Methods for analyzing adeno-associated viruses based on serotypes

Abstract

To provide a method for analyzing adeno-associated virus (AAV) included in a sample, based on a difference of serotype, while holding its structure.SOLUTION: A method for analyzing AAV includes the steps of: causing an adsorbent including an insoluble carrier and a polypeptide at least having an amino acid sequence corresponding to extracellular domain 1 and / or domain 2 of KIAA0319L immobilized to the carrier to adsorb AAV included in a sample; and eluting the AAV adsorbed to the adsorbent using an eluate.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for analyzing adeno-associated viruses (AAV), and in particular to a method for analyzing AAV contained in a sample based on its serotype. [Background technology]

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

[0003] In nature, AAV lacks the ability to replicate autonomously and depends on helper viruses such as adenoviruses and herpesviruses for replication. In the presence of the helper virus, the AAV genome replicates in the host cell, forming complete AAV particles containing the AAV genome, which are then released from the host cell. On the other hand, in the absence of the helper virus, the AAV genome is maintained in an episome or integrated into the host chromosome (latent state).

[0004] AAV is capable of infecting 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 also non-pathogenic to humans, meaning there is little risk of side effects. Furthermore, the viral particles are physically and chemically stable. For these reasons, AAV is attracting attention as a potential vector for gene transfer aimed at treating congenital genetic diseases.

[0005] Recombinant AAV vectors (hereafter simply referred to as AAV vectors) are typically produced by introducing nucleic acids encoding elements essential for AAV particle formation into cells to generate 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. The produced AAV vectors are recovered and purified from the AAV-producing cells to obtain therapeutic AAV vector formulations.

[0006] AAV vectors exist in multiple serotypes, each with different cell tropism and infectivity (Non-Patent Document 1). Therefore, analyzing the differences in serotypes while maintaining their structure is essential for understanding the properties of AAV vectors. Conventional methods for analyzing AAV vectors while maintaining their structure include transmission electron microscopy, gel filtration analysis, and dynamic light scattering. However, while these methods can provide information such as size and shape, analysis based on differences in serotype has been difficult. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Ellis B.et al.,Virology Journal,74,10,2013 Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is to provide a method for analyzing adeno-associated viruses contained in a sample based on differences in serotype while maintaining their structure. [Means for solving the problem]

[0009] As a result of extensive research, the present inventors discovered that by analyzing adeno-associated virus (AAV) contained in a sample using an adsorbent containing an insoluble carrier and an AAV-binding protein immobilized on the carrier, AAV can be analyzed based on differences in serotype, leading to the completion of the present invention.

[0010] That is, the present invention includes the following aspects.

[0011] [1] A method for analyzing adeno-associated viruses (AAV) contained in a sample based on differences in serotypes, comprising: The method is carried out by a method including a step of adsorbing the AAV to an adsorbent comprising an insoluble carrier and an AAV-binding protein immobilized on the carrier, and a step of eluting the AAV adsorbed to the adsorbent using an elution solution, and wherein the AAV-binding protein is a polypeptide selected from the following (i) to (iii): (i) a polypeptide containing at least the amino acid residues from serine at position 312 to glutamic acid at position 401 or the amino acid residues from isoleucine at position 409 to aspartic acid at position 500 in the amino acid sequence set forth in SEQ ID NO: 1; (ii) A polypeptide having an amino acid sequence comprising at least the amino acid residues from serine at position 312 to glutamic acid at position 401 or from isoleucine at position 409 to aspartic acid at position 500 of the amino acid sequence set forth in SEQ ID NO: 1, wherein the amino acid sequence comprises substitution, deletion, insertion, or addition of one or several amino acid residues at one or several positions within the amino acid residues at positions 312 to 401 or the amino acid residues at positions 409 to 500, and having AAV-binding activity; (iii) A polypeptide comprising at least the amino acid residues from serine at position 312 to glutamic acid at position 401 or from isoleucine at position 409 to aspartic acid at position 500 of the amino acid sequence set forth in SEQ ID NO: 1, wherein the polypeptide has 70% or more homology to the amino acid sequence consisting of the amino acid residues at positions 312 to 401 or the amino acid residues at positions 409 to 500, and wherein the polypeptide has AAV-binding activity.

[0012] [2] The method according to [1], wherein the AAV-binding protein is a polypeptide selected from any one of the following (iv) to (vi): (iv) a polypeptide containing at least the amino acid residues from serine at position 312 to aspartic acid at position 500 in the amino acid sequence set forth in SEQ ID NO: 1; (v) A polypeptide having an amino acid sequence containing at least the amino acid residues from serine at position 312 to aspartic acid at position 500 of the amino acid sequence set forth in SEQ ID NO: 1, provided that the amino acid sequence contains substitution, deletion, insertion, or addition of one or several amino acid residues at one or several positions within the amino acid residues at positions 312 to 500, and having AAV-binding activity; (vi) A polypeptide comprising at least the amino acid residues from serine at position 312 to aspartic acid at position 500 of the amino acid sequence set forth in SEQ ID NO: 1, wherein the polypeptide has 70% or more homology to the amino acid sequence consisting of the amino acid residues at positions 312 to 500, and wherein the polypeptide has AAV-binding activity.

[0013] The present invention will be described in detail below.

[0014] The AAV-binding protein, which is the ligand protein (protein immobilized on an insoluble carrier) of the adsorbent used in the analysis of AAV in the present invention (hereinafter also referred to as "AAV adsorbent"), is a polypeptide that has AAV-binding activity and contains at least a region corresponding to extracellular domain 1 (PKD1) (amino acid residues from serine at position 312 to glutamic acid at position 401 in SEQ ID NO: 1) or domain 2 (PKD2) (amino acid residues from isoleucine at position 409 to aspartic acid at position 500 in SEQ ID NO: 1) of KIAA0319L (UniProt accession number: Q8IZA0, SEQ ID NO: 1), an embodiment of the AAV receptor (AAVR). Specific examples include polypeptides selected from any of (i) to (iii) below. (i) a polypeptide containing at least the amino acid residues from serine at position 312 to glutamic acid at position 401 or the amino acid residues from isoleucine at position 409 to aspartic acid at position 500 in the amino acid sequence set forth in SEQ ID NO: 1; (ii) A polypeptide having an amino acid sequence comprising at least the amino acid residues from serine at position 312 to glutamic acid at position 401 or from isoleucine at position 409 to aspartic acid at position 500 of the amino acid sequence set forth in SEQ ID NO: 1, wherein the amino acid sequence comprises substitution, deletion, insertion, or addition of one or several amino acid residues at one or several positions within the amino acid residues at positions 312 to 401 or the amino acid residues at positions 409 to 500, and having AAV-binding activity; (iii) A polypeptide comprising at least the amino acid residues from serine at position 312 to glutamic acid at position 401 or from isoleucine at position 409 to aspartic acid at position 500 of the amino acid sequence set forth in SEQ ID NO: 1, wherein the polypeptide has 70% or more homology to the amino acid sequence consisting of the amino acid residues at positions 312 to 401 or the amino acid residues at positions 409 to 500, and wherein the polypeptide has AAV-binding activity.

[0015] Furthermore, preferred embodiments of AAV-binding proteins include polypeptides comprising at least regions corresponding to extracellular domain 1 (PKD1) and domain 2 (PKD2) of KIAA0319L (UniProt accession number: Q8IZA0, SEQ ID NO: 1) (amino acid residues from serine (Ser) at position 312 to aspartic acid (Asp) at position 500 of SEQ ID NO: 1), and specifically include polypeptides selected from any of (iv) to (vi) below. (iv) a polypeptide containing at least the amino acid residues from serine at position 312 to aspartic acid at position 500 in the amino acid sequence set forth in SEQ ID NO: 1; (v) A polypeptide having an amino acid sequence containing at least the amino acid residues from serine at position 312 to aspartic acid at position 500 of the amino acid sequence set forth in SEQ ID NO: 1, provided that the amino acid sequence contains substitution, deletion, insertion, or addition of one or several amino acid residues at one or several positions within the amino acid residues at positions 312 to 500, and having AAV-binding activity; (vi) A polypeptide comprising at least the amino acid residues from serine at position 312 to aspartic acid at position 500 of the amino acid sequence set forth in SEQ ID NO: 1, wherein the polypeptide has 70% or more homology to the amino acid sequence consisting of the amino acid residues at positions 312 to 500, and wherein the polypeptide has AAV-binding activity.

[0016] The polypeptides described in any of (i) to (vi) above need only contain at least the region corresponding to PKD1 and / or PKD2 of the aforementioned KIAA0319L, and may, for example, include all or part of the region corresponding to other extracellular domains located on the C-terminal side of PKD2 (domain 3 (PKD3), domain 4 (PKD4), and domain 5 (PKD5)), or may include all or part of the region corresponding to a signal sequence or 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 include all or part of the transmembrane region and intracellular region located on the N-terminal and / or C-terminal side of the extracellular region.

[0017] Examples of (ii) and (v) include a polypeptide containing at least the amino acid residues from serine at position 25 to aspartic acid at position 213 in the amino acid sequence set forth in SEQ ID NO: 4, and the AAV-binding protein disclosed in WO2021 / 106882. Examples of the substitution, deletion, insertion, or addition described in (ii) and (v) include the amino acid residue substitutions disclosed in WO2021 / 106882.

[0018] In (ii) and (v), "one or several" varies depending on the position of the amino acid substitution in the three-dimensional structure of AAVR and the type of amino acid residue, but for example, it means 1 to 50, 1 to 30, 1 to 20, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1. The substitution of "one or several" amino acid residues may occur at positions other than those of the amino acid residues disclosed in WO2021 / 106882, for example, as long as it has AAV-binding activity.

[0019] The "substitution of one or several amino acid residues" in (ii) and (v) above may include not only the amino acid substitution at the specific position described above, but also conservative substitutions in which amino acids with similar physical and / or chemical properties are substituted. Those skilled in the art know that conservative substitutions generally maintain protein function between substituted and unsubstituted amino acids. Examples of conservative substitutions include substitutions between glycine and alanine, between serine and proline, or between glutamic acid and alanine (Protein Structure and Function, Medical Science International, 9, 2005). Furthermore, the "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) due to differences in the origin of AAVR or differences in species.

[0020] The homology of the amino acid sequences in (iii) and (vi) may be 70% or more, but may be even higher (e.g., 80% or more, 85% or more, 90% or more, or 95% or more). As used herein, "homology" may refer to similarity or identity, and may particularly refer to identity. "Amino acid sequence homology" refers to homology across the entire amino acid sequence. "Identity" between amino acid sequences refers to the proportion of identical amino acid residues in those amino acid sequences (Experimental Medicine, 31(3), Yodosha). "Similarity" between amino acid sequences refers to the sum of the proportion of identical amino acid residues and the proportion of amino acid residues with similar side chain properties in those amino acid sequences (Experimental Medicine, 31(3), Yodosha). Amino acid sequence homology can be determined using alignment programs such as BLAST (Basic Local Alignment Search Tool) and FASTA.

[0021] In the present invention, the insoluble carrier used for immobilizing the AAV-binding protein is not particularly limited as long as it is insoluble in AAV-containing samples and solutions used for analysis and purification (e.g., elution solutions, equilibration solutions, and wash solutions). Examples include carriers made from polysaccharides such as agarose, alginate (alginate salts), carrageenan, chitin, cellulose, dextrin, dextran, and starch; carriers made from synthetic polymers such as polyvinyl alcohol, polymethacrylate, poly(2-hydroxyethyl methacrylate), and polyurethane; and carriers made from ceramics such as silica. Among these, carriers made from polysaccharides and synthetic polymers are preferred as insoluble carriers. Examples of preferred carriers include polymethacrylate gels with hydroxyl groups introduced, such as Toyopearl (manufactured by Tosoh Corporation), agarose gels such as Sepharose (manufactured by Cytiva), and cellulose gels such as Cellufine (manufactured by JNC). The shape of the insoluble carrier is not particularly limited, but a shape that can be packed into a column is preferred. For example, the material may be in the form of particles, monoliths, membranes, or fibers, and may be either porous or non-porous.

[0022] The AAV-binding protein can be immobilized on an insoluble carrier, for example, via a covalent bond. Specifically, for example, the AAV-binding protein can be immobilized on the insoluble carrier by covalently bonding the insoluble carrier via an active group possessed by the insoluble carrier, thereby producing the AAV adsorbent used in the present invention. Examples of the active group include an N-hydroxysuccinimide (NHS)-activated ester group, an epoxy group, a carboxy group, a maleimide group, a haloacetyl group, a tresyl group, a formyl group, and a haloacetamide group. As the insoluble carrier having an active group, for example, a commercially available insoluble carrier having an active group can be used as is, or an active group can be introduced into the insoluble carrier. Examples of commercially available supports having active groups include TOYOPEARL AF-Epoxy-650M and TOYOPEARL AF-Tresyl-650M (both manufactured by Tosoh Corporation), HiTrap NHS-activated HP Columns, NHS-activated Sepharose 4 Fast Flow, and Epoxy-activated Sepharose 6B (all manufactured by Cytiva), and SulfoLink Coupling Resin (manufactured by Thermo Fisher Scientific).

[0023] An example of a method for introducing active groups onto the support surface is to react one of a compound having two or more active sites with a hydroxy group, epoxy group, carboxy group, amino group, etc. present on the support surface.

[0024] Examples of compounds that introduce epoxy groups into hydroxyl or amino groups present on the support surface include epichlorohydrin, ethanediol diglycidyl ether, butanediol diglycidyl ether, hexanediol diglycidyl ether, and polyethylene glycol diglycidyl ether. Specific examples of polyethylene glycol diglycidyl ether include Denacol EX-810 (n=1), Denacol EX-811 (n=1), Denacol EX-850 (n=2), Denacol EX-851 (n=2), Denacol EX-821 (n=4), Denacol EX-830 (n=9), Denacol EX-832 (n=9), Denacol EX-841 (n=13), and Denacol EX-861 (n=22) (all manufactured by Nagase ChemteX Corporation).

[0025] Examples of compounds that introduce carboxy groups into epoxy groups present on the surface of the carrier include 2-mercaptoacetic acid, 3-mercaptopropionic acid, 4-mercaptobutyric acid, 6-mercaptobutyric acid, glycine, 3-aminopropionic acid, 4-aminobutyric acid, and 6-aminohexanoic acid.

[0026] Compounds that introduce maleimide groups into hydroxyl, epoxy, carboxyl, and amino groups present on the support surface include N-(ε-maleimidocaproic acid) hydrazide, N-(ε-maleimidopropionic acid) hydrazide, 4-(4-N-maleimidophenyl)acetic acid hydrazide, 2-aminomaleimide, 3-aminomaleimide, 4-aminomaleimide, 6-aminomaleimide, 1-(4-aminophenyl)maleimide, 1-(3-aminophenyl)maleimide, 4-(maleimido)phenyl isocyanate, 2-maleimidoacetic acid, and 3-maleimidopropionic acid. Examples include 4-maleimidobutyric acid, 6-maleimidohexanoic acid, N-(α-maleimidoacetoxy)succinimide ester, (m-maleimidobenzoyl)N-hydroxysuccinimide ester, succinimidyl-4-(maleimidomethyl)cyclohexane-1-carbonyl-(6-aminohexanoic acid), succinimidyl-4-(maleimidomethyl)cyclohexane-1-carboxylic acid, (p-maleimidobenzoyl)N-hydroxysuccinimide ester, (m-maleimidobenzoyl)N-hydroxysuccinimide ester, and dextran.

[0027] Furthermore, examples of compounds that introduce haloacetyl groups into hydroxy groups or amino groups present on the surface of a carrier include chloroacetic acid, bromoacetic acid, iodoacetic acid, chloroacetic acid chloride, bromoacetic acid chloride, bromoacetic acid bromide, chloroacetic acid anhydride, bromoacetic acid anhydride, iodoacetic acid anhydride, 2-(iodoacetamido)acetic acid-N-hydroxysuccinimide ester, 3-(bromoacetamido)propionic acid-N-hydroxysuccinimide ester, and 4-(iodoacetyl)aminobenzoic acid-N-hydroxysuccinimide ester.

[0028] Another example of a method for introducing active groups onto the support surface is to react hydroxyl or amino groups present on the support surface with an ω-alkenyl alkane glycidyl ether, followed by activation by halogenating the ω-alkenyl moiety with a halogenating agent. Examples of ω-alkenyl alkane glycidyl ethers include allyl glycidyl ether, 3-butenyl glycidyl ether, and 4-pentenyl glycidyl ether. Examples of halogenating agents include N-chlorosuccinimide, N-bromosuccinimide, and N-iodosuccinimide.

[0029] Another example of a method for introducing active groups onto the support surface is to use a condensing agent and an additive to introduce active groups into carboxy groups present on the support surface. Examples of condensing agents include 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), dicyclohexylcarbodiamide, and carbonyldiimidazole. Examples of additives include NHS, 4-nitrophenol, and 1-hydroxybenzotriazole.

[0030] Immobilization of an AAV-binding protein on an insoluble carrier can be carried out, for example, in a buffer solution. Examples of buffer solutions include acetate buffer, phosphate buffer, MES (2-MorpholinoEthaneSulfonic acid) buffer, HEPES (4-(2-HydroxyEthyl)-1-PiperazineEthaneSulfonic acid) buffer, Tris (Tris(hydroxymethyl)aminomethane) buffer, and borate buffer. The reaction temperature during immobilization can be appropriately set depending on various conditions, such as the reactivity of the active group and the stability of the AAV-binding protein. The reaction temperature during immobilization may be, for example, 4°C or higher and 50°C or lower, and preferably 10°C or higher and 35°C or lower.

[0031] The present invention realizes AAV analysis based on differences in serotypes by analyzing AAV contained in a sample using a method comprising the steps of adsorbing AAV contained in the sample to an AAV adsorbent prepared by the method described above (hereinafter also referred to simply as the "adsorption step") and eluting the AAV adsorbed to the adsorbent using an elution solution (hereinafter also referred to simply as the "elution step"). It is preferable that the AAV adsorbent used in the adsorption step and the elution step be packed into a column (hereinafter also referred to as an "AVR column"), as this simplifies and simplifies these steps. An example of using an AVR column will be described in detail below.

[0032] A sample containing AAV can be applied to an AVR column using a liquid delivery device such as a pump. Herein, adding a liquid to a column is also referred to as "delivering a liquid to the column." The AAV-containing sample may be solvent-exchanged with an appropriate buffer solution before application to the AVR column. Furthermore, the AVR column may be equilibrated with an appropriate buffer solution (equilibration solution) before application of the AAV-containing sample to the AVR column (i.e., before the adsorption step). This equilibration is expected to enable, for example, more accurate analysis of AAV. Examples of buffer solutions used for solvent exchange and equilibration include phosphate buffer, acetate buffer, succinate buffer, citrate buffer, Tris buffer, HEPES buffer, and MES buffer, all of which have buffering capacity in the neutral range (referring to a pH range of 4.0 to 9.0 in this specification). Such buffer solutions may further contain inorganic salts, such as sodium chloride or calcium chloride, at a concentration of 10 mM to 600 mM. The buffer solution used for solvent exchange and the equilibration solution may or may not be the same. Furthermore, if components other than AAV, such as contaminants, remain on the AVR column after passing an AAV-containing sample through the AVR column, such components may be removed (washed) from the AVR column before eluting the AAV adsorbed to the AAV adsorbent with an eluent (i.e., before the elution step). Components other than AAV can be removed from the AVR column, for example, by using an appropriate buffer as a wash solution. Regarding the wash solution, the same descriptions as for buffers used for solvent substitution and equilibration can be applied mutatis mutandis.

[0033] In the elution step, AAV is eluted based on differences in serotype using a gradient. The gradient may be changed in two or more steps (stepwise) (step gradient) or linearly (linear gradient). An example of gradient elution is a gradient that decreases the pH from the neutral range described above to the acidic range (a range more acidic than the neutral range described above). The initial pH of the eluate should be in the neutral range described above, i.e., pH 4.0 to 9.0, and preferably pH 4.5 to 6.0. The pH of the eluate after the gradient should be in the acidic range, i.e., pH 3.5 or less, and preferably pH 2.0 to 2.5. The flow rate has no lower limit, but the upper limit depends on the backpressure of the insoluble carrier used in the AAV adsorbent. Typically, a flow rate of 0.5 mL / min to 2.0 mL / min is preferred. The gradient time is sufficient as long as differences in serotype can be identified as differences in elution time. Specifically, it is preferably 5 minutes or more, more preferably 20 minutes or more, and even more preferably 45 minutes or more.

[0034] The serotypes that can be analyzed by the method of the present invention may be naturally occurring AAVs or artificially produced AAVs, such as 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), serotype 13 (AAV13), AAVrh8, and AAVrh10. [Effects of the Invention]

[0035] The present invention is characterized by a method for analyzing the serotypes of adeno-associated viruses (AAV) contained in a sample, comprising the steps of adsorbing the AAV to an adsorbent comprising an insoluble carrier and a polypeptide containing at least an amino acid sequence corresponding to extracellular domain 1 and / or domain 2 of KIAA0319L immobilized on the carrier, and eluting the AAV adsorbed to the adsorbent with an elution solution. The present invention enables analysis of the serotypes of AAV contained in a sample while maintaining their structure, something that was not possible with conventional methods. [Brief explanation of the drawings]

[0036] [Figure 1] The figures show the results (chromatographic patterns) of the analysis of AAVX-EGFP solutions using an AVRwild column: (a) AAV1-EGFP solution, (b) AAV2-EGFP solution, (c) AAV5-EGFP solution, (d) AAV8-EGFP solution, and (e) AAV9-EGFP solution. [Figure 2] The figures show the results (chromatographic patterns) of the analysis of AAVX-EGFP solutions using an AVR10s column: (a) AAV1-EGFP solution, (b) AAV2-EGFP solution, (c) AAV5-EGFP solution, (d) AAV8-EGFP solution, and (e) AAV9-EGFP solution. [Figure 3] Figure 1 shows the results (chromatographic patterns) of AAV solutions analyzed using an AVR10s column: (a) AAV8-EGFP solution, (b) commercially available AAV8 solution, and (c) commercially available AAVrh10 solution. [Figure 4] Figure 1 shows the results (particle size distribution) of AAVX-EGFP solutions analyzed using dynamic light scattering: (a) AAV1-EGFP solution, (b) AAV2-EGFP solution, (c) AAV5-EGFP solution, (d) AAV8-EGFP solution, and (e) AAV9-EGFP solution. [Figure 5]FIG. 1 shows the results (chromatographic patterns) of analysis of an AAV1-EGFP solution, an AAV2-EGFP solution, and a mixed solution thereof using an AVR10s column. [Example]

[0037] The present invention will be described in more detail below using examples and comparative examples, but the present invention is not limited to these examples.

[0038] Example 1 Preparation of Adeno-Associated Virus (AAV) Vectors (1) A nucleotide sequence (SEQ ID NO: 3) was designed in which a restriction enzyme EcoRI recognition sequence (GAATTC) was added to the 5' end of a polynucleotide encoding EGFP (Enhanced Green Fluorescent Protein) consisting of the amino acid sequence set forth in SEQ ID NO: 2, and a stop codon (TAG) and a BamHI recognition sequence (GGATTC) were added to the 3' end.

[0039] (2) A polynucleotide consisting of the sequence set forth in SEQ ID NO: 3 was totally synthesized and cloned into a plasmid (commissioned to FASMAC, named pUC-EGFP). Escherichia 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).

[0040] (3) The pUC-EGFP obtained in (2) was digested with the restriction enzymes EcoRI and BamHI, and then ligated into the expression vector pAAV-CMV (Takara Bio Inc.) which had been previously digested with the restriction enzymes EcoRI and BamHI. The ligation product was used to transform the Escherichia coli JM109 strain.

[0041] (4) The transformant obtained in (3) was cultured overnight at 37°C with shaking in a 5-L baffled flask containing 1 L of 2YT medium (1.6% (w / v) Tryptone, 1% (w / v) Yeast Extract, 0.5% (w / v) sodium chloride) containing 100 μg / mL carbenicillin. After the culture was completed, the cells were collected by centrifugation. The EGFP-expressing vector pAAV-EGFP was prepared in large quantities from the collected cells using a Plasmid Mega Kit (Qiagen).

[0042] (5) The Escherichia coli JM109 strain was transformed with a plasmid containing a polynucleotide encoding any one of the capsids of serotype 1 (AAV1), serotype 2 (AAV2), serotype 5 (AAV5), serotype 8, and serotype 9 (AAV9) (collectively referred to as "pRCX Vector") and the pHelper Vector (Takara Bio Inc.). The resulting transformants were used in the same manner as in (4) to prepare large quantities of pRCX Vector and pHelper.

[0043] (6) HEK293T cells were cultured in eight Cellstack cell culture surface-treated 5-chamber (Corning) plates containing 500 mL of D-MEM medium (Fujifilm Wako Pure Chemical Industries, Ltd.) containing 5% (v / v) bovine serum. Gene transfection was performed by adding the pAAV-EGFP prepared in (4), the pRCX Vector and pHelper prepared in (5), and a complex of polyethyleneimine (Polysciences), and the cells were cultured statically for 3 days under conditions of 5% (v / v) carbon dioxide and 37°C.

[0044] (7) To the culture medium obtained in (6), Triton X-100 (Sigma) (final concentration 0.1%) and Benzonase (Merck Millipore) (final concentration 1 U / mL) were added to a final concentration of 0.1%, and the medium was incubated at 37°C for 3 hours. After centrifugation, the supernatant was collected and concentrated using a 300 kD-cutoff membrane cassette for a tangential flow system (Pall). The supernatant was then replaced with 20 mM Tris-HCl buffer (pH 8.0) containing 0.5 M sodium chloride (hereinafter referred to as "equilibration solution A"). The resulting concentrate was passed through a 0.22 μm pore filter to remove suspended matter.

[0045] (8) The solution from which the suspended matter had been removed was applied to a 7 mL AVB Sepharose column (manufactured by Cytiva) or a POROS AAVX column (manufactured by Thermo Fisher Scientific).

[0046] (9) After washing with equilibration solution A, the column was eluted with 0.1 M acetate buffer (pH 2.5) containing 0.5 M sodium chloride. The resulting eluted fraction containing the AAV vector was neutralized with 1 / 4 volume of 1 M Tris-HCl buffer (pH 8.5) containing 20 mM magnesium chloride to obtain solutions of the AAV vectors of each serotype: AAV1-EGFP, AAV2-EGFP, AAV5-EGFP, AAV8-EGFP, and AAV9-EGFP (hereinafter collectively referred to as "AAVX-EGFP").

[0047] (10) The AAVX-EGFP concentration in the solution obtained in (9) was quantified by qPCR using the AAVpro Titration Kit (Takara Bio Inc.).

[0048] As a result, the concentration of AAV in the AAVX-EGFP solution was 8.2 × 10 12 cp / mL (cp indicates the number of AAV particles; the same applies below), and the AAV2-EGFP solution was 1.7 × 10 13 cp / mL, and the AAV5-EGFP solution was 2.5 x 10 13 cp / mL, and the AAV8-EGFP solution was 3.5 x 1013 cp / mL, and the AAV9-EGFP solution was 4.0 x 10 13 cp / mL.

[0049] In addition, the AAVX-EGFP 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. As a result, only bands corresponding to the three types of outer capsid proteins (VP1, VP2, and VP3) that make up the AAV vector were observed in all solutions, confirming that there was no problem with purity.

[0050] Example 2 Preparation of AAV-binding proteins (part 1) (1) Escherichia coli BL21(DE3) strain was transformed with the plasmid pET-AVRwild, which contains a polynucleotide (SEQ ID NO: 7) encoding a polypeptide (SEQ ID NO: 6) containing the amino acid sequence from serine at position 312 to aspartic acid at position 500 (hereinafter also referred to as "AVRwild"), which corresponds to the region of the amino acid sequence of the AAV receptor KIAA0319L set forth in SEQ ID NO: 1, corresponding to extracellular domain 1 (PKD1) and domain 2 (PKD2). The resulting transformant capable of expressing the AAV-binding protein AVRwild was inoculated into 3 mL of 2YT liquid medium containing 50 μg / mL kanamycin and pre-cultured overnight at 37°C under aerobic shaking. In SEQ ID NO: 6, the portion from the 1st methionine (Met) to the 22nd alanine (Ala) is the PelB signal peptide, the portion from the 25th serine (Ser) to the 213th aspartic acid (Asp) is the AAV binding protein AVRwild (a polypeptide consisting of amino acid residues 312 to 500 of SEQ ID NO: 1), and the portion from the 220th cysteine ​​(Cys) to the 226th glycine (Gly) is a cysteine ​​tag sequence used as a tag for immobilization.

[0051] (2) 2 mL of the preculture solution from (1) was inoculated into 200 mL of 2YT liquid medium containing 50 μg / mL of kanamycin in a 1 L baffled flask, and the mixture was cultured aerobically at 37°C with shaking.

[0052] (3) After 2.0 hours of culturing, the mixture was cooled on ice, IPTG (IsoPropyl-β-D-ThioGalactopyranoside) was added to a final concentration of 0.1 mM, and the mixture was then cultured aerobically with shaking at 37°C for 3 hours.

[0053] (4) After the cultivation was completed, the culture medium was centrifuged at 4°C and 8000 rpm for 20 minutes to collect the bacterial cells.

[0054] (5) The cells recovered in (4) were suspended in 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride and 20 mM imidazole at a concentration of 5 mL / 1 g (cells), and then disrupted using an ultrasonic generator (Insonator 201M [Kubota Shoji Co., Ltd.]) at 8°C for approximately 10 minutes at an output of approximately 150 W. The disrupted cell solution was centrifuged twice at 8000 rpm for 20 minutes at 4°C, and the supernatant was recovered from each suspension.

[0055] (6) The supernatant obtained in (5) was applied to an XK26 / 20 column (manufactured by Cytiva) packed with 50 mL of Ni Sepharose 6 Fast Flow (manufactured by Cytiva) that had been equilibrated with Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride and 20 mM imidazole (hereinafter also referred to as "equilibration solution B"). After washing with equilibration solution B, the column was eluted with 20 mM Tris-HCl buffer (pH 7.4) containing 0.5 M imidazole and 150 mM sodium chloride.

[0056] (7) Each eluate obtained in (6) was dialyzed against 20 mM Tris buffer (pH 7.4) containing 150 mM sodium chloride to prepare the amount of AVRwild required for producing the AAV adsorbent.

[0057] Example 3 Preparation of AAV-binding proteins (part 2) The AAV binding protein AVR10s was prepared in the same manner as in Example 2, except that a transformant capable of expressing AVR10s was used, which was obtained by transforming the Escherichia coli BL21(DE3) strain with the plasmid pET-AVR10s containing a polynucleotide (SEQ ID NO: 5) encoding a polypeptide containing the AAV binding protein AVR10s (SEQ ID NO: 4). In SEQ ID NO: 4, the PelB signal peptide is located from methionine (Met) at position 1 to alanine (Ala) at position 22, the AAV binding protein AVR10s is located from serine (Ser) at position 25 to aspartic acid (Asp) at position 213, and the cysteine ​​tag sequence, which is a tag for immobilization, is located from cysteine ​​(Cys) at position 220 to glycine (Gly) at position 226. Furthermore, AVR10s is a polypeptide in which the amino acid substitutions shown in (I) to (X) below have occurred relative to AVRwild (amino acid residues from serine at position 312 to aspartic acid at position 500 in SEQ ID NO: 1). (I) Valine at position 317 of SEQ ID NO: 1 (position 30 of SEQ ID NO: 4) is substituted with aspartic acid (II) Tyrosine at position 342 of SEQ ID NO: 1 (position 55 of SEQ ID NO: 4) is substituted with serine (III) Lysine at position 362 of SEQ ID NO: 1 (position 75 of SEQ ID NO: 4) is substituted with glutamic acid (IV) Lysine at position 371 of SEQ ID NO: 1 (position 84 of SEQ ID NO: 4) is substituted with asparagine (V) Valine at position 381 of SEQ ID NO: 1 (position 94 of SEQ ID NO: 4) is substituted with alanine (VI) Isoleucine at position 382 of SEQ ID NO: 1 (position 95 of SEQ ID NO: 4) is substituted with valine (VII) Glycine at position 390 of SEQ ID NO: 1 (position 103 of SEQ ID NO: 4) is substituted with serine (VIII) Lysine at position 399 of SEQ ID NO: 1 (position 112 of SEQ ID NO: 4) is substituted with glutamic acid (IX) Serine at position 476 of SEQ ID NO: 1 (position 189 of SEQ ID NO: 4) is substituted with arginine (X) Asparagine at position 487 of SEQ ID NO: 1 (position 200 of SEQ ID NO: 4) is substituted with aspartic acid Example 4 Preparation of AAV adsorbent (1) A hydrophilic vinyl polymer for separation agents (Toyopearl, manufactured by Tosoh Corporation) was prepared as an insoluble carrier, and a carrier with iodoacetamide groups was prepared by chemically modifying the hydroxyl groups on the polymer surface.

[0058] (2) To 7 g (wet weight) of the carrier prepared in (1), 14 mg of AVRwild prepared in Example 2 or AVR10s prepared in Example 3 and TCEP (Tris(2-CarboxyEthyl)Phosphine) at a final concentration of 0.3 mM as a reducing agent were added, and the mixture was allowed to react by shaking at pH 8.1 and 25°C for 3 hours. This resulted in the preparation of an AVRwild-immobilized gel, which is an AAV adsorbent in which AVRwild is immobilized, and an AVR10s-immobilized gel, which is an AAV adsorbent in which AVR10s is immobilized.

[0059] Example 5 AAV analysis using AAV adsorbent (part 1) (1) 1.25 mL of the AVRwild immobilization gel and AVR10s immobilization gel prepared in Example 4 were packed into empty stainless steel columns (φ4.6 mm × 75 mm, manufactured by Tosoh Corporation) to prepare columns (hereinafter, the column packed with the AVRwild immobilization gel is referred to as the AVRwild column, and the column packed with the AVR10s immobilization gel is referred to as the AVR10s column).

[0060] (2) Each column prepared in (1) was connected to an HPLC M40A (Shimadzu Corporation) and equilibrated with 10 mM glycine buffer (pH 4.5) containing 50 mM calcium chloride and 10 mM sodium acetate (hereinafter also referred to as "equilibration solution C").

[0061] (3) To each column equilibrated in (2), 0.01 mL of any of the AAVX-EGFP solutions obtained in Example 1 (AAV1-EGFP solution, AAV2-EGFP solution, AAV5-EGFP solution, AAV8-EGFP solution, and AAV9-EGFP solution) was applied.

[0062] (4) After washing with equilibration solution C for 10 minutes at a flow rate of 0.5 mL / min, 10 mM glycine buffer (pH 2.5) containing 50 mM calcium chloride and 10 mM sodium acetate (hereinafter also referred to as "eluent A") and equilibration solution C were pumped through each column at a flow rate of 0.5 mL / min, so as to obtain a linear gradient of eluent A concentration from 0% to 100% over 45 minutes. After the linear gradient was completed, eluent A was pumped for 3 minutes, and then equilibration solution C was pumped through each column again to re-equilibrate.

[0063] (5) AAVX-EGFP eluted from the AAV adsorbent packed in each column was detected by fluorescence intensity at 350 nm in response to excitation light at 280 nm.

[0064] The chromatographic patterns obtained with the AVRwild column are shown in Figure 1, and those obtained with the AVR10s column are shown in Figure 2. With the AVRwild column, peaks appeared for AAV1-EGFP at approximately 30 min (Figure 1(a)), AAV2-EGFP at approximately 37 min (Figure 1(b)), AAV5-EGFP at approximately 47 min (Figure 1(c)), AAV8-EGFP at approximately 30 min (Figure 1(d)), and AAV9-EGFP at approximately 32 min (Figure 1(e)). On the AVR10s column, AAV1-EGFP peaked at approximately 46 minutes (Fig. 2(a)), AAV2-EGFP peaked at approximately 38 minutes (Fig. 2(b)), AAV5-EGFP peaked at approximately 45 minutes (Fig. 2(c)), AAV8-EGFP peaked at approximately 38 minutes (Fig. 2(d)), and AAV9-EGFP peaked at approximately 41 minutes (Fig. 2(e)).

[0065] These results demonstrate that both the AVRwild column and the AVR10s column, which are columns packed with AAV adsorbents, can analyze AAV contained in samples based on differences in serotype. In the AVRwild column, the following are based on the differences between AAV1 / AAV8 / AAV9, AAV2, and AAV5: In the AVR10s column, the differences between AAV1 / AAV5, AAV2 / AAV8, and AAV9 are shown. It turns out that each can be analyzed.

[0066] Example 6 AAV analysis using AAV adsorbent (part 2) (1) The AVR10s column prepared in Example 5(1) was connected to an HPLC M40A (Shimadzu Corporation) and equilibrated with 50 mM sodium acetate buffer (pH 4.5) containing 50 mM calcium chloride (hereinafter also referred to as "equilibration solution D").

[0067] (2) The AVR10s column equilibrated in (1) was loaded with the AAV8-EGFP solution obtained in Example 1 and a commercially available AAV8 solution (concentration: 5.0 × 10 11 cp / mL) and AAVrh10 solution (concentration: 1.5 × 10 12 cp / mL) (both manufactured by VectorBuilder) was applied in an amount of 0.05 mL.

[0068] (3) After washing with equilibration solution D for 10 minutes at a flow rate of 0.5 mL / min, 20 mM glycine buffer (pH 2.0) containing 50 mM calcium chloride (hereinafter also referred to as "eluent B") and equilibration solution D were pumped through the AVR10s column (flow rate 0.5 mL / min) to achieve a linear gradient of eluent B concentration from 0% to 100% over 50 minutes. After the linear gradient was completed, eluent B was pumped for 10 minutes, and then equilibration solution D was pumped through the AVR10s column again to re-equilibrate.

[0069] (4) Each AAV eluted from the AAV adsorbent (AVR10s-immobilized gel) packed in the AVR10s column was detected by fluorescence intensity at 350 nm in response to excitation light at 280 nm.

[0070] The resulting chromatographic patterns are shown in Figure 3. A peak appears at 43.4 minutes for both the AAV8-EGFP solution (Figure 3(a)) and the commercially available AAV8 solution (Figure 3(b)), demonstrating that AAV8 can be analyzed based on its serotype properties, regardless of the production method. Furthermore, the commercially available AAVrh10 solution was detected at approximately 45 minutes (Figure 3(c)), demonstrating that the column packed with the AAV adsorbent can also analyze commercially available AAV based on its serotype.

[0071] Comparative Example 1: AAV analysis by dynamic light scattering (DLS) The AAVX-EGFP solutions obtained in Example 1 (AAV1-EGFP solution, AAV2-EGFP solution, AAV5-EGFP solution, AAV8-EGFP solution, and AAV9-EGFP solution) were each diluted to 4.0 × 10 13 The concentration was adjusted to cp / mL, and the particle size of AAVX-EGFP contained in each solution was determined by dynamic light scattering measurement using a Zetasizer Ultra (Malvern Panalytical).

[0072] The particle size distributions obtained are shown in Figure 4. AAV1, AAV2, and AAV5, which were analyzed using a column packed with AAV adsorbent (Figures 1 and 2) as distinct peak elution times, exhibit the same peak particle size in the particle size distribution (Figures 4(a), 4(b), and 4(c)), demonstrating the difficulty of analyzing serotype-based differences using DLS. Furthermore, AAV8 and AAV9 (Figures 4(d) and 4(e)) exhibit different peak particle sizes from AAV1, AAV2, and AAV5, but show a different trend from the chromatographic patterns obtained using a column packed with AAV adsorbent (Figures 1 and 2), demonstrating that analysis using a column packed with AAV adsorbent is not based on AAV particle size.

[0073] Example 7 AAV analysis using AAV adsorbent (part 3) AAVX-EGFP was analyzed in the same manner as in Example 5, except that an AVR10s column was used and 0.1 mL of any of the solutions shown below (α) to (ζ) was used as the AAVX-EGFP solution applied to the column. (α) AAV1-EGFP solution (1.0×10 12 (Adjust the concentration to cp / mL) (β)AAV2-EGFP solution (1.0×10 12 (Adjust the concentration to cp / mL) (γ) (α) 80% and (β) 20% mixed solution (δ) A mixed solution of (α) 60% and (β) 40% Mixture of (ε)(α) 40% and (β) 60% (ζ) A mixed solution of 20% (α) and 80% (β) The resulting chromatographic patterns are shown in Figure 5. The chromatographic patterns for (γ), (δ), (ε), and (ζ) (i.e., a mixture of AAV1-EGFP and AAV2-EGFP) showed two peaks: (α) AAV1-EGFP and (β) AAV2-EGFP, and the heights of these peaks roughly reflected the mixture ratio. These results demonstrate that by using a column packed with AAV adsorbent, it is possible to analyze the AAV of a given serotype from a sample containing a mixture of AAVs of different serotypes, and further, to analyze the mixture ratio of these AAVs.

Claims

[Claim 1] A method for analyzing adeno-associated viruses (AAV) contained in a sample based on differences in serotypes, comprising: The method is carried out by a method comprising the steps of: adsorbing the AAV to an adsorbent comprising an insoluble carrier and an AAV-binding protein immobilized on the carrier; and eluting the AAV adsorbed to the adsorbent using an elution solution; and wherein a polypeptide selected from any one of (i) to (iii) below is used as the AAV-binding protein: (i) a polypeptide containing at least the amino acid residues from serine at position 312 to aspartic acid at position 500 in the amino acid sequence set forth in SEQ ID NO: 1; (ii) A polypeptide having an amino acid sequence containing at least the amino acid residues from serine at position 312 to aspartic acid at position 500 of the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that the amino acid sequence contains a substitution, deletion, insertion, or addition of 1 to 10 amino acid residues in the amino acid residues at positions 312 to 500, and having AAV-binding activity; (iii) A polypeptide comprising at least the amino acid residues from the serine at position 312 to the aspartic acid at position 500 of the amino acid sequence set forth in SEQ ID NO: 1, wherein the polypeptide has 90% or more homology to the amino acid sequence consisting of the amino acid residues at positions 312 to 500, and has AAV-binding activity.

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