Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
  • C12N15/86—Viral vectors
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/70—Vectors or expression systems specially adapted for E. coli
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P21/00—Preparation of peptides or proteins
  • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
  • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
  • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
  • C12N2750/00011—Details
  • C12N2750/14011—Parvoviridae
  • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
  • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
  • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
  • C12N2750/00011—Details
  • C12N2750/14011—Parvoviridae
  • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
  • C12N2750/14151—Methods of production or purification of viral material

Definitions

• the present disclosure relates to a protein that has binding affinity to an adeno-associated virus (AAV).
• AAV adeno-associated virus
• the present disclosure relates to an improved recombinant AAV binding protein, e.g., having improved alkaline resistance.
• the present disclosure relates to a method for purifying an adeno-associated virus (AAV). In another aspect, the present disclosure relates to a method for easily purifying an AAV vector containing a gene, for example.
• AAV adeno-associated virus
• Adeno-associated virus is a non-enveloped virus classified in the Parvoviridae family and Dependovirus genus.
• AAV lacks the ability to replicate autonomously and depends on helper viruses such as adenovirus and herpes virus for replication.
• helper viruses such as adenovirus and herpes virus for replication.
• the AAV genome is replicated within the host cell, complete AAV particles containing the AAV genome are formed, and the AAV particles are released from the host cell.
• the helper virus is not present, the AAV genome is maintained in an episome or integrated into the host chromosome (latent state).
• 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 virus particles are physically and chemically stable. For these reasons, AAV is attracting attention as a vector for gene transfer to treat congenital genetic diseases.
• AAV vectors The production of recombinant AAV vectors (hereafter simply referred to as AAV vectors) is usually carried out by introducing nucleic acid encoding elements essential for AAV particle formation into cells to produce cells capable of producing AAV (hereafter also referred to as AAV-producing cells), and then culturing the cells to express the elements essential for AAV particle formation.
• AAV vector is recovered and purified from the AAV-producing cells to obtain a therapeutic AAV vector preparation.
• a method for recovering and purifying AAV vectors from AAV-producing cells is an affinity chromatography method 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 vector from a solution containing the AAV vector coexisting with impurities.
• Patent Document 1 uses a polypeptide containing the extracellular domain 1 (PKD1) and domain 2 (PKD2) of KIAA0319L (UniProt No.
• AAV-binding protein (hereinafter also referred to simply as "ligand protein") immobilized on an insoluble carrier, thereby realizing high-purity purification of AAV vectors.
• the produced AAV vectors include those that contain genes (Full AAV vectors) and those that do not contain genes (Empty AAV vectors). Of these, Empty AAV vectors may reduce the efficacy of the vector as a therapeutic drug or induce side effects due to overdosing (Non-Patent Document 2). Therefore, in order to use the vector as a therapeutic AAV vector preparation, it is necessary to purify the Full AAV vector from the produced AAV vectors to a high degree of purity.
• a method for purifying AAV vectors uses affinity chromatography with an AAV adsorbent that contains an insoluble carrier and an AAV-binding protein immobilized on the carrier (Patent Documents 1 and 2, and Non-Patent Document 2).
• affinity chromatography with an AAV adsorbent that contains an insoluble carrier and an AAV-binding protein immobilized on the carrier
• Patent Documents 1 and 2 and Non-Patent Document 2
• a known method for obtaining full AAV vectors is to ultracentrifuge a solution containing the produced AAV vectors and obtain a fraction rich in full AAV vectors.
• ultracentrifugation requires complex processes such as preparing a solution for density gradients, ultracentrifugation, and obtaining fractions, and it generally takes several days to obtain full AAV vectors.
• it is difficult to distinguish fractions rich in full AAV vectors there is a risk of obtaining layers before and after the fractions when collecting them, and there is a high risk of contamination with impurities.
• Non-Patent Document 3 A method has also been reported for separating full and empty AAV vectors using Tosoh TSKgel Q-STAT as an anion exchange gel and choline chloride as an eluent.
• the adeno-associated virus (AAV) binding protein disclosed in WO2021/106882 has improved stability against heat, acid, and alkali compared to the native form (AAV binding protein without amino acid substitutions).
• AAV binding protein without amino acid substitutions.
• an object of the present disclosure is to provide an AAV-binding protein that has improved alkali resistance compared to the AAV-binding protein disclosed in the above publication.
• AAV vectors that do not contain genes may reduce their efficacy as a therapeutic drug or may induce side effects due to overdosing. Therefore, in order to use them as therapeutic AAV vector preparations, it was necessary to highly purify AAV vectors that contain genes (Full AAV vectors) from the produced AAV vectors.
• an object of the present disclosure is to provide a method capable of purifying a full AAV vector with high purity and in a simple manner.
• an object of the present disclosure is to provide a method capable of purifying a full AAV vector with high purity and in a simple manner from a solution containing an AAV vector obtained, for example, by culturing an AAV-producing cell.
• alkaline resistance can be significantly improved by substituting specific amino acid residues that make up the adeno-associated virus (AAV) binding protein with other amino acid residues.
• AAV adeno-associated virus
• the present inventors also discovered that full AAV vectors can be purified to high purity by purifying a solution containing AAV vectors by affinity chromatography using an AAV-binding protein as a ligand, and then subjecting the solution to anion exchange chromatography.
• an AAV binding protein selected from any of the following (i) to (iii): (i) an AAV binding protein comprising at least the amino acid residues from the 25th serine to the 213th aspartic acid in the amino acid sequence set forth in SEQ ID NO: 2, with the proviso that at least one of the following amino acid substitutions (1) to (74) occurs in the amino acid residues from the 25th to the 213th, and having AAV-binding activity; (1) Threonine at position 139 of SEQ ID NO:2 is replaced with Alanine (2) Isoleucine at position 32 of SEQ ID NO:2 is replaced with Asparagine (3) Leucine at position 34 of SEQ ID NO:2 is replaced with Glutamine (4) Leucine at position 41 of SEQ ID NO:2 is replaced with Proline (5) Asparagine at position 42 of SEQ ID NO:2 is replaced with Lysine or Serine (6) Valine at position 45 of SEQ ID NO:
• AAV-binding protein according to [1] or [2], which comprises at least the amino acid residues from the 25th serine to the 213th aspartic acid in the amino acid sequence set forth in SEQ ID NO: 2, with the proviso that at least one amino acid substitution shown in any one of the following (A) to (P) occurs in the amino acid residues from the 25th to the 213th, and has AAV-binding activity;
• A Asparagine at position 42 of SEQ ID NO:2 is replaced with lysine, glutamic acid at position 114 of SEQ ID NO:2 is replaced with glycine, and threonine at position 139 of SEQ ID NO:2 is replaced with alanine.
• Asparagine at position 42 of SEQ ID NO:2 is replaced with lysine, isoleucine at position 79 of SEQ ID NO:2 is replaced with phenylalanine, valine at position 107 of SEQ ID NO:2 is replaced with alanine, glutamic acid at position 114 of SEQ ID NO:2 is replaced with glycine, threonine at position 139 of SEQ ID NO:2 is replaced with alanine, lysine at position 168 of SEQ ID NO:2 is replaced with arginine, alanine at position 174 of SEQ ID NO:2 is replaced with proline, and glutamine at position 180 of SEQ ID NO:2 is replaced with glycine.
• An AAV adsorbent comprising an insoluble carrier and an AAV binding protein according to any one of [1] to [3] immobilized on the carrier.
• a column comprising the AAV adsorbent according to [8].
• a method for purifying or analyzing AAV comprising the steps of adding a solution containing AAV to the column described in [9] to adsorb the AAV to the adsorbent, and eluting the AAV adsorbed to the adsorbent using an elution solution.
• the method for purifying or analyzing AAV described in [10] further comprising a step of washing the AAV adsorbent with an alkaline solution after the step of eluting AAV.
• a method for purifying AAV contained in a sample comprising: adding a sample containing AAV to an adsorbent comprising an insoluble carrier and an AAV-binding protein immobilized on the insoluble carrier, and allowing the AAV to be adsorbed onto the adsorbent; Eluting the AAV adsorbed to the adsorbent; adding the fraction containing the AAV eluted in the elution step to an anion exchange chromatography carrier to adsorb the AAV to the anion exchange chromatography carrier; and eluting the AAV adsorbed on the anion exchange chromatography carrier. Purification method. [13] 1.
• a method for purifying AAV contained in a sample comprising: adding a sample containing AAV to an adsorbent comprising an insoluble carrier and an AAV-binding protein immobilized on the insoluble carrier, and allowing the AAV to be adsorbed onto the adsorbent; Eluting the AAV adsorbed to the adsorbent; adding the fraction containing the AAV eluted in the elution step to an anion exchange chromatography carrier to adsorb the AAV to the anion exchange chromatography carrier; and eluting the AAV adsorbed on the anion exchange chromatography carrier,
• a purification method wherein the AAV binding protein is an AAV binding protein according to any one of [1] to [3].
• AVR29c column affinity chromatography
• GigaCapQ column anion exchange chromatography
• the chromatogram was obtained by applying each peak shown in the chromatogram of FIG. 3 to size exclusion chromatography (G6000PW XL column).
• FIG. 1 shows the results of evaluating the binding ability of AVR29c or AVR29c( ⁇ ) to VLP2.
• FIG. 1 shows the results of evaluating the elution properties of AAV8 adsorbed on an AVR29c column or an AVR29c( ⁇ ) column.
• the chromatograms are obtained by applying a fraction containing AAV8-EGFP purified using an AVR29c column to an anion exchange chromatography column (SkillPak GigaCapQ column, capacity 1.0 mL).
• the chromatograms are obtained when the mixing ratio for eluting impurities is adjusted to (a) 49.0%, (b) 48.0%, (c) 47.0%, and (d) 46.5% for solution AEX-A and solution AEX-C, which is 20 mmol/L Tris-HCl buffer (pH 9.0) containing 300 mmol/L choline chloride.
• the chromatograms are obtained by applying a fraction containing AAV8-EGFP purified with an AVR29c column to a SkillPak GigaCapQ column (volume 5.0 mL).
• (a) is a chromatogram obtained by purifying by affinity chromatography (AF) using an AVR29c column.
• FIG. 9(b) is a chromatogram obtained by purifying by anion exchange chromatography (AEX) using a SkillPak GigaCapQ column.
• FIG. 9(b) shows a comparison of the positive rates when cells were infected with the AAV8-EGFP solution obtained in Example 24(1) (peak indicated by the black arrow in FIG. 9(a)), and the eluted fractions obtained in Examples 24(3) (peak indicated by the white arrow in FIG. 9(b)), and (4) (peak indicated by the black arrow in FIG. 9(b)).
• the infection dose shown on the X-axis is expressed as the number of particles per cell in (a).
• the infection dose shown on the X-axis is expressed as the number of vector genomes (VG) per cell in (b).
• the AAV-binding protein refers to a protein that contains at least the amino acid residues from serine (S) at position 312 to aspartic acid (D) at position 500, which is the region 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, which is a native AAV-binding protein, and in which an amino acid substitution has occurred at a specific position in the amino acid residues at positions 312 to 500.
• the AAV-binding protein of the present disclosure may contain all or part of the other extracellular domains (domain 3 (PKD3), domain 4 (PKD4) and domain 5 (PKD5)) on the C-terminal side of the protein.
• the AAV-binding protein of the present disclosure may also include all or a part of a signal sequence or a cysteine-rich region, such as a MANSC (Motif At N terminus with Seven Cysteines) domain, located on the N-terminus side of PKD1.
• MANSC Motif At N terminus with Seven Cysteines
• the AAV-binding protein of the present disclosure may also include all or a part of a transmembrane region located on the N-terminus and/or C-terminus side of the extracellular region, and an intracellular region.
• the amino acid substitution at the specific position includes at least the amino acid residues from the 312th serine to the 500th aspartic acid in the amino acid sequence set forth in SEQ ID NO: 1, provided that in the amino acid residues from the 312th to the 500th V317D (this notation indicates that the 317th valine in SEQ ID NO: 1 is substituted with aspartic acid, the same applies below), N324H, V326A, A330V, Q334L, E335V, T341A, Y342S, K362E, K371N, F379Y, K380R, V381A, I382V, G390S, K399E, K467Q, S476R, S482T, N487D, and N492D amino acid substitutions are included at least, and I319N, L321Q , L328P, N329K, N329S, V332A, E(V)335A (this notation indicates that gluta
• an AAV binding protein consisting of amino acid residues from the 312th serine to the 500th aspartic acid in the amino acid sequence set forth in SEQ ID NO:1, with the following amino acid substitutions occurring in the 312th to 500th amino acid residues: V317D, N324H, V326A, A330V, Q334L, E335V, T341A, Y342S, K362E, K371N, F379Y, K380R, V381A, I382V, G390S, K399E, K467Q, S476R, S482T, N487D, and N492D, is synonymous with an AAV binding protein consisting of amino acid residues from the 25th serine to the 213th aspartic acid in the amino acid sequence set forth in SEQ ID NO:2.
• the positions of the amino acid residues in SEQ ID NO:2 correspond to the positions of the amino acid residues in SEQ ID NO:1 minus 287.
• the V317D amino acid substitution corresponds to the substitution of the amino acid residue located at position 30 in SEQ ID NO:2
• the T426A amino acid substitution corresponds to the substitution of the amino acid residue located at position 139 in SEQ ID NO:2.
• AAV-binding protein of the present disclosure there is no particular limit to the number of amino acids to be substituted. Examples include the AAV-binding proteins shown in any of (A) to (P) below. These AAV-binding proteins are more preferable in that they have improved stability against alkali.
• AAV binding protein that contains amino acid residues from serine at position 25 to aspartic acid at position 213 of the amino acid sequence set forth in SEQ ID NO:2, and in which at least the amino acid substitutions N329K, E401G, and T426A have occurred in the amino acid residues from position 25 to position 213.
• (B) An AAV binding protein that contains amino acid residues from serine at position 25 to aspartic acid at position 213 of the amino acid sequence set forth in SEQ ID NO:2, and in which at least the amino acid substitutions K338R, T426A, and A461P have occurred in the amino acid residues at positions 25 to 213.
• (C) An AAV binding protein that contains amino acid residues from serine at position 25 to aspartic acid at position 213 of the amino acid sequence set forth in SEQ ID NO:2, and in which at least the amino acid substitutions N329K, E401G, T426A, and A461P have occurred in the amino acid residues from position 25 to position 213.
• (D) An AAV binding protein that contains amino acid residues from serine at position 25 to aspartic acid at position 213 of the amino acid sequence set forth in SEQ ID NO:2, and in which at least the amino acid substitutions K338R, E401G, T426A, and A461P have occurred in the amino acid residues at positions 25 to 213.
• (E) An AAV binding protein that contains amino acid residues from serine at position 25 to aspartic acid at position 213 of the amino acid sequence set forth in SEQ ID NO:2, and in which at least the amino acid substitutions N329K, K338R, E401G, T426A, and A461P have occurred in the amino acid residues at positions 25 to 213.
• (F) An AAV binding protein that contains amino acid residues from serine at position 25 to aspartic acid at position 213 of the amino acid sequence set forth in SEQ ID NO:2, and in which at least the following amino acid substitutions have occurred in the amino acid residues at positions 25 to 213: N329K, E401G, T426A, A461P, and K(Q)467N.
• (G) An AAV binding protein that contains amino acid residues from the 25th serine to the 213th aspartic acid in the amino acid sequence set forth in SEQ ID NO:2, and that has at least the following amino acid substitutions in the 25th to 213th amino acid residues: N329K, I366F, E401G, T426A, A461P, K(Q)467N, and T474R.
• AAV binding protein comprising amino acid residues from serine at position 25 to aspartic acid at position 213 of the amino acid sequence set forth in SEQ ID NO:2, and having at least the following amino acid substitutions in the amino acid residues at positions 25 to 213: N329K, V394A, E401G, T426A, A461P, K(Q)467N, and T474R.
• AAV binding protein that contains amino acid residues from the 25th serine to the 213th aspartic acid in the amino acid sequence set forth in SEQ ID NO:2, and that has at least the following amino acid substitutions in the 25th to 213th amino acid residues: N329K, E401G, T426A, K455R, A461P, K(Q)467N, and T474R.
• An AAV binding protein comprising amino acid residues from serine at position 25 to aspartic acid at position 213 of the amino acid sequence set forth in SEQ ID NO:2, and having at least the following amino acid substitutions in the amino acid residues at positions 25 to 213: N329K, I366F, V394A, E401G, T426A, K455R, A461P, and K(Q)467N.
• (K) An AAV binding protein comprising amino acid residues from the 25th serine to the 213th aspartic acid in the amino acid sequence set forth in SEQ ID NO:2, and having at least the following amino acid substitutions in the 25th to 213th amino acid residues: N329K, I366F, V394A, E401G, T426A, K455R, A461P, K(Q)467N, and T474R.
• (L) An AAV binding protein comprising the amino acid residues from the 25th serine to the 213th aspartic acid in the amino acid sequence set forth in SEQ ID NO:2, and having at least the following amino acid substitutions in the 25th to 213th amino acid residues: G314D, N329K, I366F, V394A, E401G, T426A, K455R, A461P, and K(Q)467N.
• (M) An AAV binding protein that contains amino acid residues from the 25th serine to the 213th aspartic acid in the amino acid sequence set forth in SEQ ID NO:2, and that has at least the following amino acid substitutions in the 25th to 213th amino acid residues: N329K, I366F, V394A, E401G, T426A, K455R, A461P, K(Q)467N, and K497E.
• N An AAV binding protein that contains amino acid residues from the 25th serine to the 213th aspartic acid in the amino acid sequence set forth in SEQ ID NO:2, and that has at least the following amino acid substitutions in the 25th to 213th amino acid residues: N329K, I366F, V394A, E401G, T426A, K455R, A461P, K(Q)467N, and V499I.
• An AAV binding protein comprising amino acid residues from the 25th serine to the 213th aspartic acid in the amino acid sequence set forth in SEQ ID NO:2, and having at least the following amino acid substitutions in the 25th to 213th amino acid residues: G314D, N329K, I366F, V394A, E401G, T426A, K455R, A461P, K(Q)467N, K497E, and V499I.
• one or several means any of the following, although it varies depending on the position of the amino acid substitution in the three-dimensional structure of the AAV binding protein and the type of amino acid residue, for example, 1 to 50, 1 to 40, 1 to 30, 1 to 25, 1 to 20, 1 to 18, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1.
• substitutions, deletions, insertions, or additions described in (ii) and (v) above include the amino acid residue substitutions disclosed in WO2021/106882 and WO2023/140197.
• substitution of one or several amino acid residues may include, in addition to the amino acid substitution at the specific position described above, conservative substitutions in which substitution occurs between amino acids with similar physical and/or chemical properties. It is generally known to those skilled in the art that conservative substitutions maintain the function of a protein between a substituted and unsubstituted protein. Examples of conservative substitutions include substitutions between glycine and alanine, between serine and threonine, or between glutamic acid and aspartic acid (Protein Structure and Function, Medical Science International, 9, 2005). Another example of the amino acid substitution is a substitution for monomerizing an AAV-binding protein. Specifically, an amino acid substitution in which a cysteine residue, which is likely to form a higher-order structure, is replaced with a serine residue or a methionine residue is exemplified.
• substitution, deletion, insertion, or addition of one or several amino acid residues in (ii) and (v) above also includes naturally occurring mutations (mutants or variants) based on differences in the origin or species of AAV binding proteins.
• amino acid sequence identity refers to identity with respect to the entire amino acid sequence.
• Identity refers to the ratio of amino acid residues in those amino acid sequences that have the same type of amino acid (Experimental Medicine, 31(3), Yodosha).
• Amino acid sequence identity can be determined using an alignment program such as BLAST (Basic Local Alignment Search Tool) or FASTA.
• the present disclosure provides a method for purifying AAV contained in a sample using an adsorbent (hereinafter also referred to as "AAV adsorbent”) that includes an insoluble carrier and an AAV-binding protein immobilized on the carrier.
• AAV adsorbent an adsorbent
• the AAV-binding protein includes laminin receptors such as integrins, anti-AAV antibodies, and AAV receptors (AAVR).
• a preferred embodiment of the AAV-binding protein when it is an anti-AAV antibody is a polypeptide comprising at least a heavy chain antibody variable region (VHH) capable of binding to AAV.
• a preferred embodiment of the AAV-binding protein when it is an AAVR is a polypeptide selected from any one of the following ⁇ i> to ⁇ iii>: ⁇ i> a polypeptide comprising at least the amino acid residues from the 312th serine to the 500th aspartic acid in the amino acid sequence set forth in SEQ ID NO: 1; ⁇ ii> a polypeptide comprising at least the amino acid residues from the 312th serine to the 500th aspartic acid in the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that one or more of substitutions, deletions, insertions, and additions of one or several amino acid residues at one or several positions in the amino acid residues from the 312th to the 500th positions occur, and having AAV-binding activity; ⁇
• the amino acid sequence described in SEQ ID NO:1 is the amino acid sequence of KIAA0319L (official database: UniProt, accession number: Q8IZA0), which is one embodiment of AAVR, and the amino acid residues from serine (S) at position 312 to aspartic acid (D) at position 500 in the amino acid sequence described in SEQ ID NO:1 correspond to the extracellular domain 1 (PKD1) and domain 2 (PKD2) of KIAA0319L.
• the polypeptides shown in any of ⁇ i> to ⁇ iii> above may contain at least the regions corresponding to PKD1 and PKD2 of the above-mentioned KIAA0319L, and may, for example, contain all or a part of the regions corresponding to other extracellular 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 the region corresponding to a signal sequence or a cysteine-rich region such as the MANSC (Motif At N terminus with Seven Cysteines) domain located on the N-terminal side of PKD1, or may contain all or a part of the transmembrane region and the intracellular region located on the N-terminal and/or C-terminal side of the extracellular region.
• MANSC Motif At N terminus with Seven Cysteines
• Examples of the above ⁇ ii> include the AAV binding proteins disclosed in WO2021/106882 and WO2023/140197, and AAV binding proteins having at least one amino acid substitution shown in the following (I) to (LXIX); (I) Glycine at position 314 of SEQ ID NO:1 is replaced with aspartic acid (II) Valine at position 317 of SEQ ID NO:1 is replaced with glycine (III) Glutamine at position 318 of SEQ ID NO:1 is replaced with arginine (IV) Isoleucine at position 319 of SEQ ID NO:1 is replaced with asparagine (V) Leucine at position 321 of SEQ ID NO:1 is replaced with glutamine (VI) Proline at position 322 of SEQ ID NO:1 is replaced with alanine (VII) Glutamic acid at position 325 of SEQ ID NO:1 is replaced with valine (VIII) Leucine at position 328 of SEQ ID NO:1 is replaced with proline (IX) Aspara
• LXV leucine at position 493 of SEQ ID NO: 1 with methionine
• LXVII asparagine at position 496 of SEQ ID NO: 1 with glutamic acid
• LXVIII replacement of valine at position 499 of SEQ ID NO: 1 with isoleucine (LXIX) replacement of aspartic acid at position 500 of SEQ ID NO: 1 with glycine or asparagine.
• Examples of the substitution, deletion, insertion, or addition described in ⁇ ii> above include the substitutions of amino acid residues disclosed in WO2021/106882 and WO2023/140197, and the amino acid substitutions shown in (I) to (LXIX) above.
• “one or several” means, for example, 1 to 50, 1 to 40, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 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 and WO2023/140197 and the substitution of amino acid residues shown in (I) to (LXIX), as long as it has AAV binding activity.
• substitution of one or several amino acid residues in ⁇ ii> above may include, in addition to the amino acid substitution at the specific position described above, conservative substitutions in which substitution occurs between amino acids with similar physical and/or chemical properties. It is generally known to those skilled in the art that conservative substitutions maintain the function of a protein between those with and without substitution. Examples of conservative substitutions include substitutions between glycine and alanine, between serine and threonine, or between glutamic acid and aspartic acid (Protein Structure and Function, Medical Science International, 9, 2005). Furthermore, the "substitution, deletion, insertion, or addition of one or several amino acid residues" in ⁇ ii> above also includes naturally occurring mutations (mutants or variants) based on differences in the origin of AAVR or differences in species.
• the identity of the amino acid sequence in ⁇ iii> above may be 70% or more, and may be higher (e.g., 80% or more, 85% or more, 90% or more, or 95% or more). "Identity" between amino acid sequences means the ratio of amino acid residues of the same type in those amino acid sequences (Experimental Medicine, 31(3), Yodosha).
• the identity of amino acid sequences can be determined using an alignment program such as BLAST (Basic Local Alignment Search Tool) or FASTA.
• the AAV-binding protein of the present disclosure may further include 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.
• the oligopeptide include polyhistidine, polylysine, polyarginine, polyglutamic acid, polyaspartic acid, and the like.
• an oligopeptide containing cysteine that is useful for immobilizing the AAV-binding protein on a solid phase such as a chromatographic support may further be attached to the N-terminus or C-terminus of the AAV-binding protein.
• the length of the oligopeptide added to the N-terminus or C-terminus of the AAV-binding protein is not particularly limited as long as it does not impair the AAV-binding ability or stability of the AAV-binding protein.
• a polynucleotide encoding the oligopeptide may be prepared, and then the oligopeptide may be added to the N-terminus or C-terminus of the AAV-binding protein by genetic engineering using a method well known to those skilled in the art.
• the chemically synthesized oligopeptide may be added by chemically binding to the N-terminus or C-terminus of the AAV-binding protein.
• a signal peptide may be added to the N-terminus of the AAV-binding protein of the present disclosure to promote efficient expression in Escherichia coli used as a host.
• the signal peptide include signal peptides that cause secretion of proteins into the periplasm, such as PelB, OmpA, DsbA, DsbC, MalE, and TorT (JP Patent Publication No. 2011-097898). It is also possible not to add a signal peptide to the N-terminus. In this case, it is preferable in that the homogeneity of the protein is improved during protein preparation.
• polynucleotide of the present disclosure As an example of a method for producing a polynucleotide encoding the AAV binding protein of the present disclosure (hereinafter also referred to as the "polynucleotide of the present disclosure"), (I) A method of converting the amino acid sequence of the AAV binding protein of the present disclosure 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 or the like using a DNA amplification method such as PCR, and the prepared polynucleotides are then ligated by an appropriate method.
• 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 can introduce a desired mutation into the polynucleotide encoding the AAV binding protein.
• the concentration of the four types of substrate deoxynucleotides (dATP/dTTP/dCTP/dGTP) is made non-uniform, and MnCl2 is added to the PCR reaction solution at a concentration of 0.01 mM or more and 10 mM or less (preferably 0.1 mM or more and 1 mM or less) to perform PCR, thereby introducing a mutation into the polynucleotide.
• 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 or 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.
• the agent used as the mutagen may be any mutagenic agent commonly used by those skilled in the art, such as hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine, nitrous acid, sulfurous acid, or hydrazine.
• the polynucleotide of the present disclosure may be used as is, but it is more preferable to use an expression vector (e.g., a bacteriophage, cosmid, or plasmid typically used for transforming prokaryotic or eukaryotic cells) into which the polynucleotide of the present disclosure has been inserted at an appropriate position.
• an expression vector e.g., a bacteriophage, cosmid, or plasmid typically used for transforming prokaryotic or eukaryotic cells
• an expression vector e.g., a bacteriophage, cosmid, or plasmid typically used for transforming prokaryotic or eukaryotic cells
• an expression vector e.g., a bacteriophage, cosmid, or plasmid typically used for transforming prokaryotic or eukaryotic cells
• the expression vector include pET plasmid vector, pUC plasmid vector, pTrc
• the appropriate position may mean a position that does not destroy the replication function of the expression vector, the desired antibiotic marker, or the region involved in transmissibility.
• a functional polynucleotide such as a promoter required for expression.
• promoters include the trp promoter, tac promoter, trc promoter, lac promoter, T7 promoter, recA promoter, and lpp promoter.
• transformation may be performed by a method described in known documents such as Molecular Cloning (Cold Spring Harbor Laboratory), 256, 1992.
• the transformants obtained by transformation using the above method may be screened by an appropriate method to obtain a transformant capable of expressing the AAV binding protein of the present disclosure (hereinafter, also simply referred to as the "transformant of the present disclosure”).
• the vector may be prepared from the culture obtained by culturing the transformant of the present disclosure by alkaline extraction or using a commercially available extraction kit such as the QIAprep Spin Miniprep kit (Qiagen).
• the AAV-binding protein can be produced by culturing the transformant of the present disclosure and recovering the AAV-binding protein of the present disclosure from the resulting culture.
• the culture may include not only the cultured cells of the transformant of the present disclosure, but also the medium used for the culture.
• the transformant used in the protein production method may be cultured in a medium suitable for culturing the target host (Escherichia coli), and a preferred example of the medium is Luria-Bertani (LB) medium supplemented with necessary nutrient sources.
• LB Luria-Bertani
• the medium may contain appropriate nutrient sources and, if desired, one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine, thioglycolate, and dithiothreitol.
• the culture temperature is generally 10°C to 40°C, preferably 20°C to 37°C, and more preferably around 25°C, but may be selected depending on the characteristics of the protein to be expressed.
• the pH of the medium is 6.8 to 7.4, and preferably around 7.0.
• the vector disclosed herein contains an inducible promoter, it is preferable to induce it under conditions that allow good expression of the AAV binding protein disclosed herein.
• IPTG isopropyl- ⁇ -D-thiogalactopyranoside
• the turbidity of the culture medium (absorbance at 600 nm) is measured, and when it reaches approximately 0.5 to 1.0, an appropriate amount of IPTG is added and the culture is continued, thereby inducing expression of the AAV binding protein.
• 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.
• the AAV-binding protein of the present disclosure may be recovered by isolating/purifying it from the culture using a method suitable for the expression form of the AAV-binding protein in the transformant. For example, when the protein is expressed in the culture supernatant, the cells may be separated by centrifugation, and the AAV-binding protein may be purified from the resulting culture supernatant.
• the cells When the protein is expressed intracellularly (which may include the periplasm), the cells may be collected by centrifugation, and then the cells may be disrupted by adding an enzyme treating agent, a surfactant, or the like to extract the AAV-binding protein, which may then be purified.
• the AAV-binding protein of the present disclosure can be purified using methods known in the art, one example being separation/purification using liquid chromatography.
• liquid chromatography include ion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, affinity chromatography, and the like. By combining these chromatographic techniques to perform the purification procedure, the AAV-binding protein of the present disclosure can be prepared with high purity.
• the binding activity to AAV may be measured using an enzyme-linked immunosorbent assay method (hereinafter referred to as the ELISA method).
• the AAV used to measure the binding activity may be a VLP (virus-like particle).
• any serotype (serum type) of AAV vector and VLP may be used as long as they show binding activity to the AAV-binding protein of the present disclosure.
• the AAV-binding protein of the present disclosure can be used, for example, for purifying or analyzing AAV.
• AAV AAV to be bound
• it may be an AAV that exists in nature or an AAV that is artificially created.
• AAV AAV that exists in nature
• AAV examples include serotype 1 (AAV1), serotype 2 (AAV2), serotype 3 (AAV3), serotype 4 (AAV4), serotype 5 (AAV5), serotype 6 (AAV6), serotype 7 (AAV7), serotype 8 (AAV8), serotype 9 (AAV9), serotype 10 (AAV10), serotype 11 (AAV11), serotype 12 (AAV12), and serotype 13 (AAV13).
• Artificially produced AAVs include AAVrh8, AAVrh10, and chimeric AAVs that have characteristics (cell tropism and infectivity) of two or more of these serotypes.
• the AAV-binding protein of the present disclosure can be used, for example, by immobilizing it on an insoluble carrier. That is, AAV purification or analysis can be specifically performed, for example, using an AAV adsorbent comprising an insoluble carrier and the AAV-binding protein of the present disclosure immobilized on the insoluble carrier.
• an AAV adsorbent comprising an insoluble carrier and the AAV-binding protein of the present disclosure immobilized on the insoluble carrier is also referred to as the AAV adsorbent of the present disclosure.
• AAV purification does not only refer to purification of AAV from a solution containing impurities, but also includes purification of AAV based on structure, properties, activity, etc.
• the insoluble carrier which is a component of the AAV adsorbent of the present disclosure, is not particularly limited as long as it is insoluble in samples containing AAV and solutions used for purification (elution solution, equilibration solution, washing solution, etc.).
• insoluble carriers include carriers made from polysaccharides such as agarose, alginate (alginic acid salt), carrageenan, chitin, cellulose, dextrin, dextran, starch, etc., carriers made from synthetic polymers such as polyvinyl alcohol, polymethacrylate, poly(2-hydroxyethyl methacrylate), polyurethane, and carriers made from ceramics such as silica.
• insoluble carriers carriers made from polysaccharides and carriers made from synthetic polymers are preferred as insoluble carriers.
• 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, and may be, for example, granular, monolithic, membranous, or fibrous, and may be either porous or non-porous. Among these, a shape that can be packed into a column is preferable.
• the AAV-binding protein may be immobilized on an insoluble carrier, for example, via a covalent bond.
• the AAV-binding protein may be immobilized on an insoluble carrier by covalently bonding the AAV-binding protein to the insoluble carrier via an active group possessed by the insoluble carrier, thereby producing an AAV adsorbent.
• the active group include an N-hydroxysuccinimide (NHS) activated ester group, an epoxy group, a carboxy group, a maleimide group, a haloacetyl group, a tresyl group, a formyl group, and a haloacetamide group.
• NHS N-hydroxysuccinimide
• an insoluble carrier having an active group for example, a commercially available insoluble carrier having an active group may be used as it is, or an active group may be introduced into the insoluble carrier.
• examples of commercially available carriers 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).
• An example of a method for introducing active groups onto the carrier surface is to react one of the compounds having two or more active sites with hydroxyl groups, epoxy groups, carboxyl groups, amino groups, etc. present on the carrier surface.
• Examples of compounds that introduce epoxy groups to hydroxyl or amino groups present on the carrier surface include epichlorohydrin, ethanediol diglycidyl ether, butanediol diglycidyl ether, and hexanediol diglycidyl ether.
• Examples of compounds that introduce carboxy groups to epoxy groups present on the carrier surface include 2-mercaptoacetic acid, 3-mercaptopropionic acid, 4-mercaptobutyric acid, 6-mercaptobutyric acid, glycine, 3-aminopropionic acid, 4-aminobutyric acid, and 6-aminohexanoic acid.
• compounds that introduce maleimide groups into hydroxyl, epoxy, carboxyl, and amino groups present on the carrier surface include N-( ⁇ -maleimidocaproic acid) hydrazide, N-( ⁇ -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 pionic acid, 4-maleimidobutyric acid, 6-maleimidohexanoic acid, N-( ⁇ -maleimidoacetoxy)succinimide ester, (m-maleimidobenzoyl)N-hydroxysuccinimi
• Examples of compounds that introduce haloacetyl groups into hydroxyl or amino groups present on the surface of the 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.
• Another example of a method for introducing active groups onto the support surface is to react ⁇ -alkenylalkane glycidyl ether with hydroxyl groups or amino groups present on the support surface, and then activate the ⁇ -alkenyl moiety by halogenating it with a halogenating agent.
• ⁇ -alkenylalkane glycidyl ethers include allyl glycidyl ether, 3-butenyl glycidyl ether, and 4-pentenyl glycidyl ether.
• halogenating agents include N-chlorosuccinimide, N-bromosuccinimide, and N-iodosuccinimide.
• Another example of a method for introducing active groups onto the carrier surface is a method in which active groups are introduced onto carboxy groups present on the carrier surface using a condensation agent and an additive.
• condensation agents include 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), dicyclohexylcarbodiamide, and carbonyldiimidazole.
• additives include NHS, 4-nitrophenol, and 1-hydroxybenztriazole.
• the immobilization of the AAV-binding protein to the insoluble carrier can be carried out, for example, in a buffer solution.
• the buffer solution include acetate buffer, phosphate buffer, MES (2-MorpholinoEthaneSulfonic acid) buffer, HEPES (4-(2-HydroxyEthyl)-1-PiperazineEthaneSulfonic acid) buffer, Tris (Tris(hydroxymethyl)aminomethane) buffer, and borate buffer.
• the reaction temperature during immobilization can be appropriately set depending on various conditions, such as the reactivity of the active group and the stability of the AAV-binding protein.
• the reaction temperature during immobilization can be, for example, 4°C or higher and 50°C or lower, and preferably 10°C or higher and 35°C or lower.
• the AAV adsorbent of the present disclosure can be used, for example, in purifying or analyzing AAV by packing it into a column.
• AAV can be purified or analyzed by adding a solution containing AAV to a column packed with the AAV adsorbent of the present disclosure (hereinafter also simply referred to as the "column of the present disclosure") to adsorb the AAV to the adsorbent, and eluting the AAV adsorbed to the adsorbent.
• the present disclosure provides a method for purifying or analyzing AAV, comprising the steps of adding a solution containing AAV to the column of the present disclosure to adsorb the AAV to the adsorbent, and eluting the AAV adsorbed to the adsorbent.
• Purification of AAV using the column of the present disclosure may be carried out, for example, based on the disclosure in WO2021/106882.
• purified AAV By purifying AAV using the AAV adsorbent of the present disclosure, purified AAV can be obtained. That is, in one embodiment, the method for purifying AAV may be a method for producing AAV, and specifically, may be 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.
• AAV can also 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 purifying proteins.
• the method for washing the AAV adsorbent may be a method of washing the AAV adsorbent with an alkali after purifying AAV.
• the method may involve washing with a low pH solution and then washing with an alkali.
• An example of the alkaline solution used for washing is an aqueous solution of sodium hydroxide at 0.1 M or more and 0.5 M or less.
• the AAV purification method disclosed herein may refer to the purification of an AAV vector containing a gene (full AAV vector).
• the method of purifying AAV of the present disclosure comprises: a step of adding a sample containing AAV to an AAV adsorbent and adsorbing the AAV to the adsorbent (hereinafter also referred to as a "first adsorption step”); A step of eluting the AAV adsorbed to the adsorbent (hereinafter also referred to as the "first elution step”); a step of adding a fraction containing the AAV eluted in the first elution step to a carrier for anion exchange chromatography and adsorbing the AAV to the carrier (hereinafter also referred to as a "second adsorption step”); A step of eluting the AAV adsorbed on the anion exchange chromatography carrier (hereinafter also referred to as the "second elution step”); Incidentally, an embodiment in which the AAV adsorbent and the anion exchange chromatography carrier are packed in a column (hereinafter also
• a sample containing AAV can be added to the AAV adsorbent column using a liquid delivery means such as a pump.
• adding a liquid to a column is also referred to as "delivering a liquid to the column.”
• the sample containing AAV may be solvent-substituted using an appropriate buffer before being added to the AAV adsorbent column.
• the AAV adsorbent column may be equilibrated using an appropriate buffer (equilibration liquid). It is expected that the equilibration can purify AAV to a higher purity, for example.
• the buffer used for solvent replacement or equilibration may be a neutral buffer having buffer capacity in the neutral range (herein referring to a range of pH 5.0 to 9.0, preferably pH 5.5 to 8.0).
• a neutral buffer having buffer capacity in the neutral range
• phosphate buffer, acetate buffer, succinate buffer, citrate buffer, Tris buffer, HEPES buffer, and MES buffer can be exemplified.
• Such a buffer solution may further contain, for example, 0.1 mmol/L to 50 mmol/L of a chelating agent.
• the buffer solution and the equilibration solution used for solvent replacement may or may not be the same.
• components other than AAV such as impurities
• such components may be removed (washed) from the AAV adsorbent column before eluting the AAV adsorbed to the AAV adsorbent (i.e., before the elution step).
• Components other than AAV can be removed from the AAV adsorbent column, for example, by using an appropriate buffer solution as a washing solution.
• the washing solution for example, the description of the buffer solution used for solvent replacement and equilibration can be applied mutatis mutandis.
• the AAV adsorbed to the AAV adsorbent in the first adsorption step may be eluted using, for example, a buffer solution having a lower pH than the equilibration solution or the washing solution, a neutral buffer solution containing 800 mmol/L or more of chloride ions or anions smaller in the Hofmeister series than the chloride ions, or a buffer solution containing a chelating agent (such as ethylenediaminetetraacetic acid) adjusted to the pH used in the second adsorption step.
• a buffer solution having a lower pH than the equilibration solution or the washing solution e.g., a neutral buffer solution containing 800 mmol/L or more of chloride ions or anions smaller in the Hofmeister series than the chloride ions, or a buffer solution containing a chelating agent (such as ethylenediaminetetraacetic acid) adjusted to the pH used in the second adsorption step.
• AAV adsorbent column By passing the elution solution through an AAV adsorbent column, the interaction between AAV and AAV-binding proteins (ligands of the AAV adsorbent) in the column is weakened, and the AAV adsorbed to the AAV adsorbent is eluted, and a fraction containing AAV is obtained.
• the AAV-containing fraction obtained in the first elution step may be adjusted to a pH of about 8.0 to 9.5 using Tris, for example, or diluted with the buffer used in the second adsorption step.
• the anion exchange column can be produced, for example, by soaking an anion exchange chromatography carrier in a 1 mol/L aqueous sodium chloride solution and then packing it into a column.
• the buffer component used in the second adsorption step and the second elution step may be any buffer component that has a buffer capacity at a pH of about 8.0 to 9.5, and is preferably Tris.
• examples of preferred salts to be included in the elution solution in the second elution step include sodium chloride, sodium acetate, ammonium chloride, ammonium acetate, tetramethylammonium chloride, magnesium chloride, calcium chloride, ammonium sulfate, tetraethylammonium chloride, choline chloride, acetylcholine chloride, carnitine hydrochloride, trimethylglycine, etc., and more preferably tetraethylammonium chloride or choline chloride.
• it is preferable to include choline chloride in the elution solution since it allows full AAV with infectious capacity to be purified to a high degree of purity.
• Full AAV may mean AAV containing genes.
• the salt concentration is increased to an appropriate concentration using a linear gradient to elute the first peak, and then the salt concentration is increased using a step gradient to elute the second peak.
• the salt concentration is increased using a step gradient to elute the second peak.
• the conductivity of the eluate when eluting the first peak is 13.5 mS/cm or less
• the conductivity of the eluate when eluting the second peak is 15.0 mS/cm or more, which is preferable because it increases the proportion of full AAV contained in the second peak. It is even more preferable to set the conductivity of the eluate when eluting the first peak to 5.0 mS/cm or more and 13.5 mS/cm or less, and the conductivity of the eluate when eluting the second peak to 15.0 mS/cm or more and 50.0 mS/cm or less.
• a good method for measuring conductivity is to use a conductivity monitor built into a liquid chromatography system, such as the AKTA go (manufactured by Cytiva).
• the percentage of AAV vectors that contain genes is also referred to as the "full rate.”
• the full rate can be measured using a mass photometry method such as Refeyn Two (manufactured by Refeyn).
• Example 1 Construction of AAV vector (part 1) (1) Escherichia coli JM109 strain was transformed with plasmid pRC2-mi342 Vector (Takara Bio Inc.) and pHelper Vector (Takara Bio Inc.) containing a polynucleotide encoding the capsid of AAV serotype 2 (AAV2). The resulting transformant was cultured overnight at 37° C. with shaking in a 5-L baffled flask containing 1 L of 2 ⁇ YT medium (1.6% (w/v) tryptone, 1% (w/v) yeast extract, 0.5% (w/v) sodium chloride) containing 100 ⁇ g/mL carbenicillin.
• AAV2 AAV serotype 2
• the culture medium (1) was centrifuged to collect the bacterial cells, and then the pRC2-mi342 Vector and pHelper Vector were prepared in large quantities from the collected bacterial cells using a Plasmid Mega Kit (Qiagen).
• 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.
• the pRC2-mi342 Vector and pHelper Vector prepared in (2) were introduced into the HEK293T cells cultured in (3), and the cells were cultured statically for 3 days at 37°C under 5% carbon dioxide.
• VLP2 virus-like particle, outer protein particle of AAV serotype 2.
• Example 2 Preparation and screening of a mutant library of AAV binding proteins (part 1) Using vector pET-AVR21 capable of expressing a polypeptide (SEQ ID NO: 2) containing the AAV binding protein AVR21 as a template, random mutations were introduced into the polynucleotide portion encoding the protein by error-prone PCR.
• SEQ ID NO: 2 the portion from the 1st methionine (M) to the 22nd alanine (A) is the PelB signal peptide (the 22 amino acid residues on the N-terminal side of UniProt No.
• AVR21 is a polypeptide in which the following 21 amino acid substitutions have been made in the amino acid residues from position 312 to position 500 of SEQ ID NO: 1, which correspond to extracellular domain 1 (PKD1) and domain 2 (PKD2) of the native AAV-binding protein KIAA0319L (UniProt No.
• Valine (V) at position 317 of SEQ ID NO:1 is replaced with aspartic acid (D).
• Asparagine (N) at position 324 of SEQ ID NO:1 (position 37 in SEQ ID NO:2) is replaced with histidine (H).
• Valine (V) at position 326 of SEQ ID NO:1 (position 39 in SEQ ID NO:2) is replaced with alanine (A).
• Alanine (A) at position 330 of SEQ ID NO:1 (position 43 in SEQ ID NO:2) is replaced with valine (V).
• Glutamine (Q) at position 334 of SEQ ID NO:1 is replaced with leucine (L).
• Glutamic acid (E) at position 48 of SEQ ID NO: 1 is replaced with valine (V).
• Threonine (T) at position 341 of SEQ ID NO: 1 is replaced with alanine (A).
• Tyrosine (Y) at position 342 of SEQ ID NO: 1 (position 55 of SEQ ID NO: 2) is replaced with serine (S).
• Lysine (K) at position 362 of SEQ ID NO: 1 is replaced with glutamic acid (E).
• Lysine (K) at position 371 of SEQ ID NO: 1 is replaced with asparagine (N).
• Phenyl 379 of SEQ ID NO: 1 is replaced with phenyl 200 of SEQ ID NO: 1 (position 93 of SEQ ID NO: 2).
• Alanine (F) is replaced by tyrosine (Y).
• Lysine (K) at position 380 of SEQ ID NO:1 is replaced by arginine (R).
• Valine (V) at position 381 of SEQ ID NO:1 is replaced by alanine (A).
• Isoleucine (I) at position 382 of SEQ ID NO:1 is replaced by valine (V).
• Glycine (G) at position 390 of SEQ ID NO:1 is replaced by serine (S).
• Lysine (K) at position 399 of SEQ ID NO:1 is replaced by glutamic acid (E).
• Substitution: Lysine (K) at position 467 of SEQ ID NO:1 (position 180 in SEQ ID NO:2) is replaced with glutamine (Q).
• Serine (S) at position 476 of SEQ ID NO:1 is replaced with arginine (R).
• Serine (S) at position 482 of SEQ ID NO:1 is replaced with threonine (T).
• Asparagine (N) at position 487 of SEQ ID NO:1 is replaced with aspartic acid (D).
• Asparagine (N) at position 492 of SEQ ID NO:1 is replaced with aspartic acid (D).
• Error-prone PCR was performed using vector pET-AVR21 capable of expressing a polypeptide (SEQ ID NO:2) containing AVR21 as a template.
• Error-prone PCR was performed by preparing a reaction solution having the composition shown in Table 1, heat-treating the reaction solution at 98°C for 2 minutes, and carrying out 30 cycles of reaction in which one cycle consisted of a first step at 98°C for 30 seconds, a second step at 55°C for 20 seconds, and a third step at 72°C for 90 seconds, and finally heat-treating the reaction at 72°C for 5 minutes.
• the error-prone PCR successfully introduced mutations into the polynucleotide encoding the AAV binding protein, with the average mutation introduction rate being 1.4 amino acid mutations per molecule.
• E. coli BL21 (DE3) was transformed with the reaction mixture and cultured on LB (Luria-Bertani) plate medium containing 50 ⁇ g/mL kanamycin (37°C for 18 hours), and the colonies formed on the plate were used as a random mutant library.
• the random mutant library (transformants) prepared in (3) was inoculated into 200 ⁇ L of 2YT liquid medium containing 50 ⁇ g/mL kanamycin, and cultured overnight at 37°C with shaking in a 96-well deep well plate.
• the culture medium from (4) was centrifuged, and the resulting culture supernatant was diluted two-fold with ultrapure water. 60 ⁇ L of the diluted culture medium was mixed with 60 ⁇ L of 0.5 M aqueous sodium hydroxide solution, and alkaline treatment was performed at 30°C for 30 minutes.
• VLP2 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 ⁇ L/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.
• the residual activity was calculated by dividing the binding activity between the AAV binding protein and VLP2 when alkaline treatment was performed by the binding activity between the AAV binding protein and VLP2 when alkaline treatment was not performed.
• a random mutant library of approximately 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 AVR21 were selected from among them.
• the selected transformants were cultured, and an expression vector was prepared using a QIAprep Spin Miniprep kit (Qiagen).
• Table 2 shows the amino acid substitution positions relative to AVR21 for the AAV binding proteins expressed by the transformants selected in (7) above, as well as the residual activity [%] after alkaline treatment.
• amino acids In the amino acid sequence of SEQ ID NO:2, from the 25th serine (S) to the 213th aspartic acid (D), the following amino acids are: I319N (this notation indicates that the 319th isoleucine in SEQ ID NO:1 (32nd in SEQ ID NO:2) is replaced with asparagine, same below), L321Q, L328P, N329K, N329S, V332A, E(V)335A (this notation indicates that the 335th glutamic acid in SEQ ID NO:1 (48th in SEQ ID NO:2) is first replaced with valine in SEQ ID NO:2, and then replaced with alanine, same below), K338R, E340G, T343A, T343S, Y344F, D345G, Q347 K, T350A, E360D, Q365R, I366F, L367P, K368R, L369S, K(N)371D, K(N)
• AVR8g is a polypeptide in which the amino acid substitutions V317D, Y342C, K362E, K371N, G390S, K399E, S476R, and N487D have been made in the amino acid residues from position 312 to position 500 of SEQ ID NO: 1, which corresponds to the extracellular domain 1 (PKD1) and domain 2 (PKD2) of KIAA0319L (UniProt No. Q8IZA0) (WO2021/106882).
• Error-prone PCR was performed in the same manner as in Example 2(1), except that the plasmid pET-AVR8g, into which a polynucleotide (SEQ ID NO: 30) encoding AVR8g (SEQ ID NO: 29) was inserted, was used as a template and the concentration of the template was 10 ng/ ⁇ L. Mutations were introduced into the polynucleotide encoding AVR8g by the error-prone PCR, and the average mutation introduction rate was 2.3 amino acid mutations per molecule.
• the random mutant library (transformants) prepared in (2) was inoculated into 200 ⁇ L of 2YT liquid medium containing 50 ⁇ g/mL kanamycin, and cultured overnight at 37°C with shaking in a 96-well deep-well plate.
• the culture supernatant obtained by centrifuging the culture medium of (4) was diluted 16-fold with ultrapure water. 60 ⁇ L of the diluted culture supernatant was mixed with 60 ⁇ L of 0.1 M glycine sodium hydroxide buffer (pH 10.0) and subjected to heat and alkali treatment at 51.3°C for 15 minutes.
• the residual activity was calculated by dividing the binding activity between the AAV binding protein and VLP2 when heat treatment was performed by the binding activity between the AAV binding protein and VLP2 when heat treatment was not performed.
• a random mutant library of approximately 1,800 strains was evaluated using the method described in (6), and transformants expressing AAV-binding proteins with improved residual activity compared to the parent molecule AVR8g were selected from among them.
• the selected transformants were cultured, and expression vectors were prepared using a QIAprep Spin Miniprep kit (Qiagen).
• Table 3 shows the amino acid substitution positions relative to AVR8g and the residual activity [%] after heat and alkali treatment of the AAV binding proteins expressed by the transformants selected in (6).
• a transformant capable of expressing any one of AVR9a (SEQ ID NO: 24), AVR9b (SEQ ID NO: 25), AVR9c (SEQ ID NO: 26), and AVR9d (SEQ ID NO: 27) was inoculated into 3 mL of 2 ⁇ YT liquid medium containing 50 ⁇ g/mL kanamycin, and
• the culture solution was centrifuged at 4°C and 8,000 rpm for 20 minutes to collect each bacterial cell.
• 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 referred to as "equilibration solution A") at 5 mL/1 g (cells), and then disrupted at 4°C for approximately 10 minutes at 150 W output using an ultrasonic generator (Insonator 201M [Kubota Shoji Co., Ltd.]). The disrupted cell solution was centrifuged twice at 8,000 rpm for 20 minutes at 4°C, and each supernatant was collected.
• equilibration solution A 150 mM sodium chloride and 20 mM imidazole
• the binding activity of the AAV-binding protein prepared in (7) to the VLP2 prepared in Example 1 was measured using the ELISA method described in Example 2 (6). Based on the absorbance at 450 nm, which is the measurement result, the AAV-binding protein obtained in (7) was diluted with pure water so that the measured value was similar.
• the binding activity of the AAV binding protein to VLP2 when the treatment (9) was performed and when the treatment (9) was not performed were measured by the ELISA method described in Example 2 (6).
• the binding activity of the AAV binding protein to VLP2 when the treatment (9) was performed was divided by the binding activity of the AAV binding protein to VLP2 when the heat treatment (9) was not performed to calculate the residual activity.
• Example 3 Preparation of AVR21 Amino Acid Substitution Aggregates Among the amino acid substitutions involved in improving the alkaline stability of the AAV-binding protein clarified in Example 2, N329K, K338R, E401G, T426A and A461P were selected, and among the amino acid substitutions involved in improving the heat and alkaline stability of the AAV-binding protein clarified in Reference Examples 1 and 2, K467N was selected, and these amino acid substitutions were accumulated in AVR21 (SEQ ID NO: 2) to further improve the alkaline stability. Specifically, five types of AAV-binding proteins shown in the following (a) to (e) were designed and prepared.
• AAV binding protein having the amino acid substitutions N329K, E401G, and T426A introduced into AVR21 (SEQ ID NO:5, designated AVR24a)
• AVR24b An AAV binding protein in which the amino acid substitutions K338R, T426A, and A461P have been introduced into AVR21 (SEQ ID NO: 6, designated AVR24b).
• a PCR primer set was designed to generate mutant clusters.
• SEQ ID NOs: 10 forward and 11 (reverse) were used.
• SEQ ID NOs: 12 forward and 13 (reverse) were used.
• SEQ ID NOs: 14 forward and SEQ ID NO: 15 (reverse) were used.
• SEQ ID NOs: 16 forward and 17 (reverse) were used.
• SEQ ID NOs: 18 forward and 19 (reverse) were used. Each was designed.
• AVR24a This protein was prepared by selecting N329K, E401G and T426A from the amino acid substitutions involved in alkaline stability revealed in Example 2, and introducing these amino acid substitutions into AVR21 (SEQ ID NO: 2).
• PCR was performed by using vector pET-AVR21 capable of expressing a polypeptide containing AVR21 (SEQ ID NO: 2) as a template, oligonucleotides consisting of the sequences set forth in SEQ ID NO: 10 (5'-AGCTGAAAGTCTACGTACTGCTGGT-3') and SEQ ID NO: 11 (5'-TAGACTTTCAGCTGGGCTTCATGTT-3') as PCR primers, preparing a reaction solution with the composition shown in Table 5, and then heat-treating the reaction solution at 98°C for 5 minutes.
• (a-2) Using the PCR product obtained in (a-1) as a template and oligonucleotides consisting of the sequences described in SEQ ID NO: 14 (5'-AGCCTGGCCCGCGCAAGAATCGTCC-3') and SEQ ID NO: 15 (5'-CGCGGGCCAGGCTCAACCGTTACGT-3') as PCR primers, a reaction solution having the composition shown in Table 5 was prepared, and the reaction solution was heat-treated at 98°C for 5 minutes.
• (a-3) Using the PCR product obtained in (a-2) as a template and oligonucleotides consisting of the sequences described in SEQ ID NO: 16 (5'-CAAGTGCGGTGATCGACGGATCGCA-3') and SEQ ID NO: 17 (5'-ATCACCGCACTTGTAGTCGGGAGTG-3') as PCR primers, a reaction solution having the composition shown in Table 5 was prepared, and the reaction solution was heat-treated at 98°C for 5 minutes.
• a-4 The PCR product obtained in (a-3) was used to transform E. coli BL21 strain (DE3).
• the resulting transformant was cultured in LB medium supplemented with 50 ⁇ g/mL kanamycin, and the resulting transformant was then centrifuged to recover the bacterial cells (transformant).
• a plasmid was extracted from the bacterial cells (transformant) to obtain a plasmid (expression vector) pET-AVR24a containing a polynucleotide encoding AVR24a, which has 24 amino acid substitutions relative to the native AAV binding protein.
• the amino acid sequence of AVR24a with the signal sequence and polyhistidine tag added is shown in SEQ ID NO: 5.
• SEQ ID NO: 5 the portion from the 1st methionine (M) to the 22nd alanine (A) is the PelB signal peptide, the portion from the 25th serine (S) to the 213th aspartic acid (D) is the AAV binding protein AVR24a (corresponding to the region from the 25th to the 213th in SEQ ID NO: 2), and the portion from the 214th to the 219th histidine (H) is the tag sequence.
• the aspartic acid of V317D is at the 30th position
• the histidine of N324H is at the 37th position
• the alanine of V326A is at the 39th position
• the lysine of N329K is at the 42nd position
• the valine of A330V is at the 43rd position
• the leucine of Q334L is at the 47th position
• the valine of E335V is at the 48th position
• the alanine of T341A is at the 54th position
• the serine of Y342S is at the 55th position
• the glutamic acid of K362E is at the 75th position
• the asparagine of K371N is at the 84th position
• the tyrosine of F379Y is at the 92nd position
• the tyrosine of K380R is at the 93rd position.
• Arginine is at the 93rd position, V381A alanine at the 94th position, I382V valine at the 95th position, G390S serine at the 103rd position, K399E glutamic acid at the 112th position, E401G glycine at the 114th position, T426A alanine at the 139th position, K467Q glutamine at the 180th position, S476R arginine at the 189th position, S482T threonine at the 195th position, N487D aspartic acid at the 200th position, and N492D aspartic acid at the 205th position.
• AVR24b This protein was prepared by selecting K338R, T426A, and A461P from the amino acid substitutions involved in alkaline stability revealed in Example 2, and introducing these amino acid substitutions into AVR21 (SEQ ID NO: 2).
• (b-2) Using the PCR product obtained in (b-1) as a template and oligonucleotides consisting of the sequences set forth in SEQ ID NO: 16 (5'-CAAGTGCGGTGATCGACGGATCGCA-3') and SEQ ID NO: 17 (5'-ATCACCGCACTTGTAGTCGGGAGTG-3') as PCR primers, a reaction solution having the composition shown in Table 5 was prepared, and the reaction solution was heat-treated at 98°C for 5 minutes.
• the PCR product obtained in (b-3) was used to transform E. coli BL21 strain (DE3).
• the resulting transformant was cultured in LB medium supplemented with 50 ⁇ g/mL kanamycin, and the resulting transformant was then collected by centrifugation.
• the plasmid was extracted from the transformant to obtain the plasmid (expression vector) pET-AVR24b, which contains a polynucleotide encoding AVR24b, which has 24 amino acid substitutions relative to the native AAV binding protein.
• the amino acid sequence of AVR24b with the signal sequence and polyhistidine tag added is shown in SEQ ID NO: 6.
• SEQ ID NO: 6 the portion from the 1st methionine (M) to the 22nd alanine (A) is the PelB signal peptide, the portion from the 25th serine (S) to the 213th aspartic acid (D) is the AAV binding protein AVR24b (corresponding to the region from the 25th to the 213th in SEQ ID NO: 2), and the portion from the 214th to the 219th histidine (H) is the tag sequence.
• the aspartic acid of V317D is at the 30th position
• the histidine of N324H is at the 37th position
• the alanine of V326A is at the 39th position
• the valine of A330V is at the 43rd position
• the leucine of Q334L is at the 47th position
• the valine of E335V is at the 48th position
• the arginine of K338R is at the 51st position
• the alanine of T341A is at the 54th position
• the serine of Y342S is at the 55th position
• the glutamic acid of K362E is at the 75th position
• the asparagine of K371N is at the 84th position
• the tyrosine of F379Y is at the 92nd position
• the tyrosine of K380R is at the 93rd position.
• arginine is at position 93, V381A alanine at position 94, I382V valine at position 95, G390S serine at position 103, K399E glutamic acid at position 112, T426A alanine at position 139, A461P proline at position 174, K467Q glutamine at position 180, S476R arginine at position 189, S482T threonine at position 195, N487D aspartic acid at position 200, and N492D aspartic acid at position 205.
• AVR25a This protein was produced by selecting N329K, E401G, T426A, and A461P from the amino acid substitutions involved in alkaline stability revealed in Example 2, and introducing these amino acid substitutions into AVR21 (sequence number 2).
• (c-2) Using the PCR product obtained in (c-1) as a template and oligonucleotides consisting of the sequences set forth in SEQ ID NO: 14 (5'-AGCCTGGCCCGCGCAAGAATCGTCC-3') and SEQ ID NO: 15 (5'-CGCGGGCCAGGCTCAACCGTTACGT-3') as PCR primers, a reaction solution having the composition shown in Table 5 was prepared, and the reaction solution was heat-treated at 98°C for 5 minutes.
• the PCR product obtained in (c-4) was used to transform E. coli strain BL21 (DE3).
• the resulting transformant was cultured in LB medium supplemented with 50 ⁇ g/mL kanamycin, and the resulting transformant was then centrifuged to recover the bacterial cells (transformant).
• a plasmid was extracted from the bacterial cells (transformant) to obtain a plasmid (expression vector) pET-AVR25a containing a polynucleotide encoding AVR25a, which has 25 amino acid substitutions relative to the native AAV binding protein.
• the amino acid sequence of AVR25a with the signal sequence and polyhistidine tag added is shown in SEQ ID NO: 7.
• the portion from the 1st methionine (M) to the 22nd alanine (A) is the PelB signal peptide
• the portion from the 25th serine (S) to the 213th aspartic acid (D) is the AAV binding protein AVR25a (corresponding to the region from the 25th to the 213th in SEQ ID NO: 2)
• the portion from the 214th to the 219th histidine (H) is the tag sequence.
• the aspartic acid of V317D is at the 30th position
• the histidine of N324H is at the 37th position
• the alanine of V326A is at the 39th position
• the lysine of N329K is at the 42nd position
• the valine of A330V is at the 43rd position
• the leucine of Q334L is at the 47th position
• the valine of E335V is at the 48th position
• the alanine of T341A is at the 54th position
• the serine of Y342S is at the 55th position
• the glutamic acid of K362E is at the 75th position
• the asparagine of K371N is at the 84th position
• the tyrosine of F379Y is at the 92nd position
• the arginine of K380R is at the 93rd position.
• the alanine of V381A is at the 94th position
• the valine of I382V is at the 95th position
• the serine of G390S is at the 103rd position
• the glutamic acid of K399E is at the 112th position
• the glycine of E401G is at the 114th position
• the alanine of T426A is at the 139th position
• the proline of A461P is at the 174th position
• the glutamine of K467Q is at the 180th position
• the arginine of S476R is at the 189th position
• the threonine of S482T is at the 195th position
• the aspartic acid of N487D is at the 200th position
• the aspartic acid of N492D is at the 205th position.
• AVR25b This protein was produced by selecting K338R, E401G, T426A, and A461P from the amino acid substitutions involved in alkaline stability revealed in Example 2, and introducing these amino acid substitutions into AVR21 (sequence number 2).
• (d-2) Using the PCR product obtained in (d-1) as a template and oligonucleotides consisting of the sequences described in SEQ ID NO: 14 (5'-AGCCTGGCCCGCGCAAGAATCGTCC-3') and SEQ ID NO: 15 (5'-CGCGGGCCAGGCTCAACCGTTACGT-3') as PCR primers, a reaction solution having the composition shown in Table 5 was prepared, and the reaction solution was heat-treated at 98°C for 5 minutes.
• (d-3) Using the PCR product obtained in (d-2) as a template and oligonucleotides consisting of the sequences described in SEQ ID NO: 16 (5'-CAAGTGCGGTGATCGACGGATCGCA-3') and SEQ ID NO: 17 (5'-ATCACCGCACTTGTAGTCGGGAGTG-3') as PCR primers, a reaction solution having the composition shown in Table 5 was prepared, and the reaction solution was heat-treated at 98°C for 5 minutes, and 30 cycles of reaction consisting of a first step at 98°C for 10 seconds, a second step at 55°C for 5 seconds, and a third step at 72°C for 6 minutes were carried out, and finally heat-treated at 72°C for 5 minutes to perform PCR.
• SEQ ID NO: 16 5'-CAAGTGCGGTGATCGACGGATCGCA-3'
• SEQ ID NO: 17 5'-ATCACCGCACTTGTAGTCGGGAGTG-3'
• (d-4) Using the PCR product obtained in (d-3) as a template and oligonucleotides consisting of the sequences described in SEQ ID NO: 18 (5'-ATACCCCGATTCTTAAGCTCTCGCA-3') and SEQ ID NO: 19 (5'-AGAATCGGGGGTATCCTCGCTGATCT-3') as PCR primers, a reaction solution having the composition shown in Table 5 was prepared, and the reaction solution was heat-treated at 98°C for 5 minutes.
• the PCR product obtained in (d-4) was used to transform E. coli BL21 strain (DE3).
• the resulting transformant was cultured in LB medium supplemented with 50 ⁇ g/mL kanamycin, and the resulting transformant was then centrifuged to recover the bacterial cells (transformant).
• a plasmid was extracted from the bacterial cells (transformant) to obtain a plasmid (expression vector) pET-AVR25b containing a polynucleotide encoding AVR25b, which has 25 amino acid substitutions relative to the native AAV binding protein.
• the amino acid sequence of AVR25b with the signal sequence and polyhistidine tag added is shown in SEQ ID NO: 8.
• SEQ ID NO: 8 the portion from the 1st methionine (M) to the 22nd alanine (A) is the PelB signal peptide, the portion from the 25th serine (S) to the 213th aspartic acid (D) is the AAV binding protein AVR25b (corresponding to the region from the 25th to the 213th in SEQ ID NO: 2), and the portion from the 214th to the 219th histidine (H) is the tag sequence.
• the aspartic acid of V317D is at the 30th position
• the histidine of N324H is at the 37th position
• the alanine of V326A is at the 39th position
• the valine of A330V is at the 43rd position
• the leucine of Q334L is at the 47th position
• the valine of E335V is at the 48th position
• the arginine of K338R is at the 51st position
• the alanine of T341A is at the 54th position
• the serine of Y342S is at the 55th position
• the glutamic acid of K362E is at the 75th position
• the asparagine of K371N is at the 84th position
• the tyrosine of F379Y is at the 92nd position
• the arginine of K380R is at the 93rd position.
• V381A alanine at position 94 V381A alanine at position 94, I382V valine at position 95, G390S serine at position 103, K399E glutamic acid at position 112, E401G glycine at position 114, T426A alanine at position 139, A461P proline at position 174, K467Q glutamine at position 180, S476R arginine at position 189, S482T threonine at position 195, N487D aspartic acid at position 200, and N492D aspartic acid at position 205.
• AVR26 This protein was produced by selecting N329K, K338R, E401G, T426A, and A461P from the amino acid substitutions involved in alkaline stability identified in Example 2, and introducing these amino acid substitutions into AVR21 (sequence number 2).
• PCR was performed by using vector pET-AVR21 capable of expressing a polypeptide containing AVR21 (SEQ ID NO: 2) as a template, oligonucleotides consisting of the sequences set forth in SEQ ID NO: 10 (5'-AGCTGAAAGTCTACGTACTGCTGGT-3') and SEQ ID NO: 11 (5'-TAGACTTTCAGCTGGGCTTCATGTT-3') as PCR primers, preparing a reaction solution with the composition shown in Table 5, and then heat-treating the reaction solution at 98°C for 5 minutes.
• (e-2) Using the PCR product obtained in (e-1) as a template and oligonucleotides consisting of the sequences set forth in SEQ ID NO: 12 (5'-CACCGCGCGGGGAAGCGTCAACGTA-3') and SEQ ID NO: 13 (5'-TCCCCGCGCGGTGGAACCAGCAGTA-3') as PCR primers, a reaction solution having the composition shown in Table 5 was prepared, and the reaction solution was heat-treated at 98°C for 5 minutes.
• (e-3) Using the PCR product obtained in (e-2) as a template and oligonucleotides consisting of the sequences described in SEQ ID NO: 14 (5'-AGCCTGGCCCGCGCAAGAATCGTCC-3') and SEQ ID NO: 15 (5'-CGCGGGCCAGGCTCAACCGTTACGT-3') as PCR primers, a reaction solution having the composition shown in Table 5 was prepared, and the reaction solution was heat-treated at 98°C for 5 minutes.
• (e-4) Using the PCR product obtained in (e-3) as a template and oligonucleotides consisting of the sequences described in SEQ ID NO: 16 (5'-CAAGTGCGGTGATCGACGGATCGCA-3') and SEQ ID NO: 17 (5'-ATCACCGCACTTGTAGTCGGGAGTG-3') as PCR primers, a reaction solution having the composition shown in Table 5 was prepared, and the reaction solution was heat-treated at 98°C for 5 minutes.
• the amino acid sequence of AVR26 with the signal sequence and polyhistidine tag added is shown in SEQ ID NO: 9.
• the portion from the 1st methionine (M) to the 22nd alanine (A) is the PelB signal peptide
• the portion from the 25th serine (S) to the 213th aspartic acid (D) is the AAV binding protein AVR26 (corresponding to the region from the 25th to the 213th in SEQ ID NO: 2)
• the portion from the 214th to the 219th histidine (H) is the tag sequence.
• the aspartic acid of V317D is at the 30th position
• the histidine of N324H is at the 37th position
• the alanine of V326A is at the 39th position
• the lysine of N329K is at the 42nd position
• the valine of A330V is at the 43rd position
• the leucine of Q334L is at the 47th position
• the valine of E335V is at the 48th position
• the arginine of K338R is at the 51st position
• the alanine of T341A is at the 54th position
• the serine of Y342S is at the 55th position
• the glutamic acid of K362E is at the 75th position
• the asparagine of K371N is at the 84th position
• the tyrosine of F379Y is at the 92nd position
• the tyrosine of K380R is at the 93rd position.
• Arginine is at the 93rd position, V381A alanine at the 94th position, I382V valine at the 95th position, G390S serine at the 103rd position, K399E glutamic acid at the 112th position, E401G glycine at the 114th position, T426A alanine at the 139th position, A461P proline at the 174th position, K467Q glutamine at the 180th position, S476R arginine at the 189th position, S482T threonine at the 195th position, N487D aspartic acid at the 200th position, and N492D aspartic acid at the 205th position.
• Example 4 Evaluation of alkaline stability of AVR21 amino acid substitution aggregates (1)
• the E. coli strain BL21(DE3) was transformed with a plasmid containing a polynucleotide encoding any one of the six types of AAV-binding proteins (AVR21 amino acid substitution aggregates obtained in Example 3, specifically, AVR24a [SEQ ID NO: 5], AVR24b [SEQ ID NO: 6], AVR25a [SEQ ID NO: 7], AVR25b [SEQ ID NO: 8], and AVR26 [SEQ ID NO: 9], and AVR21 (SEQ ID NO: 2)).
• the resulting transformants were inoculated into 3 mL of 2 ⁇ YT liquid medium containing 50 ⁇ g/mL kanamycin and pre-cultured at 37° C. overnight under aerobic shaking.
• 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 referred to as "equilibration solution A") 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 each supernatant was collected.
• Example 2 Using the ELISA method described in Example 2 (6), the binding activity between the AAV-binding protein prepared in (7) and the VLP2 prepared in Example 1 was measured. Based on the absorbance at 450 nm, which is the measurement result, the AAV-binding protein obtained in (7) was diluted with pure water so that the measured value was similar.
• Each AAV-binding protein solution diluted in (8) was divided into two fractions.
• One of the fractions was mixed with an equal amount of 0.5 M aqueous sodium hydroxide solution and subjected to an alkaline treatment by leaving it at a constant temperature for a certain period of time (treatment temperature: 30°C, treatment time: 0, 30, 60 minutes), and the other fraction was not subjected to the alkaline treatment (corresponding to the "start" of the alkaline treatment).
• the residual activity was calculated by dividing the absorbance at 450 nm when the alkaline treatment in (10) was performed by the absorbance at 450 nm when the treatment time was 0 hours.
• Example 5 Construction of AAV vector (part 2) The AAV vector used as AAV in the following Examples was prepared by the following method.
• a nucleotide sequence (SEQ ID NO: 21) 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: 20, and a stop codon (TAG) and a BamHI recognition sequence (GGATTC) were added to the 3' end.
• GATTC restriction enzyme EcoRI recognition sequence
• TAG stop codon
• GGATTC BamHI recognition sequence
• a polynucleotide consisting of the sequence described in SEQ ID NO:21 was totally synthesized and cloned into a plasmid (commissioned 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).
• the E. coli JM109 strain was transformed with a plasmid (hereinafter also referred to as "pRC8 Vector") containing a polynucleotide encoding the capsid of serotype 8 (AAV8) and pHelper Vector (manufactured by Takara Bio Inc.).
• pRC8 Vector a plasmid containing a polynucleotide encoding the capsid of serotype 8 (AAV8) and pHelper Vector (manufactured by Takara Bio Inc.).
• AAV8 Vector a polynucleotide encoding the capsid of serotype 8
• pHelper Vector manufactured by Takara Bio Inc.
• HEK293T cells were cultured in 10 T-225 flasks (Thermo Fisher Scientific) containing 45 mL of D-MEM medium (Fujifilm Wako Pure Chemical Industries, Ltd.) containing 10% (v/v) bovine serum. Gene transfer was performed by adding the pAAV-EGFP prepared in (4), the pRC8 Vector and pHelper prepared in (5), and a complex of polyethyleneimine (Polysciences), and the cells were statically cultured for 3 days under conditions of 5% (v/v) carbon dioxide and 37°C. After culture, the cells were detached by centrifugation and collected, and the cells obtained from each of the five T-225 flasks were frozen and stored at -80°C.
• the AAV8-EGFP concentration in the solution obtained in (9) was quantified by qPCR using the AAVpro Titration Kit (Takara Bio). The solution was subjected to SDS-PAGE and silver stained using the Pierce Silver Stain Kit (Thermo Fisher Scientific) to confirm the purity of the AAV vector contained in the solution.
• AVR25a (SEQ ID NO: 7) was selected from the AVR21 amino acid substitution aggregates evaluated for alkaline stability in Example 4, and an AVR21 amino acid substitution aggregate (named AVR25c) was prepared by introducing the amino acid substitution K467N, which was found to be an amino acid substitution that improves thermal and alkaline stability in Reference Examples 1 and 2, into AVR25a.
• SEQ ID NOs: 29 (Forward) and 30 (Reverse) were designed as PCR primers for introducing the amino acid substitution K467N, respectively.
• PCR was performed in the same manner as in Example 3(a-1), except that vector pET-AVR25a capable of expressing a polypeptide containing AVR25a (SEQ ID NO: 7) was used as a template, and oligonucleotides consisting of the sequences described in SEQ ID NO: 29 (5'-GAGAGCTTAAGAATGGGGGTATCCTC-3') and SEQ ID NO: 30 (5'-TTCTTAAGCTCTCGAATCTGGTACC-3') were used as PCR primers.
• a plasmid (expression vector) pET-AVR25c was obtained, which contains a polynucleotide encoding AVR25c, which has 25 amino acid substitutions relative to the native AAV binding protein, in a manner similar to that of Example 3 (a-4).
• the amino acid sequence of AVR25c with the signal sequence and polyhistidine tag added is shown in SEQ ID NO: 28.
• SEQ ID NO: 28 the portion from the 1st methionine (M) to the 22nd alanine (A) is the PelB signal peptide, the portion from the 25th serine (S) to the 213th aspartic acid (D) is the AAV binding protein AVR25c (corresponding to the region from the 25th to the 213th in SEQ ID NO: 2), and the portion from the 214th to the 219th histidine (H) is the tag sequence.
• the aspartic acid of V317D is at the 30th position
• the histidine of N324H is at the 37th position
• the alanine of V326A is at the 39th position
• the lysine of N329K is at the 42nd position
• the valine of A330V is at the 43rd position
• the leucine of Q334L is at the 47th position
• the valine of E335V is at the 48th position
• the alanine of T341A is at the 54th position
• the serine of Y342S is at the 55th position
• the glutamic acid of K362E is at the 75th position
• the asparagine of K371N is at the 84th position
• the tyrosine of F379Y is at the 92nd position
• the arginine of K380R is at the 93rd position.
• the alanine of V381A is at the 94th position
• the valine of I382V is at the 95th position
• the serine of G390S is at the 103rd position
• the glutamic acid of K399E is at the 112th position
• the glycine of E401G is at the 114th position
• the alanine of T426A is at the 139th position
• the proline of A461P is at the 174th position
• the asparagine of K467N is at the 180th position
• the arginine of S476R is at the 189th position
• the threonine of S482T is at the 195th position
• the aspartic acid of N487D is at the 200th position
• the aspartic acid of N492D is at the 205th position.
• Example 7 Evaluation of Alkaline Stability of AAV-binding Protein AVR25c
• E. coli strain BL21(DE3) was transformed with a plasmid containing the polynucleotide encoding AVR25c (SEQ ID NO:28) obtained in Example 6, or with a plasmid encoding the AAV-binding protein AVR25a (SEQ ID NO:7) obtained in Example 3(c), and the resulting transformants were cultured in the same manner as in Examples 4(1) to (3). The cultured cells were then recovered by the method described in Example 4(4).
• An AAV-binding protein was prepared by the method described in Examples 4(5) to (8), and its alkaline stability was evaluated by the methods described in Examples 4(9) to (11).
• Example 8 Preparation and screening of AAV binding protein mutation library (part 3) Using the vector pET-AVR25c capable of expressing the polypeptide containing the AAV binding protein AVR25c (SEQ ID NO:28) prepared in Example 6 as a template, random mutations were introduced into the polynucleotide portion encoding the protein by error-prone PCR.
• Error-prone PCR was performed in the same manner as described in Example 2(1) except that vector pET-AVR25c capable of expressing a polypeptide containing AVR25c (SEQ ID NO:28) was used as a template.
• the error-prone PCR successfully introduced mutations into the polynucleotide encoding the AAV binding protein, with the average mutation introduction rate being 1.8 amino acid mutations per molecule.
• a random mutant library was prepared from the PCR products obtained in (1) by the methods described in Example 2 (2) and (3).
• the random mutant library (transformants) prepared in (2) was inoculated into 200 ⁇ L of 2xYT liquid medium containing 50 ⁇ g/mL kanamycin, and cultured overnight at 37°C with shaking in a 96-well deep-well plate.
• the culture medium from (3) was centrifuged, and the resulting culture supernatant was diluted 2-fold with ultrapure water. 50 ⁇ L of the diluted culture supernatant was mixed with 50 ⁇ L of 1 M aqueous sodium hydroxide solution, and then subjected to alkaline treatment at 30°C for 15 minutes.
• the binding activity of the AAV binding protein to VLP2 when the treatment (4) was performed and when the treatment (4) was not performed were evaluated by the ELISA method described in Example 2 (6).
• the binding activity of the AAV binding protein to VLP2 when the alkali treatment was performed was divided by the binding activity of the AAV binding protein to VLP2 when the alkali treatment was not performed to calculate the residual activity.
• a random mutant library of approximately 1,800 strains was evaluated using the method described in (5), and transformants expressing AAV binding proteins with improved residual activity compared to the parent molecule, AVR25c, were selected.
• the transformant selected in (6) was cultured, and an expression vector was prepared using a QIAprep Spin Miniprep kit (Qiagen), after which the sequence of the polynucleotide region encoding the AAV-binding protein inserted into the vector was analyzed by the method described in Example 2 (8) (commissioned to FASMAC) to identify the amino acid substitution sites.
• Qiagen QIAprep Spin Miniprep kit
• Table 8 shows the amino acid substitution positions relative to AVR25c for the AAV binding proteins expressed by the transformants selected in (7) above, as well as the residual activity [%] after alkaline treatment.
• amino acid residues from the 25th serine (S) to the 500th aspartic acid (D) are G314D, V(D)317G, Q318R, P322A, E325V, Q347R, T350A, E362D, H363P, I366F, E378G, F(Y)379N, G392E, V394A, V398A, E399K, R406H, Q415R, F4
• An AAV binding protein having at least one amino acid substitution of 16L, L421P, S433G, T434S, D437V, E445K, K455R, S457R, T474R, T478I, A485V, T486A, L493M, N496D, K497E, V499I, or D500G can be said to have improved alkaline stability compared to AVR25c (SEQ ID NO:28
• Example 9 Preparation of AVR25c Amino Acid Substitution Aggregates (Part 1) Among the amino acid substitutions involved in improving the alkaline stability of AAV-binding proteins clarified in Example 8, G314D, I366F, V394A, K455R, T474R, K467E, and V499I were selected, and these amino acid substitutions were accumulated in AVR25c (SEQ ID NO: 28) to further improve the alkaline stability. Specifically, nine types of AAV-binding proteins shown in (a) to (i) below were designed and prepared. (a) An AAV binding protein having the amino acid substitutions I366F and T474R introduced into AVR25c (SEQ ID NO: 31, designated AVR27b).
• AAV binding protein having the amino acid substitutions I366F, V394A, K455R, and T474R introduced into AVR25c SEQ ID NO: 35, designated AVR29a.
• An AAV binding protein having the amino acid substitutions G314D, I366F, V394A, and K455R introduced into AVR25c SEQ ID NO: 36, designated AVR29b.
• An AAV binding protein having the amino acid substitutions I366F, V394A, K455R, and K497E introduced into AVR25c SEQ ID NO: 37, designated AVR29c).
• AAV binding protein having the amino acid substitutions I366F, V394A, K455R, and V499I introduced into AVR25c (SEQ ID NO: 38, designated AVR29d).
• AVR29d An AAV binding protein in which the following amino acid substitutions were introduced into AVR25c: G314D, I366F, V394A, K455R, K497E, and V499I (SEQ ID NO: 39; designated AVR31).
• a PCR primer set was designed to generate mutant clusters.
• SEQ ID NO: 40 forward and SEQ ID NO: 41 (reverse) were used.
• SEQ ID NOs: 42 forward and 43 (reverse) were used.
• SEQ ID NOs: 44 forward and 45 (reverse) were used.
• SEQ ID NOs: 46 forward and 47 (reverse) were used.
• PCR primers for introducing the amino acid substitution T474R SEQ ID NOs: 48 (forward) and 49 (reverse) were used.
• PCR primers for introducing the amino acid substitution K497E SEQ ID NO:50 (forward) and SEQ ID NO:51 (reverse) were used.
• PCR primers for introducing the amino acid substitution of V499I SEQ ID NO:52 (forward) and SEQ ID NO:53 (reverse) were used.
• SEQ ID NOs: 54 forward) and 55 (reverse) were used. Each was designed.
• AVR27b This protein was prepared by selecting I366F and T474R from the amino acid substitutions involved in alkaline stability revealed in Example 9, and introducing I366F, one of the amino acid substitutions, into the AAV binding protein obtained in Example 8 in which the amino acid substitution T474R was introduced into AVR25c (see Table 8; hereinafter also referred to as "AVR25c_T474R").
• a-1 Using a vector capable of expressing AVR25c_T474R as a template and oligonucleotides consisting of the sequences set forth in SEQ ID NO: 42 (5'-GTCAGTTTCTGAAATTATCCAACTT-3') and SEQ ID NO: 43 (5'-TTCAGAAACTGACTGTGTTCGCCTT-3') as PCR primers, a reaction solution with the composition shown in Table 9 was prepared, and the reaction solution was heat-treated at 98°C for 5 minutes, and 30 cycles of reaction consisting of a first step at 98°C for 10 seconds, a second step at 55°C for 5 seconds, and a third step at 72°C for 6 minutes were carried out, and finally heat-treated at 72°C for 5 minutes to perform PCR.
• SEQ ID NO: 42 5'-GTCAGTTTCTGAAATTATCCAACTT-3'
• SEQ ID NO: 43 5'-TTCAGAAACTGACTGTGTTCGCCTT-3'
• a-2 The PCR product obtained in (a-1) was treated with DpnI (New England Biolabs) to digest the template strand, and the PCR product was then used to transform E. coli BL21 strain (DE3).
• the resulting transformant was cultured in LB medium supplemented with 50 ⁇ g/mL kanamycin, and the resulting transformant was centrifuged to recover the bacterial cells (transformant).
• the plasmid was extracted from the cells (transformant) to obtain the plasmid (expression vector) pET-AVR27b, which contains a polynucleotide encoding AVR27b, which has 27 amino acid substitutions relative to the native AAV binding protein.
• the amino acid sequence of AVR27b with the signal sequence and polyhistidine tag added is shown in SEQ ID NO: 31.
• SEQ ID NO: 31 the portion from the 1st methionine (M) to the 22nd alanine (A) is the PelB signal peptide, the portion from the 25th serine (S) to the 213th aspartic acid (D) is the AAV binding protein AVR27b (corresponding to the region from the 25th to the 213th in SEQ ID NO: 2), and the 214th to 219th histidines (H) are the tag sequence.
• the aspartic acid of V317D is at the 30th position
• the histidine of N324H is at the 37th position
• the alanine of V326A is at the 39th position
• the lysine of N329K is at the 42nd position
• the valine of A330V is at the 43rd position
• the leucine of Q334L is at the 47th position
• the valine of E335V is at the 48th position
• the alanine of T341A is at the 54th position
• the serine of Y342S is at the 55th position
• the glutamic acid of K362E is at the 75th position
• the phenylalanine of I366F is at the 79th position
• the asparagine of K371N is at the 84th position
• the tyrosine of F379Y is at the 92nd position
• the arginine of K380R is at the 93rd position.
• V381A alanine at position 94 V381A alanine at position 94, I382V valine at position 95, G390S serine at position 103, K399E glutamic acid at position 112, E401G glycine at position 114, T426A alanine at position 139, A461P proline at position 174, K467N asparagine at position 180, T474R arginine at position 187, S476R arginine at position 189, S482T threonine at position 195, N487D aspartic acid at position 200, and N492D aspartic acid at position 205.
• AVR27c This protein was prepared by selecting V394A and T474R from the amino acid substitutions involved in alkaline stability revealed in Example 8, and introducing V394A, one of the amino acid substitutions, into AVR25c_T474R.
• (b-1) PCR was performed in the same manner as in (a-1), except that a vector capable of expressing AVR25c_T474R was used as a template and oligonucleotides consisting of the sequences set forth in SEQ ID NO: 44 (5'-GGTATGCGAACGTAACGGTTGAGCC-3') and SEQ ID NO: 45 (5'-ACGTTCGCATACCCCTCGGAATGCG-3') were used as PCR primers.
• the amino acid sequence of AVR27c with the signal sequence and polyhistidine tag added is shown in SEQ ID NO: 32.
• SEQ ID NO: 32 the portion from the 1st methionine (M) to the 22nd alanine (A) is the PelB signal peptide, the portion from the 25th serine (S) to the 213th aspartic acid (D) is the AAV binding protein AVR27c (corresponding to the region from the 25th to the 213th in SEQ ID NO: 2), and the 214th to 219th histidines (H) are the tag sequence.
• the aspartic acid of V317D is at the 30th position
• the histidine of N324H is at the 37th position
• the alanine of V326A is at the 39th position
• the lysine of N329K is at the 42nd position
• the valine of A330V is at the 43rd position
• the leucine of Q334L is at the 47th position
• the valine of E335V is at the 48th position
• the alanine of T341A is at the 54th position
• the serine of Y342S is at the 55th position
• the glutamic acid of K362E is at the 75th position
• the asparagine of K371N is at the 84th position
• the tyrosine of F379Y is at the 92nd position
• the arginine of K380R is at the 93rd position
• the alanine of V381A is at the 94th position.
• I382V valine is at position 95
• G390S serine is at position 103
• V394A alanine is at position 107
• K399E glutamic acid is at position 112
• E401G glycine is at position 114
• T426A alanine is at position 139
• A461P proline is at position 174
• K467N asparagine is at position 180
• T474R arginine is at position 187
• S476R arginine is at position 189
• S482T threonine is at position 195
• N487D aspartic acid is at position 200
• N492D aspartic acid is at position 205.
• AVR27d This protein was produced by selecting K455R and T474R from the amino acid substitutions involved in alkaline stability revealed in Example 8, and introducing K455R, one of the amino acid substitutions, into a vector capable of expressing AVR25c_T474R as a template.
• (c-1) PCR was performed in the same manner as in (a-1), except that a vector capable of expressing AVR25c_T474R was used as a template, and oligonucleotides consisting of the sequences set forth in SEQ ID NO: 46 (5'-AAGAACGCATCAGCGAGGATACCCC-3') and SEQ ID NO: 47 (5'-CTGATGCGTTCTTCACGGAGCGGGGC-3') were used as PCR primers.
• the amino acid sequence of AVR27d with the signal sequence and polyhistidine tag added is shown in SEQ ID NO: 33.
• SEQ ID NO: 33 the portion from the 1st methionine (M) to the 22nd alanine (A) is the PelB signal peptide, the portion from the 25th serine (S) to the 213th aspartic acid (D) is the AAV binding protein AVR27d (corresponding to the region from the 25th to the 213th in SEQ ID NO: 2), and the portion from the 214th to the 219th histidine (H) is the tag sequence.
• the aspartic acid of V317D is at the 30th position
• the histidine of N324H is at the 37th position
• the alanine of V326A is at the 39th position
• the lysine of N329K is at the 42nd position
• the valine of A330V is at the 43rd position
• the leucine of Q334L is at the 47th position
• the valine of E335V is at the 48th position
• the alanine of T341A is at the 54th position
• the serine of Y342S is at the 55th position
• the glutamic acid of K362E is at the 75th position
• the asparagine of K371N is at the 84th position
• the tyrosine of F379Y is at the 92nd position
• the arginine of K380R is at the 93rd position
• the alanine of V381A is at the 94th position.
• I382V valine is at the 95th position
• G390S serine is at the 103rd position
• K399E glutamic acid is at the 112th position
• E401G glycine is at the 114th position
• T426A alanine is at the 139th position
• K455R arginine is at the 168th position
• A461P proline is at the 174th position
• K467N asparagine is at the 180th position
• T474R arginine is at the 187th position
• S476R arginine is at the 189th position
• S482T threonine is at the 195th position
• N487D aspartic acid is at the 200th position
• N492D aspartic acid is at the 205th position.
• SEQ ID NO: 34 The amino acid sequence of AVR28 with the signal sequence and polyhistidine tag added is shown in SEQ ID NO: 34.
• the portion from the 1st methionine (M) to the 22nd alanine (A) is the PelB signal peptide
• the portion from the 25th serine (S) to the 213th aspartic acid (D) is the AAV binding protein AVR28a (corresponding to the region from the 25th to the 213th in SEQ ID NO: 2)
• the portion from the 214th to the 219th histidine (H) is the tag sequence.
• the aspartic acid of V317D is at the 30th position
• the histidine of N324H is at the 37th position
• the alanine of V326A is at the 39th position
• the lysine of N329K is at the 42nd position
• the valine of A330V is at the 43rd position
• the leucine of Q334L is at the 47th position
• the valine of E335V is at the 48th position
• the alanine of T341A is at the 54th position
• the serine of Y342S is at the 55th position
• the glutamic acid of K362E is at the 75th position
• the phenylalanine of I366F is at the 79th position
• the asparagine of K371N is at the 84th position
• the tyrosine of F379Y is at the 92nd position
• the arginine of K380R is at the 93rd position
• alanine is at position 94, valine at position 95, serine at position G390S, alanine at position 103, alanine at position V394A, glutamic acid at position K399E, glycine at position E401G, alanine at position 139, arginine at position K455R, proline at position A461P, asparagine at position K467N, arginine at position S476R, threonine at position S482T, threonine at position 195, aspartic acid at position N487D, and aspartic acid at position N492D.
• AVR29a This protein was prepared by selecting T474R from the amino acid substitutions involved in alkaline stability revealed in Example 8 and introducing this amino acid substitution into AVR28 (SEQ ID NO: 34).
• (e-1) PCR was performed in the same manner as in (a-1), except that vector pET-AVR28a capable of expressing a polypeptide containing AVR28 (SEQ ID NO: 34) was used as a template, and oligonucleotides consisting of the sequences set forth in SEQ ID NO: 48 (5'-ACTACCGCTTTCGCCTGACCGTGAC-3') and SEQ ID NO: 49 (5'-CGAAAGCGGTAGTTACCTGGTACCA-3') were used as PCR primers.
• a plasmid (expression vector) pET-AVR29a was obtained, which contains a polynucleotide encoding AVR29a, which has 29 amino acid substitutions compared to the native AAV binding protein, in a manner similar to (a-2).
• the amino acid sequence of AVR29a with the signal sequence and polyhistidine tag added is shown in SEQ ID NO: 35.
• SEQ ID NO: 35 the portion from the 1st methionine (M) to the 22nd alanine (A) is the PelB signal peptide, the portion from the 25th serine (S) to the 213th aspartic acid (D) is the AAV binding protein AVR29a (corresponding to the region from the 25th to the 213th in SEQ ID NO: 2), and the portion from the 214th to the 219th histidine (H) is the tag sequence.
• the aspartic acid of V317D is at the 30th position
• the histidine of N324H is at the 37th position
• the alanine of V326A is at the 39th position
• the lysine of N329K is at the 42nd position
• the valine of A330V is at the 43rd position
• the leucine of Q334L is at the 47th position
• the valine of E335V is at the 48th position
• the alanine of T341A is at the 54th position
• the serine of Y342S is at the 55th position
• the glutamic acid of K362E is at the 75th position
• the phenylalanine of I366F is at the 79th position
• the asparagine of K371N is at the 84th position
• the tyrosine of F379Y is at the 92nd position
• the arginine of K380R is at the 93rd position
• the valine in I382V is located at the 95th position
• the serine in G390S is located at the 103rd position
• the alanine in V394A is located at the 107th position
• the glutamic acid in K399E is located at the 112th position
• the glycine in E401G is located at the 114th position
• the alanine in T426A is located at the 139th position
• the arginine in K455R is located at the 168th position
• the proline in A461P is located at the 174th position
• the asparagine in K467N is located at the 180th position
• the arginine in T474R is located at the 187th position
• the arginine in S476R is located at the 189th position
• the threonine in S482T is located at the 195th position
• the aspartic acid in N487D is located at the 200th position
• AVR29b This protein was prepared by selecting G314D from the amino acid substitutions involved in alkaline stability revealed in Example 9, and introducing this amino acid substitution into AVR28 (SEQ ID NO: 34).
• (f-1) PCR was performed in the same manner as in (e-1), except that oligonucleotides consisting of the sequences described in SEQ ID NO: 40 (5'-CTGCAGATGAAAGCGACCAAATCAC-3') and SEQ ID NO: 41 (5'-CTTTCATCTGCAGAGCCCATGGCCA-3') were used as PCR primers.
• a plasmid (expression vector) pET-AVR29b was obtained, which contains a polynucleotide encoding AVR29b, which has 29 amino acid substitutions compared to the native AAV binding protein, in a manner similar to (a-2).
• the amino acid sequence of AVR29b with the signal sequence and polyhistidine tag added is shown in SEQ ID NO: 36.
• SEQ ID NO: 36 the portion from the 1st methionine (M) to the 22nd alanine (A) is the PelB signal peptide, the portion from the 25th serine (S) to the 213th aspartic acid (D) is the AAV binding protein AVR29b (corresponding to the region from the 25th to the 213th in SEQ ID NO: 2), and the 214th to 219th histidines (H) are the tag sequence.
• the aspartic acid of G314D is at the 27th position
• the aspartic acid of V317D is at the 30th position
• the histidine of N324H is at the 37th position
• the alanine of V326A is at the 39th position
• the lysine of N329K is at the 42nd position
• the valine of A330V is at the 43rd position
• the leucine of Q334L is at the 47th position
• the valine of E335V is at the 48th position
• the alanine of T341A is at the 54th position
• the serine of Y342S is at the 55th position
• the glutamic acid of K362E is at the 75th position
• the phenylalanine of I366F is at the 79th position
• the asparagine of K371N is at the 84th position
• the tyrosine of F379Y is at the 92nd position
• V381A alanine is at position 94
• I382V valine is at position 95
• G390S serine is at position 103
• V394A alanine is at position 107
• K399E glutamic acid is at position 112
• E401G glycine is at position 114
• T426A alanine is at position 139
• K455R arginine is at position 168
• A461P proline is at position 174
• K467N asparagine is at position 180
• S476R arginine is at position 189
• S482T threonine is at position 195
• N487D aspartic acid is at position 200
• N492D aspartic acid is at position 205.
• AVR29c This protein was prepared by selecting K497E from the amino acid substitutions involved in alkaline stability revealed in Example 8 and introducing this amino acid substitution into AVR28 (SEQ ID NO: 34).
• (g-1) PCR was performed in the same manner as in (e-1), except that oligonucleotides consisting of the sequences described in SEQ ID NO: 50 (5'-TTAACGAAGCCGTCGACCATCATCA-3') and SEQ ID NO: 51 (5'-ACGGCTTCGTTAACGGTCAAGTCTG-3') were used as PCR primers.
• a plasmid (expression vector) pET-AVR29c was obtained, which contains a polynucleotide encoding AVR29c, which has 29 amino acid substitutions compared to the native AAV binding protein, in a manner similar to (a-2).
• the amino acid sequence of AVR29c with the signal sequence and polyhistidine tag added is shown in SEQ ID NO: 37.
• SEQ ID NO: 37 the portion from the 1st methionine (M) to the 22nd alanine (A) is the PelB signal peptide, the portion from the 25th serine (S) to the 213th aspartic acid (D) is the AAV binding protein AVR29c (corresponding to the region from the 25th to the 213th in SEQ ID NO: 2), and the 214th to 219th histidines (H) are the tag sequence.
• the aspartic acid of V317D is at the 30th position
• the histidine of N324H is at the 37th position
• the alanine of V326A is at the 39th position
• the lysine of N329K is at the 42nd position
• the valine of A330V is at the 43rd position
• the leucine of Q334L is at the 47th position
• the valine of E335V is at the 48th position
• the alanine of T341A is at the 54th position
• the serine of Y342S is at the 55th position
• the glutamic acid of K362E is at the 75th position
• the phenylalanine of I366F is at the 79th position
• the asparagine of K371N is at the 84th position
• the tyrosine of F379Y is at the 92nd position
• the arginine of K380R is at the 93rd position
• the valine in I382V is at the 95th position
• the serine in G390S is at the 103rd position
• the alanine in V394A is at the 107th position
• the glutamic acid in K399E is at the 112th position
• the glycine in E401G is at the 114th position
• the alanine in T426A is at the 139th position
• the arginine in K455R is at the 168th position
• the proline in A461P is at the 174th position
• the asparagine in K467N is at the 180th position
• the arginine in S476R is at the 189th position
• the threonine in S482T is at the 195th position
• the aspartic acid in N487D is at the 200th position
• the aspartic acid in N492D is at the 205th position
• the glutamic acid in K497E is at the 210
• AVR29d This protein was prepared by selecting V499I from the amino acid substitutions involved in alkaline stability revealed in Example 8, and introducing this amino acid substitution into AVR28 (SEQ ID NO: 34).
• (h-1) PCR was performed in the same manner as (e-1), except that oligonucleotides consisting of the sequences described in SEQ ID NO: 52 (5'-AAGCCATTGACCATCATCATCATCA-3') and SEQ ID NO: 53 (5'-TGGTCAATGGCTTTGTTAACGGTCA-3') were used as PCR primers.
• a plasmid (expression vector) pET-AVR29d was obtained, which contains a polynucleotide encoding AVR29d, which has 29 amino acid substitutions compared to the native AAV binding protein, in a manner similar to (a-2).
• the amino acid sequence of AVR29d with the signal sequence and polyhistidine tag added is shown in SEQ ID NO: 38.
• SEQ ID NO: 38 the portion from the 1st methionine (M) to the 22nd alanine (A) is the PelB signal peptide, the portion from the 25th serine (S) to the 213th aspartic acid (D) is the AAV binding protein AVR29d (corresponding to the region from the 25th to the 213th in SEQ ID NO: 2), and the portion from the 214th to the 219th histidine (H) is the tag sequence.
• the aspartic acid of V317D is at the 30th position
• the histidine of N324H is at the 37th position
• the alanine of V326A is at the 39th position
• the lysine of N329K is at the 42nd position
• the valine of A330V is at the 43rd position
• the leucine of Q334L is at the 47th position
• the valine of E335V is at the 48th position
• the alanine of T341A is at the 54th position
• the serine of Y342S is at the 55th position
• the glutamic acid of K362E is at the 75th position
• the phenylalanine of I366F is at the 79th position
• the asparagine of K371N is at the 84th position
• the tyrosine of F379Y is at the 92nd position
• the arginine of K380R is at the 93rd position
• the valine in I382V is located at the 95th position
• the serine in G390S is located at the 103rd position
• the alanine in V394A is located at the 107th position
• the glutamic acid in K399E is located at the 112th position
• the glycine in E401G is located at the 114th position
• the alanine in T426A is located at the 139th position
• the arginine in K455R is located at the 168th position
• the proline in A461P is located at the 174th position
• the asparagine in K467N is located at the 180th position
• the arginine in S476R is located at the 189th position
• the threonine in S482T is located at the 195th position
• the aspartic acid in N487D is located at the 200th position
• the aspartic acid in N492D is located at the 205th position
• AVR31 This protein was produced by selecting K497E and V499I from the amino acid substitutions involved in alkaline stability revealed in Example 8, and introducing these amino acid substitutions into AVR29b (SEQ ID NO: 36).
• (i-1) PCR was performed in the same manner as in (a-1), except that the vector pET-AVR29b capable of expressing a polypeptide containing AVR29b (SEQ ID NO: 36) was used as a template, and oligonucleotides consisting of the sequences described in SEQ ID NO: 54 (5'-AACGAAGCCATTGACCATCATCATCATCAT-3') and SEQ ID NO: 55 (5'-GTCAATGGCTTCGTTAACGGTCAAGTCTGC-3') were used as PCR primers.
• SEQ ID NO: 54 5'-AACGAAGCCATTGACCATCATCATCATCATCATCAT-3'
• SEQ ID NO: 55 5'-GTCAATGGCTTCGTTAACGGTCAAGTCTGC-3'
• a plasmid (expression vector) pET-AVR31 was obtained, which contains a polynucleotide encoding AVR31, which has 31 amino acid substitutions compared to the native AAV binding protein, in a manner similar to (a-2).
• the amino acid sequence of AVR31 with the signal sequence and polyhistidine tag added is shown in SEQ ID NO: 39.
• SEQ ID NO: 39 the portion from the 1st methionine (M) to the 22nd alanine (A) is the PelB signal peptide, the portion from the 25th serine (S) to the 213th aspartic acid (D) is the AAV binding protein AVR31 (corresponding to the region from the 25th to the 213th in SEQ ID NO: 2), and the portion from the 214th to the 219th histidine (H) is the tag sequence.
• the aspartic acid of G314D is at the 27th position
• the aspartic acid of V317D is at the 30th position
• the histidine of N324H is at the 37th position
• the alanine of V326A is at the 39th position
• the lysine of N329K is at the 42nd position
• the valine of A330V is at the 43rd position
• the leucine of Q334L is at the 47th position
• the valine of E335V is at the 48th position
• the alanine of T341A is at the 54th position
• the serine of Y342S is at the 55th position
• the glutamic acid of K362E is at the 75th position
• the phenylalanine of I366F is at the 79th position
• the asparagine of K371N is at the 84th position
• the tyrosine of F379Y is at the 92nd position
• the valine in I382V is located at the 95th position
• the serine in G390S is located at the 103rd position
• the alanine in V394A is located at the 107th position
• the glutamic acid in K399E is located at the 112th position
• the glycine in E401G is located at the 114th position
• the alanine in T426A is located at the 139th position
• the arginine in K455R is located at the 168th position
• the proline in A461P is located at the 174th position
• the asparagine in K467N is located at the 180th position
• the arginine in S476R is located at the 189th position
• the threonine in S482T is located at the 195th position
• the aspartic acid in N487D is located at the 200th position
• the aspartic acid in N492D is located at the 205th position
• Example 10 Preparation of AVR25c Amino Acid Substitution Aggregates (Part 2) (1)
• a transformant obtained by transforming Escherichia coli strain BL21(DE3) with a plasmid containing a polynucleotide encoding any one of the nine types of AAV-binding proteins (AVR25c amino acid substitution aggregates, specifically, AVR27b (SEQ ID NO: 31), AVR27c (SEQ ID NO: 32), AVR27d (SEQ ID NO: 33), AVR28 (SEQ ID NO: 34), AVR29a (SEQ ID NO: 35), AVR29b (SEQ ID NO: 36), AVR29c (SEQ ID NO: 37), AVR29d (SEQ ID NO: 38), and AVR31 (SEQ ID NO: 39)) obtained in Example 9, and AVR21 (SEQ ID NO: 2) and AVR25c (SEQ ID NO: 28) was used.
• the transformants were cultured in
• Example 11 Evaluation of alkaline stability of AVR25c amino acid substitution aggregate (part 1) Of the AAV binding proteins prepared in Example 10, AVR25c (SEQ ID NO: 28), AVR28 (SEQ ID NO: 34), and AVR29a (SEQ ID NO: 35) were used to evaluate alkaline stability.
• Example 10 The concentration of the AAV-binding protein solution prepared in Example 10 was measured using NanoDrop OneC (Thermo Fisher Scientific), and then diluted with 20 mM Tris-HCl buffer (pH 7.4) to a protein concentration of 5 ⁇ g/mL.
• Each AAV-binding protein solution diluted in (1) was mixed with an equal amount of 1 M aqueous sodium hydroxide solution containing 1 mM calcium chloride, and subjected to an alkaline treatment by leaving the solution at a constant temperature for a certain period of time (treatment temperature: 30°C, treatment time: 0, 30, 60 minutes).
• the residual activity was calculated by dividing the absorbance at 450 nm when the alkaline treatment in (3) was performed by the absorbance at 450 nm when the treatment time was 0 hours.
• Example 12 Evaluation of alkaline stability of AVR25c amino acid substitution aggregate (part 2) Of the AAV binding proteins prepared in Example 10, alkaline stability was evaluated using AVR25c (SEQ ID NO: 28), AVR27b (SEQ ID NO: 31), AVR27c (SEQ ID NO: 32), and AVR27d (SEQ ID NO: 33). The evaluation was performed in the same manner as in Example 11, except that the treatment time was 0, 30, or 45 minutes.
• Example 13 Evaluation of alkaline stability of AVR25c amino acid substitution aggregates (part 3) Of the AVR25c amino acid substitution aggregates prepared in Example 10, AVR25c (SEQ ID NO: 28), AVR29b (SEQ ID NO: 36), AVR29c (SEQ ID NO: 37), and AVR29d (SEQ ID NO: 38) were used to evaluate alkaline stability. The evaluation was performed in the same manner as in Example 11, except that the treatment time was 0, 20, or 45 minutes.
• Example 14 Evaluation of alkaline stability of AVR25c amino acid substitution aggregates (part 4) Of the AAV binding proteins prepared in Example 10, alkaline stability was evaluated using AVR28 (SEQ ID NO: 34), AVR29b (SEQ ID NO: 36), AVR29c (SEQ ID NO: 37), and AVR29d (SEQ ID NO: 38). The evaluation was performed in the same manner as in Example 11, except that the treatment time was 0, 20, or 60 minutes.
• AVR29b (SEQ ID NO: 36), AVR29c (SEQ ID NO: 37) and AVR29d (SEQ ID NO: 38) all have higher residual activity than AVR28 (SEQ ID NO: 34), and are found to have improved stability against alkali.
• Example 15 Evaluation of alkaline stability of AVR25c amino acid substitution aggregate (part 5) Of the AAV binding proteins prepared in Example 10, AVR25c (SEQ ID NO: 28), AVR28 (SEQ ID NO: 34), AVR29c (SEQ ID NO: 37), and AVR31 (SEQ ID NO: 39) were used to evaluate alkaline stability by the method described in Example 11.
• Example 16 Evaluation of alkaline stability of AVR25c amino acid substitution aggregate (part 6) Of the AAV binding proteins prepared in Example 10, AVR21 (SEQ ID NO: 2), AVR25c (SEQ ID NO: 28), and AVR31 (SEQ ID NO: 39) were used to evaluate alkaline stability by the method described in Example 11.
• AVR31 (SEQ ID NO: 39) has higher residual activity and improved alkali stability compared to AVR21 (SEQ ID NO: 2) and AVR25c (SEQ ID NO: 28).
• a gel was prepared by chemically modifying the hydroxyl groups on the surface of a hydrophilic vinyl polymer for separation agents (Tosoh Corporation: Toyopearl) to introduce an iodoacetamide group.
• 6 mg of AVR29c (SEQ ID NO: 37) as the AAV-binding protein prepared in Example 10 and TCEP (Tris (2-CarboxyEthyl) Phosphine) at a final concentration of 0.3 mM as a reducing agent were added to 1.0 g of the prepared gel, and the mixture was allowed to react by shaking for 3 hours under conditions of pH 7.4 and 25°C.
• AVR29c immobilized gel was prepared.
• AVR29c column 2.0 mL of the AVR29c immobilized gel prepared in (1) was packed into an empty column ( ⁇ 10 mm ⁇ 50 mm, Cytiva) to prepare an AAV adsorbent column (hereinafter also referred to as "AVR29c column").
• FIG. 1 The obtained chromatogram is shown in Figure 1.
• the peak indicated by the black arrow corresponds to the peak of the fraction containing the AAV vector (AAV8-EGFP). Since most of the impurities contained in the AAV8-EGFP solution pass through the AVR29c column, it is clear that the AAV vector contained in the solution can be purified using the AVR29c column.
• Example 18 Purification by combining affinity chromatography and anion exchange chromatography (part 1) The AAV vector solution after affinity chromatography purification was applied to an anion exchange column to attempt further purification of the AAV vector.
• Example 17(4) The AAV vector solution obtained in Example 17(4) was diluted 5-fold with 20 mM Tris-HCl buffer (pH 9.0) (hereinafter also referred to as solution AEX-A) to prepare a sample to be applied to the anion exchange column.
• solution AEX-A 20 mM Tris-HCl buffer
• solution AEX-A (hereinafter also referred to as solution AEX-B) containing 1 M choline chloride reached 14.5%, and impurities were eluted by running the solution for 60 CV while maintaining this state (state in which AEX-B was 14.5%).
• the obtained chromatogram is shown in Figure 2.
• the peak indicated by the black arrow corresponds to the peak of the fraction containing the AAV vector (AAV8-EGFP).
• the impurities remaining in the AAV vector solution obtained in Example 18 (4) were removed using an anion exchange column (the peak indicated by the white arrow in Figure 2 corresponds to the impurities). From these results, it can be seen that the purity of the AAV vector can be further improved by purifying the solution containing the AAV vector using affinity chromatography using an AAV-binding protein-immobilized gel and then further purifying it using anion exchange chromatography.
• Example 19 Measurement of the Proportion of AAV Vector Containing a Gene (Full Rate) The proportion of AAV vectors containing a gene (full rate) among the AAV vectors contained in the fraction obtained in Example 17(4) (peak indicated by the black arrow in Fig. 1 ) and in the fractions obtained in Example 18(3) (peak indicated by the white arrow in Fig. 2 ) and Example 18(4) (peak indicated by the black arrow in Fig. 2 ) was measured by the method shown below.
• Example 17(4) i.e., the purified fraction on the AVR29c column
• the fraction obtained in Example 18(3) i.e., the washing fraction in the GigaCapQ column
• the fraction obtained in Example 18(4) that is, the purified fraction on the GigaCapQ column
• FIG. 3 The results of purification using the GigaCapQ column (chromatogram) are shown in FIG. 3, and the results of analysis of each fraction obtained using TSKgel G6000PW XL are shown in FIG. 4.
• the black arrow in FIG. 4 corresponds to the peak of the fraction containing the AAV vector (AAV8-EGFP).
• peaks corresponding to the AAV vector were confirmed from peak 2 to peak 4 in FIG. 3, it can be seen that a large amount of impurities are also contained. From the above results, it can be seen that it is difficult to obtain a highly pure AAV even if a solution containing an AAV vector is purified only by anion exchange chromatography.
• Example 20 Preparation of AAV binding protein from which signal peptide sequence has been removed (1) PCR was performed in the same manner as in Example 10(a-1) except that a vector capable of expressing AVR29c (SEQ ID NO:37) was used as a template and oligonucleotides consisting of the sequences described in SEQ ID NO:56 (5'-ACATATGTCTGCAGGCGAAAGCGAC-3') and SEQ ID NO:57 (5'-CCTGCAGACATATGTATATCTCCTTC-3') were used as PCR primers.
• SEQ ID NO:37 a vector capable of expressing AVR29c
• SEQ ID NO:57 5'-CCTGCAGACATATGTATATCTCCTTC-3'
• a plasmid (expression vector) pET-AVR29c(-) was obtained in a manner similar to that of Example 9(a-2), which encodes AVR29c, which has 29 amino acid substitutions in the native AAV binding protein, and contains a polynucleotide that does not have a signal sequence on the N-terminus.
• the amino acid sequence of AVR29c(-) with a polyhistidine tag is shown in SEQ ID NO: 58.
• SEQ ID NO: 58 the sequence from the second serine (S) to the 190th aspartic acid (D) represents the AAV binding protein AVR29c(-) (corresponding to the region from the 25th to the 213th in SEQ ID NO: 2), and the 191st to 196th histidines (H) represent the tag sequence.
• the aspartic acid of V317D is at the 7th position
• the histidine of N324H is at the 14th position
• the alanine of V326A is at the 16th position
• the lysine of N329K is at the 19th position
• the valine of A330V is at the 20th position
• the leucine of Q334L is at the 24th position
• the valine of E335V is at the 25th position
• the alanine of T341A is at the 31st position
• the serine of Y342S is at the 32nd position
• the glutamic acid of K362E is at the 52nd position
• the phenylalanine of I366F is at the 56th position
• the asparagine of K371N is at the 61st position
• the tyrosine of F379Y is at the 69th position
• the arginine of K380R is at the 70th position
• the valine in I382V is at the 72nd position
• the serine in G390S is at the 80th position
• the alanine in V394A is at the 84th position
• the glutamic acid in K399E is at the 89th position
• the glycine in E401G is at the 91st position
• the alanine in T426A is at the 116th position
• the arginine in K455R is at the 145th position
• the proline in A461P is at the 151st position
• the asparagine in K467N is at the 157th position
• the arginine in S476R is at the 166th position
• the threonine in S482T is at the 172nd position
• the aspartic acid in N487D is at the 177th position
• the aspartic acid in N492D is at the 182nd position
• the glutamic acid in K497E is at the
• the transformant was cultured in the same manner as in Example 4(1) to (4), except that the transformant obtained by transforming Escherichia coli strain BL21(DE3) with a plasmid containing a polynucleotide encoding the AAV binding protein AVR29c(-) (SEQ ID NO:58) obtained in (3) was used, and the transformant was collected.
• AVR29c prepared in Example 10 and AVR29c(-) prepared in (5) were subjected to SDS-PAGE and stained with CBB (Coomassie Brilliant Blue) staining solution (FUJIFILM Wako Pure Chemical Industries, Ltd.).
• Example 21 Performance Evaluation of AAV-Binding Proteins
• AVR29c (SEQ ID NO: 37) prepared in Example 10 and AVR29c(-) prepared in Example 20 (SEQ ID NO: 58) were used to evaluate their binding to AAV.
• Example 1 The VLP2 prepared in Example 1 was immobilized and blocked in the wells of a 96-well microplate (Thermo Fisher Scientific) by the method described in Example 2 (6-1).
• Example 22 Preparation of AAV-binding protein-immobilized gel and performance evaluation (1)
• a gel was prepared in which AVR29c(-) prepared in Example 22 was immobilized on a hydrophilic vinyl polymer for a separating agent (designated AVR29c(-)-immobilized gel).
• washing solution (20 mM Tris-HCl buffer (pH 7.4) containing 0.5 M sodium chloride and 0.5 mM calcium chloride) was added and the gel was washed by stirring at 25°C for 3 minutes.
• eluent B 0.1 M acetic acid (pH 2.5) containing 0.5 M sodium chloride
• a size exclusion chromatography column TSKgel G6000PW (Tosoh Corporation), was connected to an ultra-high performance liquid chromatograph Nexera (Shimadzu Corporation), and equilibrated with eluent A (50 mM sodium acetate buffer (pH 6.0) containing 0.5 M sodium chloride, 0.01% (w/v) Tween 20 (Sigma-Aldrich), and 0.01% (w/v) Pluronic F-68 (Sigma-Aldrich)).
• eluent A 50 mM sodium acetate buffer (pH 6.0) containing 0.5 M sodium chloride, 0.01% (w/v) Tween 20 (Sigma-Aldrich), and 0.01% (w/v) Pluronic F-68 (Sigma-Aldrich)
• AAV8-EGFP of known concentration as well as fraction A obtained in (4), fraction B obtained in (5), and fraction C obtained in (6) were each applied to the column equilibrated in (7) and pumped at a flow rate of 1 mL/min to obtain a separated peak derived from AAV8-EGFP in each sample.
• the separated peak was detected by fluorescence intensity (280 nm excitation/350 nm emission).
• Example 23 Purification by combining affinity chromatography and anion exchange chromatography (part 2) (1) The AVR29c column prepared in Example 17 was equilibrated with 20 mmol/L Tris-HCl buffer (pH 7.4) containing 500 mmol/L sodium chloride (hereinafter also referred to as "equilibration solution D").
• Example 5 The AAV8-EGFP solution obtained in Example 5 was applied to an AVR29c column, washed with equilibration solution D, and then eluted with 20 mmol/L Tris-HCl buffer (pH 7.4) containing 2.0 mol/L magnesium chloride to obtain a solution of AAV8-EGFP, which is an AAV vector.
• solution AEX-C Tris-HCl buffer (pH 9.0) containing 300 mmol/L choline chloride
• the ratio of solution AEX-C was (a) 49.0% (conductivity: 13.3 mS/cm (millisiemens per centimeter)); (b) 48.0% (conductivity: 13.0 mS/cm), The mixture ratio was adjusted to either (c) 47.0% (conductivity: 12.8 mS/cm) or (d) 46.5% (conductivity: 12.7 mS/cm), and impurities were eluted by running the solution for 30 to 60 CV while maintaining this condition.
• the obtained chromatogram is shown in Figure 8.
• the fullness of the AAV vector contained in the elution fractions obtained in (5) (peak indicated by the white arrow in Figure 8) and (6) (peak indicated by the black arrow in Figure 8) was measured and the results are shown in Table 16. It can be seen that a solution containing AAV vector with a higher fullness can be obtained by setting the conductivity of the elution solution used to elute impurities to 13.5 mS/cm or less and the conductivity of the elution solution used to elute the AAV vector to 15.0 mS/cm or more.
• Example 24 Purification by combining affinity chromatography and anion exchange chromatography (part 3) (1)
• the AAV8-EGFP solution obtained in Example 5 was purified using an AVR29c column by the method described in Example 23 (1) to (3) to prepare a sample (AAV8-EGFP solution) to be applied to an anion exchange column.
• FIG 9(a) The chromatogram obtained by affinity chromatography (AF) purification (purification using an AVR29c column) is shown in Figure 9(a), and the chromatogram obtained by anion exchange chromatography (AEX) purification (purification using a SkillPak GigaCapQ column) is shown in Figure 9(b).
• Table 17 shows the results of measuring the full rate of the AAV vector contained in the AAV8-EGFP solution obtained in (1) (peak indicated by the black arrow in Figure 9(a); AF-purified fraction), (3) (peak indicated by the white arrow in Figure 9(b); Fr18), and (4) (peak indicated by the black arrow in Figure 9(b); Fr38).
• Example 25 Evaluation of infectivity after anion exchange chromatography purification
• the AF purified fraction obtained in Example 24(1), Fr18 obtained in Example 24(3), and Fr38 obtained in Example 24(4) were each dialyzed against PBS (Phosphate Buffered Saline) containing 1 mmol/L magnesium chloride, AAV-HT1080 cells (Agilent Technologies) were infected with 62,500 to 10 6 particles per cell.
• PBS Phosphate Buffered Saline
• the number of particles per cell was determined by drawing a calibration curve using a size exclusion chromatography column (G6000PW XL column).
• the cells were harvested. After suspending the solution several times with a pipette, the fluorescence intensity derived from EGFP expressed in each cell was measured using Guava easyCyte (Luminex). The proportion of cells with enhanced fluorescence intensity compared to cells to which the AAV infection solution was not added was measured to calculate the proportion of cells infected with the AAV8 mutant vector (infection rate).
• the infection amount shown on the X-axis is expressed as the number of particles per cell in Fig. 10(a), and as the number of vector genomes (VG) per cell calculated based on the Full rate measured in Example 10(5) in Fig. 10(b).
• Fig. 10(b) does not include the results of infecting cells with the particles in Fr18 obtained in Example 24(3) because this is outside the range.
• the number of vector genomes per cell was measured by qPCR.
• the PCR device was the QuantStudio3 Real-time PCR System (Thermo Fisher Scientific), and the reagents included with the AAVpro Titration Kit (Takara Bio Scientific) were used for pretreatment. DNase I treatment was performed according to the protocol, and a portion was detected by PCR.
• the PCR reagent was TaqPath qPCR Master Mix (Thermo Fisher Scientific), and the primers used were sequence numbers 59 (CTCCATCACTAGGGGTTC) and 60 (TTGGGATTCCAGGCATGC).
• the probe used was a TaqMan probe with FAM added to sequence number 61 (TCCCTTCCCTGTCCTT).
• a calibration curve was created for the vector concentration in the resulting solution using the positive standard provided with the AAVpro Titration Kit (Takara Bio).
• the adeno-associated virus (AAV) binding protein of the present disclosure 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 disclosure can have significantly improved alkaline resistance compared to conventional AAV binding proteins. Therefore, an AAV adsorbent that uses the AAV binding protein as a ligand can be washed with alkali and can be repeatedly used for AAV purification, and is therefore considered to be useful for the industrial production of AAV.
• the present disclosure is characterized by purifying AAV contained in the sample by a method including the steps of adding a sample containing AAV to an adsorbent containing an insoluble carrier and an AAV-binding protein immobilized on the carrier to adsorb the AAV to the adsorbent, eluting the AAV adsorbed to the adsorbent using an elution solution, adding a fraction containing AAV eluted in the elution step to an anion exchange chromatography carrier to adsorb the AAV to the carrier, and eluting the AAV adsorbed to the carrier using an elution solution.
• This can have the effect of easily purifying AAV contained in the sample, including AAV vectors containing a gene with a therapeutic effect (Full AAV vectors), with high purity.

Landscapes

• Life Sciences & Earth Sciences (AREA)
• Health & Medical Sciences (AREA)
• Genetics & Genomics (AREA)
• Chemical & Material Sciences (AREA)
• Organic Chemistry (AREA)
• Engineering & Computer Science (AREA)
• Zoology (AREA)
• Wood Science & Technology (AREA)
• Biotechnology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• General Engineering & Computer Science (AREA)
• General Health & Medical Sciences (AREA)
• Molecular Biology (AREA)
• Biomedical Technology (AREA)
• Biochemistry (AREA)
• Microbiology (AREA)
• Biophysics (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Physics & Mathematics (AREA)
• Plant Pathology (AREA)
• Cell Biology (AREA)
• Immunology (AREA)
• Toxicology (AREA)
• Gastroenterology & Hepatology (AREA)
• Medicinal Chemistry (AREA)
• Virology (AREA)
• Chemical Kinetics & Catalysis (AREA)
• General Chemical & Material Sciences (AREA)
• Peptides Or Proteins (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)

Priority Applications (3)

Applications Claiming Priority (8)

Publications (1)

Family

ID=93589449

Family Applications (1)

Country Status (4)

Citations (6)

• 2024
  • 2024-05-22 WO PCT/JP2024/018896 patent/WO2024242150A1/ja not_active Ceased
  • 2024-05-22 CN CN202480033591.4A patent/CN121358762A/zh active Pending
  • 2024-05-22 JP JP2025522440A patent/JPWO2024242150A1/ja active Pending
  • 2024-05-22 EP EP24811156.9A patent/EP4717706A1/en active Pending

Patent Citations (6)

Non-Patent Citations (4)

Also Published As

Similar Documents

Legal Events