Modified recombinant adeno-associated virus (AAV)-binding protein and method of analyzing AAV based on infectivity

Abstract

To provide improved adeno associated virus (AAV) binding protein with improved binding activity to AAV, and a method for analyzing AAV in a sample based on the difference in infectivity.SOLUTION: The object is achieved by substituting an amino acid residue at a specific position in regions corresponding to extracellular region domain 1 (PKD1) and domain 2 (PKD2) of KIAA0319L (UniProt No. Q8IZA0) with a different specific amino acid residue. The object is achieved by an AAV analysis method comprising a step of adsorbing AAV contained in a sample onto an adsorbent comprising an insoluble carrier and a polypeptide comprising at least an amino acid sequence corresponding to extracellular region domain 1 or domain 2 of KIAA0319L immobilized on the carrier; and a step of eluting the AAV adsorbed on the adsorbent using an eluent.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a protein having binding affinity to an adeno-associated virus (AAV) and a method for analyzing an adeno-associated virus (AAV), and more particularly, to an improved AAV-binding protein having improved binding activity to AAV and a method for analyzing AAV contained in a sample based on the strength of infectivity. [Background technology]

[0002] Adeno-associated virus (AAV) is a non-enveloped virus classified in the Parvoviridae family and Dependovirus genus. The AAV envelope particle is composed of three types of proteins (VP1, VP2, and VP3), and is formed by mixing and assembling approximately 60 protein molecules in a ratio of VP1:VP2:VP3 = 1:1:10, resulting in 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] It has been reported that the replacement of amino acid residues at specific positions in AAV with other amino acid residues results in differences in the strength of the infectivity of AAV to cells (Non-Patent Document 1). Therefore, when AAV is used as a vector for gene transfer, it is necessary to analyze the differences in the strength of the infectivity quickly and simply.

[0006] Recombinant AAV vectors (hereinafter simply referred to as "AAV vectors") are usually produced by introducing a nucleic acid encoding elements essential for AAV particle formation into cells to produce cells capable of producing AAV (hereinafter also referred to as AAV-producing cells), and then culturing the cells to express the elements essential for AAV particle formation. The produced AAV vector is recovered and purified from the AAV-producing cells to obtain a therapeutic AAV vector formulation.

[0007] As a method for recovering and purifying an AAV vector from an AAV-producing cell, there is a method using affinity chromatography based on the binding affinity with AAV, using an adsorbent containing an insoluble carrier and an AAV-binding protein immobilized on the carrier, which can recover and purify the AAV vector from a solution containing the AAV vector coexisting with impurities. As a specific example, Patent Document 1 uses a polypeptide containing the extracellular domain 1 (PKD1) and domain 2 (PKD2) of KIAA0319L (UniProt No. Q8IZA0), but with amino acid residues at specific positions in these domains replaced with other amino acid residues, which has improved stability against heat, acid, and alkali, as an AAV-binding protein (hereinafter, also simply referred to as "ligand protein") immobilized on an insoluble carrier, thereby realizing high-purity purification of an AAV vector.

[0008] It has been reported that the replacement of amino acid residues at specific positions in AAV with other amino acid residues results in differences in the strength of infectivity to cells (Non-Patent Document 1). Therefore, when AAV is used as a vector for gene transfer, it is necessary to analyze the difference in the strength of the infectivity quickly and easily. However, as disclosed in Non-Patent Document 1, the conventional analysis method uses cultured cells and requires a lot of time and effort. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] WO2021 / 106882 issue [Non-patent literature]

[0010] [Non-Patent Document 1] Lochrie, MA, et al., Journal of virology, 80(2), 821, 2006 Summary of the Invention [Problem to be solved by the invention]

[0011] As mentioned above, the adeno-associated virus (AAV) binding protein used as a ligand protein in WO2021 / 106882 has improved stability against heat, acid, and alkali compared to the native type (AAV binding protein without amino acid substitution). However, further improvement of the binding activity to AAV was required.

[0012] As described above, when AAV is used as a vector for gene transfer, it is necessary to analyze the difference in the strength of the infectivity quickly and easily. However, the conventional analysis method uses cultured cells as disclosed in Lochrie, MA, et al., Journal of Virology, 80(2), 821, 2006 (Non-Patent Document 1), and requires a lot of time and effort.

[0013] In other words, the object of the present invention is to provide an improved AAV-binding protein with improved binding activity to AAV, and to provide a method for analyzing adeno-associated virus contained in a sample based on the strength of its infectivity. [Means for solving the problem]

[0014] In order to solve the above problems, the present inventors conducted intensive research and discovered that by substituting specific amino acid residues among the amino acid residues constituting an adeno-associated virus (AAV)-binding protein with other amino acid residues, the binding activity to AAV is improved, and that by analyzing adeno-associated virus (AAV) contained in a sample using an adsorbent comprising an insoluble carrier and an AAV-binding protein immobilized on the carrier, AAV can be analyzed based on the strength of infectivity, thereby completing the present invention.

[0015] The present invention includes the following aspects [1] to

[14] : [1] An adeno-associated virus (AAV) selected from any one of the following (i) to (iii): Binding Proteins: (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 (5) has occurred in the amino acid residues at positions 312 to 500; (1) Alanine at position 330 of SEQ ID NO:1 is replaced with valine (2) Alanine at position 330 of SEQ ID NO:1 is substituted with any one of cysteine, phenylalanine, leucine, arginine, tryptophan, or tyrosine. (3) Substitution of tyrosine at position 331 of SEQ ID NO:1 with cysteine (4) Valine at position 332 of SEQ ID NO: 1 is replaced with tryptophan or tyrosine (5) The 333rd leucine in SEQ ID NO:1 is replaced with cysteine. (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 (1) to (5) has occurred in the amino acid residues at positions 312 to 500, and further comprising one or more of substitutions, deletions, insertions, and additions of one or more amino acid residues at one or more positions in addition to the amino acid substitutions shown in (1) to (5), 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 (5) has occurred, and the amino acid sequence in which at least one of the amino acid substitutions remains, and the AAV binding protein has AAV binding activity.

[0016] [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) Alanine at position 330 of SEQ ID NO:1 is replaced with valine. (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 in which the amino acid sequence set forth in SEQ ID NO:1, from the serine at position 312 to the aspartic acid at position 500, has a homology of 70% or more to the entire amino acid sequence in which at least the amino acid substitution of (1) has occurred, and in which the amino acid substitution remains, and which has AAV-binding activity.

[0017] [3] The AAV binding protein according to [1], further comprising at least the following 10 amino acid substitutions: 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 Valine 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 Asparagine at position 487 of SEQ ID NO:1 is substituted with aspartic acid.

[0018] [4] The AAV binding protein according to [3], selected from any one of the following (vii) to (ix): (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: 15 to 25. (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: 15 to 25, wherein the amino acid residues from position 25 to position 213 further include 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 contained in the amino acid sequence, and the AAV binding protein has 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: 15 to 25, wherein the AAV binding protein has 70% or more identity to the amino acid sequence from the amino acid residues at positions 25 to 213, and wherein the amino acid substitutions in the amino acid sequence remain, and the AAV binding activity is maintained.

[0019] [5] The AAV binding protein according to [3], further comprising at least one amino acid substitution selected from the following (I) to (LXXXVII): (I) Serine at position 312 of SEQ ID NO: 1 is replaced with proline (II) Alanine at position 313 of SEQ ID NO: 1 is replaced with valine (III) Glutamic acid at position 315 of SEQ ID NO: 1 is replaced with glycine or valine (IV) Substitution of glutamine at position 318 of SEQ ID NO: 1 with arginine (V) Isoleucine at position 319 of SEQ ID NO: 1 is replaced with valine (VI) Lysine at position 323 of SEQ ID NO: 1 is replaced with methionine or asparagine (VII) Asparagine at position 324 of SEQ ID NO: 1 is substituted with histidine (VIII) Substitution of lysine for glutamic acid at position 325 of SEQ ID NO:1 (IX) valine at position 326 of SEQ ID NO: 1 is substituted with glutamic acid or alanine (X) Glutamine at position 327 of SEQ ID NO: 1 is replaced with leucine (XI) Leucine at position 328 of SEQ ID NO: 1 is substituted with any one of glutamine, arginine, and proline. (XII) Asparagine at position 329 of SEQ ID NO: 1 is substituted with tyrosine or serine (XIII) valine at position 332 of SEQ ID NO: 1 is substituted with glutamic acid or alanine (XIV) Substitution of leucine at position 333 of SEQ ID NO: 1 with proline or methionine (XV) Glutamine at position 334 of SEQ ID NO: 1 is substituted with leucine or histidine (XVI) Glutamic acid at position 335 of SEQ ID NO: 1 is replaced with valine (XVII) Proline at position 337 of SEQ ID NO: 1 is replaced with glutamine (XVIII) Lysine at position 338 of SEQ ID NO: 1 is substituted with glutamic acid or arginine (XIX) Glycine at position 339 of SEQ ID NO: 1 is replaced with glutamic acid (XX) Glutamic acid at position 340 of SEQ ID NO: 1 is replaced with Aspartic acid (XXI) Substitution of threonine at position 341 of SEQ ID NO: 1 with alanine (XXII) Threonine at position 343 of SEQ ID NO: 1 is substituted with any one of proline, alanine, and serine. (XXIII) Aspartic acid at position 345 of SEQ ID NO: 1 is replaced with glycine (XXIV) Substitution of isoleucine at position 349 of SEQ ID NO: 1 with threonine (XXV) Substitution of threonine at position 350 of SEQ ID NO: 1 with alanine (XXVI) Aspartic acid at position 354 of SEQ ID NO: 1 is replaced with valine (XXVII) Tyrosine at position 355 of SEQ ID NO: 1 is replaced with histidine (XXVIII) Methionine at position 359 of SEQ ID NO: 1 is substituted with any one of isoleucine, leucine, and valine. (XXIX) Substitution of glutamic acid at position 360 of SEQ ID NO:1 with aspartic acid (XXX) Glycine at position 361 of SEQ ID NO:1 is replaced with glutamic acid (XXXI) Isoleucine at position 366 of SEQ ID NO: 1 is substituted with threonine or proline (XXXII) Leucine at position 367 of SEQ ID NO: 1 is replaced with valine (XXXIII) Lysine at position 368 of SEQ ID NO:1 is substituted with any one of asparagine, arginine, and glutamic acid (XXXIV) Leucine at position 369 of SEQ ID NO: 1 is replaced with glutamine or proline (XXXV) Leucine at position 376 of SEQ ID NO:1 is replaced with proline (XXXVI) Substitution of phenylalanine at position 379 of SEQ ID NO:1 with tyrosine (XXXVII) Lysine at position 380 of SEQ ID NO:1 is substituted with asparagine or arginine (XXXVIII) Glutamic acid at position 384 of SEQ ID NO: 1 is replaced with Valine (XXXIX) Alanine at position 388 of SEQ ID NO: 1 is replaced with threonine (XL) Histidine at position 389 of SEQ ID NO: 1 is substituted with any one of asparagine, aspartic acid, arginine, and leucine (XLI) Glycine at position 392 of SEQ ID NO:1 is replaced with arginine (XLII) Valine at position 394 of SEQ ID NO: 1 is substituted with alanine or aspartic acid (XLII) Asparagine at position 395 of SEQ ID NO: 1 is substituted with aspartic acid (XLIII) Valine at position 396 of SEQ ID NO: 1 is replaced with Glycine (XLIV) Valine at position 398 of SEQ ID NO:1 is substituted with alanine (XLV) Substitution of arginine at position 403 of SEQ ID NO:1 with serine or histidine (XLVI) Lysine at position 404 of SEQ ID NO:1 is substituted with alanine (XLVII) Substitution of arginine at position 406 of SEQ ID NO: 1 with serine (XLVIII) Substitution of isoleucine at position 409 of SEQ ID NO:1 with asparagine (XLIX) Substitution of isoleucine at position 411 of SEQ ID NO:1 with threonine or leucine (L) Valine at position 412 of SEQ ID NO: 1 is replaced with isoleucine (LI) Substitution of phenylalanine at position 416 of SEQ ID NO: 1 with serine (LII) Isoleucine at position 419 of SEQ ID NO: 1 is substituted with phenylalanine or valine (LIII) Serine at position 420 of SEQ ID NO: 1 is replaced with threonine (LIV) Leucine at position 421 of SEQ ID NO:1 is replaced with proline (LV) Proline at position 422 of SEQ ID NO:1 is replaced with serine (LVI) Substitution of threonine at position 423 of SEQ ID NO:1 with alanine (LVII) Serine at position 425 of SEQ ID NO: 1 is replaced with glycine (LVIII) Isoleucine at position 428 of SEQ ID NO: 1 is replaced with valine (LIX) Glycine at position 430 of SEQ ID NO:1 is replaced with alanine (LX) Threonine at position 434 of SEQ ID NO: 1 is replaced with Alanine (LXI) Substitution of aspartic acid at position 437 of SEQ ID NO:1 with asparagine (LXII) Valine at position 440 of SEQ ID NO: 1 is replaced with alanine (LXIII) Histidine at position 443 of SEQ ID NO: 1 is substituted with tyrosine or arginine (LXIV) Substitution of glutamic acid at position 446 of SEQ ID NO:1 with aspartic acid (LXV) Lysine at position 448 of SEQ ID NO:1 is substituted with glutamic acid or asparagine (LXVI) Leucine at position 451 of SEQ ID NO: 1 is replaced with isoleucine (LXVII) Glutamic acid at position 453 of SEQ ID NO: 1 is replaced with Lysine (LXVIII) Glutamic acid at position 454 of SEQ ID NO: 1 is replaced with glycine (LXIX) Lysine at position 455 of SEQ ID NO:1 is substituted with glutamic acid or arginine (LXX) Lysine at position 464 of SEQ ID NO:1 is substituted with asparagine or glutamic acid (LXXI) Lysine at position 467 of SEQ ID NO:1 is substituted with glutamine or glutamic acid (LXXII) Substitution of valine at position 469 of SEQ ID NO:1 with glutamic acid (LXXIII) Glycine at position 471 of SEQ ID NO:1 is replaced with cysteine (LXXIV) Asparagine at position 472 of SEQ ID NO:1 is substituted with tyrosine or aspartic acid (LXXV) Tyrosine at position 473 of SEQ ID NO:1 is replaced with histidine (LXXVI) Substitution of threonine at position 478 of SEQ ID NO:1 with alanine (LXXVII) Valine at position 479 of SEQ ID NO:1 is substituted with alanine (LXXVIII) Valine at position 480 of SEQ ID NO:1 is substituted with aspartic acid or alanine (LXXIX) Serine at position 482 of SEQ ID NO:1 is replaced with threonine (LXXX) Glycine at position 484 of SEQ ID NO:1 is replaced with serine (LXXXI) Substitution of threonine at position 486 of SEQ ID NO:1 with serine (LXXXII) Serine at position 488 of SEQ ID NO: 1 is substituted with threonine or phenylalanine (LXXXIII) Substitution of threonine at position 489 of SEQ ID NO:1 with alanine or serine (LXXXIV) Alanine at position 491 of SEQ ID NO:1 is replaced with serine (LXXXV) Asparagine at position 492 of SEQ ID NO:1 is replaced with aspartic acid (LXXXVI) Asparagine at position 496 of SEQ ID NO:1 is substituted with tyrosine or aspartic acid (LXXXVII) The 500th aspartic acid in SEQ ID NO:1 is replaced with glycine.

[0020] [6] The AAV binding protein according to [5], selected from any one of the following (x) to (xii): (x) 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: 54 to 63, 65, 83, 85, 87, 88, and 90 to 92. (xi) 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: 54 to 63, 65, 83, 85, 87, 88, and 90 to 92, provided that the amino acid residues from position 25 to position 213 further include 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 contained in the amino acid sequence, and having AAV-binding activity. (xii) 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: 54 to 63, 65, 83, 85, 87, 88, and 90 to 92, wherein the AAV binding protein has 70% or more identity to the amino acid sequence from the amino acid residues at positions 25 to 213, and wherein the amino acid substitutions in the amino acid sequence remain, and the AAV binding activity is maintained.

[0021] [7] A polynucleotide encoding an AAV binding protein described in any one of [1] to [6].

[0022] [8] An expression vector comprising the polynucleotide described in [7].

[0023] [9] A transformant obtained by transforming Escherichia coli with the expression vector described in [8].

[0024]

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

[0025]

[11] An AAV adsorbent comprising an insoluble carrier and an AAV binding protein described in any one of [1] to [6] immobilized on the carrier.

[0026]

[12] A column containing the AAV adsorbent described in

[11] .

[0027]

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

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

[0028]

[14] A method for analyzing AAV contained in a sample based on the strength of its infectivity, comprising the steps of adding a sample containing AAV to the column described in

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

[0029] The present invention will be described in detail below.

[0030] In this specification, infectivity refers to the ability of a virus such as AAV to infect a host cell. The infectivity may be expressed as a ratio or a percentage of a mutant (amino acid substitution) to a natural type using a known indicator of infectivity. Indicators of infectivity include, for example, the infection rate when a virus is infected into a host cell, the ability of the virus to bind to a receptor on the cell membrane of a host cell, the activity of an encapsulated gene or a protein encoded by the gene, such as luminescence of GFP, and the intensity of luminescence inside or outside an animal (for example, Fig. 6 in Amanda M. Dudek, et al., Journal of virology, 92(7), e02213-17, 2018), and the expression level of a marker indicating the effect of a virus such as AAV. Host cells generally refer to various cells such as HEK293 and HT-1080, animals such as mice and monkeys, and the like, and may be suspension cells, adherent cells, or living organisms, regardless of their form. The concentration at which a virus such as AAV is infected can be changed appropriately so that the difference is easily visible. Furthermore, the definition of the infectivity of an AAV mutant is not limited, but can be determined, for example, by infecting a host cell such as human HepG2 cell with an AAV mutant and a wild-type AAV to measure the infectivity, as described in Non-Patent Document 1, and calculating the ratio of the infectivity of the AAV mutant to the infectivity of the wild-type AAV, which is set to 100. Alternatively, the infectivity of the mutant may be determined as a relative value to the infectivity of the wild-type.

[0031] In the present specification, the AAV binding protein refers to 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 (UniProt No. Q8IZA0) set forth in SEQ ID NO: 1, and that has an amino acid substitution 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 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.

[0032] The amino acid substitution at the specific position is specifically, in the amino acid sequence set forth in SEQ ID NO: 1, (1) Ala330Val (this notation indicates that the 330th alanine in SEQ ID NO: 1 is replaced with valine, hereinafter the same), (2) any of Ala330Cys, Ala330Phe, Ala330Leu, Ala330Arg, Ala330Trp, and Ala330Tyr; (3) Tyr331Cys, (4) Val332Trp or Val332Tyr, and (5) At least one of the amino acid substitutions Leu333Cys is present, and the occurrence of these amino acid substitutions improves the binding activity to AAV.

[0033] Among these, (1) Ala330Val is an amino acid substitution that not only significantly improves the binding activity to AAV, but also significantly suppresses the production of degradation products derived from the AAV-binding protein when the AAV-binding protein is produced by genetic engineering techniques using recombinant E. coli, thereby significantly improving the production efficiency of the AAV-binding protein in the recombinant E. coli. Therefore, an AAV-binding protein in which at least the amino acid substitution (1) Ala330Val has occurred is a preferred embodiment of the AAV-binding protein of the present invention.

[0034] There is no limitation on the number of amino acid substitutions shown in (1) to (5) above. That is, only one of the amino acid substitutions shown in (1) to (5) above may occur, or two or more of the amino acid substitutions shown in (1) to (5) above may occur. However, since the amino acid substitutions shown in (1) and (2) above occur at the same position, even if an amino acid substitution occurs, it will be either (1) or (2).

[0035] There is no particular limitation on the AAV whose binding activity is improved, and it may be an AAV that exists in nature or an AAV that is artificially produced. Examples of AAV that exists in nature include serotype 1 (AAV1), serotype 2 (AAV2), serotype 3 (AAV3), serotype 5 (AAV5), serotype 6 (AAV6), serotype 7 (AAV7), serotype 8 (AAV8), serotype 9 (AAV9), serotype 10 (AAV10), serotype 11 (AAV11), serotype 12 (AAV12), and serotype 13 (AAV13). Examples of artificially produced AAV include AAVrh8, AAVrh10, and chimeric AAVs that have the characteristics (cell tropism and infectivity) of two or more of these serotypes.

[0036] In the above (ii), (v), (viii), and (xi), "one or several" will vary depending on the position of the amino acid substitution in the three-dimensional structure of the AAV binding protein and the type of amino acid residue, but for example, it means any of the following: 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.

[0037] Examples of the substitutions, deletions, insertions, or additions described in (ii), (v), (viii), and (xi) above include the substitutions of amino acid residues disclosed in WO2021 / 106882. Among them, at least the substitutions of Val317Asp, Tyr342Ser, Lys362Glu, Lys371Asn, Val381Ala, Ile382Val, Gly390Ser, Lys399Glu, Ser476Arg, and Asn487Asp are particularly preferred embodiments of the AAV binding protein of the present invention, since they particularly improve stability against heat, acid, and alkali.

[0038] Examples of particularly preferred embodiments include the AAV binding proteins shown in (A) to (K) below. (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, with the proviso that at least the following amino acid substitutions have occurred in the amino acid residues at positions 312 to 500: Val317Asp, Ala330Val, Tyr342Ser, Lys362Glu, Lys371Asn, Val381Ala, Ile382Val, Gly390Ser, Lys399Glu, Ser476Arg, and Asn487Asp. (B) An AAV binding protein comprising amino acid residues from serine at position 312 to aspartic acid at position 500 of the amino acid sequence set forth in SEQ ID NO:1, with the proviso that at least the following amino acid substitutions have occurred in the amino acid residues at positions 312 to 500: Val317Asp, Ala330Cys, Tyr342Ser, Lys362Glu, Lys371Asn, Val381Ala, Ile382Val, Gly390Ser, Lys399Glu, Ser476Arg, and Asn487Asp. (C) An AAV binding protein comprising amino acid residues from serine at position 312 to aspartic acid at position 500 of the amino acid sequence set forth in SEQ ID NO:1, with the proviso that at least the following amino acid substitutions have occurred in the amino acid residues at positions 312 to 500: Val317Asp, Ala330Phe, Tyr342Ser, Lys362Glu, Lys371Asn, Val381Ala, Ile382Val, Gly390Ser, Lys399Glu, Ser476Arg, and Asn487Asp. (D) An AAV binding protein comprising amino acid residues from serine at position 312 to aspartic acid at position 500 of the amino acid sequence set forth in SEQ ID NO:1, with the proviso that at least the following amino acid substitutions have occurred in the amino acid residues at positions 312 to 500: Val317Asp, Ala330Leu, Tyr342Ser, Lys362Glu, Lys371Asn, Val381Ala, Ile382Val, Gly390Ser, Lys399Glu, Ser476Arg, and Asn487Asp. (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, with the proviso that at least the following amino acid substitutions have occurred in the amino acid residues at positions 312 to 500: Val317Asp, Ala330Arg, Tyr342Ser, Lys362Glu, Lys371Asn, Val381Ala, Ile382Val, Gly390Ser, Lys399Glu, Ser476Arg, and Asn487Asp. (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, with the proviso that at least the following amino acid substitutions have occurred in the amino acid residues at positions 312 to 500: Val317Asp, Ala330Trp, Tyr342Ser, Lys362Glu, Lys371Asn, Val381Ala, Ile382Val, Gly390Ser, Lys399Glu, Ser476Arg, and Asn487Asp. (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, with the proviso that at least the following amino acid substitutions have occurred in the amino acid residues at positions 312 to 500: Val317Asp, Ala330Tyr, Tyr342Ser, Lys362Glu, Lys371Asn, Val381Ala, Ile382Val, Gly390Ser, Lys399Glu, Ser476Arg, and Asn487Asp. (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, with the proviso that at least the following amino acid substitutions have occurred in the amino acid residues at positions 312 to 500: Val317Asp, Tyr331Cys, Tyr342Ser, Lys362Glu, Lys371Asn, Val381Ala, Ile382Val, Gly390Ser, Lys399Glu, Ser476Arg, and Asn487Asp. (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, with the proviso that at least the following amino acid substitutions have occurred in the amino acid residues at positions 312 to 500: Val317Asp, Val332Trp, Tyr342Ser, Lys362Glu, Lys371Asn, Val381Ala, Ile382Val, Gly390Ser, Lys399Glu, Ser476Arg, and Asn487Asp. (J) An AAV binding protein comprising 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 the following amino acid substitutions have occurred in the amino acid residues at positions 312 to 500: Val317Asp, Val332Tyr, Tyr342Ser, Lys362Glu, Lys371Asn, Val381Ala, Ile382Val, Gly390Ser, Lys399Glu, Ser476Arg, and Asn487Asp. (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, with the proviso that at least the following amino acid substitutions have occurred in the amino acid residues at positions 312 to 500: Val317Asp, Leu333Cys, Tyr342Ser, Lys362Glu, Lys371Asn, Val381Ala, Ile382Val, Gly390Ser, Lys399Glu, Ser476Arg, and Asn487Asp.

[0039] Specific examples of the particularly preferred embodiments include the AAV binding proteins shown below. 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:19, 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: 15, 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: 16, 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:17, 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:18, 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:20, 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:21, 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:22, 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:23, 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:24, 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 213 of the amino acid sequence set forth in SEQ ID NO:25, which is one embodiment of the AAV binding protein shown in (K) above.

[0040] In addition, the "substitution of one or several amino acid residues" in (ii), (v), (viii), and (xi) may include, in addition to the amino acid substitution at the specific position described above, 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 protein having a substitution and a protein having no substitution. Examples of conservative substitutions include substitutions between glycine and alanine, between serine and proline, or between glutamic acid and alanine (Protein Structure and Function, Medical Science International, 9, 2005). 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 is 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.

[0041] Furthermore, the "substitution, deletion, insertion, or addition of one or several amino acid residues" in (ii), (v), (viii), and (xi) above also includes naturally occurring mutations (mutants or variants) based on differences in the origin or species of the AAV binding protein.

[0042] The homology of the amino acid sequences in (iii), (vi), (ix), and (xii) may be 70% or more, and may be higher (e.g., 80% or more, 85% or more, 90% or more, or 95% or more). In this specification, "homology" may mean similarity or identity, and may particularly mean identity. "Homology of amino acid sequence" means homology to the entire amino acid sequence. "Identity" between amino acid sequences refers to the ratio of amino acid residues of the same type in those amino acid sequences (Experimental Medicine, 31(3), Yodosha). "Similarity" between amino acid sequences refers to the sum of the 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, 31(3), Yodosha). Homology of amino acid sequences can be determined using alignment programs such as BLAST (Basic Local Alignment Search Tool) and FASTA.

[0043] 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 from a solution in the presence of contaminants. Examples of the oligopeptide include polyhistidine, polylysine, polyarginine, polyglutamic acid, polyaspartic acid, etc. In addition, an oligopeptide containing cysteine ​​that is useful for immobilizing the AAV-binding protein of the present invention on a solid phase such as a chromatographic support may further be attached to the N-terminus or C-terminus of the AAV-binding protein of the present invention.

[0044] 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.

[0045] 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 Escherichia coli used as a host. 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).

[0046] As an example of a method for producing a polynucleotide encoding the AAV binding protein of the present invention (hereinafter also referred to as "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.

[0047] In the above method (I), when converting an amino acid sequence to a nucleotide sequence, it is preferable to convert the sequence taking into consideration the frequency of codon usage in Escherichia coli, the host to be transformed. Specifically, 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).

[0048] 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 introduction of a desired mutation 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 mM to 10 mM (preferably 0.1 mM 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 the 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.

[0049] When transforming a host, E. coli, 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 (E. coli) to be transformed and can be replicated, and examples of the expression vector include pET plasmid vector, pUC plasmid vector, pTrc plasmid vector, and pCDF plasmid vector.

[0050] 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 inserting the polynucleotide of the present invention 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 include the trp promoter, tac promoter, trc promoter, lac promoter, T7 promoter, recA promoter, and lpp promoter.

[0051] The host Escherichia coli can be transformed with the expression vector into which the polynucleotide of the present invention is inserted, prepared by the above-mentioned method (hereinafter, also simply referred to as "the expression vector of the present invention"), by a method commonly used by those skilled in the art. Specifically, transformation can be performed by a method described in a known document such as Molecular Cloning (Cold Spring Harbor Laboratory), 256, 1992. The transformants obtained by transformation by the above-mentioned method can be screened by an appropriate method to obtain a transformant capable of expressing the AAV binding protein of the present invention (hereinafter, also simply referred to as "the transformant of the present invention").

[0052] 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).

[0053] 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.

[0054] The transformant used in the protein production method of the present invention may be cultured in a medium suitable for culturing the target host (Escherichia coli), and 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, if the vector contains a kanamycin resistance gene, kanamycin may be added to the medium.

[0055] In addition to carbon, nitrogen and inorganic salt sources, the medium may contain an appropriate nutrient source, and may contain one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine, thioglycolate and dithiothreitol, if desired. The culture temperature is generally 10°C to 40°C, preferably 20°C to 37°C, more preferably around 25°C, but may be selected depending on the characteristics of the protein to be expressed. The pH of the medium is pH 6.8 to 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.

[0056] An example of an inducer is IPTG (isopropyl-β-D-thiogalactopyranoside). The turbidity of the culture solution (absorbance at 600 nm) is measured, and when it reaches approximately 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 mM to 1.0 mM, with the range of 0.01 mM to 0.5 mM being preferred. Various conditions for IPTG induction may be those well known in the art.

[0057] 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.

[0058] The AAV-binding protein of the present invention can be purified by any method known in the art, including, for example, separation / purification using liquid chromatography, which 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.

[0059] 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.

[0060] 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 performed, 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, an 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. There are no limitations on the material, shape, etc. of the insoluble carrier, and an example is the insoluble carrier disclosed in WO2021 / 106882. In addition, the insoluble carrier may be porous or non-porous.

[0061] 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. The immobilization of the AAV binding protein of the present invention on an insoluble carrier may be carried out, for example, based on the disclosure in WO2021 / 106882.

[0062] The AAV adsorbent of the present invention can be used, for example, in the purification of AAV by packing it into a column. Specifically, for example, a solution containing AAV can be added to a column packed with the AAV adsorbent of the present invention (hereinafter also simply referred to as the "column of the present invention") to adsorb the AAV to the adsorbent, and the AAV adsorbed to the adsorbent can be eluted to purify AAV. That is, the present invention provides a method for purifying AAV, which includes, for example, a step of adding a solution containing AAV to the column of the present invention to adsorb the AAV to the adsorbent, and a step of eluting the AAV adsorbed to the adsorbent. Purification of AAV using the column of the present invention may be carried out, for example, based on the disclosure in WO2021 / 106882.

[0063] By carrying out the purification of AAV using the AAV adsorbent of the present invention, 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.

[0064] The present invention also includes the following aspects

[15] to

[17] :

[15] A method for analyzing an adeno-associated virus (AAV) contained in a sample based on the strength of infectivity, comprising: the method, in which the analysis is carried out by a method comprising the steps of adsorbing the AAV to an AAV adsorbent comprising an insoluble carrier and an AAV-binding protein immobilized on the carrier, and eluting the AAV adsorbed to the adsorbent with an elution solution, wherein the AAV-binding protein is a polypeptide selected from any one of the following (xiii) to (xv); (xiii) a polypeptide comprising at least the amino acid residues from serine at position 312 to glutamic acid at position 401 or the amino acid residues from isoleucine at position 409 to aspartic acid at position 500 in the amino acid sequence set forth in SEQ ID NO: 94; (xiv) a polypeptide having an amino acid sequence comprising at least the amino acid residues from serine at position 312 to glutamic acid at position 401 or from isoleucine at position 409 to aspartic acid at position 500 of the amino acid sequence set forth in SEQ ID NO: 94, and including substitution, deletion, insertion, or addition of one or several amino acid residues at one or several positions within these amino acid residues, and having AAV binding activity; (xv) A polypeptide comprising at least the amino acid residues from serine at position 312 to glutamic acid at position 401 or from isoleucine at position 409 to aspartic acid at position 500 of the amino acid sequence set forth in SEQ ID NO: 94, and having 70% or more homology to an amino acid sequence consisting of these amino acid residues, and having AAV binding activity.

[0065]

[16] The method according to

[15] , wherein the AAV binding protein is a polypeptide selected from any one of the following (xvi) to (xviii): (xvi) a polypeptide 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: 94; (xvii) A polypeptide having an amino acid sequence including 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: 94, provided that the amino acid sequence includes substitution, deletion, insertion, or addition of one or several amino acid residues at one or several positions in the amino acid residues at positions 312 to 500, and having AAV binding activity; (xviii) A polypeptide comprising at least the amino acid residues from serine at position 312 to aspartic acid at position 500 of the amino acid sequence set forth in SEQ ID NO: 94, wherein the amino acid sequence has 70% or more identity to the amino acid sequence consisting of the amino acid residues at positions 312 to 500, and wherein the polypeptide has AAV-binding activity.

[0066]

[17] The method according to

[15] or

[16] , wherein the AAV adsorbent is in the form of a column packed with the adsorbent.

[0067] The AAV-binding protein, which is a ligand protein (protein immobilized on an insoluble carrier) of the adsorbent used in the analysis of AAV in the present invention (hereinafter also referred to as "AAV adsorbent"), is a polypeptide that has AAV-binding activity and includes at least a region corresponding to extracellular domain 1 (PKD1) (amino acid residues from serine (Ser) at position 312 to glutamic acid (Glu) at position 401 of SEQ ID NO: 94) or domain 2 (PKD2) (amino acid residues from isoleucine (Ile) at position 409 to aspartic acid (Asp) at position 500 of SEQ ID NO: 94) of KIAA0319L (UniProt accession number: Q8IZA0, SEQ ID NO: 94), which is one embodiment of the AAV receptor (AAVR). Specifically, the polypeptide may be any of the following polypeptides (xiii) to (xv): (xiii) a polypeptide comprising at least the amino acid residues from serine at position 312 to glutamic acid at position 401 or the amino acid residues from isoleucine at position 409 to aspartic acid at position 500 in the amino acid sequence set forth in SEQ ID NO: 94; (xiv) a polypeptide having an amino acid sequence comprising at least the amino acid residues from serine at position 312 to glutamic acid at position 401 or from isoleucine at position 409 to aspartic acid at position 500 of the amino acid sequence set forth in SEQ ID NO: 94, and including substitution, deletion, insertion, or addition of one or several amino acid residues at one or several positions within these amino acid residues, and having AAV binding activity; (xv) A polypeptide comprising at least the amino acid residues from serine at position 312 to glutamic acid at position 401 or from isoleucine at position 409 to aspartic acid at position 500 of the amino acid sequence set forth in SEQ ID NO: 94, and having 70% or more homology to an amino acid sequence consisting of these amino acid residues, and having AAV binding activity.

[0068] Furthermore, preferred embodiments of the AAV-binding protein include polypeptides that include at least a region corresponding to PKD1 and PKD2 of KIAA0319L (SEQ ID NO: 94) (amino acid residues from serine (Ser) at position 312 to aspartic acid (Asp) at position 500 of SEQ ID NO: 94), and specifically include polypeptides selected from any of the following (xvi) to (xviii): (xvi) a polypeptide 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: 94; (xvii) A polypeptide having an amino acid sequence including 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: 94, provided that the amino acid sequence includes substitution, deletion, insertion, or addition of one or several amino acid residues at one or several positions in the amino acid residues at positions 312 to 500, and having AAV binding activity; (xviii) A polypeptide comprising at least the amino acid residues from serine at position 312 to aspartic acid at position 500 of the amino acid sequence set forth in SEQ ID NO: 94, wherein the amino acid sequence has 70% or more identity to the amino acid sequence consisting of the amino acid residues at positions 312 to 500, and wherein the polypeptide has AAV-binding activity.

[0069] The polypeptide shown in any of (xiii) to (xviii) above need only contain at least a region corresponding to PKD1 and / or PKD2 of the above-mentioned KIAA0319L, and may, for example, contain all or a part of a region corresponding to other extracellular region domains (domain 3 (PKD3), domain 4 (PKD4) and domain 5 (PKD5)) located on the C-terminal side of PKD2, or may contain all or a part of a region corresponding to a signal sequence such as the MANSC (Motif At N terminus with Seven Cysteines) domain located on the N-terminal side of PKD1 or a cysteine-rich region, or may contain all or a part of a transmembrane region and an intracellular region located on the N-terminal and / or C-terminal side of the extracellular region.

[0070] Examples of (xiv) and (xvii) include a polypeptide containing at least the amino acid residues from the 25th serine (Ser) to the 213th aspartic acid (Asp) in the amino acid sequence set forth in SEQ ID NO: 97, and the AAV binding protein disclosed in WO2021 / 106882. Examples of the substitutions, deletions, insertions, or additions described in (xiv) and (xvii) include the substitutions of amino acid residues disclosed in WO2021 / 106882.

[0071] In (xiv) and (xvii), "one or several" varies depending on the position of the amino acid substitution in the three-dimensional structure of AAVR and the type of amino acid residue, but for example, it means 1 to 50, 1 to 30, 1 to 20, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1. The substitution of "one or several" amino acid residues may occur at positions other than the substitution of amino acid residues disclosed in WO2021 / 106882, for example, as long as it has AAV binding activity.

[0072] In addition, the "substitution of one or several amino acid residues" in (xiv) and (xvii) may include, in addition to the amino acid substitution at the specific position described above, conservative substitution in which a substitution occurs between amino acids having similar physical and / or chemical properties. Conservative substitution is generally known to those skilled in the art to maintain the function of a protein between a substituted and unsubstituted one. Examples of conservative substitutions include substitution between glycine and alanine, between serine and proline, or between glutamic acid and alanine (Protein Structure and Function, Medical Science International, 9, 2005). In addition, the "substitution, deletion, insertion, or addition of one or several amino acid residues" in (xiv) and (xvii) also includes naturally occurring mutations (mutants or variants) based on differences in the origin of AAVR, differences in species, and the like.

[0073] The homology of the amino acid sequences in (xv) and (xviii) may be 70% or more, and may be higher (e.g., 80% or more, 85% or more, 90% or more, or 95% 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, 31(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, 31(3), Yodosha). The homology of amino acid sequences can be determined using an alignment program such as BLAST (Basic Local Alignment Search Tool) or FASTA.

[0074] In the present invention, the insoluble carrier used for immobilizing the AAV-binding protein described above is not particularly limited as long as it is insoluble in samples containing AAV and solutions used for analysis and purification (e.g., elution solution, equilibration solution, washing solution, etc.). Examples include carriers made from polysaccharides such as agarose, alginate (alginate salt), carrageenan, chitin, cellulose, dextrin, dextran, and starch, carriers made from synthetic polymers such as polyvinyl alcohol, polymethacrylate, poly(2-hydroxyethyl methacrylate), and polyurethane, and carriers made from ceramics such as silica. Among 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, but it is preferred that the shape can be packed into a column. For example, the material may be in the form of particles, monoliths, membranes or fibers, and may be either porous or non-porous.

[0075] The AAV binding protein may be immobilized on the insoluble carrier, for example, via covalent bond. Specifically, for example, the AAV binding protein may be immobilized on the insoluble carrier by covalently bonding the insoluble carrier via an active group possessed by the insoluble carrier, thereby producing the AAV adsorbent used in the present invention. Examples of the active group include N-hydroxysuccinimide (NHS) activated ester group, epoxy group, carboxy group, maleimide group, haloacetyl group, tresyl group, formyl group, and haloacetamide 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).

[0076] 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.

[0077] Examples of compounds that introduce epoxy groups into hydroxyl groups or amino groups present on the carrier surface include epichlorohydrin, ethanediol diglycidyl ether, butanediol diglycidyl ether, hexanediol diglycidyl ether, and polyethylene glycol diglycidyl ether. Specific examples of polyethylene glycol diglycidyl ether include Denacol EX-810 (n=1), Denacol EX-811 (n=1), Denacol EX-850 (n=2), Denacol EX-851 (n=2), Denacol EX-821 (n=4), Denacol EX-830 (n=9), Denacol EX-832 (n=9), Denacol EX-841 (n=13), and Denacol EX-861 (n=22) (all manufactured by Nagase ChemteX Corporation).

[0078] 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.

[0079] Compounds for introducing maleimide groups into hydroxy groups, epoxy groups, carboxy groups, or 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, Examples include succinimidyl-4-(maleimidomethyl)cyclohexane-1-carbonyl-(6-aminohexanoic acid), succinimidyl-4-(maleimidomethyl)cyclohexane-1-carboxylic acid, (p-maleimidobenzoyl)N-hydroxysuccinimide ester, (m-maleimidobenzoyl)N-hydroxysuccinimide ester, and dextran.

[0080] 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.

[0081] 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.

[0082] 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 NHS, 4-nitrophenol, and 1-hydroxybenztriazole.

[0083] The immobilization of the AAV binding protein 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 may be, for example, 4°C or higher and 50°C or lower, and preferably 10°C or higher and 35°C or lower.

[0084] The present invention is characterized in that the AAV contained in a sample is analyzed based on the strength of infectivity by a method including a step of adsorbing the AAV contained in the sample to an AAV adsorbent prepared by the above-mentioned method (hereinafter, also simply referred to as "adsorption step") and a step of eluting the AAV adsorbed to the adsorbent using an elution solution (hereinafter, also simply referred to as "elution step"). Note that an embodiment in which the AAV adsorbent used in the adsorption step and the elution step is packed in a column (hereinafter, also referred to as "AVR column") is preferable in that these steps can be performed easily. The following will explain in detail using an embodiment using an AVR column as an example.

[0085] A sample containing AAV can be added (applied) to the AVR 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 sample containing AAV may be solvent-substituted using an appropriate buffer solution before being added to the AVR column. In addition, before adding a sample containing AAV to the AVR column (i.e., before the adsorption step), the AVR column may be equilibrated using an appropriate buffer solution (equilibration solution). The equilibration is expected to enable, for example, analysis of AAV with higher accuracy. Examples of buffer solutions used for solvent replacement and equilibration include phosphate buffer solution, acetate buffer solution, succinate buffer solution, citrate buffer solution, Tris buffer solution, HEPES buffer solution, and MES buffer solution, which have buffering capacity in the neutral region (referring to a region of pH 4.0 to 9.0 in this specification). Such buffer solutions may further contain inorganic salts such as sodium chloride or calcium chloride at a concentration of 10 mM to 600 mM. The buffer solution used for solvent replacement and the equilibration solution may be the same or different. In addition, if components other than AAV, such as impurities, remain in the AVR column after passing an AAV-containing sample through the AVR column, such components may be removed (washed) from the AVR column before eluting the AAV adsorbed to the AAV adsorbent with an elution solution (i.e., before the elution step). Components other than AAV can be removed from the AVR column, for example, by using an appropriate buffer solution as a washing solution. For the washing solution, for example, the description of the buffer solution used for solvent replacement or equilibration can be applied mutatis mutandis.

[0086] In the elution step, AAV is eluted based on the strength of infectivity, so that it is eluted with a gradient. The gradient may be changed in two or more steps (stepwise) (step gradient), or may be changed with a linear gradient (linear gradient). An example of gradient elution is a gradient that lowers the pH from the neutral region described above to the acidic region (region more acidic than the neutral region described above). The pH of the elution solution at the start may be in the neutral region described above, that is, pH 4.0 to 9.0, and preferably pH 4.5 to 6.0. The acidic region, which is the pH of the elution solution after the gradient, may be pH 3.5 or less, and preferably pH 2.0 to 2.5. There is no particular limit to the lower limit of the flow rate, and the upper limit depends on the back pressure of the insoluble carrier used in the AAV adsorbent. Typically, 0.5 mL / min to 2.0 mL / min is preferable. The gradient time may be any time that the difference in the strength of infectivity can be identified as the difference in elution time, and is preferably 5 minutes or more, more preferably 20 minutes or more, and further preferably 45 minutes or more.

[0087] The AAV that can be analyzed by the method of the present invention may be an AAV that exists in nature or an AAV that has been artificially produced, such as AAV of each serotype (AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, etc.), AAV with amino acid substitutions, AAV fused with a peptide or protein, and AAV modified with a polymer.

[0088] By using the method of the present invention, for example, it is possible to easily screen for AAV amino acid substitutions (AAV mutants) having the desired infectivity. Specifically, a mutant library of plasmids encoding AAV capsids is prepared by gene modification such as error-prone PCR, and a host is transformed with other plasmids necessary for AAV formation. The AAV mutants expressed by the transformants are then applied to an AVR column, and AAV mutants having the desired infectivity can be screened based on the elution peak from the column.

[0089] According to the present invention, AAV contained in a sample can be analyzed according to the strength of infectivity. This allows, for example, purification of AAV with high infectivity and use as a highly efficient gene therapy drug. In addition, when AAV with different infectivity is mixed, the ratio can be confirmed. Furthermore, it may be possible to use it for quality control in the AAV production process to check the difference between lots and uniformity. Effect of the Invention

[0090] The adeno-associated virus (AAV) binding protein of the present invention is a protein in which amino acid residues at specific positions in the region corresponding to the extracellular domain 1 (PKD1) and domain 2 (PKD2) of KIAA0319L (UniProt No. Q8IZA0) are replaced with other amino acid residues. The AAV binding protein of the present invention has improved binding activity to AAV compared to conventional AAV binding proteins. Furthermore, when the AAV binding protein of the present invention is produced by genetic engineering techniques using recombinant E. coli, the production of degradation products derived from the protein is suppressed compared to conventional AAV binding proteins. Therefore, the AAV binding protein can be produced efficiently, and is useful for industrial production of the protein. The present invention is also characterized in that an analysis based on the infectivity of adeno-associated virus (AAV) contained in a sample is carried out by a method comprising the steps of adsorbing the AAV to an adsorbent comprising an insoluble carrier and a polypeptide containing at least an amino acid sequence corresponding to the extracellular domain 1 or domain 2 of KIAA0319L immobilized on the carrier, and eluting the AAV adsorbed to the adsorbent with an elution solution. According to the present invention, an analysis based on the infectivity of AAV contained in a sample can be carried out quickly and simply, whereas in conventional methods, it required a lot of time and effort using cultured cells. [Brief description of the drawings]

[0091] [Figure 1]A diagram summarizing the results of analysis of AAV2 (wild type and its amino acid substitutions) using an AVR10s column. AAV was eluted by applying a gradient of buffer pH from 4.5 to 2.2. The infectivity of each AAV2 (relative to the wild type) is plotted on the vertical axis, and the elution time of the AAV2 on the horizontal axis. Among the AAV2 amino acid substitutions, G383A, H271F, and D529A were not adsorbed to the AVR10s immobilized gel and passed through the AVR10s column, so they are not shown in the figure. [Diagram 2] A diagram summarizing the results of analysis of AAV2 (wild type and its amino acid substitutions) using an AAVX column. AAV was eluted by applying a gradient of buffer pH from 4.5 to 2.2. The infectivity of each AAV2 (relative to the wild type) is plotted on the vertical axis against the elution time of the AAV2 on the horizontal axis. [Diagram 3] A diagram summarizing the results of analysis of AAV2 (wild type and its amino acid substitutions) using an AVR11a column. AAV was eluted by applying a gradient of buffer pH from 6.0 to 2.0. The infectivity of each AAV2 (relative to the wild type) is plotted on the vertical axis, and the elution time of the AAV2 on the horizontal axis. Among the AAV2 amino acid substitutions, H271A, H271Q, H271T, G383A, H271F, and D529A were not adsorbed to the AVR11a immobilized gel and passed through the AVR11a column, so they are not shown in the figure. [Figure 4] A diagram summarizing the results of analysis of AAV2 (wild type and its amino acid substitutions) using an AVR21 column. AAV was eluted by applying a gradient of buffer pH from 6.0 to 2.0. The infectivity of each AAV2 (relative value to the wild type) is plotted on the vertical axis, and the elution time of the AAV2 on the horizontal axis. Among the AAV2 amino acid substitutions, G383A, H271F, and D529A were not adsorbed to the AVR21 immobilized gel and passed through the AVR21 column, so they are not shown in the figure. [Diagram 5]A diagram summarizing the results of analysis of AAV2 (wild type and its amino acid substitutions) using an AAVX column. AAV was eluted by applying a gradient of buffer pH from 6.0 to 2.0. The infectivity of each AAV2 (relative to the wild type) is plotted on the y-axis, and the elution time of the AAV2 on the x-axis. EXAMPLES

[0092] The present invention will be described in detail below using examples, but the present invention is not limited to the above examples.

[0093] Example 1: Construction of adeno-associated virus (AAV) vector (part 1) Among AAVs, serotype 5 (AAV5) was selected to prepare viral vectors to be used in the following examples.

[0094] (1) A nucleotide sequence (SEQ ID NO: 3) 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: 2, and a stop codon (TAG) and a BamHI recognition sequence (GGATCC) were added to the 3'-end.

[0095] (2) A polynucleotide consisting of the sequence set forth in SEQ ID NO:3 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).

[0096] (3) The pUC-EGFP obtained in (2) was digested with the restriction enzymes EcoRI and BamHI, and then ligated into the expression vector pAAV-CMV (Takara Bio Inc.) which had been previously digested with the restriction enzymes EcoRI and BamHI. The ligation product was used to transform the Escherichia coli JM109 strain.

[0097] (4) The transformant obtained in (3) was cultured overnight at 37°C with shaking in a 5L baffled flask containing 1L of 2xYT medium (1.6% (w / v) Tryptone, 1% (w / v) Yeast Extract, 0.5% (w / v) sodium chloride) containing 100 μg / mL carbenicillin. After the culture was completed, the cells were collected by centrifugation, and a large amount of the EGFP-expressing vector pAAV-EGFP was prepared from the collected cells using a Plasmid Mega Kit (Qiagen).

[0098] (5) The E. coli JM109 strain was transformed with a plasmid containing a polynucleotide encoding the AAV5 capsid (hereinafter referred to as "pRC5 Vector") and the pHelper Vector (Takara Bio Inc.). The obtained transformant was used to carry out the same procedure as in (4) to prepare large quantities of the pRC5 Vector and pHelper.

[0099] (6) HEK293T cells were cultured in eight Cellstack cell culture surface treated 5-chamber (Corning) plates containing 500 mL of D-MEM medium (Fujifilm Wako Pure Chemical Industries, Ltd.) containing 5% (v / v) bovine serum. Gene transfer was performed by adding the pAAV-EGFP prepared in (4), the pRC5 Vector and pHelper prepared in (5), and a complex of polyethyleneimine (Polysciences), and the cells were cultured stationarily for 3 days under conditions of 5% (v / v) carbon dioxide and 37°C.

[0100] (7) Triton X-100 (Sigma) was added to the culture medium obtained in (6) at a final concentration of 0.1% (w / v) and Benzonase (Merck Millipore) was added to a final concentration of 1 U / mL, and the culture medium was allowed to stand at 37°C for 3 hours.

[0101] (8) After standing, the solution was centrifuged to collect the supernatant, which was then concentrated using a 300 kD-cutoff membrane cassette for tangential flow systems (Pall Corporation) and solvent-substituted with 20 mM Tris-HCl buffer (pH 8.0) containing 0.5 M sodium chloride (hereinafter also referred to as "equilibration solution A"). The resulting concentrated solution was passed through a filter with a pore size of 0.22 μm to remove suspended matter.

[0102] (9) The solution from which the suspended matter had been removed was applied to a 7 mL POROS AAVX column (manufactured by Thermo Fisher Scientific).

[0103] (10) After washing with equilibration solution A, the solution was eluted with 0.1 M acetate buffer (pH 2.5) containing 0.5 M sodium chloride. The eluted fraction containing the AAV vector 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 (AAV5-EGFP) solution.

[0104] (11) The AAV vector concentration in the solution obtained in (10) was quantified by qPCR using the AAVpro Titration Kit (Takara Bio Inc.). In addition, the solution obtained in (10) was subjected to SDS-PAGE (SDS-polyacrylamide electrophoresis) and silver stained using the Pierce Silver Stain Kit (Thermo Fisher Scientific Inc.) to confirm the purity of the AAV vector contained in the solution.

[0105] As a result of qPCR, the concentration of AAV vector (AAV5-EGFP) in the solution was 2.5 × 10 13 The concentration was cp / mL (cp: number of AAV particles). In addition, the results of SDS-PAGE (silver staining) showed that only bands corresponding to the three types of outer capsid proteins (VP1, VP2, and VP3) that compose the AAV vector were observed, confirming that there was no problem with the purity.

[0106] Example 2 Site-specific amino acid substitutions in AAV binding proteins and library construction (part 1) Using the vector pET-AVR10s (WO2021 / 106882) capable of expressing a polypeptide (sequence number 4) containing the AAV binding protein AVR10s as a template, a saturated substitution library was prepared for the amino acid residues corresponding to alanine (Ala330) at position 330 (position 43 in SEQ ID NO: 4) of SEQ ID NO: 1, tyrosine (Tyr331) at position 331 (position 44 in SEQ ID NO: 4), valine (Val332) at position 332 (position 45 in SEQ ID NO: 4), and leucine (Leu333) at position 333 (position 46 in SEQ ID NO: 4). In addition, in the polypeptide consisting of the amino acid sequence described in SEQ ID NO: 4, 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, and the histidine (His) at positions 214 to 219 is the tag sequence. AVR10s is a polypeptide in which the following 10 amino acid substitutions have occurred in the amino acid residues from positions 312 to 500 of SEQ ID NO: 1, which correspond to the extracellular domain 1 (PKD1) and domain 2 (PKD2) of KIAA0319L (UniProt No. Q8IZA0). Valine at position 317 of SEQ ID NO:1 (position 30 of SEQ ID NO:4) is substituted with aspartic acid Tyrosine at position 342 of SEQ ID NO:1 (position 55 of SEQ ID NO:4) is substituted with serine Lysine at position 362 of SEQ ID NO:1 (position 75 of SEQ ID NO:4) is substituted with glutamic acid The lysine at position 371 of SEQ ID NO:1 (position 84 of SEQ ID NO:4) is substituted with asparagine Valine at position 381 of SEQ ID NO:1 (position 94 of SEQ ID NO:4) is substituted with alanine Isoleucine at position 382 of SEQ ID NO:1 (position 95 of SEQ ID NO:4) is substituted with valine Glycine at position 390 of SEQ ID NO:1 (position 103 of SEQ ID NO:4) is substituted with serine Lysine at position 399 of SEQ ID NO:1 (position 112 of SEQ ID NO:4) is substituted with glutamic acid Serine at position 476 of SEQ ID NO:1 (position 189 of SEQ ID NO:4) is substituted with arginine The asparagine at position 487 of SEQ ID NO: 1 (position 200 of SEQ ID NO: 4) is substituted with aspartic acid.

[0107] (1) A PCR primer set for preparing the above-mentioned saturated substitution library was designed. Specifically, As PCR primers for amino acid substitution for Ala330, SEQ ID NOs: 5 (forward) and 6 (reverse) were used. As PCR primers for amino acid substitution at Tyr331, SEQ ID NOs: 7 (forward) and 8 (reverse) were used. As PCR primers for amino acid substitution at Val332, SEQ ID NO: 9 (forward) and 10 (reverse) were used. As PCR primers for amino acid substitution at Leu333, SEQ ID NOs: 11 (forward) and 12 (reverse) were used. Each was designed.

[0108] (2) After preparing a reaction solution having the composition shown in Table 1, the reaction solution was heat-treated at 98° C. for 5 minutes, and PCR was performed by performing 30 cycles of reaction in which the first step was at 98° C. for 10 seconds, the second step was at 55° C. for 5 seconds, and the third step was at 72° C. for 6 minutes, and finally heat-treated at 72° C. for 5 minutes. In addition, when performing amino acid substitution for Ala330, PCR primers having the sequences shown in SEQ ID NOs: 5 and 6 were used, when performing amino acid substitution for Tyr331, PCR primers having the sequences shown in SEQ ID NOs: 7 and 8 were used, when performing amino acid substitution for Val332, PCR primers having the sequences shown in SEQ ID NOs: 9 and 10 were used, and when performing amino acid substitution for Leu333, PCR primers having the sequences shown in SEQ ID NOs: 11 and 12 were used.

[0109] [Table 1]

[0110] (3) The amplified PCR product was subjected to agarose gel electrophoresis and purified from the gel using a QIAquick Gel Extraction kit (Qiagen).

[0111] (4) The resulting PCR product was treated with the restriction enzyme DpnI (NEB) at 37° C. for 1.5 hours, and then at 80° C. for 20 minutes. The resulting polynucleotide was subjected to agarose gel electrophoresis and purified from the gel using a QIAquick Gel Extraction kit (Qiagen).

[0112] (5) A reaction solution having the composition shown in Table 2 was prepared, and the DpnI-treated PCR product purified in (4) was digested with the restriction enzyme BsaI (NEB) while a ligation reaction was carried out at 25°C for 1.0 hour using T4 DNA ligase (NEB).

[0113] [Table 2]

[0114] (6) The resulting ligation products were used to transform E. coli BL21(DE3) strain and cultured on LB plate medium containing 50 μg / mL kanamycin (37°C, 16 hours). The colonies formed on the plate were used as a site-directed mutant library.

[0115] Example 3 Screening of AVR10s amino acid substitutions (part 1) (1) The site-directed mutant library (transformant) prepared in Example 2 was inoculated into 300 μL of 2×YT liquid medium containing 50 μg / mL of kanamycin, and cultured overnight at 37° C. with shaking in a 96-deep-well plate.

[0116] (2) After cultivation, 5 μL of the culture solution from (1) was inoculated into 500 μL of 2×YT liquid medium containing 50 μg / mL kanamycin and 0.1 mM IPTG (Isopropyl-β-D-thiogalactopyranoside), and further cultured overnight at 25°C with shaking in a 96-deep-well plate.

[0117] (3) After the cultivation, the culture supernatant obtained by centrifugation was diluted 10-fold with 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride.

[0118] (4) The binding affinity between the AAV binding protein in the culture supernatant prepared in (3) and the AAV vector (AAV5-EGFP) prepared in Example 1 was evaluated by the following Enzyme-Linked Immunosorbent Assay (ELISA). (4-1) The AAV5-EGFP prepared in Example 1 was diluted 200-fold 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. (4-2) 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), the culture supernatant prepared in (3) was added and the AAV binding protein was reacted with AAV5-EGFP (at 30°C for 1 hour). (4-3) 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. (4-4) 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).

[0119] (5) Approximately 400 transformant strains were evaluated, and transformants expressing AAV binding proteins with improved binding activity to AAV5 compared to AVR10s (sequence number 4) were selected.

[0120] (6) The selected transformants were cultured, and expression vectors were prepared using a QIAprep Spin Miniprep kit (QIAGEN).

[0121] (7) Of the expression vectors prepared in (6), the nucleotide sequences of the polynucleotides encoding the AAV binding proteins and the surrounding regions were analyzed using a fully automated DNA sequencer, Genetic Analyzer 3500 (Thermo Fisher Scientific), to identify amino acid substitution sites. In this analysis, an oligonucleotide consisting of the sequence shown in SEQ ID NO: 13 (5'-TAATACGACTCACTATAGGG-3') or SEQ ID NO: 14 (5'-ATGCTAGTTATTGCTCAGCGG-3') was used as a sequencing primer.

[0122] Table 3 shows the amino acid substitution positions for AVR10s and the binding activity for AAV5 (ratio when the binding activity for AVR10s is set to 1) of the AAV binding proteins expressed by the transformants selected in (5) above.

[0123] It can be seen that an AAV binding protein having at least one amino acid substitution, among Ala330Cys (this notation indicates that alanine at position 330 in SEQ ID NO: 1 (position 43 in SEQ ID NO: 4) has been substituted with cysteine, and the same applies below), Ala330Phe, Ala330Leu, Ala330Arg, Ala330Val, Ala330Trp, Ala330Tyr, Tyr331Cys, Val332Trp, Val332Tyr, or Leu333Cys, in the amino acid residues from serine (Ser) at position 312 to aspartic acid (Asp) at position 500 in the amino acid sequence set forth in SEQ ID NO: 1 has improved binding activity to AAV5 compared to an AAV binding protein (AVR10s) without these amino acid substitutions.

[0124] In particular, AAV binding proteins having any of the amino acid substitutions Ala330Cys, Ala330Val, Ala330Tyr, and Val332Trp are found to have particularly improved binding activity to AAV5.

[0125] [Table 3]

[0126] Among the AAV-binding proteins with improved binding activity to AAV5 obtained in this example, the AAV-binding protein in which the amino acid substitution Ala330Cys was made in AVR10s was designated AVR10s_A330C (SEQ ID NO: 15); The AAV binding protein in which the amino acid substitution Ala330Phe was made in AVR10s was designated AVR10s_A330F (SEQ ID NO: 16). The AAV binding protein in which the amino acid substitution Ala330Leu was made in AVR10s was designated AVR10s_A330L (SEQ ID NO: 17). The AAV binding protein in which the amino acid substitution Ala330Arg was made in AVR10s was designated AVR10s_A330R (SEQ ID NO: 18). The AAV binding protein in which the amino acid substitution Ala330Val was generated in AVR10s was designated AVR10s_A330V (SEQ ID NO: 19). The AAV binding protein in which the amino acid substitution Ala330Trp was generated in AVR10s was designated AVR10s_A330W (SEQ ID NO: 20). The AAV binding protein in which the amino acid substitution Ala330Tyr was made in AVR10s was designated AVR10s_A330Y (SEQ ID NO: 21). The AAV binding protein in which the amino acid substitutions of Tyr331Cys were made in AVR10s was designated AVR10s_Y331C (SEQ ID NO: 22). The AAV binding protein in which the amino acid substitution Val332Trp was generated in AVR10s was designated AVR10s_V332W (SEQ ID NO: 23). The AAV binding protein in which the amino acid substitution Val332Tyr was generated in AVR10s was designated AVR10s_V332Y (SEQ ID NO: 24). The AAV binding protein in which the amino acid substitution Leu333Cys has been generated relative to AVR10s is designated AVR10s_L333C (sequence number 25).

[0127] Of the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 15 to 25, the portion from the 1st methionine (Met) to the 22nd alanine (Ala) is the PelB signal peptide, the portion from the 25th serine (Ser) to the 213th aspartic acid (Asp) is the AAV binding protein (AVR10s amino acid substitution), and the 214th to 219th histidines (His) are the tag sequence.

[0128] Example 4 Preparation of AAV binding protein and calculation of degradation ratio (1) The 11 types of AAV binding proteins (AVR10s_A330C, AVR10s_A330F, AVR10s_A330L, AVR10s_A330R, AVR10s_A330V, AVR10s_A330W, AVR10s_A330Y, AVR10s_Y331C, AVR10s_V332W, AVR10s_V332Y, and AVR10s_L333C) obtained in Example 3 and a plasmid containing a polynucleotide encoding any one of AVR10s were transformed into Escherichia coli strain BL21(DE3). The resulting transformants capable of expressing the AAV binding proteins were inoculated into 3 mL of 2×YT liquid medium containing 50 μg / mL kanamycin, and pre-cultured overnight at 37° C. under aerobic shaking.

[0129] (2) 2 mL of the preculture solution from (1) was inoculated into 200 mL of 2xYT liquid medium containing 50 μg / mL kanamycin in a 1 L baffled flask, and cultured aerobically at 37°C with shaking.

[0130] (3) After 2.0 hours from the start of the culture, the mixture was cooled on ice, IPTG was added to a final concentration of 0.1 mM, and the mixture was subsequently cultured aerobically with shaking at 25°C for 20 hours.

[0131] (4) After the cultivation was completed, the culture medium was centrifuged at 4°C and 8,000 rpm for 20 minutes to recover each bacterial cell.

[0132] (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 (hereinafter also referred to as "equilibration solution B") 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 8000 rpm for 20 minutes at 4°C, and each supernatant was collected.

[0133] (6) The supernatant obtained in (5) was applied to an open column packed with 1.5 mL of Ni Sepharose 6 Fast Flow (manufactured by Cytiva) that had been previously equilibrated with equilibration solution B. After washing with equilibration solution B, elution was performed with 20 mM Tris-HCl buffer (pH 7.4) containing 0.5 M imidazole and 150 mM sodium chloride.

[0134] (7) Each eluate obtained in (6) was dialyzed against 20 mM HEPES (2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid) buffer (pH 7.4) containing 150 mM sodium chloride to prepare AAV-binding proteins.

[0135] (8) The AAV-binding protein obtained in (7) was subjected to capillary electrophoresis (SCIEX) to calculate the percentage of decomposition products of the AAV-binding protein.

[0136] The results are shown in Table 4. In the amino acid sequence of SEQ ID NO: 1, an AAV binding protein having at least one amino acid substitution of any of Ala330Cys, Ala330Phe, Ala330Leu, Ala330Arg, Ala330Val, Ala330Trp, Ala330Tyr, Tyr331Cys, Val332Trp, Val332Tyr, and Leu333Cys in the amino acid residues from serine (Ser) at position 312 to aspartic acid (Asp) at position 500 has a reduced proportion of degradation products derived from the AAV binding protein compared to an AAV binding protein without these amino acid substitutions (AVR10s: degradation rate 30.0%), suggesting an improvement in the efficiency of AAV binding protein production using recombinant E. coli.

[0137] In particular, the AAV binding proteins with amino acid substitutions of Ala330Val or Tyr331Cys (AVR10s_A330V or AVR10s_Y331C) showed a particularly reduced proportion of degradation products derived from the AAV binding protein (AVR10s_A330V: degradation rate 1.9%, AVR10s_Y331C: degradation rate 2.0%), which is expected to significantly improve the production efficiency of AAV binding proteins using recombinant E. coli.

[0138] [Table 4]

[0139] Example 5 Site-specific amino acid substitutions in AAV binding proteins and library construction (part 2) Using the vector pET-AVR10s (WO2021 / 106882) capable of expressing a polypeptide containing the AAV binding protein AVR10s (SEQ ID NO: 4) as a template, a saturated substitution library was prepared for the amino acid residues corresponding to glutamine (Gln347) at position 347 (position 60 in SEQ ID NO: 4), leucine (Leu348) at position 348 (position 61 in SEQ ID NO: 4), and isoleucine (Ile349) at position 349 (position 62 in SEQ ID NO: 4) of SEQ ID NO: 1.

[0140] (1) A PCR primer set for preparing the above-mentioned saturated substitution library was designed. Specifically, As PCR primers for amino acid substitution at Gln347, SEQ ID NO: 26 (forward) and 27 (reverse) were used. As PCR primers for amino acid substitution at Leu348, SEQ ID NOs: 28 (forward) and 29 (reverse) were used. As PCR primers for amino acid substitution for Ile349, SEQ ID NOs: 30 (forward) and 31 (reverse) were used. Each was designed.

[0141] (2) PCR was performed in the same manner as in Example 2(2) except that PCR primers consisting of the sequences set forth in SEQ ID NOs: 26 and 27 were used when making an amino acid substitution for Gln347, PCR primers consisting of the sequences set forth in SEQ ID NOs: 28 and 29 were used when making an amino acid substitution for Leu348, and PCR primers consisting of the sequences set forth in SEQ ID NOs: 30 and 31 were used when making an amino acid substitution for Ile349.

[0142] (3) The PCR products amplified by the methods described in Examples 2(3) and (4) were purified and then subjected to a ligation reaction by the method described in Example 2(5).

[0143] (4) The resulting ligation products were used to transform E. coli BL21(DE3) strain and cultured on LB plate medium containing 50 μg / mL kanamycin (37°C, 16 hours). The colonies formed on the plate were used as a site-directed mutant library.

[0144] Example 6 Screening of AVR10s amino acid substitutions (part 2) (1) The site-directed mutant library (transformant) prepared in Example 5 was cultured by the method described in Example 3(1) and (2), and the culture supernatant obtained by centrifugation was diluted 10-fold with 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride.

[0145] (2) The binding affinity between the AAV binding protein in the diluted culture supernatant prepared in (1) and the AAV vector (AAV5-EGFP) prepared in Example 1 was evaluated by the ELISA method described in Example 3(4).

[0146] (3) Approximately 400 transformant strains were evaluated, and from among them, transformants expressing AAV binding proteins with improved binding activity to AAV5 compared to AVR10s (sequence number 4) were selected.

[0147] (4) The selected transformants were cultured, and expression vectors were prepared using a QIAprep Spin Miniprep kit (QIAGEN).

[0148] (5) Of the expression vectors prepared in (4), the nucleotide sequences of the polynucleotides encoding the AAV binding proteins and the surrounding regions were analyzed by the method described in Example 3(7) to identify amino acid substitution sites.

[0149] Table 5 shows the amino acid substitution positions for AVR10s and the binding activity for AAV5 (ratio when the binding activity for AVR10s is set to 1) of the AAV binding proteins expressed by the transformants selected in (3) above.

[0150] It has been shown that an AAV binding protein having at least one amino acid substitution, Gln347Ser, Leu348Cys, or Ile349Asn, in the amino acid residues from serine (Ser) 312 to aspartic acid (Asp) 500 in the amino acid sequence set forth in SEQ ID NO:1 has improved binding activity to AAV5 compared to an AAV binding protein (AVR10s) that does not have these amino acid substitutions.

[0151] [Table 5]

[0152] Among the AAV-binding proteins with improved binding activity to AAV5 obtained in this example, the AAV-binding protein in which the amino acid substitution Gln347Ser was made in AVR10s was designated AVR10s_Q347S (SEQ ID NO: 32); The AAV binding protein in which the amino acid substitution Leu348Cys was generated in AVR10s was designated AVR10s_L348C (SEQ ID NO: 33). The AAV binding protein in which the amino acid substitution Ile349Asn has been generated relative to AVR10s is designated AVR10s_I349N (sequence number 34).

[0153] Of the polypeptide consisting of the amino acid sequence set forth in SEQ ID NOs: 32 to 34, the portion from the 1st methionine (Met) to the 22nd alanine (Ala) is the PelB signal peptide, the portion from the 25th serine (Ser) to the 213th aspartic acid (Asp) is the AAV binding protein (AVR10s amino acid substitution), and the 214th to 219th histidines (His) are the tag sequence.

[0154] Example 7 Preparation of AAV binding protein and calculation of degradation product ratio (part 2) (1) The three types of AAV binding proteins (AVR10s_Q347S, AVR10s_L348C, and AVR10s_I349N) obtained in Example 6 and a plasmid containing a polynucleotide encoding any one of AVR10s were transformed into Escherichia coli strain BL21(DE3), and the AAV binding proteins were then prepared from the transformants capable of expressing the proteins by the methods described in Example 4(1) to (7).

[0155] (2) The AAV-binding protein obtained in (1) was subjected to capillary electrophoresis (SCIEX) to calculate the percentage of decomposition products of the AAV-binding protein.

[0156] The results are shown in Table 6. The AAV binding protein having at least one amino acid substitution of Gln347Ser, Leu348Cys, or Ile349Asn in the amino acid residues from serine (Ser) at position 312 to aspartic acid (Asp) at position 500 in the amino acid sequence set forth in SEQ ID NO: 1 had a reduced proportion of degradation products derived from the AAV binding protein compared to the AAV binding protein without these amino acid substitutions (AVR10s: degradation rate 18.4%), suggesting improved efficiency in production of the AAV binding protein using recombinant E. coli.

[0157] [Table 6]

[0158] Example 8: Construction of AAV vector (part 2) (1) The Escherichia coli JM109 strain was transformed with the plasmid pRC2-mi342 Vector (Takara Bio Inc.) and pHelper Vector (Takara Bio Inc.), which contain a polynucleotide encoding the capsid of AAV serotype 2 (AAV2). The resulting transformant was cultured by the method described in Example 1(4).

[0159] (2) The culture medium of (1) was centrifuged to recover the bacterial cells, and then the pRC2-mi342 Vector and pHelper Vector were prepared in large quantities from the recovered bacterial cells using a Plasmid Mega Kit (Qiagen).

[0160] (3) HEK293T cells were cultured in five T-225 flasks (Thermo Fisher Scientific) containing 40 mL of D-MEM medium (Fujifilm Wako Pure Chemical Industries, Ltd.) containing 10% (v / v) bovine serum.

[0161] (4) Using TransIT-ViruGEN Transfection Reagent (Takara Bio), the pRC2-mi342 Vector and pHelper Vector prepared in (2) were transfected into the HEK293T cells cultured in (3), and the cells were cultured statically for 3 days under conditions of 5% carbon dioxide and 37°C.

[0162] (5) The cells cultured in (4) were collected and extracted and purified using the AAVpro Purification Kit (Takara Bio Inc.) to obtain VLP2 (virus-like particle, outer protein particle of AAV serotype 2). Approximately 1 mL of VLP2 purified solution was prepared from five T-225 flasks.

[0163] Example 9: Preparation and screening of AAV binding protein mutation library (part 1) Among the AAV-binding proteins evaluated in Examples 2 and 3, a protein in which an amino acid substitution of Ala330Val had occurred (SEQ ID NO: 19, hereinafter also referred to as AVR11a) was selected, and mutations were randomly introduced into the polynucleotide portion encoding the protein using error-prone PCR.

[0164] (1) Error-prone PCR was performed using the vector pET-AVR11a capable of expressing the polypeptide (SEQ ID NO: 19) containing AVR11a prepared in Example 2 as a template. Error-prone PCR was performed by preparing a reaction solution having the composition shown in Table 7, 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. Mutations were successfully introduced into the polynucleotide encoding the AAV binding protein by the error-prone PCR, and the average mutation introduction rate was 1.0 amino acid mutation per molecule.

[0165] [Table 7]

[0166] (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.

[0167] (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.

[0168] (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.

[0169] (5) The culture medium from (4) was centrifuged, and the resulting culture supernatant was diluted 2-fold with ultrapure water. 60 μL of the diluted culture medium was mixed with 60 μL of 0.1 M sodium hydroxide aqueous solution and subjected to an alkali treatment at 61.2° C. for 15 minutes.

[0170] (6) The binding activity of the AAV binding protein to VLP2 when the treatment in (5) was performed and the binding activity of the AAV binding protein to VLP2 when the treatment in (5) was not performed were measured by the ELISA method described in Example 3 (4). The binding activity of the AAV binding protein to VLP2 when the heat treatment was performed was divided by the binding activity of the AAV binding protein to VLP2 when the heat treatment was not performed to calculate the residual activity.

[0171] (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 AVR11a were selected from the library. The selected transformants were cultured, and expression vectors were prepared using a QIAprep Spin Miniprep kit (Qiagen).

[0172] (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 3(7), and the amino acid mutation sites were identified.

[0173] Tables 8 and 9 show a summary of the amino acid substitution positions relative to AVR11a and the remaining activity (%) after alkali treatment of the AAV binding proteins expressed by the transformants selected in (7) above.

[0174] 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, Ala313Val, Glu315Gly, Glu315Val, Gln318Arg, Lys323Met, Asn324His, Val326Glu, Gln327Leu, Leu328Gln, Leu328Arg, Leu328Pro, Asn329Tyr, Val332Glu, Val332Ala, Leu333Pro, Gln334Leu, Gln334His, Glu 335Val, Pro337Gln, Lys338Glu, Glu340Asp, Thr341Ala, Thr343Pro, Thr343Ala, Thr343Ser, Asp345Gly, Ile349Thr, Thr350Ala, Asp354Val, Tyr355Hi s, Met359Ile, Met359Leu, Glu360Asp, Ile366Thr, Leu367Val, Lys368Asn, Leu369Gln, Leu376Pro, Phe379Tyr, Lys380Asn, Lys380Arg, Ile(Val)382As p (this notation indicates that the 382nd isoleucine in SEQ ID NO: 1 is first substituted with valine and then further substituted with aspartic acid, the same applies below), Ile(Val)382Ala, Glu384Val, Ala388Thr, His389Asp, His389Arg, His389Asn, His389Leu, Gly(Ser)390Asn, Gly392Arg, Val394Ala, Val394Asp, Asn395Asp, Val396Gly, Val398Ala, Arg403Ser, Arg403His, Lys404Ala, Arg406Ser, Ile409Asn, Ile411Thr, Ile411Leu, Val412Ile, Phe416Ser, Ile419Phe, Ile419Val, Ser420Thr, Thr423Ala, Ser425Gly, Ile428Val, Gly43 0Ala, Thr434Ala, Asp437Asn, Val440Ala, Glu446Asp, Lys448Glu, Lys448Asn, Leu451Ile, Glu453Lys, Lys455Glu, Lys455Arg, Lys464Asn, Lys464Glu,AAV binding proteins having at least one of the following amino acid substitutions, Val469Glu, Gly471Cys, Asn472Tyr, Asn472Asp, Tyr473His, Ser(Arg)476Gly, Thr478Ala, Val479Ala, Val480Asp, Val480Ala, Gly484Ser, Thr486Ser, Ser488Thr, Thr489Ala, Thr489Ser, Asn496Tyr, and Asn496Asp, have improved alkaline stability compared to AVR11a.

[0175] [Table 8]

[0176] [Table 9]

[0177] Example 10: Preparation of AVR11a amino acid substitution aggregates The alkaline stability was further improved by accumulating the amino acid substitutions involved in improving the alkaline stability of the AAV-binding protein, as revealed in Example 9, in AVR11a (SEQ ID NO: 19). Specifically, the following 11 types of AAV-binding proteins (a) to (k) were designed and produced.

[0178] (a) A protein in which the amino acid substitution Asn324His was introduced into AVR11a (SEQ ID NO: 54, named AVR12a) (b) A protein in which the amino acid substitution Glu335Val was introduced into AVR11a (SEQ ID NO: 55, named AVR12b) (c) A protein in which the amino acid substitutions Asn324His and Glu335Val were introduced into AVR11a (SEQ ID NO: 56, named AVR13) (d) A protein in which the amino acid substitutions Asn324His, Thr341Ala, and Glu335Val were introduced into AVR11a (SEQ ID NO: 57, named AVR14a) (e) A protein in which the amino acid substitutions Asn324His, Glu335Val, and Gln334Leu were introduced into AVR11a (SEQ ID NO: 58, named AVR14b) (f) A protein in which the amino acid substitutions Asn324His, Glu335Val, and Met359Ile were introduced into AVR11a (SEQ ID NO: 59, named AVR14c) (g) A protein in which the amino acid substitutions Asn324His, Glu335Val, and Phe379Tyr were introduced into AVR11a (SEQ ID NO: 60, named AVR14d) (h) A protein in which the amino acid substitutions Asn324His, Gln334Leu, Glu335Val, and Thr341Ala were introduced into AVR11a (SEQ ID NO: 61, named AVR15a) (i) A protein in which the amino acid substitutions Asn324His, Gln334Leu, Glu335Val, and Met359Ile were introduced into AVR11a (SEQ ID NO: 62, named AVR15b) (j) A protein in which the amino acid substitutions Asn324His, Gln334Leu, Glu335Val, and Phe379Tyr were introduced into AVR11a (SEQ ID NO: 63, named AVR15c) (k) A protein in which the amino acid substitutions Asn324His, Gln334Leu, Glu335Val, Thr341Ala, and Phe379Tyr were introduced into AVR11a (SEQ ID NO: 65, named AVR16).

[0179] Methods for producing the 11 types of AAV binding proteins shown in (a) to (k) above are described below. (a) AVR12a This protein was produced by selecting Asn324His from the amino acid substitutions involved in alkaline stability identified in Example 9 and introducing this amino acid substitution into AVR11a (SEQ ID NO: 19).

[0180] (a-1) PCR was performed in the same manner as in Example 2(2), except that vector pET-AVR11a capable of expressing a polypeptide containing AVR11a (SEQ ID NO: 19) was used as a template and oligonucleotides consisting of the sequences set forth in SEQ ID NO: 44 (5'-TTTT[GGTCTC]ACATTCAGTTGTACTTCGTGCTTCGGCAG-3') and SEQ ID NO: 45 (5'-TTTT[GGTCTC]AAATGTTTATGTGCTGCAGGAACCACCGAAAG-3') (the brackets in SEQ ID NOs: 44 and 45 indicate the recognition sequences for the restriction enzyme BsaI) were used as PCR primers (Forward: SEQ ID NO: 44, Reverse: SEQ ID NO: 45).

[0181] (a-2) The PCR products amplified by the methods described in Examples 2(3) and 2(4) were purified and then subjected to a ligation reaction by the method described in Example 2(5).

[0182] (a-3) The ligation product obtained was used to transform E. coli BL21 strain (DE3). The resulting transformant was cultured in LB medium supplemented with 50 μg / mL kanamycin, and the resulting transformant was then collected by centrifugation. The plasmid was extracted from the transformant to obtain a plasmid (expression vector) pET-AVR12a containing a polynucleotide encoding AVR12a, which has 12 amino acid substitutions relative to the wild-type AAV binding protein.

[0183] (a-4) The nucleotide sequence of pET-AVR12a was analyzed and confirmed in a manner similar to that described in Example 3(7).

[0184] The amino acid sequence of AVR12a with the signal sequence and polyhistidine tag added is shown in SEQ ID NO: 54. In SEQ ID NO: 54, the portion from the 1st methionine (Met) to the 22nd alanine (Ala) is the PelB signal peptide, the portion from the 25th serine (Ser) to the 213th aspartic acid (Asp) is the AAV binding protein AVR12a (corresponding to the region from the 312th to the 500th in SEQ ID NO: 1), and the portion from the 214th to the 219th histidine (His) is the tag sequence. In addition, in SEQ ID NO:54, the aspartic acid of Val317Asp is at the 30th position, the histidine of Asn324His is at the 37th position, the valine of Ala330Val is at the 43rd position, the serine of Tyr342Ser 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.

[0185] (b) AVR12b This protein was produced by selecting Glu335Val from the amino acid substitutions involved in improving alkaline stability revealed in Example 9, and introducing this amino acid substitution into AVR11a (SEQ ID NO: 19).

[0186] (b-1) PCR was performed in the same manner as in Example 2(2), except that the aforementioned pET-AVR11a was used as a template and oligonucleotides consisting of the sequences set forth in SEQ ID NO: 46 (5'-TTTT[GGTCTC]ACATTCAGTTGTACTTCGTTCTTCGGCAG-3') and SEQ ID NO: 47 (5'-TTTT[GGTCTC]AAATGTTTATGTGCTGCAGGTACCACCGAAAG-3') (the brackets in SEQ ID NOs: 46 and 47 indicate the recognition sequences for the restriction enzyme BsaI) were used as PCR primers (Forward: SEQ ID NO: 46, Reverse: SEQ ID NO: 47).

[0187] (b-2) From the amplified PCR product, a plasmid (expression vector) pET-AVR12b containing a polynucleotide encoding AVR12b in which 12 amino acid substitutions were made with respect to the wild-type AAV binding protein was obtained in a similar manner to (a-2) to (a-3), and the nucleotide sequence of the plasmid was then analyzed and confirmed in a similar manner to Example 3(7).

[0188] The amino acid sequence of AVR12b with the signal sequence and polyhistidine tag added is shown in SEQ ID NO: 55. In SEQ ID NO: 55, the portion from the 1st methionine (Met) to the 22nd alanine (Ala) is the PelB signal peptide, the portion from the 25th serine (Ser) to the 213th aspartic acid (Asp) is the AAV binding protein AVR12b (corresponding to the region from the 312th to the 500th in SEQ ID NO: 1), and the portion from the 214th to the 219th histidine (His) is the tag sequence. In addition, in SEQ ID NO:55, the aspartic acid of Val317Asp is at the 30th position, the valine of Ala330Val is at the 43rd position, the valine of Glu335Val is at the 48th position, the serine of Tyr342Ser 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.

[0189] (c)AVR13 This protein was produced by selecting Asn324His and Glu335Val from the amino acid substitutions involved in improving alkaline stability identified in Example 9, and introducing these amino acid substitutions into AVR11a (sequence number 19).

[0190] (c-1) PCR was performed in the same manner as in Example 2(2) except that the aforementioned pET-AVR11a was used as a template and oligonucleotides consisting of the sequences set forth in SEQ ID NO: 48 (5'-TTTT[GGTCTC]ACATTCAGTTGTACTTCGTGCTTCGGCAG-3') and SEQ ID NO: 49 (5'-TTTT[GGTCTC]AAATGTTTATGTGCTGCAGGTACCACCGAAAG-3') (the brackets in SEQ ID NOs: 48 and 49 indicate the recognition sequences for the restriction enzyme BsaI) were used as PCR primers (Forward: SEQ ID NO: 48, Reverse: SEQ ID NO: 49).

[0191] (c-2) From the amplified PCR product, a plasmid (expression vector) pET-AVR13 containing a polynucleotide encoding AVR13 in which 13 amino acid substitutions were made with respect to the wild-type AAV binding protein was obtained in a similar manner to (a-2) to (a-3), and the nucleotide sequence of the plasmid was then analyzed and confirmed in a similar manner to Example 3(7).

[0192] The amino acid sequence of AVR13 with the signal sequence and polyhistidine tag added is shown in SEQ ID NO: 56. In SEQ ID NO: 56, the portion from the 1st methionine (Met) to the 22nd alanine (Ala) is the PelB signal peptide, the portion from the 25th serine (Ser) to the 213th aspartic acid (Asp) is the AAV binding protein AVR13 (corresponding to the region from the 312th to the 500th in SEQ ID NO: 1), and the portion from the 214th to the 219th histidine (His) is the tag sequence. In addition, in SEQ ID NO:56, the aspartic acid of Val317Asp is at the 30th position, the histidine of Asn324His is at the 37th position, the valine of Ala330Val is at the 43rd position, the valine of Glu335Val is at the 48th position, the serine of Tyr342Ser 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.

[0193] (d)AVR14a This protein is a protein in which Asn324His, Glu335Val, and Thr341Ala were selected from the amino acid substitutions involved in improving alkaline stability revealed in Example 9, and these amino acid substitutions were accumulated in AVR11a (SEQ ID NO: 19). Specifically, AVR14a was produced by introducing mutations that cause amino acid substitutions of Asn324His and Glu335Val into a polynucleotide (SEQ ID NO: 38) encoding a protein obtained in Example 9 in which the amino acid substitution of Thr341Ala was introduced into AVR11a (designated AVR11a_T341A, SEQ ID NO: 39).

[0194] (d-1) PCR was performed in the same manner as in (c-1) except that the vector pET-AVR11a_T341A capable of expressing a polypeptide containing AVR11a_T341A (SEQ ID NO: 39) was used as a template.

[0195] (d-2) From the amplified PCR product, a plasmid (expression vector) pET-AVR14a containing a polynucleotide encoding AVR14a in which 14 amino acid substitutions were made with respect to the wild-type AAV binding protein was obtained in a similar manner to (a-2) to (a-3), and the nucleotide sequence of the plasmid was then analyzed and confirmed in a similar manner to Example 3(7).

[0196] The amino acid sequence of AVR14a with the signal sequence and polyhistidine tag added is shown in SEQ ID NO: 57. In SEQ ID NO: 57, the portion from the 1st methionine (Met) to the 22nd alanine (Ala) is the PelB signal peptide, the portion from the 25th serine (Ser) to the 213th aspartic acid (Asp) is the AAV binding protein AVR14a (corresponding to the region from the 312th to the 500th in SEQ ID NO: 1), and the portion from the 214th to the 219th histidine (His) is the tag sequence. In addition, in SEQ ID NO:57, the aspartic acid of Val317Asp is at the 30th position, the histidine of Asn324His is at the 37th position, the valine of Ala330Val is at the 43rd position, the valine of Glu335Val is at the 48th position, the alanine of Thr341Ala is at the 54th position, the serine of Tyr342Ser 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.

[0197] (e)AVR14b This protein is a protein in which Asn324His, Gln334Leu, and Glu335Val were selected from the amino acid substitutions involved in improving alkaline stability revealed in Example 9, and these amino acid substitutions were accumulated in AVR11a (SEQ ID NO: 19). Specifically, AVR14b was produced by introducing mutations that cause amino acid substitutions of Asn324His and Glu335Val into a polynucleotide (SEQ ID NO: 36) encoding a protein obtained in Example 9 in which the amino acid substitution of Gln334Leu had been introduced (designated AVR11a_Q334L, SEQ ID NO: 37).

[0198] (e-1) PCR was performed in the same manner as in (c-1) except that the vector pET-AVR_Q334L capable of expressing a polypeptide containing AVR11a_Q334L (SEQ ID NO: 37) was used as a template.

[0199] (e-2) From the amplified PCR product, a plasmid (expression vector) pET-AVR14b containing a polynucleotide encoding AVR14b in which 14 amino acid substitutions were made relative to the wild-type AAV binding protein was obtained in a similar manner to (a-2) to (a-3), and the nucleotide sequence of the plasmid was then analyzed and confirmed in a similar manner to Example 3(7).

[0200] The amino acid sequence of AVR14b with the signal sequence and polyhistidine tag added is shown in SEQ ID NO: 58. In SEQ ID NO: 58, the portion from the 1st methionine (Met) to the 22nd alanine (Ala) is the PelB signal peptide, the portion from the 25th serine (Ser) to the 213th aspartic acid (Asp) is the AAV binding protein AVR14b (corresponding to the region from the 312th to the 500th in SEQ ID NO: 1), and the portion from the 214th to the 219th histidine (His) is the tag sequence. In addition, in SEQ ID NO:58, the aspartic acid of Val317Asp is at the 30th position, the histidine of Asn324His is at the 37th position, the valine of Ala330Val is at the 43rd position, the leucine of Gln334Leu is at the 47th position, the valine of Glu335Val is at the 48th position, the serine of Tyr342Ser 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.

[0201] (f)AVR14c This protein is a protein in which Asn324His, Glu335Val, and Met359Ile were selected from the amino acid substitutions involved in improving alkaline stability revealed in Example 9, and these amino acid substitutions were accumulated in AVR11a (SEQ ID NO: 19). Specifically, AVR14c was produced by introducing mutations that cause amino acid substitutions of Asn324His and Glu335Val into the polynucleotide (SEQ ID NO: 40) encoding the protein obtained in Example 9 in which the amino acid substitution of Met359Ile was introduced into AVR11a (named AVR11a_M359I, SEQ ID NO: 41).

[0202] (f-1) PCR was performed in the same manner as in (c-1) except that the vector pET-AVR_M359I capable of expressing a polypeptide containing AVR11a_M359I (SEQ ID NO: 41) was used as a template.

[0203] (f-2) From the amplified PCR product, a plasmid (expression vector) pET-AVR14c containing a polynucleotide encoding AVR14c in which 14 amino acid substitutions were made relative to the wild-type AAV binding protein was obtained in a similar manner to (a-2) to (a-3), and the nucleotide sequence of the plasmid was then analyzed and confirmed in a similar manner to Example 3(7).

[0204] The amino acid sequence of AVR14c with the signal sequence and polyhistidine tag added is shown in SEQ ID NO: 59. In SEQ ID NO: 59, the portion from the 1st methionine (Met) to the 22nd alanine (Ala) is the PelB signal peptide, the portion from the 25th serine (Ser) to the 213th aspartic acid (Asp) is the AAV binding protein AVR14c (corresponding to the region from the 312th to the 500th in SEQ ID NO: 1), and the portion from the 214th to the 219th histidine (His) is the tag sequence. In addition, in SEQ ID NO:59, the aspartic acid of Val317Asp is at the 30th position, the histidine of Asn324His is at the 37th position, the valine of Ala330Val is at the 43rd position, the valine of Glu335Val is at the 48th position, the serine of Tyr342Ser is at the 55th position, the isoleucine of Met359Ile is at the 72nd 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.

[0205] (g)AVR14d This protein is a protein in which Asn324His, Glu335Val, and Phe379Tyr were selected from the amino acid substitutions involved in improving alkaline stability revealed in Example 9, and these amino acid substitutions were accumulated in AVR11a (SEQ ID NO: 19). Specifically, AVR14d was produced by introducing mutations that cause amino acid substitutions of Asn324His and Glu335Val into a polynucleotide (SEQ ID NO: 42) encoding a protein obtained in Example 9 in which an amino acid substitution of Phe379Tyr was introduced into AVR11a (designated AVR11a_F379Y, SEQ ID NO: 43).

[0206] (g-1) PCR was performed in the same manner as in (c-1) except that the vector pET-AVR_F379Y capable of expressing a polypeptide containing AVR11a_F379Y (SEQ ID NO: 41) was used as a template.

[0207] (g-2) From the amplified PCR product, a plasmid (expression vector) pET-AVR14d containing a polynucleotide encoding AVR14d, which has 14 amino acid substitutions relative to the wild-type AAV binding protein, was obtained in a similar manner to (a-2) to (a-3), and the nucleotide sequence of the plasmid was then analyzed and confirmed in a similar manner to Example 3(7).

[0208] The amino acid sequence of AVR14d with the signal sequence and polyhistidine tag added is shown in SEQ ID NO: 60. In SEQ ID NO: 60, the portion from the 1st methionine (Met) to the 22nd alanine (Ala) is the PelB signal peptide, the portion from the 25th serine (Ser) to the 213th aspartic acid (Asp) is the AAV binding protein AVR14d (corresponding to the region from the 312th to the 500th in SEQ ID NO: 1), and the portion from the 214th to the 219th histidine (His) is the tag sequence. In addition, in SEQ ID NO: 60, the aspartic acid of Val317Asp is at the 30th position, the histidine of Asn324His is at the 37th position, the valine of Ala330Val is at the 43rd position, the valine of Glu335Val is at the 48th position, the serine of Tyr342Ser 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 tyrosine of Phe379Tyr is at the 92nd 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.

[0209] (h)AVR15a This protein is a protein in which Asn324His, Gln334Leu, Glu335Val and Thr341Ala were selected from the amino acid substitutions involved in improving alkaline stability revealed in Example 9, and these amino acid substitutions were accumulated in AVR11a (SEQ ID NO: 19). Specifically, AVR15a was produced by introducing mutations that cause amino acid substitutions of Asn324His, Gln334Leu and Glu335Val into the polynucleotide (SEQ ID NO: 38) encoding AVR11a_T341A (SEQ ID NO: 39).

[0210] (h-1) PCR was performed in the same manner as in Example 2(2), except that the aforementioned pET-AVR_T341A was used as a template and oligonucleotides consisting of the sequences set forth in SEQ ID NO: 50 (5'-TTTT[GGTCTC]ACATTCAGTTGTACTTCGTGCTTCGGCAG-3') and SEQ ID NO: 51 (5'-TTTT[GGTCTC]AAATGTTTATGTGCTGCTGGTACCACCGAAAG-3') (the brackets in SEQ ID NOs: 50 and 51 indicate the restriction enzyme BsaI recognition sequences) were used as PCR primers (Forward: SEQ ID NO: 50, Reverse: SEQ ID NO: 51).

[0211] (h-2) From the amplified PCR product, a plasmid (expression vector) pET-AVR15a containing a polynucleotide encoding AVR15a, which has 15 amino acid substitutions relative to the wild-type AAV binding protein, was obtained in a similar manner to (a-2) to (a-3), and the nucleotide sequence of the plasmid was then analyzed and confirmed in a similar manner to Example 3(7).

[0212] The amino acid sequence of AVR15a with the signal sequence and polyhistidine tag added is shown in SEQ ID NO: 61. In SEQ ID NO: 61, the portion from the 1st methionine (Met) to the 22nd alanine (Ala) is the PelB signal peptide, the portion from the 25th serine (Ser) to the 213th aspartic acid (Asp) is the AAV binding protein AVR15a (corresponding to the region from the 312th to the 500th in SEQ ID NO: 1), and the portion from the 214th to the 219th histidine (His) is the tag sequence. In addition, in SEQ ID NO:61, the aspartic acid of Val317Asp is at the 30th position, the histidine of Asn324His is at the 37th position, the valine of Ala330Val is at the 43rd position, the leucine of Gln334Leu is at the 47th position, the valine of Glu335Val is at the 48th position, the alanine of Thr341Ala is at the 54th position, the serine of Tyr342Ser is at the 55th position, and the glutamine of Lys362Glu is at the 56th position. The phosphate in Lys371Asn is at position 75, the asparagine in Lys371Asn is at position 84, the alanine in Val381Ala is at position 94, the valine in Ile382Val is at position 95, the serine in Gly390Ser is at position 103, the glutamic acid in Lys399Glu is at position 112, the arginine in Ser476Arg is at position 189, and the aspartic acid in Asn487Asp is at position 200.

[0213] (i) AVR15b This protein is a protein in which Asn324His, Gln334Leu, Glu335Val, and Met359Ile were selected from the amino acid substitutions involved in improving alkaline stability revealed in Example 9, and these amino acid substitutions were accumulated in AVR11a (SEQ ID NO: 19). Specifically, AVR15b was produced by introducing mutations that cause amino acid substitutions of Asn324His, Gln334Leu, and Glu335Val into the polynucleotide (SEQ ID NO: 40) encoding AVR11a_M359I (SEQ ID NO: 41).

[0214] (i-1) PCR was carried out in the same manner as in (h-1) except that the above-mentioned pET-AVR11a_M359I was used as a template.

[0215] (i-2) From the amplified PCR product, a plasmid (expression vector) pET-AVR15b containing a polynucleotide encoding AVR15b in which 15 amino acid substitutions were made with respect to the wild-type AAV binding protein was obtained in a similar manner to (a-2) to (a-3), and the nucleotide sequence of the plasmid was then analyzed and confirmed in a similar manner to Example 3(7).

[0216] The amino acid sequence of AVR15b with the signal sequence and polyhistidine tag added is shown in SEQ ID NO: 62. In SEQ ID NO: 62, the portion from the 1st methionine (Met) to the 22nd alanine (Ala) is the PelB signal peptide, the portion from the 25th serine (Ser) to the 213th aspartic acid (Asp) is the AAV binding protein AVR15b (corresponding to the region from the 312th to the 500th in SEQ ID NO: 1), and the portion from the 214th to the 219th histidine (His) is the tag sequence. In addition, in SEQ ID NO: 62, the aspartic acid of Val317Asp is at the 30th position, the histidine of Asn324His is at the 37th position, the valine of Ala330Val is at the 43rd position, the leucine of Gln334Leu is at the 47th position, the valine of Glu335Val is at the 48th position, the serine of Tyr342Ser is at the 55th position, the isoleucine of Met359Ile is at the 72nd position, and the glutamate of Lys362Glu is at the 73rd position. The amino acid of Lys371Asn is at position 75, the asparagine of Lys371Asn is at position 84, the alanine of Val381Ala is at position 94, the valine of Ile382Val is at position 95, the serine of Gly390Ser is at position 103, the glutamic acid of Lys399Glu is at position 112, the arginine of Ser476Arg is at position 189, and the aspartic acid of Asn487Asp is at position 200.

[0217] (j)AVR15c This protein is a protein in which Asn324His, Gln334Leu, Glu335Val, and Phe379Tyr were selected from the amino acid substitutions involved in improving alkaline stability revealed in Example 9, and these amino acid substitutions were accumulated in AVR11a (SEQ ID NO: 19). Specifically, AVR15c was produced by introducing mutations that cause amino acid substitutions of Asn324His, Gln334Leu, and Glu335Val into the polynucleotide (SEQ ID NO: 42) encoding AVR11a_F379Y (SEQ ID NO: 43).

[0218] (j-1) PCR was carried out in the same manner as in (h-1) except that the above-mentioned pET-AVR11a_F379Y was used as a template.

[0219] (j-2) From the amplified PCR product, a plasmid (expression vector) pET-AVR15c containing a polynucleotide encoding AVR15c in which 15 amino acid substitutions were made relative to the wild-type AAV binding protein was obtained in a similar manner to (a-2) to (a-3), and the nucleotide sequence of the plasmid was then analyzed and confirmed in a similar manner to Example 3(7).

[0220] The amino acid sequence of AVR15c with the signal sequence and polyhistidine tag added is shown in SEQ ID NO: 63. In SEQ ID NO: 63, the portion from the 1st methionine (Met) to the 22nd alanine (Ala) is the PelB signal peptide, the portion from the 25th serine (Ser) to the 213th aspartic acid (Asp) is the AAV binding protein AVR15c (corresponding to the region from the 312th to the 500th in SEQ ID NO: 1), and the portion from the 214th to the 219th histidine (His) is the tag sequence. In addition, in SEQ ID NO: 63, the aspartic acid of Val317Asp is at the 30th position, the histidine of Asn324His is at the 37th position, the valine of Ala330Val is at the 43rd position, the leucine of Gln334Leu is at the 47th position, the valine of Glu335Val is at the 48th position, the serine of Tyr342Ser is at the 55th position, the glutamic acid of Lys362Glu is at the 75th position, and the aspartic acid of Lys371Asn is at the 76th position. Paragine is at position 84, tyrosine (Phe379Tyr) at position 92, alanine (Val381Ala) at position 94, valine (Ile382Val) at position 95, serine (Gly390Ser) at position 103, glutamic acid (Lys399Glu) at position 112, arginine (Ser476Arg) at position 189, and aspartic acid (Asn487Asp) at position 200.

[0221] (k)AVR16 This protein is a protein in which Asn324His, Gln334Leu, Glu335Val, Thr341Ala, and Phe379Tyr were selected from the amino acid substitutions involved in improving alkaline stability revealed in Example 9, and these amino acid substitutions were accumulated in AVR11a (SEQ ID NO: 19). Specifically, AVR16 was produced by introducing a mutation that causes the amino acid substitution of Thr341Ala into the polynucleotide (SEQ ID NO: 64) encoding AVR15c (SEQ ID NO: 63).

[0222] PCR was performed in the same manner as in Example 2(2), except that the vector pET-AVR 15c capable of expressing the polypeptide containing AVR15c (SEQ ID NO: 63) prepared in (k-1)(j) was used as a template and oligonucleotides consisting of the sequences set forth in SEQ ID NO: 52 (5'-TTTT[GGTCTC]ATTTCGGTGGTACCAGCAGCACATAAAC-3') and SEQ ID NO: 53 (5'-TTTT[GGTCTC]AGAAAGGGGAAGCCTCGACGTATG-3') (the brackets in SEQ ID NOs: 52 and 53 indicate the restriction enzyme BsaI recognition sequences) were used as PCR primers (Forward: SEQ ID NO: 52, Reverse: SEQ ID NO: 53).

[0223] (k-2) From the amplified PCR product, a plasmid (expression vector) pET-AVR16 containing a polynucleotide encoding AVR16 in which 16 amino acid substitutions were made with respect to the wild-type AAV binding protein was obtained in a similar manner to (a-2) to (a-3), and the nucleotide sequence of the plasmid was then analyzed and confirmed in a similar manner to Example 3(7).

[0224] The amino acid sequence of AVR16 with a signal sequence and a polyhistidine tag added is shown in SEQ ID NO: 65. In SEQ ID NO: 65, the portion from the 1st methionine (Met) to the 22nd alanine (Ala) is the PelB signal peptide, the portion from the 25th serine (Ser) to the 213th aspartic acid (Asp) is the AAV binding protein AVR16 (corresponding to the region from the 312th to the 500th in SEQ ID NO: 1), and the portion from the 214th to the 219th histidine (His) is the tag sequence. In addition, in SEQ ID NO:65, the aspartic acid of Val317Asp is at the 30th position, the histidine of Asn324His is at the 37th position, the valine of Ala330Val is at the 43rd position, the leucine of Gln334Leu is at the 47th position, the valine of Glu335Val is at the 48th position, the alanine of Thr341Ala is at the 54th position, the serine of Tyr342Ser is at the 55th position, the glutamic acid of Lys362Glu is at the 75th position, and the L The asparagine of ys371Asn is at position 84, the tyrosine of Phe379Tyr is at position 92, the alanine of Val381Ala is at position 94, the valine of Ile382Val is at position 95, the serine of Gly390Ser is at position 103, the glutamic acid of Lys399Glu is at position 112, the arginine of Ser476Arg is at position 189, and the aspartic acid of Asn487Asp is at position 200.

[0225] Example 11: Evaluation of alkaline stability of AVR11a amino acid substitution aggregates (1) As transformants capable of expressing AAV-binding proteins, transformants obtained by transforming Escherichia coli strain BL21(DE3) with a plasmid containing a polynucleotide encoding any one of the 11 types of AAV-binding proteins (AVR11a amino acid substitution aggregates, specifically, AVR12a [SEQ ID NO: 54], AVR12b [SEQ ID NO: 55], AVR13 [SEQ ID NO: 56], AVR14a [SEQ ID NO: 57], AVR14b [SEQ ID NO: 58], AVR14c [SEQ ID NO: 59], AVR14d [SEQ ID NO: 60], AVR15a [SEQ ID NO: 61], AVR15b [SEQ ID NO: 62], AVR15c [SEQ ID NO: 63], and AVR16 [SEQ ID NO: 65]) obtained in Example 10 and AVR11a (SEQ ID NO: 19) were used. The transformants were cultured in the same manner as in Example 4(1) to (3).

[0226] (2) After the culture was completed, each bacterial cell was recovered by the method described in Example 4(4), and an AAV binding protein was prepared from the recovered bacterial cells by the methods described in Example 4(5) to (7).

[0227] (3) Using the ELISA method described in Example 3(4), the binding activity of the AAV-binding protein prepared in (2) to the VLP2 prepared in Example 8 was measured. Based on the absorbance at 450 nm, which is the measurement result, the AAV-binding protein obtained in (2) was diluted with pure water so that the measured value became similar.

[0228] (4) Each AAV-binding protein solution diluted in (3) was divided into two fractions. One fraction was mixed with an equal amount of 0.1 M aqueous sodium hydroxide solution and subjected to an alkali treatment by leaving it at a constant temperature for a constant time (treatment temperature: 30°C, treatment time: 3 hours), while the other fraction was not subjected to the alkali treatment (corresponding to the "start" of the alkali treatment).

[0229] (5) The fraction after treatment in (4) was mixed with 0.5 M MES buffer (pH 6.0) in a ratio of 7:3 to adjust the pH to around 6, and then the binding activity with VLP2 was measured using the ELISA method described in Example 3(4).

[0230] (6) The residual activity was calculated by dividing the absorbance at 450 nm after the alkali treatment in (4) by the absorbance at 450 nm after the treatment time of 0 hours.

[0231] The results are shown in Table 10. It can be seen that all of the AVR11a amino acid substitution aggregates obtained in Example 10 have higher residual activity and improved alkali stability compared to AVR11a (SEQ ID NO: 19).

[0232] [Table 10]

[0233] Example 12: Preparation and screening of a mutant library of AAV binding proteins (part 2) Among the AAV binding proteins evaluated in Example 11, AVR16 (SEQ ID NO: 65) was selected, and random mutations were introduced into the polynucleotide portion (SEQ ID NO: 66) encoding the protein using error-prone PCR.

[0234] (1) Error-prone PCR was performed in the same manner as in Example 9(1), except that the vector pET-AVR16 capable of expressing the AVR16-containing polypeptide (SEQ ID NO: 65) prepared in Example 10(k) was used as a template. Mutations were successfully introduced into the polynucleotide encoding the AAV binding protein by the error-prone PCR, with the average mutation introduction rate being 0.8 amino acid mutations per molecule.

[0235] (2) A random mutant library was prepared from the PCR products obtained in (1) by the method described in Example 9 (2) to (3).

[0236] (3) The random mutant library (transformants) prepared in (2) 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.

[0237] (4) The culture medium from (3) 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 sodium hydroxide aqueous solution and subjected to an alkali treatment at 42.1° C. for 15 minutes.

[0238] (5) The binding activity of the AAV binding protein to VLP2 when the treatment in (4) was performed and the binding activity of the AAV binding protein to VLP2 when the treatment in (4) was not performed were measured by the ELISA method described in Example 3 (4). The binding activity of the AAV binding protein to VLP2 when the heat treatment was performed was divided by the binding activity of the AAV binding protein to VLP2 when the heat treatment was not performed to calculate the residual activity.

[0239] (6) A random mutant library of about 1,800 strains was evaluated using the method of (5), and transformants expressing AAV binding proteins with improved residual activity compared to the parent molecule AVR16 were selected from the library. The selected transformants were cultured, and expression vectors were prepared using a QIAprep Spin Miniprep kit (Qiagen).

[0240] (7) 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 3(7), and the amino acid mutation sites were identified.

[0241] Tables 11 and 12 show a summary of the amino acid substitution positions relative to AVR16 for the AAV binding proteins expressed by the transformants selected in (6) above, as well as the remaining activity (%) after alkali treatment.

[0242] 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 Val(Asp)317Asn, Ile319Val, Lys323Asn, Glu325Lys, Val326Ala, Asn329Ser, Leu333Met, Lys338Arg, Gly339Glu, Met359Val, Gly361Glu, Lys(Glu)362Asp, Ser366Pro, Lys368Arg, Lys368Glu, Leu369Pro, Lys380Arg, Val(Ala)381Thr, Ile(Val)382Asp, Lys(Glu)383Asp, Lys(Glu)384Asp, Lys(Glu)385Asp, Lys(Glu)386Asp, Lys(Glu)387Asp, Lys(Glu)388Asp, Lys(Glu)389Asp, Lys(Glu)390Asp, Lys(Glu)391Asp, Lys(Glu)392Asp, Lys(Glu)393Asp, Lys(Glu)394Asp, Lys(Glu)395Asp, Lys(Glu)396Asp, Lys(Glu)397Asp, Lys(Glu)398Asp, Lys(Glu)399Asp, Lys(Glu)399Asp, Lys(Glu)391Asp, Lys(Glu)392 ... u) An AAV binding protein having at least one amino acid substitution of any of 399Gly, Leu421Pro, Pro422Ser, Ser425Gly, His443Tyr, His443Arg, Glu454Gly, Lys455Arg, Lys467Glu, Lys467Gln, Ser(Arg)476Gly, Ser482Thr, Ser488Phe, Ala491Ser, Asn492Asp, Asn496Tyr, and Asp500Gly can be said to have improved alkaline stability, even when compared to AVR16 (sequence number 65), which has improved alkaline stability compared to AVR11a (sequence number 19).

[0243] [Table 11]

[0244] [Table 12]

[0245] Example 13: Preparation of AVR16 amino acid substitution aggregates The alkaline stability was further improved by integrating the amino acid substitutions involved in improving the alkaline stability of the AAV binding protein, which were revealed in Example 12, into AVR16 (SEQ ID NO: 65). Specifically, the following seven types of AAV binding proteins (a) to (g) were designed and prepared.

[0246] (a) A protein in which the amino acid substitutions Val326Ala, Lys467Gln, and Ser482Thr were introduced into AVR16 (SEQ ID NO: 83, designated AVR19a) (b) A protein in which the amino acid substitutions Lys467Gln, Ser482Thr, and Asn492Asp were introduced into AVR16 (SEQ ID NO: 85, named AVR19b) (c) A protein in which the amino acid substitutions Lys380Arg, Lys467Gln, and Ser482Thr were introduced into AVR16 (SEQ ID NO: 87, named AVR19c) (d) A protein in which the amino acid substitutions Val326Ala, Lys467Gln, Ser482Thr, and Asn492Asp were introduced into AVR16 (SEQ ID NO: 88, named AVR20a) (e) A protein in which the amino acid substitutions Val326Ala, Lys380Arg, Lys467Gln, and Ser482Thr were introduced into AVR16 (SEQ ID NO: 90, designated AVR20b) (f) A protein in which the amino acid substitutions Lys380Arg, Lys467Gln, Ser482Thr, and Asn492Asp were introduced into AVR16 (SEQ ID NO: 91, named AVR20c) (g) A protein in which the amino acid substitutions Val326Ala, Lys380Arg, Lys467Gln, Ser482Thr, and Asn492Asp were introduced into AVR16 (SEQ ID NO: 92, named AVR21).

[0247] Methods for producing the eight types of AAV binding proteins shown in (a) to (g) above are described below. (a)AVR19a This protein is a protein in which Val326Ala, Lys467Gln, and Ser482Thr were selected from the amino acid substitutions involved in improving alkali stability revealed in Example 12, and these amino acid substitutions were accumulated in AVR16 (SEQ ID NO: 65). Specifically, a mutation that causes an amino acid substitution of Ser482Thr was introduced into a polynucleotide (SEQ ID NO: 69) encoding a protein in which an amino acid substitution of Lys467Gln was introduced into AVR16 obtained in Example 12 (named AVR16_K467Q, SEQ ID NO: 70), and then a mutation that causes an amino acid substitution of Val326Ala was introduced into the polynucleotide encoding the AVR18 (named AVR18), thereby producing AVR19c.

[0248] (a-1) PCR was performed in the same manner as in Example 2(2), except that vector pET-AVR16_K467Q capable of expressing a polypeptide containing AVR16_K467Q (SEQ ID NO: 70) was used as a template and oligonucleotides consisting of the sequences set forth in SEQ ID NO: 73 (5'-TTTT[GGTCTC]AGTGGTCGATACCGATGGCG-3') and SEQ ID NO: 74 (5'-TTTT[GGTCTC]ACCACCGTCAGTCTGAAC-3') (the brackets in SEQ ID NOs: 73 and 74 indicate the restriction enzyme BsaI recognition sequences) were used as PCR primers (Forward: SEQ ID NO: 73, Reverse: SEQ ID NO: 74).

[0249] (a-2) The PCR products amplified by the methods described in Examples 2(3) and 2(4) were purified and then subjected to a ligation reaction by the method described in Example 2(5).

[0250] (a-3) The ligation product obtained was used to transform E. coli BL21 strain (DE3). The resulting transformant was cultured in LB medium supplemented with 50 μg / mL kanamycin, and the resulting transformant was then collected by centrifugation. A plasmid (expression vector) was extracted from the transformant to obtain plasmid pET-AVR18, which contains a polynucleotide encoding AVR18 with 18 amino acid substitutions relative to the wild-type AAV binding protein.

[0251] (a-4) The nucleotide sequence of pET-AVR18 was analyzed and confirmed in the same manner as in Example 3(7). The amino acid sequence of AVR18 to which a signal sequence and a polyhistidine tag were added is shown in SEQ ID NO:81.

[0252] (a-5) PCR was performed in the same manner as in Example 2(2), except that pET-AVR18 obtained in (a-3) was used as a template and oligonucleotides consisting of the sequences set forth in SEQ ID NO: 75 (5'-TTTT[GGTCTC]ACCGAAGCACGAAGCACAACTG-3') and SEQ ID NO: 76 (5'-TTTT[GGTCTC]ATCGGCAGGGTAATCTGATC-3') (the brackets in SEQ ID NOs: 75 and 76 indicate the recognition sequences for the restriction enzyme BsaI) were used as PCR primers (Forward: SEQ ID NO: 75, Reverse: SEQ ID NO: 76).

[0253] (a-6) From the amplified PCR product, a plasmid (expression vector) pET-AVR19a containing a polynucleotide encoding AVR19a, which has 19 amino acid substitutions relative to the wild-type AAV binding protein, was obtained in a similar manner to (a-2) to (a-3), and the nucleotide sequence of the plasmid was then analyzed and confirmed in a similar manner to Example 3(7).

[0254] The amino acid sequence of AVR19a with the signal sequence and polyhistidine tag added is shown in SEQ ID NO: 83. In SEQ ID NO: 83, the portion from the 1st methionine (Met) to the 22nd alanine (Ala) is the PelB signal peptide, the portion from the 25th serine (Ser) to the 213th aspartic acid (Asp) is the AAV binding protein AVR19a (corresponding to the region from the 312th to the 500th in SEQ ID NO: 1), and the portion from the 214th to the 219th histidine (His) is the tag sequence. In addition, in SEQ ID NO: 83, the aspartic acid of Val317Asp is at the 30th position, the histidine of Asn324His is at the 37th position, the alanine of Val326Ala is at the 39th position, the valine of Ala330Val is at the 43rd position, the leucine of Gln334Leu is at the 47th position, the valine of Glu335Val is at the 48th position, the alanine of Thr341Ala is at the 54th position, the serine of Tyr342Ser is at the 55th position, the glutamic acid of Lys362Glu is at the 75th position, and the asparagine of Lys371Asn is at the 76th position. Glycine is at position 84, tyrosine (Phe379Tyr) at position 92, alanine (Val381Ala) at position 94, valine (Ile382Val) at position 95, serine (Gly390Ser) at position 103, glutamic acid (Lys399Glu) at position 112, glutamine (Lys467Gln) at position 180, arginine (Ser476Arg) at position 189, threonine (Ser482Thr) at position 195, and aspartic acid (Asn487Asp) at position 200.

[0255] (b) AVR19b This protein is a protein in which Lys467Gln, Ser482Thr, and Asn492Asp were selected from the amino acid substitutions involved in improving alkaline stability revealed in Example 12, and these amino acid substitutions were accumulated in AVR16 (SEQ ID NO: 65). Specifically, AVR19b was produced by introducing a mutation that causes the amino acid substitution Asn492Asp into the polynucleotide (SEQ ID NO: 82) encoding AVR18 (SEQ ID NO: 81).

[0256] (b-1) PCR was performed in the same manner as in Example 2(2), except that pET-AVR18 obtained in (a-3) was used as a template and oligonucleotides consisting of the sequences set forth in SEQ ID NO: 77 (5'-TTTT[GGTCTC]ACACCGCCGATCTTACCGTC-3') and SEQ ID NO: 78 (5'-TTTT[GGTCTC]AGGTGGTGGAGTCGGTTGC-3') (the brackets in SEQ ID NOs: 77 and 78 indicate the restriction enzyme BsaI recognition sequences) were used as PCR primers (Forward: SEQ ID NO: 77, Reverse: SEQ ID NO: 78).

[0257] (b-2) From the amplified PCR product, a plasmid (expression vector) pET-AVR19b containing a polynucleotide encoding AVR19b, which has 19 amino acid substitutions relative to the wild-type AAV binding protein, was obtained in a similar manner to (a-2) to (a-3), and the nucleotide sequence of the plasmid was then analyzed and confirmed in a similar manner to Example 3(7).

[0258] The amino acid sequence of AVR19b with the signal sequence and polyhistidine tag added is shown in SEQ ID NO: 85. In SEQ ID NO: 85, the portion from the 1st methionine (Met) to the 22nd alanine (Ala) is the PelB signal peptide, the portion from the 25th serine (Ser) to the 213th aspartic acid (Asp) is the AAV binding protein AVR19b (corresponding to the region from the 312th to the 500th in SEQ ID NO: 1), and the portion from the 214th to the 219th histidine (His) is the tag sequence. In addition, in SEQ ID NO: 85, the aspartic acid of Val317Asp is at the 30th position, the histidine of Asn324His is at the 37th position, the valine of Ala330Val is at the 43rd position, the leucine of Gln334Leu is at the 47th position, the valine of Glu335Val is at the 48th position, the alanine of Thr341Ala is at the 54th position, the serine of Tyr342Ser is at the 55th position, the glutamic acid of Lys362Glu is at the 75th position, the asparagine of Lys371Asn is at the 84th position, and the tyrosine of Phe379Tyr. is at position 92, alanine at position 94, valine at position 95, serine at position 103, glutamic acid at position 112, glutamine at position 180, arginine at position 189, threonine at position 195, aspartic acid at position 200, and aspartic acid at position 205.

[0259] (c)AVR19c This protein is a protein in which Lys380Arg, Lys467Gln, and Ser482Thr were selected from the amino acid substitutions involved in improving alkaline stability revealed in Example 12, and these amino acid substitutions were accumulated in AVR16 (SEQ ID NO: 65). Specifically, AVR19c was produced by introducing a mutation that causes an amino acid substitution of Lys380Arg into the polynucleotide (SEQ ID NO: 82) encoding AVR18 (SEQ ID NO: 81).

[0260] (c-1) PCR was performed in the same manner as in Example 2(2), except that pET-AVR18 obtained in (a-3) was used as a template and oligonucleotides consisting of the sequences set forth in SEQ ID NO: 79 (5'-TTTT[GGTCTC]ATATGAATACCGTGCTGTCGTTG-3') and SEQ ID NO: 80 (5'-TTTT[GGTCTC]ACATACAGACCCGGCGTC-3') (the brackets in SEQ ID NOs: 79 and 80 indicate the recognition sequences for the restriction enzyme BsaI) were used as PCR primers (Forward: SEQ ID NO: 79, Reverse: SEQ ID NO: 80).

[0261] (c-2) From the amplified PCR product, a plasmid (expression vector) pET-AVR19c containing a polynucleotide encoding AVR19c in which 19 amino acid substitutions were made relative to the wild-type AAV binding protein was obtained in a similar manner to (a-2) to (a-3), and the nucleotide sequence of the plasmid was then analyzed and confirmed in a similar manner to Example 3(7).

[0262] The amino acid sequence of AVR19c with the signal sequence and polyhistidine tag added is shown in SEQ ID NO: 87. In SEQ ID NO: 87, the portion from the 1st methionine (Met) to the 22nd alanine (Ala) is the PelB signal peptide, the portion from the 25th serine (Ser) to the 213th aspartic acid (Asp) is the AAV binding protein AVR19c (corresponding to the region from the 312th to the 500th in SEQ ID NO: 1), and the portion from the 214th to the 219th histidine (His) is the tag sequence. In addition, in SEQ ID NO: 87, the aspartic acid of Val317Asp is at the 30th position, the histidine of Asn324His is at the 37th position, the valine of Ala330Val is at the 43rd position, the leucine of Gln334Leu is at the 47th position, the valine of Glu335Val is at the 48th position, the alanine of Thr341Ala is at the 54th position, the serine of Tyr342Ser is at the 55th position, the glutamic acid of Lys362Glu is at the 75th position, the asparagine of Lys371Asn is at the 84th position, and the tyrosine of Phe379Tyr is at the 85th position. syn is at position 92, arginine at position 93, alanine at position 94, valine at position 95, serine at position 103, glutamic acid at position 112, glutamine at position 180, arginine at position 189, threonine at position 195, and aspartic acid at position 200.

[0263] (d)AVR20a This protein is a protein in which Val326Ala, Lys467Gln, Ser482Thr, and Asn492Asp were selected from the amino acid substitutions involved in improving alkaline stability revealed in Example 12, and these amino acid substitutions were accumulated in AVR16 (SEQ ID NO: 65). Specifically, AVR20a was produced by introducing a mutation that causes the amino acid substitution Asn492Asp into the polynucleotide (SEQ ID NO: 84) encoding AVR19a (SEQ ID NO: 83).

[0264] (d-1) PCR was performed in the same manner as in (b-1) except that the vector pET-AVR19a capable of expressing the polypeptide containing AVR19a (sequence number 83) prepared in (a) was used as a template.

[0265] (d-2) From the amplified PCR product, a plasmid (expression vector) pET-AVR20a containing a polynucleotide encoding AVR20a in which 20 amino acid substitutions were made relative to the wild-type AAV binding protein was obtained in a similar manner to (a-2) to (a-3), and the nucleotide sequence of the plasmid was then analyzed and confirmed in a similar manner to Example 3(7).

[0266] The amino acid sequence of AVR20a with the signal sequence and polyhistidine tag added is shown in SEQ ID NO: 88. In SEQ ID NO: 88, the portion from the 1st methionine (Met) to the 22nd alanine (Ala) is the PelB signal peptide, the portion from the 25th serine (Ser) to the 213th aspartic acid (Asp) is the AAV binding protein AVR20a (corresponding to the region from the 312th to the 500th in SEQ ID NO: 1), and the 214th to 219th histidines (His) are the tag sequence. In SEQ ID NO: 88, the aspartic acid of Val317Asp is at the 30th position, the histidine of Asn324His is at the 37th position, the alanine of Val326Ala is at the 39th position, the valine of Ala330Val is at the 43rd position, the leucine of Gln334Leu is at the 47th position, the valine of Glu335Val is at the 48th position, the alanine of Thr341Ala is at the 54th position, the serine of Tyr342Ser is at the 55th position, the glutamic acid of Lys362Glu is at the 75th position, the asparagine of Lys371Asn is at the 84th position, and the Phe The tyrosine (379Tyr) is at position 92, the alanine (Val381Ala) is at position 94, the valine (Ile382Val) is at position 95, the serine (Gly390Ser) is at position 103, the glutamic acid (Lys399Glu) is at position 112, the glutamine (Lys467Gln) is at position 180, the arginine (Ser476Arg) is at position 189, the threonine (Ser482Thr) is at position 195, the aspartic acid (Asn487Asp) is at position 200, and the aspartic acid (Asn492Asp) is at position 205.

[0267] (e)AVR20b This protein is a protein in which Val326Ala, Lys380Arg, Lys467Gln, and Ser482Thr were selected from the amino acid substitutions involved in improving alkaline stability revealed in Example 12, and these amino acid substitutions were accumulated in AVR16 (SEQ ID NO: 65). Specifically, AVR20b was produced by introducing a mutation that causes an amino acid substitution of Lys380Arg into the polynucleotide (SEQ ID NO: 84) encoding AVR19a (SEQ ID NO: 83).

[0268] (e-1) PCR was performed in the same manner as in (c-1) except that pET-AVR19a prepared in (a) was used as a template.

[0269] (e-2) From the amplified PCR product, a plasmid (expression vector) pET-AVR20b containing a polynucleotide encoding AVR20b in which 20 amino acid substitutions have been made relative to the wild-type AAV binding protein was obtained in a similar manner to (a-2) to (a-3), and the nucleotide sequence of the plasmid was then analyzed and confirmed in a similar manner to Example 3(7).

[0270] The amino acid sequence of AVR20b with the signal sequence and polyhistidine tag added is shown in SEQ ID NO: 90. In SEQ ID NO: 90, the portion from the 1st methionine (Met) to the 22nd alanine (Ala) is the PelB signal peptide, the portion from the 25th serine (Ser) to the 213th aspartic acid (Asp) is the AAV binding protein AVR20b (corresponding to the region from the 312th to the 500th in SEQ ID NO: 1), and the portion from the 214th to the 219th histidine (His) is the tag sequence. In addition, in SEQ ID NO:90, the aspartic acid of Val317Asp is at the 30th position, the histidine of Asn324His is at the 37th position, the alanine of Val326Ala is at the 39th position, the valine of Ala330Val is at the 43rd position, the leucine of Gln334Leu is at the 47th position, the valine of Glu335Val is at the 48th position, the alanine of Thr341Ala is at the 54th position, the serine of Tyr342Ser is at the 55th position, the glutamic acid of Lys362Glu is at the 75th position, the asparagine of Lys371Asn is at the 84th position, and Ph The tyrosine in e379Tyr is at position 92, the arginine in Lys380Arg is at position 93, the alanine in Val381Ala is at position 94, the valine in Ile382Val is at position 95, the serine in Gly390Ser is at position 103, the glutamic acid in Lys399Glu is at position 112, the glutamine in Lys467Gln is at position 180, the arginine in Ser476Arg is at position 189, the threonine in Ser482Thr is at position 195, and the aspartic acid in Asn487Asp is at position 200.

[0271] (f)AVR20c This protein is a protein in which Lys380Arg, Lys467Gln, Ser482Thr, and Asn492Asp were selected from the amino acid substitutions involved in improving alkaline stability revealed in Example 12, and these amino acid substitutions were accumulated in AVR16 (SEQ ID NO: 65). Specifically, AVR20c was produced by introducing a mutation that causes an amino acid substitution of Lys380Arg into the polynucleotide (SEQ ID NO: 86) encoding AVR19b (SEQ ID NO: 85).

[0272] (f-1) PCR was performed in the same manner as in (c-1) except that the vector pET-AVR19b capable of expressing the polypeptide containing AVR19b (sequence number 85) prepared in (b) was used as a template.

[0273] (f-2) From the amplified PCR product, a plasmid (expression vector) pET-AVR20c containing a polynucleotide encoding AVR20c in which 20 amino acid substitutions were made relative to the wild-type AAV binding protein was obtained in a similar manner to (a-2) to (a-3), and the nucleotide sequence of the plasmid was then analyzed and confirmed in a similar manner to Example 3(7).

[0274] The amino acid sequence of AVR20c with the signal sequence and polyhistidine tag added is shown in SEQ ID NO: 91. In SEQ ID NO: 91, the portion from the 1st methionine (Met) to the 22nd alanine (Ala) is the PelB signal peptide, the portion from the 25th serine (Ser) to the 213th aspartic acid (Asp) is the AAV binding protein AVR20c (corresponding to the region from the 312th to the 500th in SEQ ID NO: 1), and the portion from the 214th to the 219th histidine (His) is the tag sequence. In addition, in SEQ ID NO: 91, the aspartic acid of Val317Asp is at the 30th position, the histidine of Asn324His is at the 37th position, the valine of Ala330Val is at the 43rd position, the leucine of Gln334Leu is at the 47th position, the valine of Glu335Val is at the 48th position, the alanine of Thr341Ala is at the 54th position, the serine of Tyr342Ser 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 tyrosine of Phe379Tyr is at the 92nd position, and the tyrosine of Lys379Tyr is at the 93rd position. The arginine in 80Arg is at position 93, the alanine in Val381Ala is at position 94, the valine in Ile382Val is at position 95, the serine in Gly390Ser is at position 103, the glutamic acid in Lys399Glu is at position 112, the glutamine in Lys467Gln is at position 180, the arginine in Ser476Arg is at position 189, the threonine in Ser482Thr is at position 195, the aspartic acid in Asn487Asp is at position 200, and the aspartic acid in Asn492Asp is at position 205.

[0275] (g)AVR21 This protein is a protein in which the amino acid substitutions Val326Ala, Lys380Arg, Lys467Gln, Ser482Thr, and Asn492Asp were selected from the amino acid substitutions involved in improving alkaline stability, as revealed in Example 12, and these amino acid substitutions were accumulated in AVR16 (SEQ ID NO: 65). Specifically, AVR21 was produced by introducing a mutation that causes an amino acid substitution of Lys380Arg into the polynucleotide (SEQ ID NO: 89) encoding AVR20a (SEQ ID NO: 88).

[0276] (g-1) PCR was performed in the same manner as in (c-1) except that the vector pET-AVR20a capable of expressing the polypeptide containing AVR20a (sequence number 88) prepared in (d) was used as a template.

[0277] (g-2) From the amplified PCR product, a plasmid (expression vector) pET-AVR21 containing a polynucleotide encoding AVR21 in which 21 amino acid substitutions were made relative to the wild-type AAV binding protein was obtained in a similar manner to (a-2) to (a-3), and the nucleotide sequence of the plasmid was then analyzed and confirmed in a similar manner to Example 3(7).

[0278] The sequence of the polynucleotide encoding the polypeptide containing AVR21 analyzed in (g-2) is shown in SEQ ID NO: 93, and the amino acid sequence of AVR21 to which a signal sequence and a polyhistidine tag have been added is shown in SEQ ID NO: 92. In SEQ ID NO: 92, the portion from the 1st methionine (Met) to the 22nd alanine (Ala) is the PelB signal peptide, the portion from the 25th serine (Ser) to the 213th aspartic acid (Asp) is the AAV binding protein AVR21 (corresponding to the region from the 312th to the 500th in SEQ ID NO: 1), and the portion from the 214th to the 219th histidine (His) is the tag sequence. In addition, in SEQ ID NO:92, the aspartic acid of Val317Asp is at the 30th position, the histidine of Asn324His is at the 37th position, the alanine of Val326Ala is at the 39th position, the valine of Ala330Val is at the 43rd position, the leucine of Gln334Leu is at the 47th position, the valine of Glu335Val is at the 48th position, the alanine of Thr341Ala is at the 54th position, the serine of Tyr342Ser is at the 55th position, the glutamic acid of Lys362Glu is at the 75th position, the asparagine of Lys371Asn is at the 84th position, and the tyrosine of Phe379Tyr is at the 84th position. is at position 92, the arginine of Lys380Arg is at position 93, the alanine of Val381Ala is at position 94, the valine of Ile382Val is at position 95, the serine of Gly390Ser is at position 103, the glutamic acid of Lys399Glu is at position 112, the glutamine of Lys467Gln is at position 180, the arginine of Ser476Arg is at position 189, the threonine of Ser482Thr is at position 195, the aspartic acid of Asn487Asp is at position 200, and the aspartic acid of Asn492Asp is at position 205.

[0279] Example 14: Evaluation of alkaline stability of AVR16 amino acid substitution aggregates (1) As a transformant capable of expressing an AAV binding protein, AVR16 [SEQ ID NO: 65] and five types of AAV binding proteins obtained in Example 12, which are AVR16 amino acid substitution aggregates (AVR16_K467Q [SEQ ID NO: 70], and AVR16 with Val326Ala [named AVR16_V326A, SEQ ID NO: 67], Lys380Arg [named AVR16_K380R, SEQ ID NO: 68], Ser482Thr [named AVR16_S482T, SEQ ID NO: 71], or Asn492Asp [named AVR16_N492D, SEQ ID NO: 69], were used. The cells were cultured in the same manner as in Example 4(1) to (3), except that the transformants used were obtained by transforming E. coli strain BL21(DE3) with plasmids containing polynucleotides encoding any of the seven AAV binding proteins obtained in Example 13 (AVR19a [SEQ ID NO: 83], AVR19b [SEQ ID NO: 85], AVR19c [SEQ ID NO: 87], AVR20a [SEQ ID NO: 89], AVR20b [SEQ ID NO: 90], AVR20c [SEQ ID NO: 91] and AVR21 [SEQ ID NO: 92]), and the transformants obtained in Example 13 (AVR19a [SEQ ID NO: 83], AVR19b [SEQ ID NO: 85], AVR19c [SEQ ID NO: 87], AVR20a [SEQ ID NO: 89], AVR20b [SEQ ID NO: 90], AVR20c [SEQ ID NO: 91] and AVR21 [SEQ ID NO: 92]) were cultured in the same manner as in Example 4(1) to (3).

[0280] (2) After the culture was completed, each bacterial cell was recovered by the method described in Example 4(4), and an AAV binding protein was prepared from the recovered bacterial cells by the methods described in Example 4(5) to (7).

[0281] (3) Using the ELISA method described in Example 3(4), the binding activity of the AAV-binding protein prepared in (2) to the VLP2 prepared in Example 8 was measured. Based on the absorbance at 450 nm, which is the measurement result, the AAV-binding protein obtained in (2) was diluted with pure water so that the measured value became similar.

[0282] (4) Each AAV-binding protein solution diluted in (3) was divided into two fractions: one fraction was mixed with an equal volume of 0.5 M aqueous sodium hydroxide solution and subjected to alkali treatment at 30°C for 1 hour, and the other fraction was not subjected to the alkali treatment (corresponding to the "start" of the alkali treatment).

[0283] (5) The fraction after treatment in (4) was mixed with 0.5 M MES buffer (pH 6.0) in a ratio of 8:2 to adjust the pH to around 6, and then the binding activity with VLP2 was measured using the ELISA method described in Example 3(4).

[0284] (6) The residual activity was calculated by dividing the absorbance at 450 nm after the alkali treatment in (4) by the absorbance at 450 nm after the treatment time of 0 hours.

[0285] The results are shown in Table 13. It can be seen that all of the AVR16 amino acid substitution aggregates evaluated in this example had higher residual activity and further improved alkaline stability, even when compared to AVR16 (SEQ ID NO: 65), which has improved alkaline stability compared to AVR11a (SEQ ID NO: 19).

[0286] [Table 13]

[0287] Example 15 Preparation of Adeno-Associated Virus (AAV) Vectors (1) A nucleotide sequence (SEQ ID NO: 96) 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: 95, and a stop codon (TAG) and a BamHI recognition sequence (GGATCC) were added to the 3'-end.

[0288] (2) A polynucleotide consisting of the sequence set forth in SEQ ID NO:96 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).

[0289] (3) The pUC-EGFP obtained in (2) was digested with the restriction enzymes EcoRI and BamHI, and then ligated into the expression vector pAAV-CMV (Takara Bio Inc.) which had been previously digested with the restriction enzymes EcoRI and BamHI. The ligation product was used to transform the Escherichia coli JM109 strain.

[0290] (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 (1.6% (w / v) Tryptone, 1% (w / v) Yeast Extract, 0.5% (w / v) Sodium Chloride) containing 100 μg / mL carbenicillin. After the culture was completed, the cells were collected by centrifugation, and a large amount of the EGFP-expressing vector pAAV-EGFP was prepared from the collected cells using a Plasmid Mega Kit (Qiagen).

[0291] (5) Escherichia coli JM109 strain was transformed with a plasmid (hereinafter collectively referred to as "pRC2 Vector") containing a polynucleotide encoding the capsid region VP1 (UniProt accession number: P03135, SEQ ID NO: 99) of AAV serotype 2 (AAV2) and any of the amino acid substitutions of VP1 shown in (a) to (m) below (hereinafter collectively referred to simply as "AAV2") and pHelper Vector (manufactured by Takara Bio Inc.). The same procedure as in (4) was carried out using the obtained transformant to prepare large quantities of pRC2 Vector and pHelper. (a) Substitution of glutamine at position 263 of SEQ ID NO: 99 with alanine (hereinafter also referred to as "AAV2 Q263A") (b) Substitution of serine at position 264 of SEQ ID NO: 99 with alanine (hereinafter also referred to as "AAV2 S264A") (c) Substitution of serine at position 267 of SEQ ID NO: 99 with alanine or threonine (hereinafter also referred to as "AAV2 S267A" and "AAV2 S267T", respectively) (d) Histidine at position 271 of SEQ ID NO: 99 is replaced with any of alanine, phenylalanine, glutamine, and threonine (hereinafter, also referred to as "AAV2 H271A", "AAV2 H271F", "AAV2 H271Q", and "AAV2 H271T", ​​respectively). (e) Substitution of asparagine at position 382 of SEQ ID NO: 99 with alanine (hereinafter also referred to as "AAV2 N382A") (f) Substitution of glycine at position 383 of SEQ ID NO: 99 with alanine (hereinafter also referred to as "AAV2 G383A") (g) Substitution of serine at position 384 of SEQ ID NO: 99 with alanine (hereinafter also referred to as "AAV2 S384A") (h) Substitution of glutamine at position 385 of SEQ ID NO: 99 with alanine (hereinafter also referred to as "AAV2 Q385A") (i) Substitution of arginine at position 471 of SEQ ID NO: 99 with alanine (hereinafter also referred to as "AAV2 R471A") (j) Substitution of glutamic acid at position 499 of SEQ ID NO: 99 with alanine (hereinafter also referred to as "AAV2 E499A") (k) Substitution of threonine at position 503 of SEQ ID NO: 99 with serine (hereinafter also referred to as "AAV2 T503S") (l) Substitution of aspartic acid at position 528 of SEQ ID NO: 99 with alanine (hereinafter also referred to as "AAV2 D528A") (m) Substitution of aspartic acid at position 529 of SEQ ID NO: 99 with alanine or glutamic acid (hereinafter also referred to as "AAV2 D529A" and "AAV2 D529E", respectively) The infectivity of the amino acid substitutions shown in (a) to (m) relative to the native type (sequence number 99) is summarized in Table 14 (Source: Lochrie, MA, et al., Journal of Virology, 80(2), 821).

[0292] [Table 14]

[0293] (6) In a 250 mL Erlenmeyer flask (Corning) containing 60 mL of Viral Production medium (Thermo Fisher Scientific), Viral Production Cells (Thermo Fisher Scientific) were incubated at 3.0 × 10 in 8% (v / v) carbon dioxide at 37 °C. 6 The culture was shaken at 120 rpm until the cell density reached 100 cells / mL.

[0294] (7) A mixture of AAV-MAX Enhancer (Thermo Fisher Scientific), pAAV-EGFP prepared in (4), pRC2 Vector and pHelper prepared in (5), and AAV-MAX Transfection Reagent, Viral Plex Complexation, and AAV-MAX Transfection Booster (all manufactured by Thermo Fisher Scientific) was added to the culture medium obtained in (6) to carry out gene transfer, and the cells were cultured with shaking at 120 rpm under conditions of 8% (v / v) carbon dioxide and 37°C for 3 days.

[0295] (8) To the culture medium obtained in (7), AAV-MAX Lysis Buffer (Thermo Fisher Scientific) was added in an amount of 1 / 10 of the medium, Benzonase (Merck Millipore) was added at a final concentration of 1 U / mL, and magnesium chloride was added at a final concentration of 2 mM, and the mixture was then lysed by shaking at 120 rpm at 37°C for 2.0 hours.

[0296] (9) The solution after the lysis treatment was centrifuged at 8,000 × g and 4 ° C for 10 minutes, and the supernatant was subjected to a filter with a pore size of 0.22 μm to remove floating matter, thereby obtaining a solution containing AAV2.

[0297] (10) The concentration of AAV2 in the solution obtained in (9) was quantified using an AAV2 Titration ELISA Kit (PROGEN).

[0298] As a result, both solutions had a concentration of 8.2 × 10 9The solution obtained contained more than cp / mL (cp indicates the number of AAV particles) of AAV2, and a sufficient peak was observed when detected at fluorescence intensity of 350 nm with excitation light of 280 nm using HPLC M40A (Shimadzu Corporation).

[0299] Example 16 Preparation of AAV binding protein (part 1) (1) A transformant capable of expressing AVR10s, obtained by transforming Escherichia coli BL21(DE3) strain with a plasmid pET-AVR10s containing a polynucleotide (SEQ ID NO: 98) encoding a polypeptide containing the AAV binding protein AVR10s consisting of the amino acid sequence set forth in SEQ ID NO: 97, was inoculated into 3 mL of 2YT liquid medium containing 50 μg / mL kanamycin, and precultured overnight at 37° C. under aerobic shaking. In SEQ ID NO: 97, the sequence from the 1st methionine (Met) to the 22nd alanine (Ala) is the PelB signal peptide, the sequence from the 25th serine (Ser) to the 213th aspartic acid (Asp) is the AAV binding protein AVR10s, and the sequence from the 220th cysteine ​​(Cys) to the 226th glycine (Gly) is the cysteine ​​tag sequence, which is a tag for immobilization. In addition, AVR10s has the following binding sites for the extracellular domain 1 (PKD1) and domain 2 (PKD2) (amino acid residues from serine 312 to aspartic acid 500 of SEQ ID NO: 94) of the AAV receptor KIAA0319L: from <x>It is a polypeptide in which the amino acid substitutions shown in are produced. Valine at position 317 of SEQ ID NO:94 (position 30 of SEQ ID NO:97) is substituted with aspartic acid <ii>Tyrosine at position 342 of SEQ ID NO:94 (position 55 of SEQ ID NO:97) is substituted with serine <iii>Lysine at position 362 of SEQ ID NO:94 (position 75 of SEQ ID NO:97) is substituted with glutamic acid <iv>The lysine at position 371 of SEQ ID NO:94 (position 84 of SEQ ID NO:97) is substituted with asparagine <v>Valine at position 381 of SEQ ID NO:94 (position 94 of SEQ ID NO:97) is substituted with alanine <vi>Isoleucine at position 382 of SEQ ID NO:94 (position 95 of SEQ ID NO:97) is substituted with valine <vii>Glycine at position 390 of SEQ ID NO:94 (position 103 of SEQ ID NO:97) is substituted with serine <viii>The lysine at position 399 of SEQ ID NO:94 (position 112 of SEQ ID NO:97) is substituted with glutamic acid <ix>Serine at position 476 of SEQ ID NO:94 (position 189 of SEQ ID NO:97) is substituted with arginine <x>Asparagine at position 487 of SEQ ID NO:94 (position 200 of SEQ ID NO:97) is substituted with aspartic acid

[0300] (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.

[0301] (3) After 2.0 hours from the start of the culture, the medium was cooled on ice, IPTG (IsoPropyl-β-D-ThioGalactopyranoside) was added to a final concentration of 0.1 mM, and the medium was subsequently cultured aerobically with shaking at 25° C. overnight.

[0302] (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.

[0303] (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.

[0304] (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 B"). After washing with equilibration solution B, elution was performed with 20 mM Tris-HCl buffer (pH 7.4) containing 0.5 M imidazole and 150 mM sodium chloride.

[0305] (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 required for producing the AAV adsorbent.

[0306] Example 17 Preparation of AAV adsorbent (part 1) (1) A hydrophilic vinyl polymer for separating agents (Toyopearl, manufactured by Tosoh Corporation) was prepared as an insoluble carrier, and a carrier was prepared by introducing iodoacetamide groups by chemically modifying the hydroxyl groups on the polymer surface.

[0307] (2) To 7 g (wet weight) of the carrier prepared in (1), 14 mg of the AAV binding protein AVR10s prepared in Example 16 and TCEP (Tris(2-CarboxyEthyl)Phosphine) as a reducing agent at a final concentration of 0.3 mM were added, and the mixture was allowed to react by shaking for 3 hours under conditions of pH 8.1 and 25° C. In this way, an AVR10s-immobilized gel, which is an AAV adsorbent in which AVR10s is immobilized, was prepared.

[0308] Example 18 AAV analysis using AAV adsorbent (part 1) (1) 1.25 mL of the AAV adsorbent AVR10s immobilized gel prepared in Example 17 was packed into an empty stainless steel column (φ4.6 mm × 75 mm, Tosoh Corporation) to prepare a column (named AVR10s column).

[0309] (2) The AVR10s column was connected to an HPLC M40A (Shimadzu Corporation) and equilibrated with 10 mM glycine buffer (pH 4.5) containing 50 mM calcium chloride and 15 mM sodium acetate (hereinafter also referred to as "equilibration solution C").

[0310] (3) 0.01 mL of the solution containing AAV2 obtained in Example 15 was applied to the AVR10s column equilibrated in (2).

[0311] (4) After washing with equilibration solution C at a flow rate of 0.5 mL / min for 30 minutes, 10 mM glycine buffer (pH 2.2) containing 50 mM calcium chloride and 15 mM sodium acetate (hereinafter also referred to as "elution solution A") and equilibration solution C were pumped to the AVR10s column (flow rate 0.5 mL / min) so as to obtain a linear gradient in which the elution solution A concentration went from 0% to 100% in 60 minutes. After the linear gradient was completed, elution solution A was pumped for 5 minutes, and then equilibration solution C was pumped again to the AVR10s column for re-equilibration.

[0312] (5) AAV2 eluted from the AVR10s column was detected by fluorescence intensity at 350 nm in response to excitation light at 280 nm, and the time at the apex of the elution peak corresponding to AAV2 was determined as the elution time of the AAV2.

[0313] The elution time of each AAV2 from the AVR10s column is shown in Table 15. The infectivity of each AAV2 (relative value to natural AAV2 (SEQ ID NO: 99)) is plotted on the vertical axis against the elution time of the AAV2 on the horizontal axis, and the results are shown in Figure 1. Note that AAV2 (AAV2 G383A, AAV2 H271F, and AAV2 D529A) that was not adsorbed to the AVR10s immobilized gel and passed through the AVR10s column without adsorption are excluded from the plot in Figure 1.

[0314] From Table 15 and Figure 1, it can be seen that AAV with strong infectivity has a delayed elution time (i.e., strong binding to the AVR10s immobilized gel). This shows that AAV contained in a sample can be analyzed based on the strength of infectivity by analyzing it using an AAV adsorbent containing an insoluble carrier and a polypeptide containing at least an amino acid sequence corresponding to PKD1 or PKD2 of KIAA0319L immobilized on the carrier.

[0315] [Table 15]

[0316] Comparative Example 1 AAV analysis using AAV adsorbent (part 2) AAV2 was analyzed in the same manner as in Example 18, except that 1.25 mL of POROS CaptureSelect AAVX Affinity Resin (Thermo Fisher Scientific), an AAV adsorbent different from the AVR10s immobilized gel, was used as the gel packed into the stainless steel column in Example 18(1).

[0317] The elution time of each AAV2 using a stainless steel column packed with POROS CaptureSelect AAVX Affinity Resin (hereinafter also referred to as "AAVX column") is shown in Table 16. The infectivity of each AAV2 (relative value to natural AAV2 (SEQ ID NO: 99)) is plotted on the vertical axis against the elution time of the AAV2 on the horizontal axis, and the results are shown in Figure 2.

[0318] From Table 16 and Figure 2, it can be seen that AAV2 is eluted at a nearly constant elution time regardless of the strength of infectivity. These results show that AAV cannot be analyzed based on the strength of infectivity with the AAVX column.

[0319] [Table 16]

[0320] Example 19 Preparation of AAV binding proteins An AAV binding protein was prepared in the same manner as in Example 16, except that any of the following was used as a plasmid containing a polynucleotide encoding a polypeptide containing an AAV binding protein. A plasmid pET-AVR11a comprising a polynucleotide (SEQ ID NO: 101) encoding a polypeptide comprising the AAV binding protein AVR11a consisting of the amino acid sequence set forth in SEQ ID NO: 100; A plasmid pET-AVR21 comprising a polynucleotide (SEQ ID NO: 103) encoding a polypeptide comprising the AAV binding protein AVR21 consisting of the amino acid sequence set forth in SEQ ID NO: 102; In SEQ ID NOs: 100 and 102, the PelB signal peptide is from the 1st methionine (Met) to the 22nd alanine (Ala), the AAV binding protein (AVR11a or AVR21) is from the 25th serine (Ser) to the 213th aspartic acid (Asp), and the 220th cysteine ​​(Cys) to the 226th glycine (Gly) is a cysteine ​​tag sequence that is a tag for immobilization. AVR11a binds to the extracellular domain 1 (PKD1) and domain 2 (PKD2) of the AAV receptor KIAA0319L (amino acid residues from the 312th serine to the 500th aspartic acid in SEQ ID NO: 94) as follows: from <xi>AVR21 is a polypeptide in which the amino acid substitutions shown in the following have been made with respect to PKD1 and PKD2: from <xxi>It is a polypeptide in which the amino acid substitutions shown in are produced. Valine at position 317 of SEQ ID NO:94 (position 30 of SEQ ID NO:100 and 102) is substituted with aspartic acid <ii>Tyrosine at position 342 of SEQ ID NO:94 (position 55 of SEQ ID NO:100 and 102) is substituted with serine <iii>The lysine at position 362 of SEQ ID NO:94 (position 75 of SEQ ID NO:100 and 102) is substituted with glutamic acid <iv>The lysine at position 371 of SEQ ID NO:94 (position 84 of SEQ ID NO:100 and 102) is substituted with asparagine <v>Valine at position 381 of SEQ ID NO:94 (position 94 of SEQ ID NO:100 and 102) is substituted with alanine <vi>Isoleucine at position 382 of SEQ ID NO:94 (position 95 of SEQ ID NO:100 and 102) is substituted with valine <vii>Glycine at position 390 of SEQ ID NO:94 (position 103 of SEQ ID NO:100 and 102) is substituted with serine <viii>Lysine at position 399 of SEQ ID NO:94 (position 112 of SEQ ID NO:100 and 102) is substituted with glutamic acid <ix>The serine at position 476 of SEQ ID NO:94 (position 189 of SEQ ID NO:100 and 102) is substituted with arginine <x>Asparagine at position 487 of SEQ ID NO:94 (position 200 of SEQ ID NO:100 and 102) is substituted with aspartic acid <xi>Alanine at position 330 of SEQ ID NO:94 (position 43 of SEQ ID NO:100 and 102) is substituted with valine <xii>Asparagine at position 324 of SEQ ID NO:94 (position 37 of SEQ ID NO:102) is substituted with histidine <xiii>Valine at position 326 of SEQ ID NO:94 (position 39 of SEQ ID NO:102) is substituted with alanine <xiv>Glutamine at position 334 of SEQ ID NO:94 (position 47 of SEQ ID NO:102) is replaced with leucine <xv>Glutamic acid at position 335 of SEQ ID NO:94 (position 48 of SEQ ID NO:102) is replaced with valine <xvi>The threonine at position 341 of SEQ ID NO:94 (position 54 of SEQ ID NO:102) is replaced with alanine <xvii>Phenylalanine at position 379 of SEQ ID NO:94 (position 92 of SEQ ID NO:102) is replaced with tyrosine <xviii>The lysine at position 380 of SEQ ID NO:94 (position 93 of SEQ ID NO:102) is substituted with arginine <xix>Lysine at position 467 of SEQ ID NO:94 (position 180 of SEQ ID NO:102) is substituted with glutamine <xx>Serine at position 482 of SEQ ID NO:94 (position 195 of SEQ ID NO:102) is substituted with threonine <xxi>Asparagine at position 492 of SEQ ID NO:94 (position 205 of SEQ ID NO:102) is substituted with aspartic acid

[0321] Example 20 Preparation of AAV adsorbent (part 2) An AAV adsorbent was prepared in the same manner as in Example 17, except that AAV11a or AAV21 prepared in Example 19 was used as the AAV-binding protein. The AAV adsorbent on which AVR11a is immobilized is named AVR11a-immobilized gel, and the AAV adsorbent on which AVR21 is immobilized is named AVR21-immobilized gel, respectively.

[0322] Example 21 AAV analysis using AAV adsorbent (part 3) (1) 1.25 mL of the AAV adsorbent (AVR11a immobilized gel or AVR21 immobilized gel) prepared in Example 20 was packed into an empty stainless steel column (φ4.6 mm×75 mm, Tosoh Corporation) to prepare a column. The column packed with the AVR11a immobilized gel is named the AVR11a column, and the column packed with the AVR21 immobilized gel is named the AVR21 column.

[0323] (2) Use AVR11a or AVR21 column as the column and equilibration solution C. AAV2 was analyzed in the same manner as in Example 18(2) to (5), except that 10 mM glycine buffer (pH 6.0) containing 50 mM calcium chloride and 15 mM sodium acetate was used as eluent A, and 10 mM glycine buffer (pH 2.0) containing 50 mM calcium chloride and 15 mM sodium acetate was used as eluent B.

[0324] Table 17 shows the elution times of each AAV2 from the AVR11a column and the AVR21 column. The infectivity of each AAV2 (relative value to the natural AAV2 (SEQ ID NO: 99)) is plotted on the vertical axis against the elution time of the AAV2 on the horizontal axis, and the results are shown in Figures 3 and 4. Note that AAV2 (AAV2 H271A, AAV2 H271Q, AAV2 H271T, AAV2 G383A, AAV2 H271F, and AAV2 D529A) that was not adsorbed to the AVR11a immobilized gel and passed through the AVR11a column are excluded from the plot in Figure 3, and AAV2 (AAV2 G383A, AAV2 H271F, and AAV2 D529A) that was not adsorbed to the AVR21 immobilized gel and passed through the AVR21 column are excluded from the plot in Figure 4.

[0325] From Table 17, Figures 3 and 4, it can be seen that AAV with strong infectivity has a delayed elution time (i.e., strong binding strength to AVR11a-immobilized gel or AVR21-immobilized gel). This shows that AAV contained in a sample can be analyzed based on the strength of infectivity by analyzing it using an AAV adsorbent containing an insoluble carrier and a polypeptide containing at least an amino acid sequence corresponding to PKD1 or PKD2 of KIAA0319L immobilized on the carrier.

[0326] [Table 17]

[0327] Comparative Example 2 AAV analysis using AAV adsorbent (part 4) In Example 21(1), AAV2 was analyzed in the same manner as in Example 20, except that 1.25 mL of POROS CaptureSelect AAVX Affinity Resin (Thermo Fisher Scientific), an AAV adsorbent different from the AVR11a immobilization gel and AVR21 immobilization gel, was used as the gel packed into the stainless steel column.

[0328] The elution time of each AAV2 using a stainless steel column packed with POROS CaptureSelect AAVX Affinity Resin (hereinafter also referred to as "AAVX column") is shown in Table 18. The infectivity of each AAV2 (relative value to natural AAV2 (SEQ ID NO: 99)) is plotted on the vertical axis against the elution time of the AAV2 on the horizontal axis, and the results are shown in Figure 5.

[0329] From Table 18 and Figure 5, it can be seen that AAV2 is eluted at a nearly constant elution time regardless of the strength of infectivity. These results show that AAVX column cannot be used to analyze AAV based on the strength of infectivity.

[0330] [Table 18] < / xxi> < / xx> < / xix> < / xviii> < / xvii> < / xvi> < / xv> < / xiv> < / xiii> < / xii> < / xi> < / x> < / ix> < / viii> < / vii> < / vi> < / v> < / iv> < / iii> < / ii> < / xxi> < / xi> < / x> < / ix> < / viii> < / vii> < / vi> < / v> < / iv> < / iii> < / ii> < / x>

Claims

1. The amino acid sequence of SEQ ID NO: 1 includes at least the amino acid residues from the 312th serine to the 500th aspartic acid, provided that the amino acid residues from the 312th to the 500th are as follows: 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 substituted with glutamic acid Lysine at position 371 of SEQ ID NO:1 is substituted with asparagine Valine 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 substituted with glutamic acid Serine at position 476 of SEQ ID NO:1 is replaced with arginine Asparagine at position 487 of SEQ ID NO:1 is substituted with aspartic acid all amino acid substitutions in An adeno-associated virus (AAV) binding protein having at least one amino acid substitution selected from the following (1) to (5): (1) Alanine at position 330 of SEQ ID NO:1 is replaced with valine (2) Alanine at position 330 of SEQ ID NO:1 is substituted with any one of cysteine, phenylalanine, leucine, arginine, tryptophan, and tyrosine. (3) Substitution of tyrosine at position 331 of SEQ ID NO:1 with cysteine (4) Valine at position 332 of SEQ ID NO: 1 is substituted with tryptophan or tyrosine (5) The 333rd leucine in SEQ ID NO:1 is replaced with cysteine.

2. The AAV binding protein according to claim 1, which comprises at least the amino acid residues from the 312th serine to the 500th aspartic acid of the amino acid sequence set forth in SEQ ID NO:1, with the proviso that at least the following amino acid substitution (1) occurs in the amino acid residues from the 312th to the 500th: (1) Alanine at position 330 of SEQ ID NO:1 is replaced with valine.

3. An adeno-associated virus (AAV) binding protein comprising at least the amino acid residues from the 25th serine to the 213th aspartic acid in any one of the amino acid sequences set forth in SEQ ID NOs: 15 to 25, 54 to 63, 65, 83, 85, 87, 88, and 90 to 92.

4. The AAV binding protein according to claim 2 further comprises any one of the following amino acid substitutions or combinations of amino acid substitutions (I) to (CXVIII): (I) Substitution of alanine at position 313 of SEQ ID NO:1 with valine (II) Substitution of glutamine at position 334 of SEQ ID NO:1 with leucine (III) Substitution of threonine at position 341 of SEQ ID NO:1 with alanine (IV) Substitution of methionine at position 359 of SEQ ID NO:1 with isoleucine (V) Substitution of isoleucine at position 366 of SEQ ID NO:1 with threonine (VI) substitution of leucine at position 367 of SEQ ID NO:1 with valine (VII) Substitution of leucine at position 369 of SEQ ID NO:1 with glutamine (VIII) Substitution of phenylalanine at position 379 of SEQ ID NO: 1 with tyrosine (IX) Substitution of lysine at position 380 of SEQ ID NO:1 with asparagine (X) Substitution of valine at position 394 of SEQ ID NO: 1 with alanine (XI) Substitution of valine at position 398 of SEQ ID NO: 1 with alanine (XII) Substitution of lysine at position 404 of SEQ ID NO: 1 with alanine (XIII) Substitution of isoleucine at position 419 of SEQ ID NO:1 with phenylalanine (XIV) Substitution of valine at position 440 of SEQ ID NO: 1 with alanine (XV) Substitution of glutamic acid at position 446 of SEQ ID NO:1 with aspartic acid (XVI) substitution of lysine at position 448 of SEQ ID NO: 1 with glutamic acid (XVII) Substitution of glutamic acid at position 453 of SEQ ID NO: 1 with lysine (XVIII) Substitution of lysine at position 464 of SEQ ID NO:1 with asparagine (XIX) Substitution of lysine at position 464 of SEQ ID NO: 1 with glutamic acid (XX) Substitution of asparagine at position 472 of SEQ ID NO:1 with tyrosine (XXI) Substitution of threonine at position 478 of SEQ ID NO: 1 with alanine (XXII) Substitution of valine at position 480 of SEQ ID NO: 1 with aspartic acid (XXIII) Substitution of glutamine at position 318 of SEQ ID NO: 1 with arginine and substitution of serine at position 420 of SEQ ID NO: 1 with threonine (XXIV) Substitution of leucine at position 328 of SEQ ID NO:1 with glutamine and substitution of threonine at position 343 of SEQ ID NO:1 with proline (XXV) Substitution of valine at position 332 of SEQ ID NO:1 with glutamic acid and substitution of valine at position 412 of SEQ ID NO:1 with isoleucine (XXVI) Substitution of valine at position 332 of SEQ ID NO:1 with alanine and substitution of tyrosine at position 473 of SEQ ID NO:1 with histidine (XXVII) Substitution of glutamine at position 334 of SEQ ID NO: 1 with histidine and substitution of serine at position 476 of SEQ ID NO: 1 with glycine (XXVIII) Substitution of glutamic acid at position 340 of SEQ ID NO:1 with aspartic acid and substitution of leucine at position 369 of SEQ ID NO:1 with glutamine (XXIX) Substitution of threonine at position 350 of SEQ ID NO:1 with alanine and substitution of serine at position 476 of SEQ ID NO:1 with glycine (XXX) Substitution of methionine at position 359 of SEQ ID NO:1 with leucine and substitution of serine at position 425 of SEQ ID NO:1 with glycine (XXXI) Substitution of leucine at position 376 of SEQ ID NO:1 with proline and substitution of serine at position 488 of SEQ ID NO:1 with threonine (XXXII) Substitution of lysine at position 380 of SEQ ID NO:1 with arginine and substitution of histidine at position 389 of SEQ ID NO:1 with leucine (XXXIII) A substitution of aspartic acid for isoleucine at position 382 of SEQ ID NO:1 and a substitution of tyrosine for asparagine at position 496 of SEQ ID NO:1 (XXXIV) Substitution of glutamic acid at position 384 of SEQ ID NO:1 with valine and substitution of valine at position 479 of SEQ ID NO:1 with alanine (XXXV) Substitution of histidine at position 389 of SEQ ID NO:1 with aspartic acid and substitution of lysine at position 455 of SEQ ID NO:1 with glutamic acid (XXXVI) Substitution of asparagine at position 395 of SEQ ID NO:1 with aspartic acid and substitution of glycine at position 430 of SEQ ID NO:1 with alanine (XXXVII) Substitution of valine at position 396 of SEQ ID NO:1 with glycine and substitution of isoleucine at position 411 of SEQ ID NO:1 with threonine (XXXVIII) Substitution of isoleucine at position 409 of SEQ ID NO:1 with asparagine and substitution of isoleucine at position 428 of SEQ ID NO:1 with valine (XXXIX) Substitution of phenylalanine at position 416 of SEQ ID NO:1 with serine and substitution of threonine at position 434 of SEQ ID NO:1 with alanine (XL) Substitution of threonine at position 423 of SEQ ID NO:1 with alanine and substitution of valine at position 480 of SEQ ID NO:1 with aspartic acid (XLI) Substitution of lysine at position 448 of SEQ ID NO:1 with glutamic acid and substitution of threonine at position 478 of SEQ ID NO:1 with alanine (XLII) Substitution of glycine at position 484 of SEQ ID NO:1 with serine and substitution of threonine at position 486 of SEQ ID NO:1 with serine (XLIII) Substitution of serine at position 312 of SEQ ID NO:1 with proline, substitution of asparagine at position 329 of SEQ ID NO:1 with tyrosine, and substitution of valine at position 469 of SEQ ID NO:1 with glutamic acid (XLIV) Substitution of glutamic acid at position 315 of SEQ ID NO:1 with glycine, substitution of isoleucine at position 411 of SEQ ID NO:1 with leucine, and substitution of lysine at position 448 of SEQ ID NO:1 with asparagine (XLV) Substitution of asparagine at position 324 of SEQ ID NO:1 with histidine, substitution of glutamic acid at position 335 of SEQ ID NO:1 with valine, and substitution of lysine at position 464 of SEQ ID NO:1 with glutamic acid (XLVI) Substitution of valine at position 326 of SEQ ID NO:1 with glutamic acid, substitution of arginine at position 403 of SEQ ID NO:1 with serine, and substitution of serine at position 425 of SEQ ID NO:1 with glycine (XLVII) Substitution of glutamine at position 327 of SEQ ID NO:1 with leucine, substitution of lysine at position 338 of SEQ ID NO:1 with glutamic acid, and substitution of leucine at position 451 of SEQ ID NO:1 with isoleucine (XLVIII) Substitution of leucine at position 328 of SEQ ID NO:1 with arginine, substitution of glutamic acid at position 360 of SEQ ID NO:1 with aspartic acid, and substitution of arginine at position 403 of SEQ ID NO:1 with histidine (XLIX) Substitution of threonine at position 343 of SEQ ID NO:1 with alanine, substitution of aspartic acid at position 345 of SEQ ID NO:1 with glycine, and substitution of valine at position 394 of SEQ ID NO:1 with aspartic acid (LI) Substitution of tyrosine at position 355 of SEQ ID NO:1 with histidine, substitution of asparagine at position 472 of SEQ ID NO:1 with aspartic acid, and substitution of valine at position 479 of SEQ ID NO:1 with alanine (LII) Substitution of lysine at position 368 of SEQ ID NO:1 with asparagine, substitution of glycine at position 390 of SEQ ID NO:1 with asparagine, and substitution of valine at position 479 of SEQ ID NO:1 with alanine (LIII) Substitution of isoleucine at position 382 of SEQ ID NO:1 with alanine, substitution of alanine at position 388 of SEQ ID NO:1 with threonine, and substitution of glycine at position 471 of SEQ ID NO:1 with cysteine (LIV) Substitution of histidine at position 389 of SEQ ID NO:1 with arginine, substitution of arginine at position 403 of SEQ ID NO:1 with histidine, and substitution of arginine at position 406 of SEQ ID NO:1 with serine (LV) Substitution of histidine at position 389 of SEQ ID NO:1 with asparagine, substitution of valine at position 480 of SEQ ID NO:1 with alanine, and substitution of asparagine at position 496 of SEQ ID NO:1 with aspartic acid (LVI) Substitution of lysine at position 448 of SEQ ID NO:1 with glutamic acid, substitution of lysine at position 455 of SEQ ID NO:1 with arginine, and substitution of valine at position 479 of SEQ ID NO:1 with alanine (LVII) Substitution of glutamic acid at position 315 of SEQ ID NO:1 with valine, substitution of lysine at position 323 of SEQ ID NO:1 with methionine, substitution of alanine at position 330 of SEQ ID NO:1 with alanine, and substitution of isoleucine at position 419 of SEQ ID NO:1 with valine (LVIII) Substitution of threonine at position 341 of SEQ ID NO:1 with alanine, substitution of threonine at position 343 of SEQ ID NO:1 with serine, substitution of isoleucine at position 419 of SEQ ID NO:1 with phenylalanine, and substitution of threonine at position 489 of SEQ ID NO:1 with alanine (LIX) Leucine at position 328 of SEQ ID NO:1 is replaced with proline, proline at position 337 of SEQ ID NO:1 is replaced with glutamine, isoleucine at position 349 of SEQ ID NO:1 is replaced with threonine, aspartic acid at position 354 of SEQ ID NO:1 is replaced with valine, and aspartic acid at position 437 of SEQ ID NO:1 is replaced with asparagine (LX) SEQ ID NO: 1, valine at position 332 is replaced with alanine, leucine at position 333 is replaced with proline, glycine at position 392 is replaced with arginine, threonine at position 486 is replaced with serine, and threonine at position 489 is replaced with serine in SEQ ID NO: 1 (LXI) Substitution of asparagine at position 324 of SEQ ID NO: 1 with histidine (LXII) Substitution of glutamic acid at position 335 of SEQ ID NO:1 with valine (LXIII) Substitution of asparagine at position 324 of SEQ ID NO:1 with histidine and substitution of glutamic acid at position 335 of SEQ ID NO:1 with valine (LXIV) Asparagine at position 324 of SEQ ID NO:1 is replaced with histidine, glutamic acid at position 335 of SEQ ID NO:1 is replaced with valine, and threonine at position 341 of SEQ ID NO:1 is replaced with alanine (LXV) Asparagine at position 324 of SEQ ID NO:1 is replaced with histidine, glutamic acid at position 335 of SEQ ID NO:1 is replaced with valine, and glutamine at position 334 of SEQ ID NO:1 is replaced with leucine (LXVI) Substitution of asparagine at position 324 of SEQ ID NO:1 with histidine, substitution of glutamic acid at position 335 of SEQ ID NO:1 with valine, and substitution of methionine at position 359 of SEQ ID NO:1 with isoleucine (LXVII) Asparagine at position 324 of SEQ ID NO:1 is replaced with histidine, glutamic acid at position 335 of SEQ ID NO:1 is replaced with valine, and phenylalanine at position 379 of SEQ ID NO:1 is replaced with tyrosine (LXVIII) Asparagine at position 324 of SEQ ID NO:1 is replaced with histidine, glutamic acid at position 335 of SEQ ID NO:1 is replaced with valine, glutamine at position 334 of SEQ ID NO:1 is replaced with leucine, and threonine at position 341 of SEQ ID NO:1 is replaced with alanine (LXIX) Asparagine at position 324 of SEQ ID NO:1 is replaced with histidine, glutamic acid at position 335 of SEQ ID NO:1 is replaced with valine, glutamine at position 334 of SEQ ID NO:1 is replaced with leucine, and methionine at position 359 of SEQ ID NO:1 is replaced with isoleucine (LXX) Asparagine at position 324 of SEQ ID NO:1 is replaced with histidine, glutamic acid at position 335 of SEQ ID NO:1 is replaced with valine, glutamine at position 334 of SEQ ID NO:1 is replaced with leucine, and phenylalanine at position 379 of SEQ ID NO:1 is replaced with tyrosine (LXXI) Asparagine at position 324 of SEQ ID NO:1 is replaced with histidine, Glutamine at position 334 of SEQ ID NO:1 is replaced with leucine, Glutamic acid at position 335 of SEQ ID NO:1 is replaced with valine, Threonine at position 341 of SEQ ID NO:1 is replaced with alanine, and Phenylalanine at position 379 of SEQ ID NO:1 is replaced with tyrosine (LXXII) Asparagine at position 324 of SEQ ID NO:1 is replaced with histidine, glutamine at position 334 of SEQ ID NO:1 is replaced with leucine, glutamic acid at position 335 of SEQ ID NO:1 is replaced with valine, threonine at position 341 of SEQ ID NO:1 is replaced with alanine, phenylalanine at position 379 of SEQ ID NO:1 is replaced with tyrosine, and valine at position 317 of SEQ ID NO:1 is replaced with asparagine (LXXIII) Asparagine at position 324 of SEQ ID NO:1 is replaced with histidine, glutamine at position 334 of SEQ ID NO:1 is replaced with leucine, glutamic acid at position 335 of SEQ ID NO:1 is replaced with valine, threonine at position 341 of SEQ ID NO:1 is replaced with alanine, phenylalanine at position 379 of SEQ ID NO:1 is replaced with tyrosine, and glutamic acid at position 325 of SEQ ID NO:1 is replaced with lysine (LXXIV) Asparagine at position 324 of SEQ ID NO:1 is replaced with histidine, glutamine at position 334 of SEQ ID NO:1 is replaced with leucine, glutamic acid at position 335 of SEQ ID NO:1 is replaced with valine, threonine at position 341 of SEQ ID NO:1 is replaced with alanine, phenylalanine at position 379 of SEQ ID NO:1 is replaced with tyrosine, and valine at position 326 of SEQ ID NO:1 is replaced with alanine (LXXV) Asparagine at position 324 of SEQ ID NO:1 is replaced with histidine, glutamine at position 334 of SEQ ID NO:1 is replaced with leucine, glutamic acid at position 335 of SEQ ID NO:1 is replaced with valine, threonine at position 341 of SEQ ID NO:1 is replaced with alanine, phenylalanine at position 379 of SEQ ID NO:1 is replaced with tyrosine, and leucine at position 333 of SEQ ID NO:1 is replaced with methionine (LXXVI) Asparagine at position 324 of SEQ ID NO:1 is replaced with histidine, glutamine at position 334 of SEQ ID NO:1 is replaced with leucine, glutamic acid at position 335 of SEQ ID NO:1 is replaced with valine, threonine at position 341 of SEQ ID NO:1 is replaced with alanine, phenylalanine at position 379 of SEQ ID NO:1 is replaced with tyrosine, and glycine at position 339 of SEQ ID NO:1 is replaced with glutamic acid (LXXVII) Asparagine at position 324 of SEQ ID NO:1 is replaced with histidine, glutamine at position 334 of SEQ ID NO:1 is replaced with leucine, glutamic acid at position 335 of SEQ ID NO:1 is replaced with valine, threonine at position 341 of SEQ ID NO:1 is replaced with alanine, phenylalanine at position 379 of SEQ ID NO:1 is replaced with tyrosine, and methionine at position 359 of SEQ ID NO:1 is replaced with valine (LXXVIII) Asparagine at position 324 of SEQ ID NO:1 is replaced with histidine, glutamine at position 334 of SEQ ID NO:1 is replaced with leucine, glutamic acid at position 335 of SEQ ID NO:1 is replaced with valine, threonine at position 341 of SEQ ID NO:1 is replaced with alanine, phenylalanine at position 379 of SEQ ID NO:1 is replaced with tyrosine, and glycine at position 361 of SEQ ID NO:1 is replaced with glutamic acid (LXXIX) Asparagine at position 324 of SEQ ID NO:1 is replaced with histidine, glutamine at position 334 of SEQ ID NO:1 is replaced with leucine, glutamic acid at position 335 of SEQ ID NO:1 is replaced with valine, threonine at position 341 of SEQ ID NO:1 is replaced with alanine, phenylalanine at position 379 of SEQ ID NO:1 is replaced with tyrosine, and serine at position 366 of SEQ ID NO:1 is replaced with proline (LXXX) Asparagine at position 324 of SEQ ID NO:1 is replaced with histidine, glutamine at position 334 of SEQ ID NO:1 is replaced with leucine, glutamic acid at position 335 of SEQ ID NO:1 is replaced with valine, threonine at position 341 of SEQ ID NO:1 is replaced with alanine, phenylalanine at position 379 of SEQ ID NO:1 is replaced with tyrosine, and lysine at position 380 of SEQ ID NO:1 is replaced with arginine (LXXXI) Asparagine at position 324 of SEQ ID NO:1 is replaced with histidine, glutamine at position 334 of SEQ ID NO:1 is replaced with leucine, glutamic acid at position 335 of SEQ ID NO:1 is replaced with valine, threonine at position 341 of SEQ ID NO:1 is replaced with alanine, phenylalanine at position 379 of SEQ ID NO:1 is replaced with tyrosine, and valine at position 381 of SEQ ID NO:1 is replaced with threonine (LXXXII) Asparagine at position 324 of SEQ ID NO:1 is replaced with histidine, glutamine at position 334 of SEQ ID NO:1 is replaced with leucine, glutamic acid at position 335 of SEQ ID NO:1 is replaced with valine, threonine at position 341 of SEQ ID NO:1 is replaced with alanine, phenylalanine at position 379 of SEQ ID NO:1 is replaced with tyrosine, and lysine at position 399 of SEQ ID NO:1 is replaced with glycine (LXXXIII) Asparagine at position 324 of SEQ ID NO:1 is replaced with histidine, glutamine at position 334 of SEQ ID NO:1 is replaced with leucine, glutamic acid at position 335 of SEQ ID NO:1 is replaced with valine, threonine at position 341 of SEQ ID NO:1 is replaced with alanine, phenylalanine at position 379 of SEQ ID NO:1 is replaced with tyrosine, and leucine at position 421 of SEQ ID NO:1 is replaced with proline (LXXXIV) Asparagine at position 324 of SEQ ID NO:1 is replaced with histidine, glutamine at position 334 of SEQ ID NO:1 is replaced with leucine, glutamic acid at position 335 of SEQ ID NO:1 is replaced with valine, threonine at position 341 of SEQ ID NO:1 is replaced with alanine, phenylalanine at position 379 of SEQ ID NO:1 is replaced with tyrosine, and proline at position 422 of SEQ ID NO:1 is replaced with serine (XC) Asparagine at position 324 of SEQ ID NO:1 is replaced with histidine, glutamine at position 334 of SEQ ID NO:1 is replaced with leucine, glutamic acid at position 335 of SEQ ID NO:1 is replaced with valine, threonine at position 341 of SEQ ID NO:1 is replaced with alanine, phenylalanine at position 379 of SEQ ID NO:1 is replaced with tyrosine, and serine at position 425 of SEQ ID NO:1 is replaced with glycine (XCI) Asparagine at position 324 of SEQ ID NO:1 is replaced with histidine, glutamine at position 334 of SEQ ID NO:1 is replaced with leucine, glutamic acid at position 335 of SEQ ID NO:1 is replaced with valine, threonine at position 341 of SEQ ID NO:1 is replaced with alanine, phenylalanine at position 379 of SEQ ID NO:1 is replaced with tyrosine, and glutamic acid at position 454 of SEQ ID NO:1 is replaced with glycine (XCII) Asparagine at position 324 of SEQ ID NO:1 is replaced with histidine, glutamine at position 334 of SEQ ID NO:1 is replaced with leucine, glutamic acid at position 335 of SEQ ID NO:1 is replaced with valine, threonine at position 341 of SEQ ID NO:1 is replaced with alanine, phenylalanine at position 379 of SEQ ID NO:1 is replaced with tyrosine, and lysine at position 467 of SEQ ID NO:1 is replaced with glutamic acid (XCIII) Asparagine at position 324 of SEQ ID NO:1 is replaced with histidine, glutamine at position 334 of SEQ ID NO:1 is replaced with leucine, glutamic acid at position 335 of SEQ ID NO:1 is replaced with valine, threonine at position 341 of SEQ ID NO:1 is replaced with alanine, phenylalanine at position 379 of SEQ ID NO:1 is replaced with tyrosine, and lysine at position 467 of SEQ ID NO:1 is replaced with glutamine (XCIV) Asparagine at position 324 of SEQ ID NO:1 is replaced with histidine, glutamine at position 334 of SEQ ID NO:1 is replaced with leucine, glutamic acid at position 335 of SEQ ID NO:1 is replaced with valine, threonine at position 341 of SEQ ID NO:1 is replaced with alanine, phenylalanine at position 379 of SEQ ID NO:1 is replaced with tyrosine, and serine at position 476 of SEQ ID NO:1 is replaced with glycine (XCV) Asparagine at position 324 of SEQ ID NO:1 is replaced with histidine, Glutamine at position 334 of SEQ ID NO:1 is replaced with leucine, Glutamic acid at position 335 of SEQ ID NO:1 is replaced with valine, Threonine at position 341 of SEQ ID NO:1 is replaced with alanine, Phenylalanine at position 379 of SEQ ID NO:1 is replaced with tyrosine, and Serine at position 482 of SEQ ID NO:1 is replaced with threonine (XCVI) Substitution of asparagine at position 324 of SEQ ID NO:1 with histidine, substitution of glutamine at position 334 of SEQ ID NO:1 with leucine, substitution of glutamic acid at position 335 of SEQ ID NO:1 with valine, substitution of threonine at position 341 of SEQ ID NO:1 with alanine, substitution of phenylalanine at position 379 of SEQ ID NO:1 with tyrosine, and substitution of alanine at position 491 of SEQ ID NO:1 with serine (XCVII) Asparagine at position 324 of SEQ ID NO:1 is replaced with histidine, glutamine at position 334 of SEQ ID NO:1 is replaced with leucine, glutamic acid at position 335 of SEQ ID NO:1 is replaced with valine, threonine at position 341 of SEQ ID NO:1 is replaced with alanine, phenylalanine at position 379 of SEQ ID NO:1 is replaced with tyrosine, and asparagine at position 492 of SEQ ID NO:1 is replaced with aspartic acid (XCVIII) Asparagine at position 324 of SEQ ID NO:1 is replaced with histidine, glutamine at position 334 of SEQ ID NO:1 is replaced with leucine, glutamic acid at position 335 of SEQ ID NO:1 is replaced with valine, threonine at position 341 of SEQ ID NO:1 is replaced with alanine, phenylalanine at position 379 of SEQ ID NO:1 is replaced with tyrosine, and asparagine at position 496 of SEQ ID NO:1 is replaced with tyrosine (XCIX) Asparagine at position 324 of SEQ ID NO:1 is replaced with histidine, glutamine at position 334 of SEQ ID NO:1 is replaced with leucine, glutamic acid at position 335 of SEQ ID NO:1 is replaced with valine, threonine at position 341 of SEQ ID NO:1 is replaced with alanine, phenylalanine at position 379 of SEQ ID NO:1 is replaced with tyrosine, and aspartic acid at position 500 of SEQ ID NO:1 is replaced with glycine (C) Asparagine at position 324 of SEQ ID NO:1 is replaced with histidine, glutamine at position 334 of SEQ ID NO:1 is replaced with leucine, glutamic acid at position 335 of SEQ ID NO:1 is replaced with valine, threonine at position 341 of SEQ ID NO:1 is replaced with alanine, phenylalanine at position 379 of SEQ ID NO:1 is replaced with tyrosine, isoleucine at position 319 of SEQ ID NO:1 is replaced with valine, and lysine at position 368 of SEQ ID NO:1 is replaced with arginine (CI) Asparagine at position 324 of SEQ ID NO:1 is replaced with histidine, glutamine at position 334 of SEQ ID NO:1 is replaced with leucine, glutamic acid at position 335 of SEQ ID NO:1 is replaced with valine, threonine at position 341 of SEQ ID NO:1 is replaced with alanine, phenylalanine at position 379 of SEQ ID NO:1 is replaced with tyrosine, lysine at position 323 of SEQ ID NO:1 is replaced with asparagine, and lysine at position 368 of SEQ ID NO:1 is replaced with glutamic acid, (CII) Asparagine at position 324 of SEQ ID NO:1 is replaced with histidine, glutamine at position 334 of SEQ ID NO:1 is replaced with leucine, glutamic acid at position 335 of SEQ ID NO:1 is replaced with valine, threonine at position 341 of SEQ ID NO:1 is replaced with alanine, phenylalanine at position 379 of SEQ ID NO:1 is replaced with tyrosine, asparagine at position 329 of SEQ ID NO:1 is replaced with serine, and lysine at position 338 of SEQ ID NO:1 is replaced with arginine (CIII) Substitution of asparagine at position 324 of SEQ ID NO:1 with histidine, substitution of glutamine at position 334 of SEQ ID NO:1 with leucine, substitution of glutamic acid at position 335 of SEQ ID NO:1 with valine, substitution of threonine at position 341 of SEQ ID NO:1 with alanine, substitution of phenylalanine at position 379 of SEQ ID NO:1 with tyrosine, substitution of lysine at position 362 of SEQ ID NO:1 with aspartic acid, substitution of histidine at position 443 of SEQ ID NO:1 with tyrosine, and substitution of lysine at position 455 of SEQ ID NO:1 with arginine (CIV) Substitution of asparagine at position 324 of SEQ ID NO:1 with histidine, substitution of glutamine at position 334 of SEQ ID NO:1 with leucine, substitution of glutamic acid at position 335 of SEQ ID NO:1 with valine, substitution of threonine at position 341 of SEQ ID NO:1 with alanine, substitution of phenylalanine at position 379 of SEQ ID NO:1 with tyrosine, substitution of lysine at position 368 of SEQ ID NO:1 with glutamic acid, and substitution of histidine at position 443 of SEQ ID NO:1 with arginine. (CV) Asparagine at position 324 of SEQ ID NO:1 is replaced with histidine, glutamine at position 334 of SEQ ID NO:1 is replaced with leucine, glutamic acid at position 335 of SEQ ID NO:1 is replaced with valine, threonine at position 341 of SEQ ID NO:1 is replaced with alanine, phenylalanine at position 379 of SEQ ID NO:1 is replaced with tyrosine, lysine at position 368 of SEQ ID NO:1 is replaced with arginine, and isoleucine at position 382 of SEQ ID NO:1 is replaced with aspartic acid (CVI) Substitution of asparagine at position 324 of SEQ ID NO:1 with histidine, substitution of glutamine at position 334 of SEQ ID NO:1 with leucine, substitution of glutamic acid at position 335 of SEQ ID NO:1 with valine, substitution of threonine at position 341 of SEQ ID NO:1 with alanine, substitution of phenylalanine at position 379 of SEQ ID NO:1 with tyrosine, substitution of leucine at position 369 of SEQ ID NO:1 with proline, and substitution of serine at position 488 of SEQ ID NO:1 with phenylalanine (CVII) Asparagine at position 324 of SEQ ID NO:1 is replaced with histidine, glutamine at position 334 of SEQ ID NO:1 is replaced with leucine, glutamic acid at position 335 of SEQ ID NO:1 is replaced with valine, threonine at position 341 of SEQ ID NO:1 is replaced with alanine, phenylalanine at position 379 of SEQ ID NO:1 is replaced with tyrosine, and valine at position 326 of SEQ ID NO:1 is replaced with alanine (CVIII) Asparagine at position 324 of SEQ ID NO:1 is replaced with histidine, glutamine at position 334 of SEQ ID NO:1 is replaced with leucine, glutamic acid at position 335 of SEQ ID NO:1 is replaced with valine, threonine at position 341 of SEQ ID NO:1 is replaced with alanine, phenylalanine at position 379 of SEQ ID NO:1 is replaced with tyrosine, and lysine at position 380 of SEQ ID NO:1 is replaced with arginine (CVIV) Substitution of asparagine at position 324 of SEQ ID NO:1 with histidine, substitution of glutamine at position 334 of SEQ ID NO:1 with leucine, substitution of glutamic acid at position 335 of SEQ ID NO:1 with valine, substitution of threonine at position 341 of SEQ ID NO:1 with alanine, substitution of phenylalanine at position 379 of SEQ ID NO:1 with tyrosine, and substitution of lysine at position 467 of SEQ ID NO:1 with glutamine (CX) Asparagine at position 324 of SEQ ID NO:1 is replaced with histidine, glutamine at position 334 of SEQ ID NO:1 is replaced with leucine, glutamic acid at position 335 of SEQ ID NO:1 is replaced with valine, threonine at position 341 of SEQ ID NO:1 is replaced with alanine, phenylalanine at position 379 of SEQ ID NO:1 is replaced with tyrosine, and serine at position 482 of SEQ ID NO:1 is replaced with threonine (CXI) Substitution of asparagine at position 324 of SEQ ID NO:1 with histidine, substitution of glutamine at position 334 of SEQ ID NO:1 with leucine, substitution of glutamic acid at position 335 of SEQ ID NO:1 with valine, substitution of threonine at position 341 of SEQ ID NO:1 with alanine, substitution of phenylalanine at position 379 of SEQ ID NO:1 with tyrosine, and substitution of asparagine at position 492 of SEQ ID NO:1 with aspartic acid (CXII) Asparagine at position 324 of SEQ ID NO:1 is replaced with histidine, glutamine at position 334 of SEQ ID NO:1 is replaced with leucine, glutamic acid at position 335 of SEQ ID NO:1 is replaced with valine, threonine at position 341 of SEQ ID NO:1 is replaced with alanine, phenylalanine at position 379 of SEQ ID NO:1 is replaced with tyrosine, lysine at position 467 of SEQ ID NO:1 is replaced with glutamine, serine at position 482 of SEQ ID NO:1 is replaced with threonine, and valine at position 326 of SEQ ID NO:1 is replaced with alanine (CXIII) Asparagine at position 324 of SEQ ID NO:1 is replaced with histidine, glutamine at position 334 of SEQ ID NO:1 is replaced with leucine, glutamic acid at position 335 of SEQ ID NO:1 is replaced with valine, threonine at position 341 of SEQ ID NO:1 is replaced with alanine, phenylalanine at position 379 of SEQ ID NO:1 is replaced with tyrosine, lysine at position 467 of SEQ ID NO:1 is replaced with glutamine, serine at position 482 of SEQ ID NO:1 is replaced with threonine, and asparagine at position 492 of SEQ ID NO:1 is replaced with aspartic acid. (CXIV) Asparagine at position 324 of SEQ ID NO:1 is replaced with histidine, glutamine at position 334 of SEQ ID NO:1 is replaced with leucine, glutamic acid at position 335 of SEQ ID NO:1 is replaced with valine, threonine at position 341 of SEQ ID NO:1 is replaced with alanine, phenylalanine at position 379 of SEQ ID NO:1 is replaced with tyrosine, lysine at position 467 of SEQ ID NO:1 is replaced with glutamine, serine at position 482 of SEQ ID NO:1 is replaced with threonine, and lysine at position 380 of SEQ ID NO:1 is replaced with arginine (CXV) Asparagine at position 324 of SEQ ID NO:1 is replaced with histidine, glutamine at position 334 of SEQ ID NO:1 is replaced with leucine, glutamic acid at position 335 of SEQ ID NO:1 is replaced with valine, threonine at position 341 of SEQ ID NO:1 is replaced with alanine, phenylalanine at position 379 of SEQ ID NO:1 is replaced with tyrosine, lysine at position 467 of SEQ ID NO:1 is replaced with glutamine, serine at position 482 of SEQ ID NO:1 is replaced with threonine, valine at position 326 of SEQ ID NO:1 is replaced with alanine, and asparagine at position 492 of SEQ ID NO:1 is replaced with aspartic acid (CXVI) Asparagine at position 324 of SEQ ID NO:1 is replaced with histidine, glutamine at position 334 of SEQ ID NO:1 is replaced with leucine, glutamic acid at position 335 of SEQ ID NO:1 is replaced with valine, threonine at position 341 of SEQ ID NO:1 is replaced with alanine, phenylalanine at position 379 of SEQ ID NO:1 is replaced with tyrosine, lysine at position 467 of SEQ ID NO:1 is replaced with glutamine, serine at position 482 of SEQ ID NO:1 is replaced with threonine, valine at position 326 of SEQ ID NO:1 is replaced with alanine, and lysine at position 380 of SEQ ID NO:1 is replaced with arginine (CXVII) Asparagine at position 324 of SEQ ID NO:1 is replaced with histidine, glutamine at position 334 of SEQ ID NO:1 is replaced with leucine, glutamic acid at position 335 of SEQ ID NO:1 is replaced with valine, threonine at position 341 of SEQ ID NO:1 is replaced with alanine, phenylalanine at position 379 of SEQ ID NO:1 is replaced with tyrosine, lysine at position 467 of SEQ ID NO:1 is replaced with glutamine, serine at position 482 of SEQ ID NO:1 is replaced with threonine, lysine at position 380 of SEQ ID NO:1 is replaced with arginine, and asparagine at position 492 of SEQ ID NO:1 is replaced with aspartic acid (CXVIII) Asparagine at position 324 of SEQ ID NO:1 is replaced with histidine, glutamine at position 334 of SEQ ID NO:1 is replaced with leucine, glutamic acid at position 335 of SEQ ID NO:1 is replaced with valine, threonine at position 341 of SEQ ID NO:1 is replaced with alanine, phenylalanine at position 379 of SEQ ID NO:1 is replaced with tyrosine, lysine at position 467 of SEQ ID NO:1 is replaced with glutamine, serine at position 482 of SEQ ID NO:1 is replaced with threonine, valine at position 326 of SEQ ID NO:1 is replaced with alanine, lysine at position 380 of SEQ ID NO:1 is replaced with arginine, and asparagine at position 492 of SEQ ID NO:1 is replaced with aspartic acid.

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

6. An expression vector comprising the polynucleotide of claim 5.

7. A transformant obtained by transforming Escherichia coli with the expression vector according to claim 6.

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

9. An AAV adsorbent comprising an insoluble carrier and an AAV binding protein according to any one of claims 1 to 4 immobilized on the carrier.

10. A column comprising the AAV adsorbent of claim 9.

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

12. 11. A method for analyzing AAV contained in a sample based on the strength of its infectivity, comprising the steps of: adding a sample containing AAV to the column described in claim 10 to adsorb the AAV to the adsorbent; and eluting the AAV adsorbed to the adsorbent using an elution solution.

Images