Adeno-associated virus (AAV) adsorbent, and method for isolating AAV using the AAV adsorbent.

Abstract

To provide a novel adeno-associated virus (AAV) adsorbent. [Means of Solution] The above problem is solved by providing an AAV adsorbent comprising an insoluble carrier and an adeno-associated virus (AAV) binding protein immobilized on the insoluble carrier, wherein the AAV binding protein is GPR108 (G protein-coupled receptor 108).

Description

[Technical Field]

[0001] This disclosure relates to an adeno-associated virus (AAV) adsorbent and a method for isolating AAV using the AAV adsorbent. [Background technology]

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

[0003] In nature, AAV lacks the ability to replicate independently, and its replication depends on helper viruses such as adenoviruses and herpesviruses. When these helper viruses are present, the AAV genome replicates within the host cell, forming complete AAV particles containing the AAV genome, which are then released from the host cell. On the other hand, when these helper viruses are absent, the AAV genome remains maintained in the episome or is integrated into the host chromosome (latent state).

[0004] AAV is attracting attention as a potential gene transfer vector for treating congenital genetic disorders because it can infect cells of a wide range of species, including humans, and can infect non-dividing cells that have completed differentiation, such as blood cells, muscle cells, and nerve cells; it is not pathogenic to humans, so there is little concern about side effects; and the viral particles are physicochemically stable.

[0005] GPR108 (G protein-coupled receptor 108) has been reported to be important for AAV infection (Non-Patent Literature 1). GPR108 is thought to be localized in the Golgi apparatus and is believed to play a role in one of the pathways necessary for various AAVs that have entered the cell to translocate into the nucleus and express their genes.

[0006] The production of recombinant AAV vectors (hereinafter also simply referred to as "AAV vectors") is typically carried out by introducing nucleic acids encoding elements essential for AAV particle formation into cells to create cells capable of producing AAV (hereinafter also referred to as AAV-producing cells), and then culturing these cells to express the elements essential for AAV particle formation. The manufactured AAV vectors are recovered and purified from the AAV-producing cells to obtain therapeutic AAV vector preparations.

[0007] The recovered and purified AAV vectors are evaluated for their performance using various analytical methods. For example, monodispersity in solution can be analyzed using size exclusion chromatography or dynamic light scattering; AAV vector concentration using quantitative PCR or ELISA; the full ratio of the AAV vector (the ratio of vectors containing the gene within the capsid) using anion exchange chromatography or analytical ultracentrifugation; and the infectivity of the AAV vector can be evaluated using cell infection assays. By analyzing these methods, the quality of the manufactured AAV vectors can be evaluated from various perspectives.

[0008] Furthermore, the performance of AAV vectors can also be evaluated by affinity chromatography using an AAV adsorbent containing an insoluble carrier and an AAV-binding protein immobilized on the carrier. Specifically, the infectivity of an AAV vector can be analyzed using an AAV adsorbent containing an insoluble carrier and a polypeptide containing extracellular domain 1 (PKD1) and domain 2 (PKD2) of KIAA0319L (UniProt No. Q8IZA0) immobilized on the carrier (Patent Document 1). [Prior art documents] [Patent Documents]

[0009] [Patent Document 1] WO2023 / 140197 [Non-Patent Document]

[0010] [Non-Patent Document 1] Amanda M.Dudek et.al.,Mol.Ther,Vol.28,No.2,367 - 381,2020 [Summary of the Invention] [[ID=!8]] [Problems to be Solved by the Invention]

[0011] The problem of the present disclosure is to provide a novel adeno-associated virus (AAV) adsorbent. Another problem in one aspect is to provide a method for separating AAV using the AAV adsorbent. Another problem in one aspect is to provide a novel AAV adsorbent capable of analyzing the quality of AAV and a method for analyzing the quality of AAV using the AAV adsorbent. Another problem in another aspect is to provide a novel AAV adsorbent capable of analyzing the degree of degradation and / or infectivity of AAV and a method for analyzing the degree of degradation and / or infectivity of AAV using the AAV adsorbent. Another problem in another aspect is to provide a novel AAV adsorbent capable of analyzing the presence or absence of a gene in the capsid of AAV and a method for analyzing the presence or absence of a gene in the capsid of AAV using the AAV adsorbent. [Means for Solving the Problems]

[0012] As a result of intensive studies to solve the above problems, the present inventors have found an adeno-associated virus (AAV) adsorbent in which GPR108 (G protein-coupled receptor 108) is immobilized on an insoluble carrier.

[0013] That is, the present disclosure includes the following aspects [1] to

[11] . [1] An AAV adsorbent comprising an insoluble carrier and an adeno-associated virus (AAV)-binding protein immobilized on the insoluble carrier, An AAV adsorbent in which the AAV-binding protein is GPR108 (G protein-coupled receptor 108). [2] The AAV adsorbent according to [1], wherein GPR108 is a polypeptide selected from any of the following (i) to (iii); (i) A polypeptide comprising at least the amino acid residues from the 33rd arginine to the 262nd lysine in the amino acid sequence set forth in SEQ ID NO: 1, (ii) A polypeptide comprising at least the amino acid residues from the 33rd arginine to the 262nd lysine in the amino acid sequence set forth in SEQ ID NO: 1, provided that in the amino acid residues from the 33rd to the 262nd, it has an amino acid sequence containing substitution, deletion, insertion, or addition of one or several amino acid residues at one or several positions, and has AAV-binding activity, (iii) A polypeptide comprising at least the amino acid residues from the 33rd arginine to the 262nd lysine in the amino acid sequence set forth in SEQ ID NO: 1, provided that it has an identity of 70% or more with the amino acid sequence consisting of the amino acid residues from the 33rd to the 262nd, and has AAV-binding activity. [3] A method for separating AAV contained in a sample, comprising a step of contacting the AAV adsorbent according to [1] or [2] with a sample containing AAV to adsorb the AAV to the AAV adsorbent, and a step of desorbing the AAV adsorbed to the AAV adsorbent from the AAV adsorbent. [4] A column filled with the AAV adsorbent according to [1] or [2]. [5] A method for separating AAV contained in a sample, comprising a step of adding a sample containing AAV to the column according to [4] to adsorb the AAV to the AAV adsorbent, and a step of eluting the AAV adsorbed to the AAV adsorbent using an eluent. [6] A method for analyzing AAV contained in a sample, comprising the steps of contacting an AAV adsorbent described in [1] or [2] with a sample containing AAV to adsorb the AAV onto the AAV adsorbent, and detaching the AAV adsorbed onto the AAV adsorbent from the AAV adsorbent. A method for analyzing AAV contained in a sample, comprising the steps of adding a sample containing AAV to the column described in [7] [4], adsorbing the AAV onto the AAV adsorbent, and eluting the AAV adsorbed onto the AAV adsorbent using an eluent. [8] The method according to [6] for analyzing the degree of degradation of AAV, the infectivity of AAV, and / or the presence or absence of genes in the capsid of AAV. [9] The method according to [7] for analyzing the degree of degradation of AAV, the infectivity of AAV, and / or the presence or absence of genes in the capsid of AAV.

[10] A method for analyzing AAV by surface plasmon resonance (SPR) using the AAV adsorbent described in [1] or [2].

[11] The method described in

[10] for analyzing the degree of degradation of AAV, the infectivity of AAV, or / and the presence or absence of genes in the capsid of AAV. [Effects of the Invention]

[0014] The adeno-associated virus (AAV) adsorbent described herein is characterized by having GPR108 (G protein-coupled receptor 108) immobilized on an insoluble carrier.

[0015] This disclosure provides a novel AAV adsorbent. According to this disclosure, AAVs can be separated using this adsorbent. Furthermore, according to one aspect of this disclosure, the quality of AAVs can be analyzed by separating them from a sample using this adsorbent. Also according to one aspect of this disclosure, a novel AAV adsorbent capable of analyzing the quality of AAVs, and a method for analyzing the quality of AAVs using this adsorbent are provided. Furthermore, according to one aspect of this disclosure, the degree of degradation and / or infectivity of AAVs can be analyzed by analyzing AAVs contained in a sample using this adsorbent. Also according to one aspect of this disclosure, the presence or absence of genes within the AAV capsid can be analyzed using this adsorbent. Therefore, the AAV adsorbent of this disclosure is useful for the quality control and improvement of industrially manufactured AAVs. [Brief explanation of the drawing]

[0016] [Figure 1] This figure shows the purity of the adeno-associated virus (AAV) binding protein GPR wild, purified in Example 2, as confirmed by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). [Figure 2] This figure shows the purity of the AAV-binding protein AVR wild, purified in Comparative Example 1, as confirmed by SDS-PAGE. [Figure 3] This figure shows the results of measuring the change in binding response when various AAVs were added to a sensor chip immobilized with an AAV-binding protein, using surface plasmon resonance. (a) shows the results when GPR wild was used as the AAV-binding protein (Example 3), and (b) shows the results when AVR wild was used (Comparative Example 2). [Figure 4] This figure shows the results of measuring the GFP positivity rate (infection rate) when various AAVs infected HeLa cells in Example 4. [Figure 5]This figure shows the results of measuring the Full percentage of the fraction obtained by ultracentrifugation purification in Example 5 using the Mass Photometry method. (a) shows the results for Fr1, and (b) shows the results for Fr2. [Figure 6] This figure shows the results of measuring the binding response change using surface plasmon resonance when Fr1 (fraction of AAV containing the gene in the capsid) or Fr2 (fraction of AAV not containing the gene in the capsid), obtained in Example 5, was added to a sensor chip immobilized with an AAV-binding protein. (a) shows the results when GPR wild was used as the AAV-binding protein (Example 6), and (b) shows the results when AVR wild was used (Comparative Example 3). The solid line represents the results for Fr1, and the dotted line represents the results for Fr2. [Figure 7] This figure shows the separation results (chromatographic patterns) obtained when AAV prepared in Example 1 was added to a column packed with a gel immobilized with AAV-binding protein, bound to the protein, and then eluted using the eluate. [Modes for carrying out the invention]

[0017] The details of this disclosure are described below.

[0018] This disclosure is characterized by the use of GPR108 (G protein-coupled receptor 108) as an AAV-binding protein constituting an adeno-associated virus (AAV) adsorbent. The amino acid sequence of GPR108 is disclosed in UniProt No. Q9NPR9 (SEQ ID NO: 1), but the AAV-binding protein may use the full-length amino acid sequence of GPR108 or a portion of the sequence. Furthermore, the AAV-binding protein may have mutations in the full-length or partial amino acid sequence described in SEQ ID NO: 1. Here, a mutation means that at least one or more amino acid residues are substituted, deleted, inserted, or added at one or more positions in the amino acid sequence. When a protein having mutations in the full-length or partial amino acid sequence described in SEQ ID NO: 1 is used as the AAV-binding protein, the protein may have AAV-binding activity.

[0019] A particularly preferred protein for use as an AAV-binding protein is a polypeptide containing at least the N-terminal extramembrane region of GPR108. In this specification, the N-terminal extramembrane region of GPR108 corresponds to the region from arginine (R) at position 33 to lysine (K) at position 262 in the amino acid sequence described in SEQ ID NO: 1.

[0020] The AAV-binding protein constituting the AAV adsorbent of this disclosure may specifically be a polypeptide selected from any of the following (i) to (iii); (i) A polypeptide comprising at least the amino acid residues from the 33rd arginine to the 262nd lysine of the amino acid sequence described in Sequence ID No. 1, (ii) A polypeptide having an amino acid sequence that includes at least the amino acid residues from the 33rd arginine to the 262nd lysine of the amino acid sequence described in Sequence ID No. 1, wherein the amino acid sequence includes substitution, deletion, insertion, or addition of one or more amino acid residues at one or more positions within the 33rd to 262nd amino acid residues, and which has AAV binding activity. (iii) A polypeptide comprising at least the amino acid residues from the 33rd arginine to the 262nd lysine of the amino acid sequence described in Sequence ID No. 1, provided that it has 70% or more identity with the amino acid sequence consisting of the 33rd to the 262nd amino acid residues, and has AAV binding activity.

[0021] In (ii) above, "one or several" means, for example, one to 40, one to 30, one to 25, one to 22, one to 20, one to 15, one to 10, one to 9, one to 8, one to 7, one to 6, one to 5, one to 4, one to 3, one or two, or one.

[0022] Furthermore, the "substitution of one or more amino acid residues" in (ii) above may include not only the amino acid substitutions at specific positions mentioned above, but also conservative substitutions that occur between amino acids with similar physical and / or chemical properties. In the case of conservative substitutions, it is generally known to those skilled in the art that the function of the protein is maintained between the substituted and unsubstituted parts. Examples of conservative substitutions include substitutions between glycine and alanine, serine and threonine, or glutamic acid and aspartic acid (Protein Structure and Function, Medical Science International, 9, 2005).

[0023] Furthermore, the "substitution, deletion, insertion, or addition of one or more amino acid residues" in (ii) above also includes naturally occurring mutations (mutants or variants) based on differences in the origin of AAV-binding proteins or differences in species.

[0024] The "identity" of amino acid sequences in (iii) above refers to the percentage obtained by aligning the two amino acid sequences to be compared so that as many amino acid residues as possible match, and then dividing the number of matching amino acid residues by the total number of amino acid residues. When aligning the sequences, gaps are inserted into one or both of the two sequences to be compared as needed. The method of aligning sequences is not particularly limited, but can be done using well-known sequence comparison programs such as BLAST (Basic Local Alignment Search Tool), FASTA, or CLUSTALW. When gaps are inserted, the total number of amino acid residues is the number of residues counted with each gap as one amino acid residue. If the total number of amino acid residues counted in this way differs between the two sequences to be compared, the identity is calculated by dividing the number of matching amino acid residues by the total number of amino acid residues of the longer sequence. Identity should be at least 70%, may be 80% or more, may be 90% or more, or may be 95% or more.

[0025] Furthermore, the AAV-binding protein may be GPR108, and may include a portion of the sequence at its N-terminal and / or C-terminal end.

[0026] Furthermore, the AAV-binding protein may have an oligopeptide added to its N-terminus or C-terminus, which is useful for separating it from a solution in the presence of contaminants. Examples of such oligopeptides include polyhistidine, polylysine, polyarginine, polyglutamic acid, and polyaspartic acid. Additionally, a cysteine-containing oligopeptide, useful for immobilizing the AAV-binding protein onto an insoluble carrier, may be added to the N-terminus or C-terminus of the AAV-binding protein.

[0027] The length of the oligopeptide to be attached to the N-terminus or C-terminus of the AAV-binding protein is not particularly limited, as long as it does not impair the AAV-binding ability or stability of the AAV-binding protein. When attaching the oligopeptide to the AAV-binding protein, the polynucleotide encoding the oligopeptide may be prepared and then genetically engineered to attach to the N-terminus or C-terminus of the AAV-binding protein using a method well known to those skilled in the art, or the oligopeptide may be chemically synthesized and chemically bonded to the N-terminus or C-terminus of the AAV-binding protein.

[0028] Furthermore, a signal peptide may be added to the N-terminus of the AAV-binding protein to promote efficient expression in the host. Examples of such signal peptides when the host is Escherichia coli include signal peptides that induce protein secretion into the periplasm, such as PelB, OmpA, DsbA, DsbC, MalE, and TorT (Japanese Patent Publication No. 2011-097898), and the translation amplification sequence (TEE) at the N-terminus of the CspA protein.

[0029] The material of the insoluble carrier constituting the AAV adsorbent of this disclosure is not particularly limited as long as it is insoluble in the sample containing AAV and the solution used for analysis (running buffer, eluent, equilibration solution, washing solution, etc.). Examples of such materials include polysaccharides such as agarose, alginate (alginate), carrageenan, chitin, cellulose, dextrin, dextran, and starch; synthetic polymers such as polyvinyl alcohol, polymethacrylate, poly(2-hydroxyethyl methacrylate), and polyurethane; ceramics such as silica; and metals such as gold. Examples of preferred insoluble carriers include polymethacrylate gels with introduced hydroxyl groups such as Toyopal (manufactured by Tosoh Corporation), agarose gels such as Sepharose (manufactured by Cytiva Corporation), cellulose gels such as Cellfine (manufactured by JNC Corporation), and sensor chips (manufactured by Cytiva Corporation) with introduced carboxymethyl (CM) groups and immobilized dextran.

[0030] The shape of the insoluble carrier is not particularly limited and may be particulate, thin film, porous, or non-porous.

[0031] In preparing the AAV adsorbent according to this disclosure, the AAV-binding protein may be immobilized on an insoluble carrier by, for example, covalent bonding. Specifically, the AAV-binding protein can be immobilized on an insoluble carrier by covalent bonding between the AAV-binding protein and the insoluble carrier via an active group present on the insoluble carrier. That is, the insoluble carrier may have an active group on its surface or elsewhere. Examples of such active groups include N-hydroxysuccinimide (NHS) activated ester group, epoxy group, carboxyl group, maleimide group, haloacetyl group, tresyl group, formyl group, and haloacetamide. As the insoluble carrier having an active group, for example, a commercially available insoluble carrier having an active group may be used as is, or an insoluble carrier may be used after introducing an active group. Examples of commercially available insoluble carriers containing active groups include TOYOPEARL AF-Epoxy-650M, TOYOPEARL AF-Tresyl-650M, TOYOPEARL AF-Formyl-650M (all 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).

[0032] One example of a method for introducing active groups to the surface of an insoluble support is to react one of two or more active sites of a compound with hydroxyl groups, epoxy groups, carboxyl groups, amino groups, etc., present on the surface of the insoluble support.

[0033] Examples of compounds that introduce epoxy groups to hydroxyl or amino groups present on the surface of an insoluble carrier include epichlorohydrin, ethanediol diglycidyl ether, butanediol diglycidyl ether, and hexanediol diglycidyl ether.

[0034] Examples of compounds that introduce carboxyl groups to epoxy groups present on the surface of an insoluble 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.

[0035] Furthermore, compounds that introduce maleimide groups to hydroxyl groups, epoxy groups, carboxyl groups, and amino groups present on the surface of an insoluble carrier 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-(maleimide)phenylisocyanate, 2-maleimidoacetic acid, and 3-maleimide. Examples include propionic acid, 4-maleimidobutyric acid, 6-maleimidohexanoic acid, N-(α-maleimidoacetoxy)succinimide ester, (m-maleimidobenzoyl)N-hydroxysuccinimide ester, succinimidyl-4-(maleimidomethyl)cyclohexane-1-carbonyl-(6-aminohexanoic acid), succinimidyl-4-(maleimidomethyl)cyclohexane-1-carboxylic acid, (p-maleimidobenzoyl)N-hydroxysuccinimide ester, and (m-maleimidobenzoyl)N-hydroxysuccinimide ester.

[0036] Examples of compounds that introduce haloacetyl groups to hydroxyl or amino groups present on the surface of an insoluble 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-(iodoacetamide)acetic acid-N-hydroxysuccinimide, 3-(bromoacetamide)propionic acid-N-hydroxysuccinimide, and 4-(iodoacetyl)aminobenzoic acid-N-hydroxysuccinimide.

[0037] Another method for introducing active groups to the surface of an insoluble carrier is to react an ω-alkenylalkaneglycidyl ether with hydroxyl groups or amino groups present on the surface of the insoluble carrier, and then activate it by halogenating the ω-alkenyl moiety with a halogenating agent. Examples of ω-alkenylalkaneglycidyl ethers include allylglycidyl ether, 3-butenylglycidyl ether, and 4-pentenylglycidyl ether. Examples of halogenating agents include N-chlorosuccinimide, N-bromosuccinimide, and N-iodosuccinimide.

[0038] Furthermore, as a method for introducing active groups to the surface of an insoluble carrier, an example can be given of introducing active groups to carboxyl groups present on the surface of the insoluble carrier using a condensing agent and an additive. Examples of condensing agents include 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), dicyclohexylcarbodiamide, and carbonyldiimidazole. Examples of additives include N-hydroxysuccinimide (NHS), 4-nitrophenol, and 1-hydroxybenztriazole.

[0039] The immobilization of the AAV-binding protein of this disclosure onto an insoluble carrier can be carried out, for example, in a buffer. Examples of buffers 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 according to various conditions, such as the reactivity of the active group and the stability of the AAV-binding protein of this disclosure. The reaction temperature during immobilization may be, for example, 5°C to 50°C, and preferably 10°C to 35°C.

[0040] In preparing the AAV adsorbent according to this disclosure, the AAV-binding protein may be immobilized on the insoluble carrier by amine coupling, for example, by reacting it with an insoluble carrier on which dextran is immobilized. Examples of insoluble carriers on which dextran is immobilized include Sensor Chip CM3, CM4, CM5, and CM7 (all manufactured by Cytiva).

[0041] The AAV-binding protein may have a polyhistidine tag, in which case the AAV-binding protein having the polyhistidine tag may be immobilized on the insoluble carrier by reacting the insoluble carrier with an immobilized nitrilotriacetic acid (NTA). An example of an insoluble carrier with immobilized NTA is Sensor Chip NTA (manufactured by Cytiva).

[0042] AAV may be analyzed by surface plasmon resonance (SPR) using the AAV adsorbent of this disclosure. Furthermore, according to one aspect of this disclosure, the degree of degradation, the infectivity of AAV, and / or the presence or absence of genes within the AAV capsid may be analyzed by SPR using the AAV adsorbent of this disclosure. In this case, the AAV adsorbent is preferably one on which AAV-binding proteins are immobilized on a thin gold film. For analysis by SPR, instruments such as Biacore 3000, Biacore T-200, Biacore X100, and Biacore 8K (all manufactured by Cytiva) may be used. In analysis by SPR, an AAV solution may be flowed onto the surface of the AAV adsorbent as an analyte, and the binding response due to the binding of AAV to AAV-binding proteins may be observed.

[0043] The AAV adsorbent disclosed herein can be used to separate AAV contained in a sample. The method for separating AAV using the AAV adsorbent is as follows: <1> The process involves bringing an AAV adsorbent into contact with a sample containing AAV, thereby adsorbing the AAV onto the AAV adsorbent (AAV adsorption process), <2> The process may include a step of detaching the AAV adsorbed onto the AAV adsorbent from the AAV adsorbent (AAV detachment step).

[0044] In the AAV separation method described above, for example, a column packed with the AAV adsorbent (also called an AAV adsorbent column) may be used. Specifically, the AAV may be separated by a method that includes the steps of adding a sample containing AAV to a column packed with the AAV adsorbent of this disclosure, adsorbing the AAV onto the AAV adsorbent, and eluting the AAV adsorbed on the AAV adsorbent using an eluent. <1> AAV adsorption process A sample containing AAV may be added to the AAV adsorbent column using, for example, a liquid delivery means such as a pump. In this specification, adding a liquid to a column is also referred to as "delivering a liquid to the column." Before adding the AAV-containing sample to the AAV adsorbent column, the solvent may be replaced with an appropriate buffer solution. Alternatively, the AAV adsorbent column may be equilibrated using an appropriate buffer solution (equilibrium solution) before adding the AAV-containing sample to the AAV adsorbent column. This equilibration is expected to allow for, for example, higher purity purification of AAV.

[0045] Any neutral buffer with buffering capacity in the neutral range (in this specification, pH 5.0 to 9.0, preferably pH 5.5 to 8.0) can be used for solvent substitution or equilibration. Examples include phosphate buffer, acetate buffer, succinate buffer, citrate buffer, Tris buffer, HEPES buffer, and MES buffer. For example, salts or surfactants may be added to such buffers. The buffer used for solvent substitution and the equilibration solution may be the same or different.

[0046] Furthermore, if non-AAV components, such as contaminants, remain on the AAV adsorbent column after passing a sample containing AAV through it, such components may be removed (washed) from the AAV adsorbent column before eluting the AAV adsorbed on the AAV (i.e., before the AAV desorption step). Non-AAV components can be removed from the AAV adsorbent column, for example, by using an appropriate buffer solution as a washing solution. For such washing solutions, the descriptions for buffer solutions used for solvent replacement and equilibration can be applied mutatis mutandis. <2> AAV Desorption Process When separating AAV using an AAV adsorbent column, the AAV desorption step may be a step of eluting the AAV adsorbed on the AAV adsorbent using an eluent.

[0047] In the AAV desorption step, the AAV adsorbed onto the AAV adsorbent can be eluted using, for example, a buffer with a pH lower than that of the equilibration solution or washing solution, a buffer containing a different salt than that of the equilibration solution or washing solution, or a buffer containing more salt than that of the equilibration solution or washing solution. By passing the aforementioned elution solution through the AAV adsorbent column, the interaction between AAV and GPR108 within the column is weakened, so the AAV adsorbed onto the AAV adsorbent is eluted, and a fraction containing AAV is obtained.

[0048] The eluent delivery method may be gradient or isocratic, but gradient elution is more preferable in terms of separation performance. The gradient may be changed in two or more steps (stepwise gradient) or with a linear gradient.

[0049] There is no particular lower limit to the flow rate when delivering the liquid to the column, and the upper limit depends on the back pressure of the AAV adsorbent column of this disclosure, so it can be set as appropriate.

[0050] A method for separating AAV using an AAV adsorbent or a method for separating AAV using an AAV adsorbent column may also be a method for analyzing AAV contained in a sample. Using a method for separating AAV using an AAV adsorbent or an AAV adsorbent column, the degree of degradation of AAV contained in the sample, the infectivity of AAV, and / or the presence or absence of genes in the AAV capsid may be analyzed.

[0051] The AAVs isolated or analyzed in this disclosure may be naturally occurring AAVs or artificially created AAVs. Examples of naturally occurring AAVs include serotype 1 (AAV1), serotype 2 (AAV2), serotype 3 (AAV3), serotype 4 (AAV4), serotype 5 (AAV5), serotype 6 (AAV6), serotype 7 (AAV7), serotype 8 (AAV8), serotype 9 (AAV9), serotype 10 (AAV10), serotype 11 (AAV11), serotype 12 (AAV12), and serotype 13 (AAV13). Examples of artificially created AAVs include AAVrh8, AAVrh10, and chimeric AAVs possessing two or more characteristics (cell-specificity or infectivity) from these serotypes.

[0052] The AAV analysis method using the AAV adsorbent described herein allows for the evaluation of the quality of the AAV. Furthermore, according to one aspect of this disclosure, the AAV adsorbent can be used to evaluate the degree of heat-induced degradation of AAV and the infectivity of AAV to cells. Heat-induced degradation of AAV is thought to be due to the denaturation and / or degradation of the AAV capsid protein caused by heat. Furthermore, according to one aspect of this disclosure, the AAV analysis method using the AAV adsorbent can analyze the presence or absence of genes within the AAV capsid.

[0053] For measuring the percentage of AAV vectors containing genes (Full rate), a mass photometry method such as the Refeyn Two (manufactured by Refeyn) is recommended. [Examples]

[0054] The present disclosure will be described in further detail below using examples and comparative examples, but the present disclosure is not limited to these examples.

[0055] Example 1: Preparation of an adeno-associated virus (AAV) vector (1) A nucleotide sequence (SEQ ID NO: 3) was designed by adding the restriction enzyme EcoRI recognition sequence (GAATTC) to the 5' end of a polynucleotide encoding EGFP (Enhanced Green Fluorescent Protein) consisting of the amino acid sequence described in SEQ ID NO: 2, and a stop codon (TAG) and a BamHI recognition sequence (GGATTC) to the 3' end.

[0056] (2) A polynucleotide consisting of the sequence described in Sequence ID No. 3 was totally synthesized and cloned into a plasmid (commissioned to FASMAC, named pUC-EGFP). Escherichia coli strain JM109 was transformed with pUC-EGFP, and the resulting transformants were cultured. pUC-EGFP was extracted from the culture medium using the QIAprep Spin Miniprep kit (Qiagen).

[0057] (3) The pUC-EGFP obtained in (2) was digested with restriction enzymes EcoRI and BamHI, and then ligated into the expression vector pAAV-CMV (manufactured by Takara Bio Inc.), which had been previously digested with restriction enzymes EcoRI and BamHI. The ligation product was then used to transform E. coli strain JM109.

[0058] (4) The transformants obtained in (3) were cultured overnight at 37°C with shaking in a 5L baffled flask containing 1L of 2×YT medium (1.6%(w / v)Tryptone, 1%(w / v)Yeast Extract, 0.5%(w / v)Sodium Chloride) containing 100 μg / mL of carbenicillin. After the culture was complete, the cells were collected by centrifugation. A large amount of pAAV-EGFP was prepared from the collected cells using Plasmid Mega Kit (Qiagen).

[0059] (5) Escherichia coli strain JM109 was transformed using the plasmid pRC8 Vector (Takara Bio Inc.) and pHelper Vector (Takara Bio Inc.), which contain polynucleotides encoding the capsid of AAV serotype 8 (AAV8). The resulting transformants were cultured overnight at 37°C in a 5L baffled flask containing 1L of 2×YT medium with 100 μg / mL of carbenicillin, with shaking.

[0060] (6) After recovering the bacterial cells from the culture solution of (5) by centrifugation, large quantities of pRC8 Vector and pHelper Vector were prepared from the recovered bacterial cells using Plasmid Mega Kit (Qiagen).

[0061] (7) Viral Production Cells 2.0 (Thermo Fisher Scientific) were cultured in a 500 mL flask containing 120 mL of Viral Production Medium (Thermo Fisher Scientific).

[0062] (8) Using the AAV-MAX Transfection Kit (Thermo Fisher Scientific), the pAAV-EGFP prepared in (4), and the pRC8 Vector and pHelper Vector prepared in (6) were introduced into Viral Production Cells 2.0 cultured in (7), and the cells were cultured with shaking for 3 days under conditions of 8% carbon dioxide, 120 rpm, and 37°C.

[0063] The culture medium prepared in (9)(8) was collected, and after adding AAV-MAX Lysis Buffer (Thermo Fisher Scientific), magnesium chloride (final concentration 2 mM), and DNase I (final concentration 2.5 units / mL), the cells were disrupted by shaking at 120 rpm and 37°C for 3 hours.

[0064] After collecting the cell lysates obtained in (10)(9), the supernatant was collected by centrifugation at 12000×g at 4°C for 10 minutes. The obtained supernatant was filtered through a 0.22 μm filter to obtain a clarified AAV8 solution.

[0065] The AAV8 solution obtained in (11)(10) was added to a column packed with 5 mL of POROS CaptureSelect AAVX Affinity Resin (Thermo Fisher Scientific), and AAV8 was adsorbed onto the Resin.

[0066] The columns from (12) and (11) were washed with 10 Column Volume (CV) washing buffer (20 mM Tris hydrochloride buffer (pH 7.4) containing 500 mM sodium chloride) to remove impurities.

[0067] (13) The column washed with (12) was treated with 5 CV of elution buffer (100 mM acetate buffer (pH 2.0) containing 500 mM sodium chloride) to elute the AAV8 adsorbed on Resin.

[0068] The AAV8 eluted in (14) and (13) was sealed in a dialysis membrane tube with an exclusion limit molecular weight of 50 kDa, and immersed in 100 times the volume of dialysis buffer A (50 mM sodium acetate buffer (pH 6.0) containing 150 mM sodium chloride, 10 mM calcium chloride, 0.01% (w / v) Tween 20 (trade name) and 0.01% (w / v) Pluronic F-68 (trade name)) and dialyzed overnight at 4°C.

[0069] AAV8 was collected after dialyzing in (15)(14) and clarified through a 0.22 μm filter to obtain a purified AAV8 solution. The particle concentration of AAV8 was measured by multi-angle dynamic light scattering and was found to be 5.9 × 10⁻⁶. 13 The full capsid (vector containing the gene) rate of AAV8 was measured at cp(capsid) / mL using the Mass Photometry method and found to be 29.2%. The gene concentration of AAV8 calculated from this full capsid rate was 1.7 × 10⁻⁶.13 The value was vg (vector genome) / mL.

[0070] Example 2: Preparation of GPR wild (1) A polynucleotide encoding an AAV-binding protein (named GPR wild) consisting of the amino acid sequence described in Sequence ID No. 4 was inserted between the cspA 5'UTR (untranslated region) sequence and the cspA 3'UTR sequence of pCold III (Takara Bio Inc.) using the Gibson Assembly method to create an expression plasmid (named pCold-GPR wild). In Sequence ID No. 4, the sequence from the first methionine (M) to the fifth valine (V) is the translation amplification sequence (TEE), the sequence from the sixth arginine (R) to the 235th lysine (K) is the N-terminal extramembrane region of the AAV-binding protein GPR108 (UniProt No. Q9NPR9) (i.e., the amino acid residues from the 33rd arginine (R) to the 262nd lysine (K) in Sequence ID No. 1), the histidine (H) from the 236th to the 241st histidine (H) is the histidine tag, and the sequence from the 242nd cysteine ​​(C) to the 248th glycine (G) is the immobilization tag.

[0071] (2) The transformants obtained by transforming E. coli strain BL21(DE3) with the pCold-GPR wild prepared in (1) were inoculated into 3 mL of 2×YT medium containing 50 μg / mL ampicillin and pre-cultured by aerobic shaking at 37°C overnight.

[0072] (3) 2 mL of the pre-culture solution from (2) was inoculated into a 5 L baffled flask containing 1 L of 2 × YT medium with 50 μg / mL ampicillin, and the culture was performed aerobically with shaking at 37°C.

[0073] (4) Three hours after the start of culture, the culture was cooled on ice, and IPTG (Isopropyl β-D-thiogalactopyranoside) was added to a final concentration of 0.1 mM. The culture was then continued at 15°C for 24 hours with shaking.

[0074] (5) After the culturing was complete, the cultured cells (transformed organisms) were collected by centrifuging the culture solution at 4°C and 8000 rpm for 20 minutes.

[0075] (6) To 1 g of the bacterial cells recovered in (5), 10 times the amount of solubilization buffer (20 mM Tris hydrochloride buffer (pH 7.4) containing 150 mM sodium chloride, 20 mM Imidazole, 0.01% (w / v) Tween 20 (trade name), 0.5% (w / v) Triton X-100 (trade name), 2 mM magnesium chloride, 2.5 units / mL DNase I, and 0.2 mg / mL lysozyme) was added to suspend the bacterial cells, and the mixture was further stirred at room temperature for 1 hour to solubilize the cells. The solubilized bacterial solution after stirring was centrifuged at 15000 × g for 30 minutes at 4 °C, and the precipitate was collected after removing the supernatant.

[0076] (7) The precipitate recovered in (6) was suspended in acetone and centrifuged at 15,000 × g for 30 minutes at 4°C, and the supernatant was removed. The precipitate was suspended in ultrapure water and centrifuged at 15,000 × g for 30 minutes at 4°C, and the supernatant was removed to recover the inclusions.

[0077] (8) To 1 g of the inclusion body obtained in (7), 10 times the amount of denaturation buffer A (20 mM Tris hydrochloride buffer (pH 7.4) containing 150 mM sodium chloride, 20 mM imizole, and 6 M guanidine hydrochloride) was added, and the inclusion body was solubilized by stirring overnight at 4°C. After stirring, the solubilized solution was centrifuged at 15000 × g for 30 minutes at 4°C, and the supernatant was collected.

[0078] (9) 5 mL of Ni-NTA agarose (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was packed into an open column, equilibrated with denaturation buffer A, and the supernatant obtained in (8) was applied to adsorb GPR wild onto the Ni-NTA agarose.

[0079] After washing off impurities by applying 10 CV of denaturation buffer A to column (10)(9), 5 CV of denaturation buffer B (20 mM Tris-HCl (pH 7.4) containing 150 mM sodium chloride, 500 mM imiazole, and 6 M guanidine hydrochloride) was added, and GPR wild adsorbed on Ni-NTA agarose was eluted.

[0080] (11) Dithiothreitol (DTT) was added to the GPR wild solution obtained in (10) to a final concentration of 10 mM, and then the solution was sealed in dialysis membrane tubes with an exclusion limit molecular weight of 6 kDa to 8 kDa.

[0081] The dialysis tubes from (12) and (11) were immersed in 100 times the volume of dialysis buffer B (20 mM Tris hydrochloride buffer (pH 8.0) containing 1 M guanidine hydrochloride, 0.4 M arginine hydrochloride, and 1 mM DTT) and dialyzed by stirring overnight at 4°C.

[0082] (13) Prepare 500 mL of dialysis buffer C (20 mM Tris hydrochloride buffer (pH 8.0) containing 1 M guanidine hydrochloride, 0.4 M arginine hydrochloride, and 1 mM L(-)-cystine dihydrochloride), transfer the dialysis tube from (12) to the buffer, and perform dialysis by stirring at 4°C for 3 hours.

[0083] (14) Prepare 5 L of dialysis buffer D (20 mM Tris hydrochloride buffer (pH 7.4)), transfer the dialysis tube from (13) into it, and perform dialysis by stirring at 4°C for 4 hours.

[0084] The solution in the dialysis tube used in (15)(14) was removed, centrifuged at 4°C and 12000×g for 10 minutes, and the supernatant was collected. The supernatant was then clarified by filtering through a 0.22 μm filter.

[0085] Figure 1 shows the results of confirming the purity of the obtained GPR wild purified product by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). It can be seen that the GPR wild expressed in the inclusion bodies was obtained in high purity by solubilization and refolding purification.

[0086] Comparative Example 1: Preparation of AVR wild (1) A plasmid (named pET-AVR wild) was constructed by inserting a polynucleotide encoding an AAV-binding protein (named AVR wild) consisting of the amino acid sequence described in Sequence ID No. 5 into a pET-26b(+) vector (Merck). In Sequence ID No. 5, the first methionine (M) to the 22nd alanine (A) is the PelB signal peptide (the N-terminal 22 residues of UniProt No. P0C1C1), the 25th serine (S) to the 213th aspartic acid (D) are the extracellular domains 1 (PKD1) and 2 (PKD2) of the AAV-binding protein KIAA0319L (amino acid residues from the 312th serine (S) to the 500th aspartic acid (D) of UniProt No. Q8IZA0), the 214th to the 219th histidine (H) is the histidine tag, and the 220th cysteine ​​(C) to the 226th glycine (G) is the immobilization tag.

[0087] (2) The pET-AVR wild prepared in (1) was cultured using the method described in Examples 2(2) to (5), and the cultured cells (transformed cells) were collected.

[0088] (3) Ten times the amount of solubilization buffer (described in Example 1(6)) was added to 1 g of the bacterial cells recovered in (2) to suspend the bacterial cells, and the mixture was stirred at room temperature for 1 hour to solubilize them. The solubilized bacterial solution after stirring was centrifuged at 15000 × g for 30 minutes at 4°C, and the supernatant after centrifugation was collected.

[0089] The supernatant obtained in (4)(3) was purified using a column packed with Ni-NTA agarose (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) by the method described in Examples 2(9) to (10) to obtain an AVR wild solution.

[0090] (5) The AVR wild solution obtained in (4) was sealed in a dialysis membrane tube with an exclusion limit molecular weight of 6 kDa to 8 kDa, then immersed in 5 L of dialysis buffer E (20 mM Tris hydrochloride buffer (pH 7.4) containing 150 mM sodium chloride) and dialyzed by stirring overnight at 4°C.

[0091] (6) The solution in the dialysis tube that was dialyzed in (5) was removed and clarified using the method described in Example 2(15).

[0092] Figure 2 shows the results of confirming the purity of the obtained AVR wild purified product by SDS-PAGE. It can be seen that the AVR wild expressed in the solubilized supernatant was purified to a high degree of purity.

[0093] Example 3: Evaluation of coupling between GPR wild and AAV using surface plasmon resonance (SPR). (1) AAV8 purified solution prepared in Example 1 (5.9 × 10 13 cp / mL, 1.7 × 10 13 100 μL each of the vg / mL solution was dispensed into five PCR tubes. One tube was stored at 4°C, and the remaining four tubes were heat-treated at 50°C for 1 hour, 3 hours, 6 hours, or 9 hours.

[0094] (2) After collecting the heat-treated or untreated tubes from (1), add 490 μL of running buffer (20 mM Tris hydrochloride buffer (pH 7.4) containing 150 mM sodium chloride, 1 mM calcium chloride, and 0.01% (w / v) Tween 20 (trade name)) to increase the AAV8 particle concentration to 1.0 × 10⁻⁶. 13 Each solution was diluted to cp / mL.

[0095] (3) An NTA sensor chip (manufactured by Cytiva) was attached to a Biacore T-200 (manufactured by Cytiva), and equilibration was performed with a dedicated running buffer.

[0096] (4) After adding an aqueous solution of 0.5 mM nickel(II) sulfate to the sensor chip equilibrated in (3) to coordinate nickel ions on the chip, the GPR wild purified product prepared in Example 2 (concentration adjusted to 0.15 mg / mL with the running buffer) was added to immobilize GPR wild on the chip.

[0097] (5) Each AAV8 solution diluted in (2) was further diluted 2-fold, 4-fold, 8-fold, and 16-fold with the running buffer (concentrations: 5.0×10 12 cp / mL, 2.5×10 12 cp / mL, 1.3×10 12 cp / mL, and 6.3×10 11 cp / mL). Starting from the lowest concentration, they were continuously added to the chip immobilized with GPR wild, and the binding responses with GPR wild were observed respectively (Single Cycle Kinetics method).

[0098] Comparative Example 2 Evaluation of the binding property between AVR wild and AAV using SPR The binding property between AVR wild and AAV was evaluated using SPR by the method described in Example 3, except that the AVR wild purified product prepared in Comparative Example 1 was used as the AAV-binding protein immobilized on the sensor chip in Example 3(4).

[0099] The results obtained in Example 3 are shown in Figure 3(a), and the results obtained in Comparative Example 2 are shown in Figure 3(b). Compared to the response obtained by binding GPR wild to an AAV8 solution stored at 4°C, the binding affinity of the AAV8 solution heated at 50°C decreased in proportion to the heating time (Figure 3(a)). This indicates that the degradation of AAV8 caused by heating affects its binding affinity to GPR wild. From these results, it can be inferred that by using a polypeptide containing the N-terminal extramembrane region of GPR108 as an AAV-binding protein constituting an AAV adsorbent, it may be possible to analyze AAV based on the degree of degradation.

[0100] On the other hand, the AAV8 solutions heated at 50°C showed a similar binding response to the AAV8 solution stored at 4°C bound to AVR wild (Figure 3(b)). This indicates that the degradation of AAV8 caused by heating had little effect on its binding ability to AVR wild. From these results, it can be inferred that if a polypeptide containing PKD1 and PKD2 of KIAA0319L is used as the AAV-binding protein constituting the AAV adsorbent, it will not be possible to analyze AAV based on the degree of degradation.

[0101] Example 4: Measurement of cell infectivity of AAV8 (1) AAV8 purified solution prepared in Example 1 (5.9 × 10 13 cp / mL, 1.7 × 10 13 The solution (vg / mL) was dispensed into 5 PCR tubes, 100 μL each. One tube was stored at 4°C, and the remaining four tubes were heat-treated at 50°C for 1 hour, 3 hours, 6 hours, or 9 hours.

[0102] (2) Seed HeLa cells into a 24-well plate, with 2.4 × 10⁶ cells per well. 5 The cells were cultured to achieve a cell-per-well ratio.

[0103] (3) Add 10 μL (5.9 × 10) of the heat-treated or untreated AAV8 solution (1) to the HeLa cells cultured in (2) in (3) (3)11 cp, 1.7 × 10 11 (vg) Add each (7.1 × 10 per cell) 5 The cells were infected by (vg) and cultured at 37°C for 4 days (N=5).

[0104] (4) The HeLa cells infected in (3) were detached by trypsin treatment and then suspended in phosphate-buffered saline (PBS) containing 2% (w / v) fetal bovine serum (FBS) and 1 mM ethylenediaminetetraacetic acid (EDTA).

[0105] (5) The HeLa cells suspended in (4) were subjected to Guava easyCyte (manufactured by Scitec Biosciences), and the percentage of HeLa cells infected with AAV8 (GFP positivity rate) was calculated by measuring the green fluorescence derived from EGFP expressed by the cells.

[0106] The results obtained are shown in Figure 4. In Figure 4, "NC" indicates the results for HeLa cells uninfected with AAV8. Compared to HeLa cells infected with untreated (4°C) AAV8, HeLa cells infected with AAV8 that had been heat-treated at 50°C showed a lower GFP positivity rate. Furthermore, the GFP positivity rate decreased as the heat treatment time increased. From these results, it can be inferred that the infectivity of AAV to HeLa cells can be analyzed by using a polypeptide containing the N-terminal extramembrane region of GPR108 as an AAV-binding protein constituting an AAV adsorbent.

[0107] Example 5: Full / Empty Purification of AAV8 by Ultracentrifugation (1) After preparing buffers 1 to 4 with the compositions shown in Table 1, 6 mL each of buffer 1, buffer 2, buffer 3, and buffer 4 were layered in the following order in a 32.4 mL volume Optiseal tube (Beckman Coulter).

[0108] [Table 1]

[0109] (2) Place the AAV8 purified solution prepared in Example 1 (5.9 × 10) on top of the layered solution in (1). 13 cp / mL, 1.7 × 10 13 Gently pour in the solution (vg / mL) to fill the tube, then seal it tightly.

[0110] (3) The tubes prepared in (2) were set on a fixed-angle ultracentrifuge rotor Ti70 (manufactured by Beckman Coulter) while being careful not to disturb the concentration gradient inside the tubes.

[0111] (4) The rotors from (3) were set in an Optima XE-100 (manufactured by Beckman Coulter) and centrifuged at 63,000 × g at 18°C ​​for 2 hours.

[0112] (5) The centrifugation tubes from (4) were carefully removed and placed in a fraction recovery system (Beckman Coulter) to extract the solution from inside the tubes by puncturing the bottom of the tubes. The solution inside the tubes was recovered using multiple fractions.

[0113] (6) The AAV8 in each fraction recovered in (5) was measured for fullness (the percentage of viruses containing the gene within the capsid) using the Mass Photometry method with Refeyn Two (Refeyn). Fractions showing a fullness of 80% or more were collected as Fr1, and fractions showing a fullness of 5% or less were collected as Fr2. The AAV8 contained in Fr1 is also referred to as Full-AAV8, and the AAV8 contained in Fr2 is also referred to as Empty-AAV8.

[0114] (7) The Fr1 and Fr2 recovered in (6) were respectively sealed in dialysis membrane tubes with an exclusion limit molecular weight of 50 kDa, immersed in 100 times the volume of dialysis buffer F (PBS containing 1 mM magnesium chloride), and dialyzed overnight at 4°C.

[0115] (8)(7) The dialysis membrane tubes were transferred to a newly prepared 100-fold volume of dialysis buffer F and dialysis was performed overnight at 4°C.

[0116] The solution in the dialysis tube that was dialyzed in (9)(8) was removed and clarified using the method described in Example 2(15). The particle concentration of AAV8 was measured by multi-angle dynamic light scattering. As a result, Fr1 was 1.2 × 10⁻⁶. 13 The value is cp / mL, and Fr2 is 5.7 × 10⁻⁶. 13 The result was cp / mL.

[0117] The full rate (the percentage of viruses containing the gene within the capsid) of AAV8 contained in Fr1 and Fr2 obtained in (10)(9) was evaluated by the Mass Photometry method using Refeyn Two (Refeyn).

[0118] The evaluation results by mass photometry are shown in Figure 5. The fullness of Fr1 was 84.9% (Figure 5(a)) and the fullness of Fr2 was 2.4% (Figure 5(b)). Compared to the AAV8 solution obtained in Example 2, it can be seen that the full-AAV8 was concentrated towards Fr1 and the empty-AAV8 was concentrated towards Fr2.

[0119] Example 6: Evaluation of the compatibility of GPR Wild with Full or Empty AAV using SPR (1) Fr1 and Fr2 obtained in Example 5(9) are used when the AAV8 particle concentration is 1.0 × 10 13 The solution was diluted with dialysis buffer F to achieve a concentration of cp / mL.

[0120] (2) An NTA sensor chip (manufactured by Cytiva) was attached to a Biacore T-200 (manufactured by Cytiva), and equilibration was performed with a dedicated running buffer (20 mM Tris hydrochloride buffer (pH 7.4) containing 150 mM sodium chloride, 1 mM calcium chloride, and 0.01% (w / v) Tween 20 (product name)).

[0121] (3) A 0.5 mM nickel(II) sulfate aqueous solution was added to the sensor chip equilibrated in (2) to coordinate nickel ions onto the chip, and then the GPR wild purified product prepared in Example 2 (concentration adjusted to 0.2 mg / mL with the running buffer) was added to immobilize the GPR wild onto the chip.

[0122] (4) After further diluting Fr1 and Fr2 diluted in (1) with the running buffer (concentration: 5.0 × 10 12 cp / mL, 2.5 × 10 12 cp / mL, 1.3 × 10 12 cp / mL and 6.3 × 10⁻⁶ 11 The GPR wild was immobilized on a chip and added sequentially to different concentrations (cp / mL), starting with the lowest concentration. The binding response to the GPR wild was then observed (Single Cycle Kinetics method).

[0123] Comparative Example 3: Evaluation of compatibility between AVR wild and Full or Empty AAV using SPR Except for the AVR wild purified product prepared in Comparative Example 1 being used as the AAV-binding protein immobilized on the sensor chip in Example 6(3), the binding affinity between AVR wild and AAV was evaluated using SPR according to the method in Example 6.

[0124] The results obtained in Example 6 are shown in Figure 6(a), and the results obtained in Comparative Example 3 are shown in Figure 6(b). Note that, taking into account the difference in molecular weight (Full-AAV8 (Fr1): 4.5 MDa, Empty-AAV8 (Fr2): 3.6 MDa), the Fr1 response is calculated by multiplying the actual response by 0.8 (Normalized Response [RU]).

[0125] The response obtained from the binding of Empty-AAV8 contained in Fr2 to GPR wild is significantly larger than the response obtained from the binding of Full-AAV8 contained in Fr1 to GPR wild (Figure 6(a)). Furthermore, using Biacore T200 / S200 evaluation software, kinetic analysis was performed on each of the obtained responses as a 1:1 binding model, and the binding affinity between Full-AAV8 contained in Fr1 and Empty-AAV8 contained in Fr2 was determined by the dissociation constant (K). D A significant difference was also observed in the results calculated using (K D =106.5nM(Fr1), K D =0.015nM(Fr2)). This indicates that the binding affinity of Full-AAV8 to GPR wild differs significantly from that of Empty-AAV8. From these results, it can be inferred that by using a polypeptide containing the N-terminal extramembrane region of GPR108 as an AAV-binding protein constituting an AAV adsorbent, the presence or absence of genes within the capsid can be analyzed.

[0126] On the other hand, the response obtained by binding to Full-AAV8 contained in Fr1 and the response to Empty-AAV8 contained in Fr2 were similar for AVR wild (Figure 6(b)). Furthermore, the binding affinity with Full-AAV8 contained in Fr1 and Empty-AAV8 contained in Fr2 was determined using a method similar to that used for GPR wild, by the dissociation constant (K D The result calculated using (K) was also an equivalent value. D =0.245nM(Fr1), K D =0.154nM(Fr2)). In other words, the binding affinity to Full-AAV8 and Empty-AAV8 is almost the same for AVR wild. From these results, it can be inferred that when a polypeptide containing PKD1 and PKD2 of KIAA0319L is used as the AAV-binding protein constituting the AAV adsorbent, it is difficult to analyze the presence or absence of genes within the capsid.

[0127] Example 7: Preparation of GPR wild-immobilized gel (1) A gel was prepared by introducing iodoacetamide groups to the hydroxyl groups on the surface of a hydrophilic vinyl polymer for separation agents (Tosoh Corporation: Toyopearl) through chemical modification, and this was used as an insoluble carrier to immobilize AAV-binding proteins.

[0128] (2) To 1 g of the gel prepared in (1), 10 mg of GPR wild prepared in Example 2 and TCEP (Tris(2-carboxyethyl)phosphine) at a final concentration of 0.3 mmol / L as a reducing agent were added and the mixture was reacted by shaking for 3 hours under conditions of pH 8.1 and 25°C. This prepared a gel immobilized with GPR wild, which is an AAV adsorbent (hereinafter also referred to as GPR wild immobilized gel).

[0129] Example 8: Separation of AAV using GPR wild-immobilized gel (1) 0.5 mL of the GPR wild immobilized gel prepared in Example 7 was packed into a Tricorn 5 / 20 column (manufactured by Cytiva) to prepare an AAV adsorbent column (hereinafter also referred to as the GPR wild column).

[0130] (2) The prepared GPR wild column was connected to an HPLC M30A (Shimadzu Corporation) and equilibrated with eluent A (20mM Tris hydrochloride buffer (pH 7.4) containing 150mM sodium chloride and 0.005% (w / v) Tween 20 (trade name)), and then the AAV8 purified solution (5.9 × 10) prepared in Example 1 was added. 13 cp / mL, 1.7 × 10 13 A 0.05 mL solution (vg / mL) was applied at a flow rate of 0.2 mL / min.

[0131] (3) After washing with eluent A for 25 minutes, eluent B (20 mM Tris hydrochloride buffer (pH 7.4) containing 2 M magnesium chloride and 0.005% (w / v) Tween 20 (trade name)) was infused into the GPR wild column for 25 minutes. After infusion, the GPR wild column was re-equilibrated by infusing eluent A into the column again for 25 minutes.

[0132] (4) AAV8 eluted from the GPR wild column was detected by fluorescence intensity at 350 nm in response to excitation light at 280 nm.

[0133] The obtained chromatographic pattern is shown in Figure 7. An elution peak for AAV8 was observed around 30 minutes of elution. From these results, it can be seen that AAV can be separated by using a polypeptide containing the N-terminal extramembrane region of GPR108 as an AAV-binding protein constituting the AAV adsorbent.

Claims

1. An AAV adsorbent comprising an insoluble carrier and an adeno-associated virus (AAV) binding protein immobilized on the insoluble carrier, An AAV adsorbent in which the AAV-binding protein is GPR108 (G protein-coupled receptor 108).

2. The AAV adsorbent according to claim 1, wherein GPR108 is a polypeptide selected from any of (i) to (iii) below; (i) A polypeptide comprising at least the amino acid residues from the 33rd arginine to the 262nd lysine of the amino acid sequence described in Sequence ID No. 1, (ii) A polypeptide having an amino acid sequence that includes at least the amino acid residues from the 33rd arginine to the 262nd lysine of the amino acid sequence described in Sequence ID No. 1, wherein the amino acid sequence includes substitution, deletion, insertion, or addition of one or more amino acid residues at one or more positions within the 33rd to 262nd amino acid residues, and which has AAV binding activity. (iii) A polypeptide comprising at least the amino acid residues from the 33rd arginine to the 262nd lysine of the amino acid sequence described in Sequence ID No. 1, provided that it has 70% or more identity with the amino acid sequence consisting of the 33rd to the 262nd amino acid residues, and has AAV binding activity.

3. A method for separating AAV contained in a sample, comprising the steps of: contacting an AAV adsorbent according to claim 1 or 2 with a sample containing AAV to adsorb the AAV onto the AAV adsorbent; and detaching the AAV adsorbed onto the AAV adsorbent from the AAV adsorbent.

4. A column packed with the AAV adsorbent according to claim 1 or 2.

5. A method for separating AAV contained in a sample, comprising the steps of: adding a sample containing AAV to the column described in claim 4; adsorbing the AAV onto the AAV adsorbent; and eluting the AAV adsorbed onto the AAV adsorbent using an eluent.

6. A method for analyzing AAV contained in a sample, comprising the steps of: contacting an AAV adsorbent according to claim 1 or 2 with a sample containing AAV to adsorb the AAV onto the AAV adsorbent; and detaching the AAV adsorbed onto the AAV adsorbent from the AAV adsorbent.

7. A method for analyzing AAV contained in a sample, comprising the steps of: adding a sample containing AAV to the column described in claim 4; adsorbing the AAV onto the AAV adsorbent; and eluting the AAV adsorbed onto the AAV adsorbent using an eluent.

8. The method according to claim 6, comprising analyzing the degree of degradation of AAV, the infectivity of AAV, and / or the presence or absence of genes within the AAV capsid.

9. The method according to claim 7, comprising analyzing the degree of degradation of AAV, the infectivity of AAV, and / or the presence or absence of genes within the AAV capsid.

10. A method for analyzing AAV by surface plasmon resonance (SPR) using the AAV adsorbent described in claim 1 or 2.

11. The method according to claim 10, comprising analyzing the degree of degradation of AAV, the infectivity of AAV, and / or the presence or absence of genes within the AAV capsid.

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