cycle consisting of a first step at 98°C for 10 seconds, a second step at 55°C for 5 seconds, and a third step at 72°C for 6 minutes, and finally heat-treated at 72°C for 5 minutes to perform PCR.

[0153] (c-2) The amplified PCR product was purified in the same manner as in (a-2). The purified PCR product was named 5bp.

[0154] (c-3) The 5 bp fragment obtained in (c-2) was treated with the restriction enzyme DpnI (NEB) at 37° C. for 1.5 hours, and then treated at 80° C. for 20 minutes. The resulting gene was subjected to agarose gel electrophoresis and purified from the gel using a QIAquick Gel Extraction kit (Qiagen).

[0155] (c-4) The DpnI treatment product obtained in (c-3) was digested with the restriction enzyme BsaI (NEB) in a reaction solution having the composition shown in Table 10, and ligated with T4 DNA ligase (NEB).

[0156] [Table 10] (c-5) The ligation product of (c-4) was used to transform E. coli BL21 strain (DE3). The resulting transformant was cultured in LB medium supplemented with 50 μg / mL kanamycin. Plasmids were extracted from the recovered cells (transformants) to obtain plasmid pET-AVR5b, which contains a polynucleotide encoding AVR5b with five amino acid substitutions relative to the wild-type AAV binding protein.

[0157] (c-6) The nucleotide sequence of pET-AVR5b was analyzed in the same manner as in Example 1(5). The amino acid sequence of AVR5b with a signal sequence and a polyhistidine tag added is shown in SEQ ID NO: 42, and the sequence of the polynucleotide encoding AVR5b is shown in SEQ ID NO: 43. In SEQ ID NO: 42, the 1st 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 AVR5b (corresponding to the region from the 312th to the 500th in SEQ ID NO: 1), and the 214th to 219th histidine (His) is the tag sequence. In SEQ ID NO: 42, the phenylalanine of Ile319Phe is at the 32nd position, the glutamic acid of Lys323Glu is at the 36th position, the cysteine ​​of Tyr342Cys is at the 55th position, the serine of Gly390Ser is at the 103rd position, and the arginine of Ser476Arg is at the 189th position.

[0158] (d)AVR5e Among the amino acid substitutions involved in improving stability to heat and acid, which were revealed in Example 6, Lys399Glu and Asn487Asp were selected, and AVR5e was prepared by integrating them into AVR3 (SEQ ID NO: 32). Specifically, AVR5e was prepared by introducing a mutation to generate Lys399Glu into the polynucleotide (SEQ ID NO: 39) encoding AVR4d (SEQ ID NO: 38) prepared in (b).

[0159] (d-1) A plasmid pET-AVR5e containing a polynucleotide encoding AVR5e with five amino acid substitutions relative to the wild-type AAV binding protein was obtained in the same manner as in (c-1) to (c-5), except that pET-AVR4d obtained in (b-5) was used as a template and oligonucleotides consisting of the sequences set forth in SEQ ID NO: 44 (5'-TTTT[GGTCTC]ACTTGCGTGGCTCCGGTTCCACGGTAACG-3') and SEQ ID NO: 45 (5'-TTTT[GGTCTC]ACAAGAACCGTCCTCCGATCGCCATTG-3') (the brackets in SEQ ID NOs: 44 and 45 indicate the restriction enzyme BsaI recognition sequence) were used as PCR primers.

[0160] (d-2) The nucleotide sequence of pET-AVR5e was analyzed in the same manner as in Example 1(5). The amino acid sequence of AVR5e to which a signal sequence and a polyhistidine tag have been added is shown in SEQ ID NO: 46, and the sequence of a polynucleotide encoding the AVR5e is shown in SEQ ID NO: 47. In SEQ ID NO: 46, the portion from methionine (Met) at position 1 to alanine (Ala) at position 22 is the PelB signal peptide, the portion from serine (Ser) at position 25 to aspartic acid (Asp) at position 213 is the AAV binding protein AVR5e (corresponding to the region from positions 312 to 500 in SEQ ID NO: 1), and the histidines (His) at positions 214 to 219 are the tag sequence. In addition, in SEQ ID NO: 46, the cysteine ​​of Tyr342Cys is at the 55th position, the serine of Gly390Ser is at the 103rd position, the glutamic acid of Lys399Glu is at the 112th position, the arginine of Ser476Arg is at the 189th position, and the aspartic acid of Asn487Asp is at the 200th position.

[0161] Example 8: Evaluation of acid stability of mutant AAV binding proteins (part 2) (1) Transformants expressing the mutant AAV binding protein AVR3 obtained in Example 6 and the mutant AAV binding proteins (AVR4a, AVR4d, AVR5b, and AVR5e) prepared in Example 7 were cultured by the methods described in Examples 3(4) and (5), respectively, and the culture medium was centrifuged to obtain culture supernatants containing each of the expressed mutant AAV binding proteins.

[0162] (2) The binding activity of the mutant AAV binding protein in the culture supernatant obtained in (1) to VLP2 was measured using the ELISA method described in Example 2(6). Based on the absorbance at 450 nm, which is the measurement result, the culture supernatant obtained in (1) was diluted with pure water so that the measured value was similar.

[0163] (3) Each diluted mutant AAV binding protein solution was divided into four portions and mixed with an equal amount of 0.1 M glycine hydrochloride buffer (pH 3.0). Three of the four samples were heated at 51.5°C, 60.1°C, and 66.1°C for 15 minutes, and the remaining sample was left to stand at room temperature (25°C) for 15 minutes. The sample was then mixed with 0.5 M MES buffer (pH 6.0) in a ratio of 3:2 to adjust the pH to around 6, and the binding activity with VLP2 was measured by the ELISA method described in Example 2(6).

[0164] (4) The residual activity was calculated by dividing the absorbance at 450 nm after the heat and acid treatment described in (3) by the absorbance at 450 nm after standing at room temperature. The results are shown in Table 11. The mutant AAV binding proteins prepared in Example 7 (AVR3, AVR4a, AVR4d, AVR5b, AVR5e) had higher residual activity than AVR3 (No. 75 in Table 7) regardless of the temperature at which they were treated. This shows that the five mutant AAV binding proteins have significantly improved stability to heat treatment under acidic conditions, i.e., to acid and heat, compared to the wild-type AAV binding protein.

[0165] [Table 11]

[0166] Example 9: Construction of mutant AAV binding proteins with improved C-terminus (1) Using pET-AVR5e prepared in Example 7(d) as a template and oligonucleotides consisting of the sequences described in SEQ ID NO: 3 and SEQ ID NO: 48 (5'-AT[CTCGAG]TCATCCGCAGGTATCGTTGCGGCAATGATGATGATGATGATGGTCTAC-3') as PCR primers (the brackets in SEQ ID NO: 48 indicate the restriction enzyme XhoI recognition sequence), a reaction solution having the composition shown in Table 12 was prepared. After heat-treating the reaction solution at 94°C for 2 minutes, PCR was performed by repeating 25 cycles of a first step at 98°C for 10 seconds, a second step at 52°C for 30 seconds, and a third step at 68°C for 1.5 minutes.

[0167] [Table 12]

[0168] (2) The PCR product obtained in (1) was subjected to agarose gel electrophoresis, purified from the gel using a QIAquick Gel Extraction kit (Qiagen), digested with restriction enzymes NcoI and XhoI, and ligated into an expression vector pET26b (Merck Millipore) that had been previously digested with restriction enzymes NcoI and XhoI, and the ligation product was used to transform Escherichia coli BL21 strain (DE3).

[0169] (3) The obtained transformant was cultured in LB medium containing 50 μg / mL kanamycin, and then the plasmid pET-AVR5eHC encoding a protein (named AVR5eHC) having an immobilization tag at the C-terminus of AVR5e was obtained using a QIAprep Spin Miniprep kit (Qiagen).

[0170] (4) The nucleotide sequence of the obtained pET-AVR5eHC was analyzed in the same manner as in Example 1(5). The nucleotide sequence of AVR5eHC is shown in SEQ ID NO: 49, and the amino acid sequence is shown in SEQ ID NO: 50. In SEQ ID NO: 50, the portion from methionine (Met) at position 1 to alanine (Ala) at position 22 is the PelB signal peptide, the portion from serine (Ser) at position 25 to aspartic acid (Asp) at position 213 is the AAV binding protein AVR5e, the portion from histidine (His) at positions 214 to 219 is a histidine tag, and the portion from cysteine ​​(Cys) at position 220 to glycine (Gly) at position 226 is a cysteine ​​tag sequence that is a tag for immobilization.

[0171] Example 10 Large-scale preparation of mutant AAV binding proteins with improved C-terminus (1) The transformant capable of expressing AVR5eHC obtained by transforming the Escherichia coli BL21(DE3) strain with the pET-AVR5eHC prepared in Example 9(3) was inoculated into a 200 mL baffled flask containing 40 mL of liquid medium (phytone peptone 16 g / L, yeast extract 10 g / L, sodium chloride 5 g / L) containing 50 μg / mL kanamycin, and pre-cultured by aerobically shaking at 37° C. for 10 hours.

[0172] (2) 10 mL of the preculture solution from (1) was inoculated into a 3 L fermenter (Bio-T) containing 0.9 L of liquid medium containing 20 g / L glucose, 40 g / L yeast extract, 6 g / L trisodium phosphate dodecahydrate, 18 g / L disodium hydrogen phosphate dodecahydrate, 2 g / L ammonium chloride, 2 g / L magnesium sulfate, 2 g / L ferrous sulfate, 10 g / L manganese chloride, and 50 mg / L kanamycin sulfate. The main culture was started under the conditions of a set temperature of 30°C, pH 6.9 to 7.1, and aeration of 3 VVM. The pH was controlled using 50% (v / v) phosphoric acid as an acid and 14% (w / v) ammonia water as an alkali, and the dissolved oxygen was controlled by changing the stirring speed within the range of lower limit 500 rpm and upper limit 1000 rpm. After the start of culture, when the glucose concentration could no longer be measured, a feed medium (glucose 425 g / L, yeast extract 140 g / L, magnesium sulfate heptahydrate 12 g / L) was added while controlling the dissolved oxygen.

[0173] (3) When the absorbance at 600 nm reached approximately 100, the set temperature was controlled to be lowered to 25° C. After confirming that the set temperature had been reached, 1.1 mL of 0.5 mM IPTG was added, and the culture was continued at 25° C. Approximately 42 hours after the start of the culture, the culture was stopped, and the culture solution was centrifuged at 4° C. and 8000 rpm for 20 minutes to collect the bacterial cells.

[0174] (4) The cells collected in (3) were suspended in 20 mM Tris-HCl buffer (pH 7.4) containing 0.5 M sodium chloride and 20 mM imidazole at 5 mL / 1 g (cells), and then disrupted using an ultrasonic generator (Insonator 201M [Kubota Shoji Co., Ltd.]) at 8° C. for about 10 minutes at an output of about 150 W. The disrupted cell solution was centrifuged twice at 8,000 rpm for 20 minutes at 4° C., and the supernatant was collected.

[0175] (5) The supernatant obtained in (4) was applied to an XK26 / 20 column (manufactured by Cytiva) packed with 50 mL of Ni Sepharose 6 Fast Flow (manufactured by Cytiva) that had been previously equilibrated with Tris-HCl buffer (pH 7.4) containing 0.5 M sodium chloride and 20 mM imidazole (hereinafter also referred to as "equilibration solution A"). After washing with equilibration solution A, the column was eluted with 20 mM Tris-HCl buffer (pH 7.4) containing 0.5 M imidazole and 150 mM sodium chloride.

[0176] (6) The eluate obtained in (5) was dialyzed against 20 mM Tris buffer (pH 7.4) containing 4 mM imidazole and 150 mM sodium chloride, and then applied to an XK26 / 20 column (Cytiva) packed with 50 mL of TALON Metal Affinity Resin (Takara Bio) equilibrated with the same buffer. After washing with the buffer used for equilibration, the column was eluted with 20 mM Tris-HCl buffer (pH 7.4) containing 0.5 M imidazole and 150 mM sodium chloride.

[0177] (7) The eluate obtained in (6) was dialyzed against 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride to prepare approximately 200 mg of AVR5eHC protein.

[0178] Example 11: Construction of a viral vector (AAV2-EGFP) (1) A nucleotide sequence was designed in which a restriction enzyme EcoRI recognition sequence (GAATTC) was added to the 5'-end of a polynucleotide encoding EGFP (enhanced green fluorescent protein) consisting of the amino acid sequence set forth in SEQ ID NO:51, and a stop codon (TAG) and a BamHI recognition sequence (GGATTC) were added to the 3'-end (SEQ ID NO:52).

[0179] (2) A polynucleotide consisting of the sequence set forth in SEQ ID NO:52 was totally synthesized and cloned into a plasmid (entrusted to FASMAC, named pUC-EGFP). E. coli JM109 strain was transformed with pUC-EGFP, and the resulting transformant was cultured. pUC-EGFP was extracted from the culture medium using a QIAprep Spin Miniprep kit (Qiagen).

[0180] (3) The pUC-EGFP obtained in (2) was digested with the restriction enzymes EcoRI and BamHI, and then ligated to an expression vector pAAV-CMV (Takara Bio) previously digested with the restriction enzymes EcoRI and BamHI, and the ligation product was used to transform E. coli JM109 strain. The obtained transformant was cultured in LB medium containing 100 μg / mL carbenicillin, and the EGFP-expressing vector pAAV-EGFP was extracted using a QIAprep Spin Miniprep kit (Qiagen).

[0181] (4) The transformant obtained in (3) was cultured overnight at 37°C with shaking in a 5 L baffled flask containing 1 L of 2YT medium containing 100 μg / mL carbenicillin. After the culture was completed, the cells were collected by centrifugation. A large amount of pAAV-EGFP was prepared from the collected cells using a Plasmid Mega Kit (Qiagen).

[0182] (5) Preparation of AAV2-EGFP (5-1) The E. coli JM109 strain was transformed with the pRC2-mi342 Vector (Takara Bio) and the pHelper Vector (Takara Bio). The resulting transformant was used in the same manner as in (4) to prepare large quantities of pRC2-mi342 and pHelper.

[0183] (5-2) HEK293T cells were cultured in 10 T-225 flasks (Thermo Fisher Scientific) containing 45 mL of D-MEM medium (Fujifilm Wako Pure Chemical Industries, Ltd.) containing 10% (v / v) bovine serum. Gene transfer was performed by adding the pAAV-EGFP prepared in (4), the pRC2-mi342 and pHelper prepared in (5-1), and a complex of polyethyleneimine (Polysciences), and the cells were cultured for 3 days under conditions of 5% (v / v) carbon dioxide and 37°C. After the culture, the cells were detached by centrifugation and collected, and the cells obtained from each of the five T-225 flasks were frozen and stored at -80°C.

[0184] (5-3) The frozen cells obtained in (5-2) were thawed and suspended in 10 mL of 20 mM Tris-HCl buffer (pH 7.4) containing 0.5 M sodium chloride, 4 mM magnesium chloride, and 0.01% (w / v) Tween 20 (trade name). 1 / 20 volume of Benzonase (Merck Millipore) was added, and the mixture was left to stand at 37°C for 1 hour, and then centrifuged at 13,000 x g and 4°C for 10 minutes to obtain a supernatant. Ammonium sulfate was added to the obtained supernatant to 15% saturation, and centrifuged again under the same conditions. The obtained supernatant was passed through a filter with a pore size of 0.45 μm to remove floating matter.

[0185] (5-4) The supernatant from which the floating matter had been removed was applied to a 5 mL AVB Sepharose column (manufactured by Cytiva) previously equilibrated with 20 mM Tris-HCl buffer (pH 8.0) containing 0.5 M sodium chloride (hereinafter also referred to as "equilibration solution B"). After washing with equilibration solution B, elution was performed with 0.1 M acetate buffer (pH 2.5) containing 0.5 M sodium chloride. The obtained eluate was neutralized by adding 1 / 4 volume of 1 M Tris-HCl buffer (pH 8.5) containing 20 mM magnesium chloride to obtain an AAV vector AAV2-EGFP solution. The AAV2-EGFP concentration in the solution was quantified by qPCR using the AAVpro Titration Kit (manufactured by Takara Bio).

[0186] Example 12 Preparation of AAV-binding protein immobilized gel (part 1) (1) A gel was prepared by chemically modifying the hydroxyl groups on the surface of a hydrophilic vinyl polymer for separation (Tosoh Corporation: Toyopearl) to introduce maleimide groups. 28 mg of the AVR5eHC protein prepared in Example 10 and TCEP (Tris (2-CarboxyEthyl) Phosphine) at a final concentration of 0.1 mM as a reducing agent were added to 3.5 g of the prepared gel, and the mixture was allowed to react by shaking at pH 7.4 and 4°C for 15 hours. As a result, a gel in which AVR5e was immobilized (named AVR5e immobilized gel) was prepared.

[0187] (2) 2 mL of the AVR5e-immobilized gel prepared in (1) was packed into a Tricorn 10 / 50 column (manufactured by Cytiva) to prepare a column (named AVR5e column). The AVR5e column was connected to AKTAprime plus (manufactured by Cytiva) and equilibrated with 50 mM phosphate buffer (pH 7.4) containing 150 mM sodium chloride and 0.05% (w / v) Tween 20 (trade name) (hereinafter also referred to as "equilibration solution C"), and 10 mL of the cell extract containing AAV2-EGFP obtained in Example 11 (5-3) was applied at a flow rate of 1 mL / min.

[0188] (3) After washing with equilibration solution C, AAV2-EGFP was eluted from the AVR5e column with 0.1 M acetate buffer (pH 2.5) containing 0.5 M sodium chloride. The eluate was collected in 1 mL portions and collected as fractions. The collected fractions were neutralized by adding 250 μL of 1 M Tris-HCl buffer (pH 8.5) containing 20 mM magnesium chloride.

[0189] (4) To compare the purity of the eluate obtained in (3) with that of the cell extract obtained in Example 10 (5-3), which is the unpurified sample, SDS-PAGE was performed and silver stained using the Pierce Silver Stain Kit (Thermo Fisher Scientific). The chromatographic pattern obtained by the AVR5e column in (3) is shown in FIG. 2, and the results of SDS-PAGE in (4) are shown in FIG. 3. The AAV2-EGFP peak appeared at an elution time of about 20 minutes (FIG. 2). When the fractions corresponding to the peaks (Fr6 to Fr9) were subjected to SDS-PAGE, only bands corresponding to VP1, VP2, and VP3 constituting AAV were confirmed (FIG. 3). On the other hand, in addition to the above bands, numerous bands derived from impurities were confirmed in the cell extract applied to the AVR5e column (FIG. 3). From the above results, it can be seen that AAV can be highly purified by the AAV adsorbent containing a gel and the AAV-binding protein of the present invention immobilized on the gel.

[0190] Example 13: Construction and screening of a mutant library of AAV binding proteins (part 3) Among the mutant AAV-binding proteins evaluated in Example 7, AVR5e (Tyr342Cys, Gly390Ser, Lys399Glu, Ser476Arg and Asn487Asp substitutions; sequence number 46) was selected, and mutations were randomly introduced into the polynucleotide portion encoding the protein by error-prone PCR.

[0191] (1) A reaction solution having the composition shown in Table 6 was prepared using the plasmid pET-AVR5e as a template, into which a polynucleotide (SEQ ID NO: 47) encoding AVR5e (SEQ ID NO: 46) was inserted, and error-prone PCR was then carried out under the same temperature conditions as in Example 3(1). Mutations were introduced into the polynucleotide encoding AVR5e by the error-prone PCR, with the average mutation introduction rate being 1.7 amino acid mutations per molecule.

[0192] (2) After purifying the PCR product obtained in (1), a random mutant library was prepared by the method described in Example 6 (2) and (3), and the library (transformants) was cultured by the method described in Example 3 (4) and (5).

[0193] (3) The culture medium from (2) was centrifuged, and the resulting culture supernatant was diluted 32-fold with ultrapure water. 60 μL of the diluted culture medium was mixed with 60 μL of 0.1 M glycine-HCl buffer (pH 3.0), and subjected to heat and acid treatment at 61.0° C. for 15 minutes.

[0194] (4) Binding activity of AAV binding protein to VLP2 after treatment in (3) and the binding of AAV binding protein to VLP2 without the treatment of (3). The combined activity was measured by the ELISA method described in Example 2(6). The remaining activity was calculated by dividing the binding activity of the AAV binding protein to VLP2 when heat treatment was performed by the binding activity of the AAV binding protein to VLP2 when heat treatment was not performed.

[0195] (5) A random mutant library of about 1,800 strains was evaluated using the method of (4), and transformants expressing AAV binding proteins with improved residual activity compared to the parent molecule AVR5e were selected from the library. The selected transformants were cultured, and expression vectors were prepared using a QIAprep Spin Miniprep kit (Qiagen).

[0196] (6) The sequence of the polynucleotide region encoding the AAV binding protein inserted into the obtained expression vector was analyzed for nucleotide sequence by the method described in Example 1(5), and the amino acid mutation sites were identified. Table 13 summarizes the amino acid substitution positions relative to AVR5e (Tyr342Cys, Gly390Ser, Lys399Glu, Ser476Arg and Asn487Asp substitutions) of the AAV binding proteins expressed by the transformants selected in (5) above, as well as the residual activity (%) after heat and acid treatment.

[0197] In the amino acid sequence of SEQ ID NO: 1, the amino acid residues from the 312th serine (Ser) to the 500th aspartic acid (Asp) are Val317Asp, Gln318Pro, Thr320Ala, Asn324Asp, Val326Ala, Val326Glu, Leu328Pro, Asn329His, Val332Ala, Lys362Glu, Lys362Asn, Lys371Glu, Lys371 ... An AAV binding protein having at least one of the following amino acid substitutions, eu376Pro, Glu378Gly, Glu378Val, Phe379Ser, Phe379Tyr, Val381Asp, Gly392Cys, Thr397Ser, Glu401Val, Arg406His, Asn492Asp, or Val499Glu, can be said to have improved heat and acid stability compared to the wild-type AAV binding protein.

[0198] [Table 13]

[0199] Example 14 Accumulation of stabilizing amino acid substitutions (part 2) The amino acid substitutions involved in improving the thermal and acid stability of the AAV binding protein as revealed in Example 13 were accumulated in AVR5e (SEQ ID NO: 46) to further improve stability. Specifically, among the (mutated) AAV binding proteins with amino acid substitutions shown in Table 13, No. 93 (AVR5e with Asn324Asp and Lys362Glu amino acid substitutions accumulated) (named AVR7a) and No. 91 (AVR5e with Val317Asp and Lys371Asn amino acid substitutions accumulated) (named AVR7b) were selected, and the following amino acid substitutions (a) or (b) were accumulated in the proteins. In addition, in the amino acid sequence of AVR7a (SEQ ID NO: 53), the Asn324Asp substitution is located at the 37th position in SEQ ID NO: 53, and the Lys362Glu substitution is located at the 75th position in SEQ ID NO: 53. In addition, in the amino acid sequence of AVR7b (SEQ ID NO:55), the Val317Asp substitution is located at position 30 in SEQ ID NO:55, and the Lys371Asn substitution is located at position 84 in SEQ ID NO:55. (a) Introducing Val317Asp into AVR7a (named AVR8a) (b) Lys362Glu was introduced into AVR7b (named AVR8g). The methods for producing each AAV binding protein are described in detail below.

[0200] (a)AVR8a Among the amino acid substitutions involved in improving stability to heat and acid, which were revealed in Example 13, Val317Asp was selected and integrated into AVR7a (SEQ ID NO: 53) to produce AVR8a. Specifically, AVR8a was produced by introducing a mutation that generates Val317Asp into the polynucleotide (SEQ ID NO: 54) encoding AVR7a (SEQ ID NO: 53).

[0201] (a-1) A reaction solution having the composition shown in Table 12 was prepared using the plasmid pET-AVR7a into which a polynucleotide encoding AVR7a (SEQ ID NO: 54) was inserted as a template and oligonucleotides consisting of the sequences set forth in SEQ ID NO: 57 (5'-TTTT[GGTCTC]AGCAGGCGAGTCGGATCAGATTACCC-3') and SEQ ID NO: 58 (5'-TTTT[GGTCTC]ACTGCAGAGCCCATGGCCATCGCCGG-3') (the brackets in SEQ ID NO: 57 and 58 indicate the restriction enzyme BsaI recognition sequence) as PCR primers. The reaction solution was heat-treated at 98°C for 5 minutes, and 30 cycles of reaction were carried out, each cycle consisting of a first step at 98°C for 10 seconds, a second step at 55°C for 5 seconds, and a third step at 72°C for 6 minutes, and finally heat-treated at 72°C for 5 minutes, thereby carrying out PCR.

[0202] (a-2) The amplified PCR product was subjected to agarose gel electrophoresis and purified from the gel using a QIAquick Gel Extraction kit (Qiagen). The purified PCR product was named 8ap.

[0203] (a-3) The 8ap obtained in (a-2) was treated with the restriction enzyme DpnI (NEB) at 37° C. for 1.5 hours, and then treated at 80° C. for 20 minutes. The obtained gene was subjected to agarose gel electrophoresis and purified from the gel using a QIAquick Gel Extraction kit (Qiagen).

[0204] (a-4) The DpnI treatment product obtained in (a-3) was digested with the restriction enzyme BsaI (NEB) in a reaction solution having the composition shown in Table 10, and ligated with T4 DNA ligase (NEB).

[0205] (a-5) The ligation product of (a-4) was used to transform E. coli BL21 strain (DE3). The resulting transformant was cultured in LB medium supplemented with 50 μg / mL kanamycin. Plasmids were extracted from the collected cells (transformants) to obtain plasmid pET-AVR8a, which contains a polynucleotide encoding AVR8a with eight amino acid substitutions relative to the wild-type AAV binding protein.

[0206] (a-6) The nucleotide sequence of pET-AVR8a was analyzed in the same manner as in Example 1(5). The amino acid sequence of AVR8a to which a signal sequence and a polyhistidine tag have been added is shown in SEQ ID NO: 59, and the sequence of a polynucleotide encoding the AVR8a is shown in SEQ ID NO: 60. In SEQ ID NO: 59, the portion from methionine (Met) at position 1 to alanine (Ala) at position 22 is the PelB signal peptide, the portion from serine (Ser) at position 25 to aspartic acid (Asp) at position 213 is the AAV binding protein AVR8a (domain 1 and domain 2, corresponding to the region from positions 312 to 500 in SEQ ID NO: 1), and the portion from histidine (His) at positions 214 to 219 is the tag sequence. In addition, in SEQ ID NO:59, the aspartic acid of Val317Asp is at the 30th position, the aspartic acid of Asn324Asp is at the 37th position, the cysteine ​​of Tyr342Cys is at the 55th position, the glutamic acid of Lys362Glu is at the 75th position, the serine of Gly390Ser is at the 103rd position, the glutamic acid of Lys399Glu is at the 112th position, the arginine of Ser476Arg is at the 189th position, and the aspartic acid of Asn487Asp is at the 200th position.

[0207] (b)AVR8g Lys362Glu was selected from the amino acid substitutions involved in improving stability to heat and acid, as revealed in Example 13, and was integrated into AVR7b (SEQ ID NO: 55) to produce AVR8g. Specifically, AVR8g was produced by introducing a mutation into the polynucleotide (SEQ ID NO: 56) encoding AVR7b (SEQ ID NO: 55) to generate Lys362Glu.

[0208] (b-1) Plasmid pET-AVR7b into which a polynucleotide encoding AVR7b (SEQ ID NO: 56) was inserted was used as a template, and oligonucleotides consisting of the sequences set forth in SEQ ID NO: 61 (5'-TTTT[GGTCTC]ACGAGATGGAAGGAACATTCGCAG-3') and SEQ ID NO: 62 (5'-TTTT[GGTCTC]ACTCGCCGCTGTAGTCGCGAGG-3') (the brackets in SEQ ID NOs: 61 and 62 indicate the restriction enzyme BsaI recognition sequence) were used as PCR primers. Except for this, a plasmid pET-AVR8g containing a polynucleotide encoding AVR8g in which eight amino acid substitutions were made with respect to the wild-type AAV binding protein was obtained in the same manner as in (a-1) to (a-5).

[0209] (b-2) The nucleotide sequence of pET-AVR8g was analyzed in the same manner as in Example 1(5). The amino acid sequence of AVR8g to which a signal sequence and a polyhistidine tag have been added is shown in SEQ ID NO: 63, and the sequence of a polynucleotide encoding the AVR8g is shown in SEQ ID NO: 64. In SEQ ID NO: 63, the portion from methionine (Met) at position 1 to alanine (Ala) at position 22 represents the PelB signal peptide, serine (Ser) at position 25 to aspartic acid (Asp) at position 213 represents the AAV binding protein AVR8g (domain 1 and domain 2, corresponding to the region from positions 312 to 500 in SEQ ID NO: 1), and histidine (His) at positions 214 to 219 represents the tag sequence. In addition, in SEQ ID NO:63, the aspartic acid of Val317Asp is at the 30th position, the cysteine ​​of Tyr342Cys is at the 55th position, the glutamic acid of Lys362Glu is at the 75th position, the aspartic acid of Lys371Asn is at the 84th position, the serine of Gly390Ser is at the 103rd position, the glutamic acid of Lys399Glu is at the 112th position, the arginine of Ser476Arg is at the 189th position, and the aspartic acid of Asn487Asp is at the 200th position.

[0210] Example 15: Evaluation of acid stability of mutant AAV binding proteins (part 3) (1) Transformants expressing the mutant AAV binding protein AVR7a obtained in Example 13 and the mutant AAV binding proteins (AVR8a and AVR8g) prepared in Example 14 were cultured by the methods described in Examples 3(4) and (5), respectively, and the culture medium was centrifuged to obtain culture supernatants containing each of the expressed mutant AAV binding proteins.

[0211] (2) The binding activity of the mutant AAV binding protein in the culture supernatant obtained in (1) to VLP2 was measured using the ELISA method described in Example 2(6). Based on the absorbance at 450 nm, which is the measurement result, the culture supernatant obtained in (1) was diluted with pure water so that the measured value was similar.

[0212] (3) Each diluted mutant AAV binding protein solution was divided into three portions and mixed with an equal amount of 0.1 M glycine hydrochloride buffer (pH 3.0). Two of the three samples were heated at 80.8°C and 89.8°C for 15 minutes, and the remaining sample was left to stand at room temperature (25°C) for 15 minutes. The sample was then mixed with 0.5 M MES buffer (pH 6.0) in a ratio of 3:2 to adjust the pH to around 6, and the binding activity with VLP2 was measured by the ELISA method described in Example 2(6).

[0213] (4) The residual activity was calculated by dividing the absorbance at 450 nm after the heat and acid treatment described in (3) by the absorbance at 450 nm after standing at room temperature. The results are shown in Table 14. The mutant AAV binding proteins (AVR8a, AVR8g) prepared in Example 14 had higher residual activity than AVR7a (No. 93 in Table 14) when treated at 80.8° C. This indicates that the two mutant AAV binding proteins have significantly improved stability to heat treatment under acidic conditions, i.e., to acid and heat, compared to the wild-type AAV binding protein.

[0214] [Table 14]

[0215] Example 16: Construction and screening of a mutant library of AAV binding proteins (part 4) Of the mutant AAV-binding proteins evaluated in Example 15, AVR8g (Val317Asp, Tyr342Cys, Lys362Glu, Lys371Asn, Gly390Ser, Lys399Glu, Ser476Arg and Asn487Asp substitutions; sequence number 63) was selected, and mutations were randomly introduced into the polynucleotide portion encoding the protein by error-prone PCR.

[0216] (1) A reaction solution having the composition shown in Table 6 was prepared using the plasmid pET-AVR8g, into which a polynucleotide (SEQ ID NO: 64) encoding AVR8g (SEQ ID NO: 63) was inserted, as a template, and error-prone PCR was then carried out under the same temperature conditions as in Example 3(1). Mutations were introduced into the polynucleotide encoding AVR8g by the error-prone PCR, with the average mutation introduction rate being 2.3 amino acid mutations per molecule.

[0217] (2) After purifying the PCR product obtained in (1), a random mutant library was prepared by the method described in Example 6 (2) and (3), and the library (transformants) was cultured by the method described in Example 3 (4) and (5).

[0218] (3) The culture medium from (2) was centrifuged, and the resulting culture supernatant was diluted 16-fold with ultrapure water. 60 μL of the diluted culture medium was mixed with 60 μL of 0.1 M glycine sodium hydroxide buffer (pH 10.0) and subjected to heat and alkali treatment at 41.2°C for 15 minutes.

[0219] (4) Binding activity of AAV binding protein to VLP2 after treatment in (3) and the binding of AAV binding protein to VLP2 without the treatment of (3). The combined activity was measured by the ELISA method described in Example 2(6). The remaining activity was calculated by dividing the binding activity of the AAV binding protein to VLP2 when heat treatment was performed by the binding activity of the AAV binding protein to VLP2 when heat treatment was not performed.

[0220] (5) A random mutant library of about 1,800 strains was evaluated using the method of (4), and transformants expressing AAV binding proteins with improved residual activity compared to the parent molecule AVR8g were selected from the library. The selected transformants were cultured, and expression vectors were prepared using a QIAprep Spin Miniprep kit (Qiagen).

[0221] (6) The sequence of the polynucleotide region encoding the AAV binding protein inserted into the obtained expression vector was analyzed for nucleotide sequence by the method described in Example 1(5), and the amino acid mutation sites were identified.

[0222] (7) Table 15 shows the amino acid substitution positions relative to AVR8g (Val317Asp, Tyr342Cys, Lys362Glu, Lys371Asn, Gly390Ser, Lys399Glu, Ser476Arg and Asn487Asp substitutions) of the AAV binding proteins expressed by the transformants selected in (6) and the residual activity (%) after heat and alkali treatment.

[0223] In the amino acid sequence of SEQ ID NO:1, the amino acid residues from the 312th serine (Ser) to the 500th aspartic acid (Asp) are: Ser312Pro, Ala313Ser, Lys323Arg, Asn324Ser, Gln327His, Gln327Leu, Asn329Ile, Glu335Gly, Glu335Val, Glu340Val, Thr341Pro, Thr343Ser, Tyr 344Phe, Trp346Cys, Arg353Cys, Tyr355Cys, Ser356Cys, Gly357Cys, Met359Leu, Glu360Lys, Glu360Val, Gly361Cy s, Lys(Glu)362Gly, Ser364Leu, Ile366Thr, Leu369Gln, Lys(Asn)371Asp, Leu372Pro, Leu372Gln, Thr373Ala, Pro 374Leu, Leu376Pro, Glu378Gly, Glu378Val, Phe379Cys, Phe379Ser, Lys380Glu, Val381Ala, Ile382Val, Val383Al a, Glu384Val, Gln386Arg, His389Arg, Tyr393Cys, Val394Asp, Val394Ile, Asn395Ser, Val396Ala, Arg403His, Gln AAV binding proteins containing at least one of the following amino acid substitutions, 415Arg, Thr426Ala, Gln432Leu, Gln432Arg, Gln441Arg, His443Leu, Lys448Glu, Ile456Val, Asp483Asn, Ser488Leu, and Val499Ile, can be said to have improved heat and alkali stability compared to the wild-type AAV binding protein.

[0224] [Table 15]

[0225] Example 17 Monomerization of mutant AAV binding proteins (part 1) From the mutant AAV binding proteins with improved stability to heat and alkali as revealed in Example 16, No. 143 (AVR8g with Val381Ala and Ile382Val amino acid substitutions) (named AVR10) was selected, and the amino acid substitution Tyr(Cys)342Ser was introduced to monomerize the protein (named AVR10s). Specifically, AVR10s was produced by introducing a mutation to generate Tyr(Cys)342Ser into the polynucleotide (SEQ ID NO: 66) encoding AVR10 (SEQ ID NO: 65). Note that in the amino acid sequence of AVR10 (SEQ ID NO: 65), the Val381Ala substitution is located at the 94th position in SEQ ID NO: 65, and the Ile382Val substitution is located at the 95th position in SEQ ID NO: 65.

[0226] (1) A plasmid pET-AVR10s containing a polynucleotide encoding AVR10s with 10 amino acid substitutions relative to the wild-type AAV binding protein was obtained in the same manner as in Example 14(a-1) to (a-5), except that pET26b-AVR10 into which a polynucleotide encoding AVR10 (SEQ ID NO: 66) had been inserted was used as a template, and oligonucleotides consisting of the sequences set forth in SEQ ID NO: 67 (5'-TTTT[GGTCTC]AGAAACCTCTACGTATGACTG-3') and SEQ ID NO: 68 (5'-TTTT[GGTCTC]ATTTCCCCTTTCGGTGG-3') (the brackets in SEQ ID NOs: 67 and 68 indicate the restriction enzyme BsaI recognition sequence) were used as PCR primers.

[0227] (2) The nucleotide sequence of pET-AVR10s was analyzed in the same manner as in Example 1(5). The amino acid sequence of AVR10s to which a signal sequence and a polyhistidine tag have been added is shown in SEQ ID NO: 69, and the sequence of a polynucleotide encoding the AVR10s is shown in SEQ ID NO: 70. In SEQ ID NO: 69, the portion from methionine (Met) at position 1 to alanine (Ala) at position 22 is the PelB signal peptide, the portion from serine (Ser) at position 25 to aspartic acid (Asp) at position 213 is the AAV binding protein AVR10s (corresponding to the region from positions 312 to 500 in SEQ ID NO: 1), and the histidines (His) at positions 214 to 219 are the tag sequence. In addition, in SEQ ID NO:69, the aspartic acid of Val317Asp is at the 30th position, the serine of Tyr(Cys)342Ser is at the 55th position, the glutamic acid of Lys362Glu is at the 75th position, the asparagine of Lys371Asn is at the 84th position, the alanine of Val381Ala is at the 94th position, the valine of Ile382Val is at the 95th position, the serine of Gly390Ser is at the 103rd position, the glutamic acid of Lys399Glu is at the 112th position, the arginine of Ser476Arg is at the 189th position, and the aspartic acid of Asn487Asp is at the 200th position.

[0228] Example 18 Monomerization of mutant AAV binding proteins (part 2) In order to monomerize the mutant AAV binding protein AAV5eHC prepared in Example 9, an amino acid substitution of Tyr(Cys)342Met was introduced, and PKD3 (amino acid residues from tyrosine at position 501 to lysine at position 597 in SEQ ID NO: 1) was added to prepare a mutant AAV binding protein (named AVR5eMHCD3). Specifically, a mutation was introduced into the polynucleotide (SEQ ID NO: 49) encoding AVR5eHC (SEQ ID NO: 50) to generate Tyr(Cys)342Met, and then PKD3 was added to prepare AVR5eMHCD3.

[0229] (1) Using pET26b-AVR5eHC as a template and oligonucleotides consisting of the sequences set forth in SEQ ID NO: 71 (5'-TTTT[GGTCTC]AGAAACCATGACGTATGACTG-3') and SEQ ID NO: 68 (the brackets in SEQ ID NOs: 71 and 68 indicate the restriction enzyme BsaI recognition sequences) as PCR primers, a plasmid pET-AVR5eMHC containing a polynucleotide encoding a mutant AAV binding protein in which the amino acid substitution Tyr(Cys)342Met was introduced into AVR5eHC was obtained in a manner similar to that of Example 14(a-1) to (a-5).

[0230] (2) PCR was performed in the same manner as in Example 14(a-1), except that pUC-AAVR was used as a template and oligonucleotides consisting of the sequences set forth in SEQ ID NO: 72 (5'-TTTT[GGTCTC]AGATGTTTGTTATTTTCGGGCTGAACAATC-3') and SEQ ID NO: 73 (5'-TTTT[GGTCTC]AAGACTATCCGCCGGTAGCGAACGC-3') (the brackets in SEQ ID NOs: 72 and 73 indicate the recognition sequences for the restriction enzyme BsaI) were used as PCR primers.

[0231] (3) PCR was performed in the same manner as in Example 14(a-1) using pET26b-AVR5eMHC prepared in (1) as a template and oligonucleotides consisting of the sequences set forth in SEQ ID NO: 74 (5'-TTTT[GGTCTC]AGTCTACCGCTTTGTTG-3') and SEQ ID NO: 75 (5'-TTTT[GGTCTC]ACATCATCATCATCATCAT-3') (the brackets in SEQ ID NOs: 74 and 75 indicate the recognition sequences for the restriction enzyme BsaI) as PCR primers.

[0232] (4) The PCR products amplified in (2) and (3) were subjected to agarose gel electrophoresis and purified from the gel using a QIAquick Gel Extraction kit (Qiagen). The purified PCR product in (2) was named D3p, and the PCR product in (3) was named 5MVp.

[0233] (5) The D3p and 5Mvp obtained in (4) were treated with the restriction enzyme DpnI (NEB) at 37° C. for 1.5 hours, and then treated at 80° C. for 20 minutes. The obtained genes were subjected to agarose gel electrophoresis and purified from the gel using a QIAquick Gel Extraction kit (Qiagen).

[0234] (6) The DpnI-treated product obtained in (5) was digested with the restriction enzyme BsaI (NEB) in a reaction solution having the composition shown in Table 16, while being ligated with T4 DNA ligase (NEB).

[0235] [Table 16]

[0236] (7) The ligation product of (6) was used to transform E. coli BL21 strain (DE3). The resulting transformant was cultured in LB medium supplemented with 50 μg / mL kanamycin. Plasmids were extracted from the recovered cells (transformants) to obtain plasmid pET-AVR5eMHCD3, which contains a polynucleotide encoding a mutant AAV binding protein in which AVR5eHD was monomerized and further PKD3 was added.

[0237] (8) The nucleotide sequence of pET-AVR5eMHCD3 was analyzed in the same manner as in Example 1(5). The amino acid sequence of AVR5eMHCD3 is shown in SEQ ID NO: 76, and the polynucleotide sequence is shown in SEQ ID NO: 77. In SEQ ID NO: 76, the region from the 1st methionine (Met) to the 22nd alanine (Ala) is the PelB signal peptide, the region from the 25th serine (Ser) to the 213th aspartic acid (Asp) is the AAV binding protein AVR5eMHC (corresponding to the region from the 312th to the 500th of SEQ ID NO: 1), the region from the 214th tyrosine (Tyr) to the 310th lysine (Lys) is the AAV extracellular domain 3 (PKD3) (the region from the 501st to the 597th of SEQ ID NO: 1), the 311th to the 316th histidine (His) is the tag sequence, and the region from the 317th cysteine ​​(Cys) to the 323rd glycine (Gly) is the cysteine ​​tag, which is a tag for immobilization. In addition, in SEQ ID NO: 76, the methionine of Tyr(Cys)342Met is at the 55th position, the serine of Gly390Ser is at the 103rd position, the glutamic acid of Lys399Glu is at the 112th position, the arginine of Ser476Arg is at the 189th position, and the aspartic acid of Asn487Asp is at the 200th position.

[0238] Example 19 Preparation of monomeric mutant AAV binding protein (part 1) (1) A transformant capable of expressing AVR10s obtained by transforming E. coli strain BL21(DE3) with pET-AVR10s prepared in Example 17, or a transformant capable of expressing AVR5eMHCD3 obtained by transforming E. coli strain BL21(DE3) with pET-AVR5eMHCD3 prepared in Example 18, was inoculated into 3 mL of 2YT liquid medium containing 50 μg / mL kanamycin and pre-cultured overnight at 37° C. under aerobic shaking.

[0239] (2) 2 mL of the preculture solution from (1) was inoculated into 200 mL of 2YT liquid medium containing 50 μg / mL kanamycin in a 1 L baffled flask, and cultured aerobically at 37°C with shaking.

[0240] (3) 2.0 hours after the start of culture, the medium was cooled on ice, IPTG was added to a final concentration of 0.1 mM, and culture was continued overnight at 25°C under aerobic shaking.

[0241] (4) After the cultivation was completed, the culture medium was centrifuged at 8,000 rpm at 4° C. for 20 minutes to collect the bacterial cells.

[0242] (5) The cells collected in (4) were suspended in 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride and 20 mM imidazole at 5 mL / 1 g (cells), and then disrupted using an ultrasonic generator (Insonator 201M [Kubota Shoji Co., Ltd.]) at 8° C. for about 10 minutes at an output of about 150 W. The disrupted cell solution was centrifuged twice at 8,000 rpm for 20 minutes at 4° C., and the supernatant was collected.

[0243] (6) The supernatant obtained in (5) was applied to an XK26 / 20 column (manufactured by Cytiva) packed with 50 mL of Ni Sepharose 6 Fast Flow (manufactured by Cytiva) that had been previously equilibrated with Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride and 20 mM imidazole (hereinafter also referred to as "equilibration solution D"). After washing with equilibration solution D, the column was eluted with 20 mM Tris-HCl buffer (pH 7.4) containing 0.5 M imidazole and 150 mM sodium chloride.

[0244] (7) The eluate obtained in (6) was dialyzed against 20 mM Tris buffer (pH 7.4) containing 150 mM sodium chloride to prepare the amount of AVR10s protein or AVR5eMHCD3 protein required for thermal stability evaluation.

[0245] Example 20: Evaluation of thermal stability of mutant AAV binding proteins (part 4) (1) The binding activity of AVR10s prepared in Example 18 to VLP2 was measured using the ELISA method described in Example 2(6). Based on the absorbance at 450 nm, which is the measurement result, the AVR10s solution was diluted with pure water so that the measured value was the same.

[0246] (2) The diluted AVR10s solution was divided into four portions, and an equal amount of 0.1 M glycine hydrochloride buffer (pH 3.0) was mixed. Three of the four samples were heated at 31.7°C, 41.2°C, and 50.0°C for 15 minutes, and the remaining sample was left to stand at room temperature (25°C) for 15 minutes. The sample was then mixed with 0.5 M MES buffer (pH 6.0) in a ratio of 3:2 to adjust the pH to around 6, and the binding activity with VLP2 was measured by the ELISA method described in Example 2(6).

[0247] (3) The residual activity was calculated by dividing the absorbance at 450 nm after the heat treatment described in (2) by the absorbance at 450 nm after standing at room temperature.

[0248] (4) For comparison, the thermal stability of the wild-type AAV binding protein (SEQ ID NO: 7) was evaluated using a method similar to (1) to (3). The results are shown in Table 17. The mutant AAV binding protein AVR10s prepared in Example 17 had higher residual activity than the wild-type AAV binding protein regardless of the temperature at which it was treated. This indicates that AVR10s has significantly improved stability to heat treatment under acidic conditions, i.e., to acid and heat, compared to the wild-type AAV binding protein.

[0249] [Table 17]

[0250] Example 21: Evaluation of thermal stability of mutant AAV binding proteins (part 5) (1) The binding activity of AVR5eMHCD3 prepared in Example 18 to VLP2 was measured using the ELISA method described in Example 2(6). Based on the absorbance at 450 nm, which is the measurement result, AVR5eMHCD3 was diluted with pure water so that the measurement value was the same.

[0251] (2) The diluted AVR5eMHCD3 solution was divided into four portions and diluted 2-fold with pure water. Three of the four samples were heated at 51.7°C, 66.4°C, and 70.0°C for 15 minutes, and the remaining sample was left to stand at room temperature (25°C) for 15 minutes. The sample was then mixed with 20 mM Tris buffer (pH 7.4) containing 150 mM sodium chloride in a ratio of 3:2, and the binding activity with VLP2 was measured by the ELISA method described in Example 2(6).

[0252] (3) The residual activity was calculated by dividing the absorbance at 450 nm after the heat treatment described in (2) by the absorbance at 450 nm after standing at room temperature.

[0253] (4) For comparison, the thermal stability of the wild-type AAV binding protein (SEQ ID NO: 7) was evaluated using a method similar to (1) to (3). The results are shown in Table 18. The mutant AAV binding protein AVR5eMHCD3 prepared in Example 18 had higher residual activity than the wild-type AAV binding protein regardless of the temperature at which it was treated. This indicates that AVR5eMHCD3 has significantly improved heat stability compared to the wild-type AAV binding protein.

[0254] [Table 18]

[0255] Example 22 Preparation of AAV-binding protein immobilized gel (part 2) (1) A wild-type AAV binding protein immobilized gel and an AVR5eHC immobilized gel were prepared in the same manner as in Example 12. Specifically, 10 mg of AVR5eHC or the wild-type AAV binding protein prepared in Example 10 and TCEP at a final concentration of 0.1 mM as a reducing agent were added to 1 mL of the gel into which a maleimide group had been introduced, and the mixture was allowed to react by shaking at pH 7.4 and 4°C for 15 hours.

[0256] (2) The amount of each protein immobilized on the gel was estimated by measuring the amount of unimmobilized protein in the filtrate after immobilization in (1) using a spectrophotometer. The amount of AVR5eHC immobilized on the AVR5eHC immobilized gel was 9 mg / mL (gel), and the amount of wild-type AAV binding protein immobilized on the wild-type AAV binding protein immobilized gel was 10 mg / mL (gel).

[0257] (3) 0.065 mL of each of the immobilization gels prepared in (1) was placed in a Cosmos Spin Filter G (Nacalai Tesque) to prepare mini-columns (named AVR5eHC immobilization gel mini-column and wild-type AAV binding protein immobilization gel mini-column, respectively). Each mini-column was equilibrated with 50 mM phosphate buffer (pH 7.4) containing 150 mM sodium chloride and 0.05% (w / v) Tween 20 (trade name) (hereinafter also referred to as "Solution A"), and 2.9 × 10 purified AAV2-EGFP product obtained in Example 11 was added. 10 VG (vector genome number) was applied.

[0258] (4) Using a shaking culture apparatus M·BR-022UP (manufactured by Taitec), the mixture was shaken at 1600 rpm for 5 minutes, then centrifuged at 1000 G for 2 minutes to collect the filtrate. 200 μL of solution A was added to the mini-column again, and the mixture was shaken for 1 minute and centrifuged in the same manner. 400 μL was collected together with the previous filtrate (hereinafter referred to as "Flow Through").

[0259] (5) The column was washed twice with 400 μL each with solution A, and each filtrate was collected (the filtrates collected in the first and second washes are referred to as "Wash 1" and "Wash 2", respectively). 200 μL of 50 mM phosphate buffer (pH 2.5) containing 150 mM sodium chloride and 0.05% (w / v) Tween 20 (trade name) (hereinafter also referred to as "solution B") was added to each mini-column, and the column was shaken for 5 minutes and then centrifuged to collect the eluate. 200 μL of solution B was added to the mini-column again, and the column was shaken for 1 minute in the same manner, followed by centrifugation, and 400 μL was collected together with the previous eluate (hereinafter also referred to as "Elute 1").

[0260] (6) Furthermore, 400 μL of solution B was added, and the mixture was centrifuged to recover the eluate (hereinafter also referred to as "Elute 2"). Elute 1 and Elute 2 were neutralized by adding 100 μL of 1 M Tris-HCl buffer (pH 8.5) containing 20 mM magnesium chloride.

[0261] (7) The number of AAV2-EGFP vector genomes contained in the Flow Through obtained in (3), Wash 1, Wash 2, and Elute 1 obtained in (5), and Elute 2 obtained in (6) was quantified by qPCR using the AAVpro Titration Kit (Takara Bio). The results are shown in Figure 4. In the case of the wild-type AAV binding protein immobilized gel minicolumn, most of the AAV applied was confirmed in the flow-through fraction, whereas in the case of the AVR5eHC immobilized gel minicolumn, almost the entire amount of AAV applied was adsorbed by the gel, and elution from the gel was confirmed in the elute 1 fraction. These results demonstrate that AAV can be purified more efficiently by using an AAV adsorbent comprising a gel and the AAV binding protein of the present invention immobilized on the gel, compared to the case of using wild-type AVR.

[0262] Example 23 Preparation of AAV-binding protein immobilized gel (part 3) (1) Using pET-AVR3 and pET-AVR8g as templates, pET-AVR3HC and pET-AVR8gHC were obtained in the same manner as in Example 9. Furthermore, AVR3HC (a protein having a tag for immobilization at the C-terminus of AVR3) and AVR8gHC (a protein having a tag for immobilization at the C-terminus of AVR8g) were prepared in the same manner as in Example 10. Using the prepared proteins, AVR3HC immobilized gel and AVR8gHC immobilized gel were prepared in the same manner as in Example 22. Alternatively, wild-type AAV binding protein immobilized gel was prepared by the method described in Example 22.

[0263] (2) The amount of each protein immobilized in the gel was estimated by measuring the amount of unimmobilized protein in the filtrate after immobilization in (1) using an absorptiometer. The amount of AVR3HC immobilized in the AVR3HC immobilized gel was 3.8 mg / mL (gel), and the amount of AVR8gHC immobilized in the AVR8gHC immobilized gel was 5.7 mg / mL (gel).

[0264] (3) 0.065 mL of the wild-type AAV binding protein immobilized gel and AVR3HC immobilized gel prepared in (1), and 0.018 mL of the AVR8gHC immobilized gel were packed in a Cosmos Spin Filter G (manufactured by Nacalai Tesque) to prepare mini-columns (named wild-type AAV binding protein immobilized gel mini-column, AVR3HC immobilized gel mini-column, and AVR8gHC immobilized gel mini-column, respectively). After equilibration with 150 mM sodium chloride and 50 mM acetate buffer (pH 6.0) (hereinafter also referred to as "Solution A"), the AAV2-EGFP purified product obtained in Example 11 was applied to the FlowThrough fraction in multiple batches until sufficient leakage of AAV2-EGFP was confirmed. The amount of AAV2-EGFP finally applied per column was 2.9 × 10 for the wild-type AAV binding protein immobilized gel mini-column. 10 VG, AVR3HC immobilized gel mini column is 3.9 x 10 10 VG, AVR8gHC immobilized gel mini column is 2.4 x 10 11 It became VG.

[0265] (4) To elute and recover AAV2-EGFP bound to the gel, the column was washed twice with 200 μL of solution A, and after each filtrate was recovered (hereinafter also referred to as "Wash 1"), 200 μL of 150 mM sodium chloride and 50 mM acetate buffer (pH 3.0) (hereinafter also referred to as "solution B") was added to each mini-column, shaken for 5 minutes, and centrifuged to recover the eluate. 200 μL of solution B was added to the mini-column again, shaken for 1 minute in the same manner, centrifuged, and 400 μL was recovered together with the previous eluate (hereinafter also referred to as "Elute 1").

[0266] (5) Furthermore, 400 μL of solution B was added, and the mixture was centrifuged to recover the eluate (hereinafter also referred to as "Elute 2"). Elute 1 and Elute 2 were neutralized by adding 100 μL of 1 M Tris-HCl buffer (pH 8.5) containing 20 mM magnesium chloride.

[0267] (6) The number of AAV2-EGFP vector genomes contained in the Flow Through, Wash 1, Elute 1, and Elute 2 obtained in (3) to (5) was quantified by qPCR using the AAVpro Titration Kit (Takara Bio). The results are shown in Figure 5. It was found that most of the AAV2-EGFP bound to the Elute1 fraction was eluted and recovered. The amount of AAV2-EGFP bound to each mini-column was converted from the amount of AAV2-EGFP recovered by elution to the amount of AAV2-EGFP bound to each mini-column per mL of immobilized gel. The wild-type AAV-binding protein immobilized gel contained 8.6 × 10 9 VG / mL (gel), AVR3HC immobilized gel is 2.8×10 12 VG / mL (gel), AVR8gHC immobilized gel is 1.2×10 13 It became VG / mL (gel).

Claims

1. An AAV binding protein selected from any of the following (i) to (iii): (i) an AAV binding protein comprising at least the amino acid residues from serine at position 312 to aspartic acid at position 500 of the amino acid sequence set forth in SEQ ID NO:1, provided that any one of the following amino acid substitutions or combinations of amino acid substitutions (1) to (158) has occurred in the amino acid residues at positions 312 to 500; (1) Substitution of asparagine at position 324 of SEQ ID NO:1 with aspartic acid (2) Substitution of tyrosine at position 331 of SEQ ID NO:1 with cysteine (3) Substitution of lysine at position 338 of SEQ ID NO:1 with glutamic acid (4) Tyrosine at position 344 of SEQ ID NO: 1 is substituted with histidine (5) Substitution of aspartic acid at position 345 of SEQ ID NO:1 with asparagine (6) Tryptophan at position 346 of SEQ ID NO:1 is substituted with leucine (7) Substitution of leucine at position 348 of SEQ ID NO:1 with proline (8) Substitution of histidine at position 351 of SEQ ID NO:1 with leucine (9) Proline at position 352 of SEQ ID NO: 1 is replaced with leucine (10) Arginine at position 353 of SEQ ID NO: 1 is substituted with cysteine (11) Tyrosine at position 355 of SEQ ID NO: 1 is substituted with asparagine (12) Serine at position 356 of SEQ ID NO: 1 is substituted with cysteine (13) Glycine at position 357 of SEQ ID NO: 1 is substituted with cysteine (14) Substitution of arginine for glutamine at position 365 of SEQ ID NO:1 (15) Substitution of leucine at position 367 of SEQ ID NO:1 with proline (16) Leucine at position 369 of SEQ ID NO: 1 is replaced with glutamine (17) Substitution of phenylalanine at position 379 of SEQ ID NO:1 with serine (18) Tyrosine at position 393 of SEQ ID NO: 1 is substituted with cysteine (19) Valine at position 396 of SEQ ID NO: 1 is substituted with alanine (20) Lysine at position 399 of SEQ ID NO:1 is substituted with glutamic acid (21) Tyrosine at position 442 of SEQ ID NO: 1 is substituted with phenylalanine (22) Leucine at position 477 of SEQ ID NO: 1 is substituted with proline (23) Substitution of asparagine at position 487 of SEQ ID NO: 1 with aspartic acid (24) Asparagine at position 492 of SEQ ID NO: 1 is substituted with aspartic acid (25) Asparagine at position 324 of SEQ ID NO: 1 is substituted with aspartic acid, and tyrosine at position 355 is substituted with histidine (26) Asparagine at position 329 of SEQ ID NO:1 is substituted with tyrosine, and lysine at position 399 is substituted with arginine. (27) SEQ ID NO: 1, in which tyrosine at position 331 is replaced by cysteine ​​and leucine at position 376 is replaced by proline (28) SEQ ID NO: 1, in which tyrosine at position 331 is replaced by cysteine ​​and glutamic acid at position 384 is replaced by glycine (29) The 333rd leucine in SEQ ID NO:1 is replaced with a valine, and the 342nd tyrosine is replaced with a cysteine. (30) SEQ ID NO: 1, in which glutamic acid at position 340 is replaced with glycine and tyrosine at position 342 is replaced with cysteine (31) SEQ ID NO: 1, in which tyrosine at position 342 is substituted with histidine and isoleucine at position 349 is substituted with threonine (32) SEQ ID NO: 1, in which tyrosine at position 342 is substituted with cysteine ​​and glycine at position 390 is substituted with serine (33) Tyrosine at position 344 of SEQ ID NO: 1 is replaced with histidine, and histidine at position 363 is replaced with arginine (34) Tryptophan at position 346 of SEQ ID NO:1 is replaced with arginine, and glutamic acid at position 458 is replaced with glycine (35) SEQ ID NO: 1, in which glutamine at position 347 is substituted with leucine and threonine at position 373 is substituted with alanine (36) SEQ ID NO: 1, in which tyrosine at position 355 is substituted with histidine and asparagine at position 387 is substituted with serine (37) SEQ ID NO: 1, in which glutamine at position 365 is replaced by arginine and histidine at position 389 is replaced by glutamine (38) The 365th glutamine in SEQ ID NO:1 is replaced with arginine, and the 415th glutamine is replaced with leucine. (39) The 366th isoleucine in SEQ ID NO:1 is replaced with threonine, and the 441st glutamine is replaced with leucine. (40) SEQ ID NO: 1, in which the 379th phenylalanine is substituted with serine and the 455th lysine is substituted with arginine (41) SEQ ID NO: 1, in which glutamic acid at position 453 is replaced with glycine and serine at position 476 is replaced with arginine (42) The 319th isoleucine of SEQ ID NO:1 is replaced by phenylalanine, the 322nd proline is replaced by leucine, and the 448th lysine is replaced by arginine. (43) The 321st leucine of SEQ ID NO:1 is substituted with proline, the 342nd tyrosine is substituted with asparagine, and the 461st alanine is substituted with serine. (44) SEQ ID NO: 1, in which glutamine at position 327 is replaced by arginine, lysine at position 338 is replaced by arginine, and leucine at position 348 is replaced by proline (45) SEQ ID NO: 1, in which the asparagine at position 329 is replaced by aspartic acid, the tyrosine at position 342 is replaced by histidine, and the tyrosine at position 355 is replaced by cysteine (46) SEQ ID NO: 1, in which the asparagine at position 329 is substituted with aspartic acid, the isoleucine at position 366 is substituted with threonine, and the lysine at position 368 is substituted with arginine (47) SEQ ID NO: 1, in which asparagine at position 329 is substituted with tyrosine, tyrosine at position 377 is substituted with cysteine, and arginine at position 406 is substituted with serine. (48) SEQ ID NO: 1, in which alanine at position 330 is substituted with glycine, glutamine at position 347 is substituted with proline, and lysine at position 371 is substituted with glutamic acid (49) SEQ ID NO: 1, in which tyrosine at position 331 is replaced by cysteine, threonine at position 343 is replaced by methionine, and tyrosine at position 355 is replaced by cysteine (50) SEQ ID NO: 1, in which the 332nd valine is substituted with glutamine, the 342nd tyrosine is substituted with cysteine, and the 412th valine is substituted with alanine. (51) In SEQ ID NO: 1, leucine at position 333 is replaced by proline, proline at position 337 is replaced by leucine, and threonine at position 350 is replaced by methionine. (52) The 348th leucine in SEQ ID NO:1 is substituted with proline, the 376th leucine is substituted with proline, and the 396th valine is substituted with alanine. (53) SEQ ID NO: 1, in which the aspartic acid at position 354 is replaced with glycine, the leucine at position 369 is replaced with glutamine, and the lysine at position 455 is replaced with arginine (54) The 363rd histidine of SEQ ID NO:1 is replaced with leucine, the 364th serine is replaced with proline, and the 389th histidine is replaced with leucine. (55) SEQ ID NO: 1, in which glutamine at position 365 is replaced by arginine, serine at position 370 is replaced by alanine, and glutamic acid at position 391 is replaced by lysine (56) In SEQ ID NO: 1, asparagine at position 324 is substituted with serine, threonine at position 341 is substituted with alanine, leucine at position 369 is substituted with proline, and phenylalanine at position 416 is substituted with serine. (57) In SEQ ID NO: 1, glutamine at position 334 is substituted with arginine, valine at position 381 is substituted with alanine, histidine at position 389 is substituted with arginine, and tyrosine at position 393 is substituted with cysteine. (58) SEQ ID NO: 1, in which tyrosine at position 342 is substituted with cysteine, glycine at position 390 is substituted with serine, and isoleucine at position 319 is substituted with phenylalanine (59) SEQ ID NO: 1, in which tyrosine at position 342 is substituted with cysteine, glycine at position 390 is substituted with serine, and isoleucine at position 319 is substituted with asparagine (60) SEQ ID NO: 1, in which tyrosine at position 342 is replaced by cysteine, glycine at position 390 is replaced by serine, and lysine at position 323 is replaced by glutamic acid (61) SEQ ID NO: 1, in which tyrosine at position 342 is replaced by cysteine, glycine at position 390 is replaced by serine, and leucine at position 328 is replaced by glutamine (62) SEQ ID NO: 1, in which tyrosine at position 342 is replaced by cysteine, glycine at position 390 is replaced by serine, and leucine at position 328 is replaced by proline (63) SEQ ID NO: 1, in which tyrosine at position 342 is substituted with cysteine, glycine at position 390 is substituted with serine, and tyrosine at position 331 is substituted with histidine (64) SEQ ID NO: 1, in which tyrosine at position 342 is substituted with cysteine, glycine at position 390 is substituted with serine, and valine at position 332 is substituted with alanine (65) SEQ ID NO: 1, in which tyrosine at position 342 is substituted with cysteine, glycine at position 390 is substituted with serine, and valine at position 332 is substituted with glutamic acid (66) SEQ ID NO: 1, in which tyrosine at position 342 is replaced by cysteine, glycine at position 390 is replaced by serine, and leucine at position 333 is replaced by proline (67) SEQ ID NO: 1, in which tyrosine at position 342 is replaced by cysteine, glycine at position 390 is replaced by serine, and proline at position 337 is replaced by glutamine (68) The 390th glycine of SEQ ID NO:1 is substituted with serine, and the 342nd tyrosine is substituted with arginine. (69) SEQ ID NO: 1, in which tyrosine at position 342 is replaced by cysteine, glycine at position 390 is replaced by serine, and threonine at position 350 is replaced by serine. (70) SEQ ID NO: 1, in which tyrosine at position 342 is substituted with cysteine, glycine at position 390 is substituted with serine, and valine at position 383 is substituted with alanine (71) SEQ ID NO: 1, in which tyrosine at position 342 is replaced by cysteine, glycine at position 390 is replaced by serine, and histidine at position 389 is replaced by aspartic acid (72) SEQ ID NO: 1, in which tyrosine at position 342 is substituted with cysteine, glycine at position 390 is substituted with serine, and valine at position 394 is substituted with alanine (73) SEQ ID NO: 1, in which tyrosine at position 342 is substituted with cysteine, glycine at position 390 is substituted with serine, and valine at position 396 is substituted with alanine (74) SEQ ID NO: 1, in which tyrosine at position 342 is replaced by cysteine, glycine at position 390 is replaced by serine, and serine at position 476 is replaced by glycine (75) SEQ ID NO: 1, in which tyrosine at position 342 is replaced by cysteine, glycine at position 390 is replaced by serine, and serine at position 476 is replaced by arginine (76) SEQ ID NO: 1, in which tyrosine at position 342 is replaced by cysteine, glycine at position 390 is replaced by serine, and asparagine at position 492 is replaced by aspartic acid (77) In SEQ ID NO: 1, tyrosine at position 342 is substituted with cysteine, glycine at position 390 is substituted with serine, leucine at position 321 is substituted with proline, and isoleucine at position 409 is substituted with valine. (78) In SEQ ID NO: 1, tyrosine at position 342 is substituted with cysteine, glycine at position 390 is substituted with serine, leucine at position 328 is substituted with proline, and isoleucine at position 366 is substituted with phenylalanine. (79) SEQ ID NO: 1, in which tyrosine at position 342 is substituted with cysteine, glycine at position 390 is substituted with serine, lysine at position 338 is substituted with asparagine, and asparagine at position 487 is substituted with aspartic acid. (80) SEQ ID NO: 1, in which tyrosine at position 342 is replaced with cysteine, glycine at position 390 is replaced with serine, glutamine at position 365 is replaced with arginine, and glycine at position 392 is replaced with cysteine (81) In SEQ ID NO: 1, tyrosine at position 342 is replaced by cysteine, glycine at position 390 is replaced by serine, phenylalanine at position 379 is replaced by serine, and serine at position 476 is replaced by glycine. (82) SEQ ID NO: 1, in which tyrosine at position 342 is substituted with cysteine, glycine at position 390 is substituted with serine, serine at position 476 is substituted with arginine, and threonine at position 490 is substituted with serine. (83) In SEQ ID NO: 1, tyrosine at position 342 is substituted with cysteine, glycine at position 390 is substituted with serine, isoleucine at position 319 is substituted with serine, threonine at position 320 is substituted with isoleucine, and lysine at position 399 is substituted with glutamic acid. (84) In SEQ ID NO: 1, tyrosine at position 342 is substituted with cysteine, glycine at position 390 is substituted with serine, proline at position 336 is substituted with glutamine, glutamine at position 386 is substituted with arginine, and asparagine at position 487 is substituted with aspartic acid. (85) In SEQ ID NO: 1, tyrosine at position 342 is substituted with cysteine, glycine at position 390 is substituted with serine, lysine at position 399 is substituted with glutamic acid, serine at position 476 is substituted with arginine, asparagine at position 487 is substituted with aspartic acid, and valine at position 317 is substituted with aspartic acid. (86) In SEQ ID NO: 1, tyrosine at position 342 is substituted with cysteine, glycine at position 390 is substituted with serine, lysine at position 399 is substituted with glutamic acid, serine at position 476 is substituted with arginine, asparagine at position 487 is substituted with aspartic acid, and lysine at position 371 is substituted with glutamic acid. (87) In SEQ ID NO: 1, tyrosine at position 342 is substituted with cysteine, glycine at position 390 is substituted with serine, lysine at position 399 is substituted with glutamic acid, serine at position 476 is substituted with arginine, asparagine at position 487 is substituted with aspartic acid, and lysine at position 371 is substituted with asparagine. (88) In SEQ ID NO: 1, tyrosine at position 342 is substituted with cysteine, glycine at position 390 is substituted with serine, lysine at position 399 is substituted with glutamic acid, serine at position 476 is substituted with arginine, asparagine at position 487 is substituted with aspartic acid, and leucine at position 376 is substituted with proline. (89) In SEQ ID NO: 1, tyrosine at position 342 is replaced by cysteine, glycine at position 390 is replaced by serine, lysine at position 399 is replaced by glutamic acid, serine at position 476 is replaced by arginine, asparagine at position 487 is replaced by aspartic acid, and glutamic acid at position 378 is replaced by glycine. (90) In SEQ ID NO: 1, tyrosine at position 342 is replaced by cysteine, glycine at position 390 is replaced by serine, lysine at position 399 is replaced by glutamic acid, serine at position 476 is replaced by arginine, asparagine at position 487 is replaced by aspartic acid, and glycine at position 392 is replaced by cysteine. (91) In SEQ ID NO: 1, tyrosine at position 342 is replaced by cysteine, glycine at position 390 is replaced by serine, lysine at position 399 is replaced by glutamic acid, serine at position 476 is replaced by arginine, asparagine at position 487 is replaced by aspartic acid, valine at position 317 is replaced by aspartic acid, and lysine at position 371 is replaced by asparagine. (92) In SEQ ID NO: 1, tyrosine at position 342 is replaced by cysteine, glycine at position 390 is replaced by serine, lysine at position 399 is replaced by glutamic acid, serine at position 476 is replaced by arginine, asparagine at position 487 is replaced by aspartic acid, threonine at position 320 is replaced by alanine, and valine at position 381 is replaced by aspartic acid. (93) In SEQ ID NO: 1, tyrosine at position 342 is replaced by cysteine, glycine at position 390 is replaced by serine, lysine at position 399 is replaced by glutamic acid, serine at position 476 is replaced by arginine, asparagine at position 487 is replaced by aspartic acid, asparagine at position 324 is replaced by aspartic acid, and lysine at position 362 is replaced by glutamic acid. (94) In SEQ ID NO: 1, tyrosine at position 342 is substituted with cysteine, glycine at position 390 is substituted with serine, lysine at position 399 is substituted with glutamic acid, serine at position 476 is substituted with arginine, asparagine at position 487 is substituted with aspartic acid, valine at position 326 is substituted with glutamic acid, and asparagine at position 329 is substituted with histidine. (95) In SEQ ID NO: 1, tyrosine at position 342 is substituted with cysteine, glycine at position 390 is substituted with serine, lysine at position 399 is substituted with glutamic acid, serine at position 476 is substituted with arginine, asparagine at position 487 is substituted with aspartic acid, valine at position 326 is substituted with alanine, and lysine at position 362 is substituted with glutamic acid. (96) In SEQ ID NO: 1, tyrosine at position 342 is replaced by cysteine, glycine at position 390 is replaced by serine, lysine at position 399 is replaced by glutamic acid, serine at position 476 is replaced by arginine, asparagine at position 487 is replaced by aspartic acid, valine at position 326 is replaced by glutamic acid, and phenylalanine at position 379 is replaced by serine. (97) In SEQ ID NO: 1, tyrosine at position 342 is substituted with cysteine, glycine at position 390 is substituted with serine, lysine at position 399 is substituted with glutamic acid, serine at position 476 is substituted with arginine, asparagine at position 487 is substituted with aspartic acid, valine at position 332 is substituted with alanine, and lysine at position 371 is substituted with glutamic acid. (98) In SEQ ID NO: 1, tyrosine at position 342 is replaced by cysteine, glycine at position 390 is replaced by serine, lysine at position 399 is replaced by glutamic acid, serine at position 476 is replaced by arginine, asparagine at position 487 is replaced by aspartic acid, leucine at position 376 is replaced by proline, and asparagine at position 492 is replaced by aspartic acid. (99) In SEQ ID NO: 1, tyrosine at position 342 is replaced by cysteine, glycine at position 390 is replaced by serine, lysine at position 399 is replaced by glutamic acid, serine at position 476 is replaced by arginine, asparagine at position 487 is replaced by aspartic acid, glutamine at position 318 is replaced by proline, phenylalanine at position 379 is replaced by tyrosine, and glutamic acid at position 401 is replaced by valine. (100) In SEQ ID NO: 1, tyrosine at position 342 is replaced by cysteine, glycine at position 390 is replaced by serine, lysine at position 399 is replaced by glutamic acid, serine at position 476 is replaced by arginine, asparagine at position 487 is replaced by aspartic acid, asparagine at position 324 is replaced by aspartic acid, lysine at position 362 is replaced by glutamic acid, and valine at position 499 is replaced by glutamic acid. (101) In SEQ ID NO: 1, tyrosine at position 342 is replaced by cysteine, glycine at position 390 is replaced by serine, lysine at position 399 is replaced by glutamic acid, serine at position 476 is replaced by arginine, asparagine at position 487 is replaced by aspartic acid, valine at position 326 is replaced by glutamic acid, asparagine at position 329 is replaced by histidine, and glutamic acid at position 378 is replaced by valine. (102) In SEQ ID NO: 1, tyrosine at position 342 is replaced by cysteine, glycine at position 390 is replaced by serine, lysine at position 399 is replaced by glutamic acid, serine at position 476 is replaced by arginine, asparagine at position 487 is replaced by aspartic acid, leucine at position 328 is replaced by proline, lysine at position 362 is replaced by asparagine, threonine at position 397 is replaced by serine, and arginine at position 406 is replaced by histidine. (103) In SEQ ID NO: 1, valine at position 317 is replaced with aspartic acid, tyrosine at position 342 is replaced with cysteine, lysine at position 362 is replaced with glutamic acid, lysine at position 371 is replaced with asparagine, glycine at position 390 is replaced with serine, lysine at position 399 is replaced with glutamic acid, serine at position 476 is replaced with arginine, asparagine at position 487 is replaced with aspartic acid, and glutamic acid at position 340 is replaced with valine. (104) In SEQ ID NO: 1, valine at position 317 is substituted with aspartic acid, tyrosine at position 342 is substituted with cysteine, lysine at position 362 is substituted with glutamic acid, lysine at position 371 is substituted with asparagine, glycine at position 390 is substituted with serine, lysine at position 399 is substituted with glutamic acid, serine at position 476 is substituted with arginine, asparagine at position 487 is substituted with aspartic acid, and tyrosine at position 344 is substituted with phenylalanine. (105) In SEQ ID NO: 1, valine at position 317 is substituted with aspartic acid, tyrosine at position 342 is substituted with cysteine, lysine at position 362 is substituted with glutamic acid, lysine at position 371 is substituted with asparagine, glycine at position 390 is substituted with serine, lysine at position 399 is substituted with glutamic acid, serine at position 476 is substituted with arginine, asparagine at position 487 is substituted with aspartic acid, and arginine at position 353 is substituted with cysteine. (106) In SEQ ID NO: 1, valine at position 317 is substituted with aspartic acid, tyrosine at position 342 is substituted with cysteine, lysine at position 362 is substituted with glutamic acid, lysine at position 371 is substituted with asparagine, glycine at position 390 is substituted with serine, lysine at position 399 is substituted with glutamic acid, serine at position 476 is substituted with arginine, asparagine at position 487 is substituted with aspartic acid, and tyrosine at position 355 is substituted with cysteine. (107) In SEQ ID NO: 1, valine at position 317 is substituted with aspartic acid, tyrosine at position 342 is substituted with cysteine, lysine at position 362 is substituted with glutamic acid, lysine at position 371 is substituted with asparagine, glycine at position 390 is substituted with serine, lysine at position 399 is substituted with glutamic acid, serine at position 476 is substituted with arginine, asparagine at position 487 is substituted with aspartic acid, and serine at position 356 is substituted with cysteine. (108) In SEQ ID NO: 1, valine at position 317 is substituted with aspartic acid, tyrosine at position 342 is substituted with cysteine, lysine at position 362 is substituted with glutamic acid, lysine at position 371 is substituted with asparagine, glycine at position 390 is substituted with serine, lysine at position 399 is substituted with glutamic acid, serine at position 476 is substituted with arginine, asparagine at position 487 is substituted with aspartic acid, and methionine at position 359 is substituted with leucine. (109) In SEQ ID NO: 1, valine at position 317 is substituted with aspartic acid, tyrosine at position 342 is substituted with cysteine, lysine at position 362 is substituted with glutamic acid, lysine at position 371 is substituted with asparagine, glycine at position 390 is substituted with serine, lysine at position 399 is substituted with glutamic acid, serine at position 476 is substituted with arginine, asparagine at position 487 is substituted with aspartic acid, and glutamic acid at position 360 is substituted with lysine. (110) In SEQ ID NO: 1, valine at position 317 is replaced with aspartic acid, tyrosine at position 342 is replaced with cysteine, lysine at position 362 is replaced with glutamic acid, lysine at position 371 is replaced with asparagine, glycine at position 390 is replaced with serine, lysine at position 399 is replaced with glutamic acid, serine at position 476 is replaced with arginine, asparagine at position 487 is replaced with aspartic acid, and glutamic acid at position 360 is replaced with valine. (111) In SEQ ID NO: 1, valine at position 317 is substituted with aspartic acid, tyrosine at position 342 is substituted with cysteine, lysine at position 371 is substituted with asparagine, glycine at position 390 is substituted with serine, lysine at position 399 is substituted with glutamic acid, serine at position 476 is substituted with arginine, asparagine at position 487 is substituted with aspartic acid, and lysine at position 362 is substituted with glycine. (112) In SEQ ID NO: 1, valine at position 317 is substituted with aspartic acid, tyrosine at position 342 is substituted with cysteine, lysine at position 362 is substituted with glutamic acid, lysine at position 371 is substituted with asparagine, glycine at position 390 is substituted with serine, lysine at position 399 is substituted with glutamic acid, serine at position 476 is substituted with arginine, asparagine at position 487 is substituted with aspartic acid, and serine at position 364 is substituted with leucine. (113) In SEQ ID NO: 1, valine at position 317 is substituted with aspartic acid, tyrosine at position 342 is substituted with cysteine, lysine at position 362 is substituted with glutamic acid, lysine at position 371 is substituted with asparagine, glycine at position 390 is substituted with serine, lysine at position 399 is substituted with glutamic acid, serine at position 476 is substituted with arginine, asparagine at position 487 is substituted with aspartic acid, and isoleucine at position 366 is substituted with threonine. (114) In SEQ ID NO: 1, valine at position 317 is substituted with aspartic acid, tyrosine at position 342 is substituted with cysteine, lysine at position 362 is substituted with glutamic acid, lysine at position 371 is substituted with asparagine, glycine at position 390 is substituted with serine, lysine at position 399 is substituted with glutamic acid, serine at position 476 is substituted with arginine, asparagine at position 487 is substituted with aspartic acid, and leucine at position 372 is substituted with proline. (115) In SEQ ID NO: 1, valine at position 317 is substituted with aspartic acid, tyrosine at position 342 is substituted with cysteine, lysine at position 362 is substituted with glutamic acid, lysine at position 371 is substituted with asparagine, glycine at position 390 is substituted with serine, lysine at position 399 is substituted with glutamic acid, serine at position 476 is substituted with arginine, asparagine at position 487 is substituted with aspartic acid, and leucine at position 372 is substituted with glutamine. (116) In SEQ ID NO: 1, valine at position 317 is substituted with aspartic acid, tyrosine at position 342 is substituted with cysteine, lysine at position 362 is substituted with glutamic acid, lysine at position 371 is substituted with asparagine, glycine at position 390 is substituted with serine, lysine at position 399 is substituted with glutamic acid, serine at position 476 is substituted with arginine, asparagine at position 487 is substituted with aspartic acid, and threonine at position 373 is substituted with alanine. (117) In SEQ ID NO: 1, valine at position 317 is substituted with aspartic acid, tyrosine at position 342 is substituted with cysteine, lysine at position 362 is substituted with glutamic acid, lysine at position 371 is substituted with asparagine, glycine at position 390 is substituted with serine, lysine at position 399 is substituted with glutamic acid, serine at position 476 is substituted with arginine, asparagine at position 487 is substituted with aspartic acid, and leucine at position 376 is substituted with proline. (118) In SEQ ID NO: 1, valine at position 317 is substituted with aspartic acid, tyrosine at position 342 is substituted with cysteine, lysine at position 362 is substituted with glutamic acid, lysine at position 371 is substituted with asparagine, glycine at position 390 is substituted with serine, lysine at position 399 is substituted with glutamic acid, serine at position 476 is substituted with arginine, asparagine at position 487 is substituted with aspartic acid, and glutamic acid at position 378 is substituted with glycine. (119) In SEQ ID NO: 1, valine at position 317 is substituted with aspartic acid, tyrosine at position 342 is substituted with cysteine, lysine at position 362 is substituted with glutamic acid, lysine at position 371 is substituted with asparagine, glycine at position 390 is substituted with serine, lysine at position 399 is substituted with glutamic acid, serine at position 476 is substituted with arginine, asparagine at position 487 is substituted with aspartic acid, and phenylalanine at position 379 is substituted with serine. (120) In SEQ ID NO: 1, valine at position 317 is substituted with aspartic acid, tyrosine at position 342 is substituted with cysteine, lysine at position 362 is substituted with glutamic acid, lysine at position 371 is substituted with asparagine, glycine at position 390 is substituted with serine, lysine at position 399 is substituted with glutamic acid, serine at position 476 is substituted with arginine, asparagine at position 487 is substituted with aspartic acid, and lysine at position 380 is substituted with glutamic acid. (121) In SEQ ID NO: 1, valine at position 317 is substituted with aspartic acid, tyrosine at position 342 is substituted with cysteine, lysine at position 362 is substituted with glutamic acid, lysine at position 371 is substituted with asparagine, glycine at position 390 is substituted with serine, lysine at position 399 is substituted with glutamic acid, serine at position 476 is substituted with arginine, asparagine at position 487 is substituted with aspartic acid, and valine at position 381 is substituted with alanine. (122) In SEQ ID NO: 1, valine at position 317 is substituted with aspartic acid, tyrosine at position 342 is substituted with cysteine, lysine at position 362 is substituted with glutamic acid, lysine at position 371 is substituted with asparagine, glycine at position 390 is substituted with serine, lysine at position 399 is substituted with glutamic acid, serine at position 476 is substituted with arginine, asparagine at position 487 is substituted with aspartic acid, and glutamine at position 386 is substituted with arginine. (123) In SEQ ID NO: 1, valine at position 317 is substituted with aspartic acid, tyrosine at position 342 is substituted with cysteine, lysine at position 362 is substituted with glutamic acid, lysine at position 371 is substituted with asparagine, glycine at position 390 is substituted with serine, lysine at position 399 is substituted with glutamic acid, serine at position 476 is substituted with arginine, asparagine at position 487 is substituted with aspartic acid, and tyrosine at position 393 is substituted with cysteine. (124) In SEQ ID NO: 1, valine at position 317 is substituted with aspartic acid, tyrosine at position 342 is substituted with cysteine, lysine at position 362 is substituted with glutamic acid, lysine at position 371 is substituted with asparagine, glycine at position 390 is substituted with serine, lysine at position 399 is substituted with glutamic acid, serine at position 476 is substituted with arginine, asparagine at position 487 is substituted with aspartic acid, and glutamine at position 441 is substituted with arginine. (125) In SEQ ID NO: 1, valine at position 317 is substituted with aspartic acid, tyrosine at position 342 is substituted with cysteine, lysine at position 362 is substituted with glutamic acid, lysine at position 371 is substituted with asparagine, glycine at position 390 is substituted with serine, lysine at position 399 is substituted with glutamic acid, serine at position 476 is substituted with arginine, asparagine at position 487 is substituted with aspartic acid, and histidine at position 443 is substituted with leucine. (126) In SEQ ID NO: 1, valine at position 317 is substituted with aspartic acid, tyrosine at position 342 is substituted with cysteine, lysine at position 362 is substituted with glutamic acid, lysine at position 371 is substituted with asparagine, glycine at position 390 is substituted with serine, lysine at position 399 is substituted with glutamic acid, serine at position 476 is substituted with arginine, asparagine at position 487 is substituted with aspartic acid, and lysine at position 448 is substituted with glutamic acid. (127) In SEQ ID NO: 1, valine at position 317 is substituted with aspartic acid, tyrosine at position 342 is substituted with cysteine, lysine at position 362 is substituted with glutamic acid, lysine at position 371 is substituted with asparagine, glycine at position 390 is substituted with serine, lysine at position 399 is substituted with glutamic acid, serine at position 476 is substituted with arginine, asparagine at position 487 is substituted with aspartic acid, and aspartic acid at position 483 is substituted with asparagine. (128) In SEQ ID NO: 1, valine at position 317 is substituted with aspartic acid, tyrosine at position 342 is substituted with cysteine, lysine at position 362 is substituted with glutamic acid, lysine at position 371 is substituted with asparagine, glycine at position 390 is substituted with serine, lysine at position 399 is substituted with glutamic acid, serine at position 476 is substituted with arginine, asparagine at position 487 is substituted with aspartic acid, serine at position 312 is substituted with proline, and proline at position 374 is substituted with leucine. (129) In SEQ ID NO: 1, valine at position 317 is substituted with aspartic acid, tyrosine at position 342 is substituted with cysteine, lysine at position 362 is substituted with glutamic acid, lysine at position 371 is substituted with asparagine, glycine at position 390 is substituted with serine, lysine at position 399 is substituted with glutamic acid, serine at position 476 is substituted with arginine, asparagine at position 487 is substituted with aspartic acid, and alanine at position 313 is substituted with serine and glutamic acid at position 378 is substituted with glycine. (130) In SEQ ID NO: 1, valine at position 317 is replaced with aspartic acid, tyrosine at position 342 is replaced with cysteine, lysine at position 362 is replaced with glutamic acid, lysine at position 371 is replaced with asparagine, glycine at position 390 is replaced with serine, lysine at position 399 is replaced with glutamic acid, serine at position 476 is replaced with arginine, asparagine at position 487 is replaced with aspartic acid, lysine at position 323 is replaced with arginine, and glutamic acid at position 384 is replaced with valine. (131) In SEQ ID NO: 1, valine at position 317 is replaced with aspartic acid, tyrosine at position 342 is replaced with cysteine, lysine at position 362 is replaced with glutamic acid, lysine at position 371 is replaced with asparagine, glycine at position 390 is replaced with serine, lysine at position 399 is replaced with glutamic acid, serine at position 476 is replaced with arginine, asparagine at position 487 is replaced with aspartic acid, asparagine serine at position 324 is replaced with asparagine, and glycine at position 357 is replaced with cysteine. (132) In SEQ ID NO: 1, valine at position 317 is substituted with aspartic acid, tyrosine at position 342 is substituted with cysteine, lysine at position 362 is substituted with glutamic acid, lysine at position 371 is substituted with asparagine, glycine at position 390 is substituted with serine, lysine at position 399 is substituted with glutamic acid, serine at position 476 is substituted with arginine, asparagine at position 487 is substituted with aspartic acid, glutamine at position 327 is substituted with histidine, and glutamic acid at position 378 is substituted with valine. (133) In SEQ ID NO: 1, valine at position 317 is substituted with aspartic acid, tyrosine at position 342 is substituted with cysteine, lysine at position 362 is substituted with glutamic acid, glycine at position 390 is substituted with serine, lysine at position 399 is substituted with glutamic acid, serine at position 476 is substituted with arginine, asparagine at position 487 is substituted with aspartic acid, glutamine at position 327 is substituted with leucine, and lysine at position 371 is substituted with aspartic acid. (134) In SEQ ID NO: 1, valine at position 317 is substituted with aspartic acid, tyrosine at position 342 is substituted with cysteine, lysine at position 362 is substituted with glutamic acid, lysine at position 371 is substituted with asparagine, glycine at position 390 is substituted with serine, lysine at position 399 is substituted with glutamic acid, serine at position 476 is substituted with arginine, asparagine at position 487 is substituted with aspartic acid, asparagine at position 329 is substituted with isoleucine, and valine at position 499 is substituted with isoleucine. (135) In SEQ ID NO: 1, valine at position 317 is replaced with aspartic acid, tyrosine at position 342 is replaced with cysteine, lysine at position 362 is replaced with glutamic acid, lysine at position 371 is replaced with asparagine, glycine at position 390 is replaced with serine, lysine at position 399 is replaced with glutamic acid, serine at position 476 is replaced with arginine, asparagine at position 487 is replaced with aspartic acid, glutamic acid at position 335 is replaced with glycine, and glutamine at position 386 is replaced with arginine. (136) In SEQ ID NO: 1, valine at position 317 is substituted with aspartic acid, tyrosine at position 342 is substituted with cysteine, lysine at position 362 is substituted with glutamic acid, lysine at position 371 is substituted with asparagine, glycine at position 390 is substituted with serine, lysine at position 399 is substituted with glutamic acid, serine at position 476 is substituted with arginine, asparagine at position 487 is substituted with aspartic acid, glutamic acid at position 335 is substituted with valine, and threonine at position 343 is substituted with serine. (137) In SEQ ID NO: 1, valine at position 317 is substituted with aspartic acid, tyrosine at position 342 is substituted with cysteine, lysine at position 362 is substituted with glutamic acid, lysine at position 371 is substituted with asparagine, glycine at position 390 is substituted with serine, lysine at position 399 is substituted with glutamic acid, serine at position 476 is substituted with arginine, asparagine at position 487 is substituted with aspartic acid, threonine at position 341 is substituted with proline, and phenylalanine at position 379 is substituted with serine. (138) In SEQ ID NO: 1, valine at position 317 is substituted with aspartic acid, tyrosine at position 342 is substituted with cysteine, lysine at position 362 is substituted with glutamic acid, lysine at position 371 is substituted with asparagine, lysine at position 399 is substituted with glutamic acid, serine at position 476 is substituted with arginine, asparagine at position 487 is substituted with aspartic acid, and glycine at position 361 is substituted with cysteine. (139) In SEQ ID NO: 1, valine at position 317 is substituted with aspartic acid, tyrosine at position 342 is substituted with cysteine, lysine at position 362 is substituted with glutamic acid, lysine at position 371 is substituted with asparagine, glycine at position 390 is substituted with serine, lysine at position 399 is substituted with glutamic acid, serine at position 476 is substituted with arginine, asparagine at position 487 is substituted with aspartic acid, leucine at position 369 is substituted with glutamine, and proline at position 374 is substituted with leucine. (140) In SEQ ID NO: 1, valine at position 317 is substituted with aspartic acid, tyrosine at position 342 is substituted with cysteine, lysine at position 362 is substituted with glutamic acid, lysine at position 371 is substituted with asparagine, glycine at position 390 is substituted with serine, lysine at position 399 is substituted with glutamic acid, serine at position 476 is substituted with arginine, asparagine at position 487 is substituted with aspartic acid, leucine at position 372 is substituted with proline, and valine at position 396 is substituted with alanine. (141) In SEQ ID NO: 1, valine at position 317 is substituted with aspartic acid, tyrosine at position 342 is substituted with cysteine, lysine at position 362 is substituted with glutamic acid, lysine at position 371 is substituted with asparagine, lysine at position 399 is substituted with glutamic acid, serine at position 476 is substituted with arginine, asparagine at position 487 is substituted with aspartic acid, and leucine at position 376 is substituted with proline. (142) In SEQ ID NO: 1, valine at position 317 is substituted with aspartic acid, tyrosine at position 342 is substituted with cysteine, lysine at position 362 is substituted with glutamic acid, lysine at position 371 is substituted with asparagine, glycine at position 390 is substituted with serine, lysine at position 399 is substituted with glutamic acid, serine at position 476 is substituted with arginine, asparagine at position 487 is substituted with aspartic acid, glutamic acid at position 378 is substituted with glycine, and arginine at position 403 is substituted with histidine. (143) In SEQ ID NO: 1, valine at position 317 is substituted with aspartic acid, tyrosine at position 342 is substituted with cysteine, lysine at position 362 is substituted with glutamic acid, lysine at position 371 is substituted with asparagine, glycine at position 390 is substituted with serine, lysine at position 399 is substituted with glutamic acid, serine at position 476 is substituted with arginine, asparagine at position 487 is substituted with aspartic acid, valine at position 381 is substituted with alanine, and isoleucine at position 382 is substituted with valine. (144) In SEQ ID NO: 1, valine at position 317 is substituted with aspartic acid, tyrosine at position 342 is substituted with cysteine, lysine at position 362 is substituted with glutamic acid, lysine at position 371 is substituted with asparagine, glycine at position 390 is substituted with serine, lysine at position 399 is substituted with glutamic acid, serine at position 476 is substituted with arginine, asparagine at position 487 is substituted with aspartic acid, valine at position 383 is substituted with alanine, and glutamine at position 415 is substituted with arginine. (145) In SEQ ID NO: 1, valine at position 317 is replaced with aspartic acid, tyrosine at position 342 is replaced with cysteine, lysine at position 362 is replaced with glutamic acid, lysine at position 371 is replaced with asparagine, glycine at position 390 is replaced with serine, lysine at position 399 is replaced with glutamic acid, serine at position 476 is replaced with arginine, asparagine at position 487 is replaced with aspartic acid, tryptophan at position 346 is replaced with cysteine, arginine at position 353 is replaced with cysteine, and histidine at position 389 is replaced with arginine. (146) In SEQ ID NO: 1, valine at position 317 is replaced with aspartic acid, tyrosine at position 342 is replaced with cysteine, lysine at position 362 is replaced with glutamic acid, lysine at position 371 is replaced with asparagine, glycine at position 390 is replaced with serine, lysine at position 399 is replaced with glutamic acid, serine at position 476 is replaced with arginine, asparagine at position 487 is replaced with aspartic acid, leucine at position 372 is replaced with glutamine, leucine at position 376 is replaced with proline, and valine at position 394 is replaced with aspartic acid. (147) In SEQ ID NO: 1, valine at position 317 is replaced with aspartic acid, tyrosine at position 342 is replaced with cysteine, lysine at position 362 is replaced with glutamic acid, lysine at position 371 is replaced with asparagine, glycine at position 390 is replaced with serine, lysine at position 399 is replaced with glutamic acid, serine at position 476 is replaced with arginine, asparagine at position 487 is replaced with aspartic acid, phenylalanine at position 379 is replaced with cysteine, glutamine at position 432 is replaced with leucine, and serine at position 488 is replaced with leucine. (148) In SEQ ID NO: 1, valine at position 317 is substituted with aspartic acid, tyrosine at position 342 is substituted with cysteine, lysine at position 362 is substituted with glutamic acid, lysine at position 371 is substituted with asparagine, glycine at position 390 is substituted with serine, lysine at position 399 is substituted with glutamic acid, serine at position 476 is substituted with arginine, asparagine at position 487 is substituted with aspartic acid, glutamic acid at position 384 is substituted with valine, glutamine at position 432 is substituted with arginine, and isoleucine at position 456 is substituted with valine. (149) In SEQ ID NO: 1, valine at position 317 is substituted with aspartic acid, tyrosine at position 342 is substituted with cysteine, lysine at position 362 is substituted with glutamic acid, lysine at position 371 is substituted with asparagine, glycine at position 390 is substituted with serine, lysine at position 399 is substituted with glutamic acid, serine at position 476 is substituted with arginine, asparagine at position 487 is substituted with aspartic acid, valine at position 394 is substituted with isoleucine, aspartic acid at position 395 is substituted with serine, and threonine at position 426 is substituted with alanine. (150) In SEQ ID NO: 1, valine at position 317 is substituted with aspartic acid, tyrosine at position 342 is substituted with cysteine, lysine at position 362 is substituted with glutamic acid, lysine at position 371 is substituted with asparagine, glycine at position 390 is substituted with serine, lysine at position 399 is substituted with glutamic acid, serine at position 476 is substituted with arginine, asparagine at position 487 is substituted with aspartic acid, valine at position 396 is substituted with alanine, glutamine at position 441 is substituted with arginine, and lysine at position 448 is substituted with glutamic acid. (151) In SEQ ID NO: 1, the 319th isoleucine is substituted with phenylalanine, the 342nd tyrosine is substituted with cysteine, the 390th glycine is substituted with serine, and the 476th serine is substituted with arginine. (152) In SEQ ID NO: 1, tyrosine at position 342 is substituted with cysteine, glycine at position 390 is substituted with serine, serine at position 476 is substituted with arginine, and asparagine at position 487 is substituted with aspartic acid. (153) In SEQ ID NO: 1, the 319th isoleucine is substituted with phenylalanine, the 323rd lysine is substituted with glutamic acid, the 342nd tyrosine is substituted with cysteine, the 390th glycine is substituted with serine, and the 476th serine is substituted with arginine. (154) In SEQ ID NO: 1, tyrosine at position 342 is substituted with cysteine, glycine at position 390 is substituted with serine, lysine at position 399 is substituted with glutamic acid, serine at position 476 is substituted with arginine, and asparagine at position 487 is substituted with aspartic acid. (155) In SEQ ID NO: 1, valine at position 317 is substituted with aspartic acid, asparagine at position 324 is substituted with aspartic acid, tyrosine at position 342 is substituted with cysteine, lysine at position 362 is substituted with glutamic acid, glycine at position 390 is substituted with serine, lysine at position 399 is substituted with glutamic acid, serine at position 476 is substituted with arginine, and asparagine at position 487 is substituted with aspartic acid. (156) In SEQ ID NO: 1, valine at position 317 is substituted with aspartic acid, tyrosine at position 342 is substituted with cysteine, lysine at position 362 is substituted with glutamic acid, lysine at position 371 is substituted with asparagine, glycine at position 390 is substituted with serine, lysine at position 399 is substituted with glutamic acid, serine at position 476 is substituted with arginine, and asparagine at position 487 is substituted with aspartic acid. (157) In SEQ ID NO: 1, valine at position 317 is substituted with aspartic acid, tyrosine at position 342 is substituted with serine, lysine at position 362 is substituted with glutamic acid, lysine at position 371 is substituted with asparagine, valine at position 381 is substituted with alanine, isoleucine at position 382 is substituted with valine, glycine at position 390 is substituted with serine, lysine at position 399 is substituted with glutamic acid, serine at position 476 is substituted with arginine, and asparagine at position 487 is substituted with aspartic acid. (158) In SEQ ID NO: 1, tyrosine at position 342 is replaced by methionine, glycine at position 390 is replaced by serine, lysine at position 399 is replaced by glutamic acid, serine at position 476 is replaced by arginine, and asparagine at position 487 is replaced by aspartic acid. (ii) An AAV binding protein comprising at least the amino acid residues from serine at position 312 to aspartic acid at position 500 of the amino acid sequence set forth in SEQ ID NO:1, provided that the amino acid residues from position 312 to position 500 have any one of the amino acid substitutions or combination of amino acid substitutions of (1) to (158) above, and further comprising substitutions, deletions, insertions and / or additions of 1 to 10 amino acid residues other than the amino acid substitutions or combination of amino acid substitutions, and which has AAV-binding activity and has improved stability against heat, acid, or alkali compared to the wild-type AAV binding protein. (iii) An AAV binding protein comprising an amino acid sequence having 90% or more identity to the entire amino acid sequence in which any one of the amino acid substitutions or combination of amino acid substitutions (1) to (158) has occurred in the amino acid sequence set forth in SEQ ID NO: 1, from the serine at position 312 to the aspartic acid at position 500, and in which the amino acid substitution or combination of amino acid substitutions remains, the AAV binding protein has AAV binding activity and has improved stability against heat, acid, or alkali compared to a wild-type AAV binding protein.

2. An AAV binding protein selected from any of (vii) to (ix) below: (vii) an AAV binding protein comprising at least the amino acid residues from serine at position 25 to aspartic acid at position 213 in the amino acid sequence set forth in any one of SEQ ID NOs: 18, 32, 34, 38, 42, 46, 53, 55, 59, 63, 69, and 76; (viii) an AAV binding protein comprising at least amino acid residues from serine at position 25 to aspartic acid at position 213 of the amino acid sequence set forth in any one of SEQ ID NOs: 18, 32, 34, 38, 42, 46, 53, 55, 59, 63, 69, and 76, provided that in the amino acid residues from position 25 to position 213, substitutions, deletions, insertions, and / or additions of 1 to 10 amino acid residues are further caused in addition to the amino acid substitution or combination of amino acid substitutions in the amino acid sequence from serine at position 312 to aspartic acid at position 500 of the amino acid sequence set forth in SEQ ID NO: 1, which is contained in the amino acid sequence set forth in any one of SEQ ID NOs: 18, 32, 34, 38, 42, 46, 53, 55, 59, 63, 69, and 76, and which has AAV binding activity and improved stability against heat, acid, or alkali compared to a wild-type AAV binding protein; (ix) An AAV binding protein which comprises at least the amino acid residues from serine at position 25 to aspartic acid at position 213 of the amino acid sequence set forth in any one of SEQ ID NOs: 18, 32, 34, 38, 42, 46, 53, 55, 59, 63, 69, and 76, and which has 90% or more identity to the amino acid sequence from the 25th to the 213th, and which retains an amino acid substitution or a combination of amino acid substitutions in the amino acid sequence from serine at position 312 to aspartic acid at position 500 of the amino acid sequence set forth in SEQ ID NO: 1, as contained in the amino acid sequence set forth in any one of SEQ ID NOs: 18, 32, 34, 38, 42, 46, 53, 55, 59, 63, 69, and 76, and which has AAV-binding activity and improved stability against heat, acid, or alkali compared to a wild-type AAV binding protein.

3. A polynucleotide encoding the AAV binding protein of claim 1 or 2.

4. An expression vector comprising the polynucleotide of claim 3.

5. A transformant obtained by transforming a host with the expression vector according to claim 4.

6. The transformant according to claim 5 , wherein the host is Escherichia coli.

7. A method for producing an AAV binding protein, comprising the steps of expressing the AAV binding protein by culturing the transformant described in claim 5 or 6, and recovering the expressed AAV binding protein from the resulting culture.

8. An AAV adsorbent comprising an insoluble carrier and an AAV binding protein according to claim 1 or 2 immobilized on the carrier.

9. A column packed with the AAV adsorbent described in claim 8.

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

Images