Patent Application
20250236657
A sequence listing in electronic ST.26 (XML file) format is filed with this application and incorporated herein by reference. The name of the ST.26 file is “P220611WO.xml”; the file was created on Aug. 15, 2024; the size of the file is 155,379 bytes.
The present invention relates to a protein having a binding activity to an adeno-associated virus (AAV) and a method for analyzing AAV. More specifically, the present invention relates to an improved AAV-binding protein having an enhanced binding activity to AAV and a method for analyzing AAV contained in a sample based on the strength of the infectivity.
Adeno-associated virus (AAV) is a non-enveloped virus classified into the genus Dependoparvovirus of the family Parvoviridae. AAV capsid particles are composed of three types of proteins (VP1, VP2, and VP3), in which about 60 protein molecules are mixed in a ratio of VP1:VP2:VP3=about 1:1:10, collectively forming the shape of a regular icosahedron with a diameter of from 20 nm to 30 nm.
Natural AAV lacks autonomous proliferation capacity and replication depends on helper viruses such as adenoviruses and herpesviruses. In the presence of the helper viruses, the AAV genome is replicated within host cells to form complete AAV particles containing the AAV genome, and the AAV particles are released from the host cells. On the other hand, in the absence of the helper viruses, the AAV genome is in a state of being maintained in the episome or integrated into the host chromosome (latent state).
AAV can infect cells of a wide variety of species, including human, and also infect non-dividing cells that have been differentiated such as blood cells, muscles, and nerve cells, AAV is less concerned about side effects because it is not pathogenic to humans, AAV particles are physicochemically stable, and the like. Therefore, AAV is attracting attention for its utility value as a vector for gene introduction for the treatment of congenital genetic diseases.
It is reported that a substitution of an amino acid residue at specific position in AAV with a different amino acid residue makes a difference in the strength of the infectivity to cells (NPL 1). Therefore, when AAV is used as a vector for gene introduction, it is necessary to quickly and easily analyze differences in the strength of the infectivity thereof.
In general, production of gene recombinant AAV vector (hereinafter, also simply referred to as “AAV vector”) is carried out by introducing nucleic acids that encode essential elements for AAV particle formation into cells to prepare cells capable of producing AAV (hereinafter also referred to as “AAV-producing cells”) and culturing the cells to express the essential elements for AAV particle formation. The produced AAV vector is recovered and purified from AAV producing cells to obtain a therapeutic AAV vector formulation.
As a method for recovering and purifying an AAV vector from AAV producing cells, there is a method with affinity chromatography based on the binding affinity with AAV using an adsorbent comprising an insoluble carrier and an AAV-binding protein immobilized on the carrier, by which the vector can be recovered and purified from the solution comprising the AAV vector in which contaminants coexist. As a specific example, PL 1 achieves purification of the AAV vector with high purify by using a polypeptide with enhanced stability to heat, acid and alkali, comprising the extracellular domain 1 (PKD1) and domain 2 (PKD2) of KIAA0319L (UniProt No. Q8IZA0), wherein an amino acid residue at a specific position within these domains is substituted with a different amino acid residue, as an AAV-binding protein immobilized on the insoluble carrier (hereinafter, also simply referred to as “ligand protein”).
It is reported that a substitution of an amino acid residue at a specific position in AAV with a different amino acid residue makes a difference in the strength of the infectivity to cells (NPL 1). Therefore, when AAV is used as a vector for gene introduction, it is necessary to quickly and easily analyze differences in the strength of the infectivity thereof. However, as disclosed in NPL 1, conventional analysis methods use cultured cells, which require a lot of time and effort.
As described above, the adeno-associated virus (AAV)-binding protein used as a ligand protein in WO2021/106882 has enhanced stability to heat, acid, and alkali than the wild type (AAV-binding protein without amino acid substitution). On the other hand, further enhancement of the binding activity to AAV is necessary.
In addition, as described above, when AAV is used as a vector for gene introduction, it is necessary to quickly and easily analyze differences in the strength of the infectivity thereof. However, as disclosed in Lochrie, M. A., et al., Journal of virology, 80(2), 821, 2006 (NPL 1), conventional analysis methods use cultured cells, which require a lot of time and effort.
Therefore, the object of the invention is to provide a modified AAV-binding protein having an enhanced binding activity to an AAV, as well as providing a method for analyzing an adeno-associated virus contained in a sample based on the strength of the infectivity.
In order to solve the above problems, the present inventors conducted intensive studies. As a result, the inventors found that a substitution of a specific amino acid residue in the amino acid residues constituting an adeno-associated virus (AAV)-binding protein with a different amino acid residue enhances a binding activity to AAV, and analysis of an adeno-associated virus (AAV) contained in a sample using an adsorbent comprising an insoluble carrier and an AAV-binding protein immobilized on the carrier enables the analysis of the AAV based on the strength of the infectivity. This has led to the completion of the invention.
The present invention encompasses the following aspects of [1] to [14].
Hereinafter, the invention will be described in detail.
The term infectivity as used herein refers to the ability of a virus such as AAV to infect host cells. Infectivity may be expressed by using a known index indicating the ability to infect, as a proportion or ratio of the index of the variant (amino acid substitution product) to that of the wild type. Examples of the index indicating the ability to infect include the infection rate when a virus infects host cells, the ability of a virus to bind to a receptor on the cell membrane of host cells, the level of the activity of a gene contained therein, the intensity of luminescence inside or outside an animal of the protein encoded by the gene, such as GFP (for example, Fig. 6 of Amanda M. Dudek, et al., Journal of virology, 92(7), e02213-17, 2018), and such expression levels of markers indicating the effectiveness of a virus such as AAV. Host cells generally refer to various cells such as HEK293 and HT-1080, cells of animals such as mice and monkeys, which can take any form, including floating cells, adherent cells, and living cells. The concentration for infecting a virus such as AAV can be changed as appropriate so that the difference can be easily seen. Although the infectivity of an AAV variant is not limited, for example, as described in NPL 1, the infectivity can be obtained by infecting host cells such as human HepG2 cells with the AAV variant and the wild type AAV, measuring the infection rates thereof, and calculating the ratio of the infection rate of the AAV variant relative to the infection rate of the wild type AAV being set to 100. Alternatively, the infectivity of the variant may also be determined as a relative value to the infectivity of the wild type.
The AAV binding protein described herein is a protein comprising at least amino acid residues from serine (Ser) at position 312 to aspartic acid (Asp) at position 500, which are the regions corresponding to the extracellular domain 1 (PKD1) and domain 2 (PKD2) in the amino acid sequence of KIAA0319L (UniProt No. Q8IZA0) set forth in SEQ ID NO: 1, wherein amino acid substitution(s) is(are) present at specific position(s) of the amino acid residues from position 312 to position 500. Therefore, the AAV-binding protein of the invention may comprise: all or part of the other extracellular domains (domain 3 (PKD3), domain 4 (PKD4), and domain 5 (PKD5)) on the C-terminal side of the protein; all or part of a signal sequence such as the MANSC (motif at N terminus with seven cysteines) domain or cysteine-rich region on the N-terminal side of PKD1; and all or part of the transmembrane and intracellular domains on the N-terminal and/or C-terminal side of the extracellular domain.
The amino acid substitutions at specific position(s) is(are) specifically at least any one of amino acid substitutions of:
Among them, (1) Ala330Val is an amino acid substitution which significantly enhances the binding activity to AAV, and significantly suppresses the generation of decomposition products derived from an AAV-binding protein at the time of the production of the AAV-binding protein with genetic engineering techniques using recombinant E. coli, and significantly improves the production efficiency of the AAV-binding protein using the recombinant E. coli. For that reason, preferable aspect of the AAV-binding protein of the invention is an AAV binding protein wherein at least the amino acid substitution of (1) Ala330Val is present.
The number of amino acid substitutions set forth in (1) to (5) is not particularly limited. In other words, only any one of the amino acid substitutions set forth in the (1) to (5) may be present, or two or more of the amino acid substitutions set forth in the (1) to (5) may be present. However, since the amino acid substitutions set forth in (1) and (2) are substitutions that occur at the same position, even if two or more amino acid substitutions occur, either (1) or (2) can occur.
The AAV to enhance the binding activity is not particularly limited, and it may be a naturally occurring AAV or an artificially produced AAV. Examples of the naturally occurring AAV include serotype 1 (AAV1), serotype 2 (AAV2), serotype 3 (AAV3), serotype 5 (AAV5), serotype 6 (AAV6), serotype 7 (AAV7), serotype 8 (AAV8), serotype 9 (AAV9), serotype 10 (AAV10), serotype 11 (AAV11), serotype 12 (AAV12), and serotype 13 (AAV13). In addition, examples of the artificially produced AAV include AAVrh8, AAVrh10, and chimeric AAVs having two or more characteristics (cell tropism and infectivity) of these serotypes.
In (ii), (v), (viii), and (xi) described above, the expression “one or several” refers to, for example, from 1 to 50, from 1 to 30, from 1 to 20, from 1 to 10, from 1 to 9, from 1 to 8, from 1 to 7, from 1 to 6, from 1 to 5, from 1 to 4, from 1 to 3, from 1 to 2, or 1, although it depends on positions of amino acid substitutions and types of amino acid residues in the protein conformation of the AAV-binding protein.
Examples of the substitutions, deletions, insertions, or additions described in (ii), (v), (viii), and (xi) above include substitutions of amino acid residues disclosed in WO2021/106882. Among them, particularly preferable aspect of the AAV-binding protein of the invention is an AAV-binding protein wherein at least amino acid substitutions of Val317Asp, Tyr342Ser, Lys362Glu, Lys371Asn, Val381Ala, Ile382Val, Gly390Ser, Lys399Glu, Ser476Arg and Asn487Asp are present, as the stability to heat, acid and alkali is particularly enhanced.
Examples of the particularly preferable aspect described above include AAV-binding proteins set forth in the following (A) to (K).
Specific examples of the particularly preferable aspect described above include AAV-binding proteins as follows.
In (ii), (v), (viii), and (xi) described above, the expression “substitutions of one or several amino acid residues” may include a conservative substitution between amino acids having similar physical and/or chemical properties, in addition to the amino acid substitution(s) at specific position(s) described above. Those skilled in the art know that in the case of conservative substitution, the protein function is usually maintained between a protein having a substitution and a protein lacking the substitution. One example of a conservative substitution is a substitution that occurs between glycine and alanine, serine and proline, or glutamic acid and alanine (Protein Structure and Function, Medical Sciences International, Ltd., 9, 2005). Another example of the amino acid substitution is a substitution for monomerizing the AAV-binding protein of the invention. Specific examples thereof include amino acid substitutions in which a cysteine residue that easily constitutes a higher-order structure is substituted with a serine residue or a methionine residue.
Further, the expression “one or more substitutions, deletions, insertions, and additions” in (ii), (v), (viii), and (xi) described above may also include naturally occurring mutations due to the difference in the origin of the AAV-binding protein, the difference in species, and the like (mutants or variants).
In (iii), (vi), (ix), and (xii) described above, the homology of the amino acid sequences may be 70% or more, and the homology may be more than that (e.g., 80% or more, 85% or more, 90% or more, or 95% or more). The term “homology” used herein may refer to similarity or identity and may particularly refer to identity. The “amino acid sequence homology” means homology to the entire amino acid sequence.
The “identity” between amino acid sequences means the proportion of amino acid residues of the same type in those amino acid sequences (Experimental Medicine, February 2013, Vol. 31, No. 3, YODOSHA CO., LTD.). The “similarity” between amino acid sequences means the sum of the proportion of amino acid residues of the same type in those amino acid sequences and the proportion of amino acid residues having similar side chain properties (Experimental Medicine, February 2013, Vol. 31, No. 3, YODOSHA CO., LTD.). The homology of the amino acid sequence can be determined by using an alignment program such as BLAST (Basic Local Alignment Search Tool) or FASTA.
It is also possible to further add a useful oligopeptide to the N-terminal side or the C-terminal side of the AAV-binding protein of the invention for separation from a solution with the presence of contaminants. Examples of the oligopeptide include polyhistidine, polylysine, polyarginine, polyglutamic acid, and polyaspartic acid. In addition, a cysteine-containing oligopeptide useful for immobilizing the AAV-binding protein of the invention to a solid phase such as a support for chromatography may be further added to the N-terminal side or the C-terminal side of the AAV-binding protein of the invention.
The length of an oligopeptide to be added to the N-terminal side or the C-terminal side of the AAV-binding protein is not particularly limited as long as the AAV-binding property and stability of the AAV-binding protein of the invention are not impaired. When adding the oligopeptide to the AAV-binding protein of the invention, it is possible to prepare a polynucleotide encoding the oligopeptide, and then, add the polynucleotide to the N-terminal side or the C-terminal side of the AAV-binding protein by a genetic engineering method known to those skilled in the art. Alternatively, it is also possible to chemically synthesize the oligopeptide and add the oligopeptide to the N-terminal side or the C-terminal side of the AAV-binding protein of the invention via chemical binding.
It is also possible to further add a signal peptide for promoting efficient expression in Escherichia coli (E. coli) used as a host, to the N-terminal side of the AAV-binding protein of the invention. Examples of the signal peptide can include signal peptides that promotes protein secretion in a periplasmic space, such as PelB, OmpA, DsbA, DsbC, MalE, and TorT (Japanese Unexamined Patent Publication (Kokai) No. 2011-097898).
Examples of a method for producing a polynucleotide encoding the AAV-binding protein of the invention (hereinafter referred to as “polynucleotide of the invention”) can include: (I) a method for artificially synthesizing a polynucleotide comprising a nucleotide sequence that is converted from the amino acid sequence of the AAV-binding protein of the invention; and (II) a method in which a polynucleotide comprising the entire or partial sequence of the AAV-binding protein is prepared directly in an artificial manner or by a DNA amplification method such as a PCR method using cDNA or the like of the AAV-binding protein, and the prepared polynucleotide is ligated in a suitable manner.
In the method (I) described above, when converting from an amino acid sequence to a nucleotide sequence, it is preferable to perform the conversion in consideration of the frequency of use of codons in E. coli which is the host to be transformed. Specifically, the conversion may be performed to avoid codons such as AGA/AGG/CGG/CGA for arginine (Arg), ATA for isoleucine (Ile), CTA for leucine (Leu), GGA for glycine (Gly), and CCC for proline (Pro) because those codons are used infrequently (they are so-called rare codons). Analysis of the frequency of codon usage is also possible by using a public database (e.g., the Codon Usage Database on the website of Kazusa DNA Research Institute).
When introducing a mutation into the polynucleotide of the invention, the error-prone PCR method can be used. The reaction conditions in the error-prone PCR method are not particularly limited as long as the desired mutation can be introduced into the polynucleotide encoding the AAV-binding protein. For example, the mutation can be introduced by, making the concentration of four kinds of deoxynucleotides (dATP/dTTP/dCTP/dGTP) serving as substrates different, adding MnCl2 at a concentration of from 0.01 to 10 mM (preferably from 0.1 to 1 mM) to a PCR reaction solution, and performing PCR. In addition, examples of a mutagenesis method other than the error prone PCR method include a method for producing a mutant by allowing an agent serving as a mutagen to come into contact with or act on a polynucleotide comprising the entire or partial sequence of an AAV-binding protein or irradiating the polynucleotide with ultraviolet rays to introduce a mutation into the polynucleotide. As an agent used as a mutagen in the method, a mutagenic agent commonly used by those skilled in the art such as hydroxylamine, N-methyl-N′-nitro-N-nitrosoguanidine, nitrite, sulfurous acid, or hydrazine may be used.
When transforming E. coli as the host with the polynucleotide of the invention, the polynucleotide of the invention itself may be used. However, it is more preferable to use a polynucleotide obtained by inserting the polynucleotide of the invention at an appropriate position in an expression vector (e.g., bacteriophage, cosmid, plasmid, or the like commonly used for transformation of prokaryotic or eukaryotic cells). The expression vector is not particularly limited as long as it exists stably and can be replicated in the host to be transformed (E. coli). Examples of the expression vector can include a pET plasmid vector, a pUC plasmid vector, a pTrc plasmid vector, and a pCDF plasmid vector.
In addition, the appropriate position means a position that does not destroy the replication function of the expression vector, the desired antibiotic marker, or the transmissible region. When inserting the polynucleotide of the invention into the expression vector, it is preferable to insert it in a state of being linked to a functional polynucleotide such as a promoter required for expression. Examples of the promoter include a trp promoter, a tac promoter, a trc promoter, a lac promoter, a T7 promoter, a recA promoter, and an lpp promoter.
E. coli as the host may be transformed using the expression vector produced by the method in which the polynucleotide of the invention is inserted (hereinafter referred to as “expression vector of the invention”), by a method usually used by those skilled in the art. Specifically, E. coli may be transformed by a method described in a known document (e.g., Molecular Cloning, Cold Spring Harbor Laboratory, 256, 1992) or the like. Transformants obtained via transformation by the method described above are screened by an appropriate method such that a transformant capable of expressing the AAV-binding protein of the invention (hereinafter referred to as “transformant of the invention”) can be obtained.
The expression vector of the invention from the transformant of the invention may be prepared from the culture obtained by culturing the transformant of the invention by an alkaline extraction method or a commercially available extraction kit such as a QIAprep Spin Miniprep kit (manufactured by QIAGEN).
The AAV-binding protein of the invention can be produced by culturing the transformant of the invention and recovering the AAV-binding protein of the invention from the obtained culture. The culture described herein includes not only the cultured cells of the transformant of the invention itself, but also the medium used for the culture.
The transformant used in the method for producing the protein of the invention may be cultured in a medium suitable for culturing a target host (E. coli). Examples of a preferable medium include LB (Luria-Bertani) medium supplemented with necessary nutrient sources. To selectively grow the transformant of the invention depending on whether or not the vector of the invention is introduced, it is preferable to add a drug corresponding to a drug resistance gene contained in the vector to the medium and culture the transformant. For example, when the vector contains a kanamycin resistance gene, kanamycin may be added to the medium.
In addition to carbon, nitrogen, and inorganic salt sources, suitable nutrient sources may be added to the medium. The medium may contain one or more reducing agents optionally selected from the group consisting of glutathione, cysteine, cystamine, thioglycolate, and dithiothreitol. The culture temperature is generally from 10° C. to 40° C., preferably from 20° C. to 37° C., more preferably around 25° C., but may be selected depending on the characteristics of the protein to be expressed. The pH of the medium is pH 6.8 to pH 7.4, preferably around pH 7.0. When the vector of the invention contains an inducible promoter, it is preferable to induce the vector under conditions that allow favorable expression of the AAV-binding protein of the invention.
As the inducer, isopropyl-β-D-thiogalactopyranoside (IPTG) can be exemplified. The turbidity of the culture solution (absorbance at 600 nm) is measured, and when it becomes about 0.5 to 1.0, an appropriate amount of IPTG is added and then the culture is continued, thereby achieving the expression of the AAV-binding protein. The concentration of IPTG added may be appropriately selected from a range of from 0.005 to 1.0 mM, preferably a range of from 0.01 to 0.5 mM. Various conditions relating to IPTG induction may be performed under conditions well known in the art.
To recover the AAV-binding protein of the invention from the culture obtained by culturing the transformant of the invention, the AAV-binding protein of the invention may be recovered by separation/purification from the culture by a method suitable for the expression form of the AAV-binding protein of the invention in the transformant of the invention. For example, when the AAV-binding protein of the invention is expressed in the culture supernatant, the cells may be separated by centrifugation and the AAV-binding protein may be purified from the obtained culture supernatant. In addition, when the AAV-binding protein of the invention is expressed intracellularly (in a periplasmic space), after collecting the cells by centrifugation, the cells are disrupted by adding an enzyme treatment agent, a surfactant, or the like to extract the AAV-binding protein, and then the AAV-binding protein may be purified.
To purify the AAV-binding protein of the invention, a method known in the art may be used, and one example thereof is separation/purification using liquid chromatography. Examples of liquid chromatography include on ion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, and affinity chromatography. By performing a purification operation in combination with these chromatography methods, the AAV-binding protein of the invention can be prepared with high purity.
As a method for measuring the binding activity of the obtained AAV-binding protein of the invention to AAV, for example, the binding activity to AAV may be measured by using an enzyme-linked immunosorbent assay method (hereinafter referred to as “ELISA method”), a surface plasmon resonance method, or the like. The AAV used for measuring the binding activity may be an AAV vector or a VLP (virus-like particle). In addition, any serotype of the AAV vector and VLP may be used as long as it exhibits the binding activity to the AAV-binding protein of the invention.
The AAV-binding protein of the invention can be used, for example, for purification or analysis of AAV. The AAV-binding protein of the invention can be used, for example, by immobilizing it on an insoluble carrier. In other words, purification or analysis of AAV can be specifically performed using, an AAV adsorbent comprising, for example, an insoluble carrier and the AAV-binding protein of the invention immobilized on the insoluble carrier. The AAV adsorbent of the invention described herein also refers to an AAV adsorbent comprising the insoluble carrier and the AAV-binding protein of the invention immobilized on the insoluble carrier. The purification of AAV is not limited to the purification of AAV from a solution in which contaminants coexist, but also includes the purification of AAV based on its structure, properties, activity, and the like. The material, shape, and the like of the insoluble carrier are not particularly limited, examples of which include those disclosed in WO2021/106882. Further, the insoluble carrier may be, for example, porous or non-porous.
The AAV-binding protein of the invention can be immobilized on an insoluble carrier, for example, via a covalent bond. Specifically, the AAV-binding protein of the invention can be immobilized on an insoluble carrier by covalently binding the AAV-binding protein of the invention and the insoluble carrier, for example, via an active group of the insoluble carrier. The immobilization of the AAV-binding protein of the invention on the insoluble carrier may be performed, for example, based on the disclosure in WO2021/106882.
The AAV adsorbent of the invention can be used, for example, by filling it into a column to purify AAV. Specifically, for example, by adding a solution containing AAV to a column filled with the AAV adsorbent of the invention (hereinafter, also simply referred to as “the column of the invention”), the AAV is adsorbed by the adsorbent, and the AAV adsorbed by the adsorbent is eluted such that the AAV can be purified. In other words, the present invention provides a method for purifying AAV comprising a step of adding a solution containing AAV to a column filled with the AAV adsorbent of the invention to allow the AAV to be adsorbed by the adsorbent and a step of eluting the AAV adsorbed by the adsorbent. The AAV purification using the column of the invention may be performed, for example, based on the disclosure in WO2021/106882.
By purifying AAV with the AAV adsorbent of the invention, for example, purified AAV can be obtained. In other words, the method for purifying AAV may be, in one aspect, a method for producing AAV, and specifically, a method for producing purified AAV. AAV is obtained, for example, as an elution fraction containing AAV. This means that the fraction containing the eluted AAV can be obtained. For example, the AAV fraction can be obtained by a conventional method. As a method for obtaining the AAV fraction, a method comprising replacing a collection container every fixed time or fixed capacity, a method comprising replacing a collection container according to the shape of an eluate chromatogram, and a method using an automated fraction collector such as autosampler can be exemplified. Furthermore, AAV can also be recovered from the fraction containing AAV. Recovery of AAV from the fraction containing AAV can be performed, for example, by known methods used for protein purification.
The present invention also encompasses the following aspects [15] to [17]:
The AAV-binding protein which is a ligand protein (a protein to be immobilized on the insoluble carrier) for the adsorbent for use in the AAV analysis in the present invention (hereinafter, also referred to as “AAV adsorbent”) is a polypeptide comprising at least a region corresponding to the extracellular domain 1 (PKD1) (amino acid residues from serine (Ser) at position 312 to glutamic acid (Glu) at position 401 of SEQ ID NO: 94) or the extracellular domain 2 (PKD2) (amino acid residues from isoleucine (Ile) at position 409 to aspartic acid (Asp) at position 500 of SEQ ID NO: 94) in KIAA0319L (UniProt; Accession Number: Q8IZA0, SEQ ID NO: 94) which is one aspect of the AAV receptor (AAVR), having AAV binding activity. Specific examples thereof include a polypeptide selected from any of the following (xiii) to (xv):
In addition, preferable aspects of the AAV-binding protein includes a polypeptide comprising at least regions corresponding to the PKD1 and the PKD2 (amino acid residues from serine (Ser) at position 312 to aspartic acid (Asp) at position 500 of SEQ ID NO: 94) in KIAA0319L (SEQ ID NO: 94). Specific examples thereof include a polypeptide selected from any of the following (xvi) to (xviii):
The polypeptide described in any of (xiii) to (xviii) described above may suffice as long as it comprises at least a region corresponding to PKD1 and/or PKD2 in KIAA0319L. For example, it may comprise: all or part of a region corresponding to the other extracellular domains (domain 3 (PKD3), domain 4 (PKD4), and domain 5 (PKD5)) on the C-terminal side of PKD2; all or part of a region corresponding to a signal sequence such as the MANSC (motif at N terminus with seven cysteines) domain on the N-terminal side of PKD1 or cysteine-rich region; and all or part of the transmembrane and intracellular domains on the N-terminal and/or C-terminal side of the extracellular domain.
Examples of the (xiv) and (xvii) described above includes polypeptides comprising at least amino acid residues from serine at position 25 (Ser) to aspartic acid (Asp) at position 213 of the amino acid sequence set forth in SEQ ID NO: 97, and AAV-binding proteins disclosed in WO2021/106882. Examples of the substitutions, deletions, insertions, or additions described in (xiv) and (xvii) described above include substitutions of amino acid residues disclosed in WO2021/106882.
In (xiv) and (xvii) described above, the expression “one or several” refers to, for example, from 1 to 50, from 1 to 30, from 1 to 20, from 1 to 10, from 1 to 9, from 1 to 8, from 1 to 7, from 1 to 6, from 1 to 5, from 1 to 4, from 1 to 3, from 1 to 2, or 1, although it depends on positions of amino acid substitutions and types of amino acid residues in the conformation of the AAVR. Substitutions of “one or several” amino acid residues may be present at positions other than the substitutions of the amino acid residues disclosed in WO2021/106882, as long as it has AAV binding activity.
In the “substitutions of one or several amino acid residues” in (xiv) and (xvii) described above, a conservative substitution between amino acids having similar physical and/or chemical properties may be present in addition to the substitution(s) of amino acid residue(s) at specific position(s) described above. Those skilled in the art know that in the case of conservative substitution, the protein function is usually maintained between a protein having a substitution and a protein lacking the substitution. One example of a conservative substitution is a substitution that occurs between glycine and alanine, serine and proline, or glutamic acid and alanine (Protein Structure and Function, Medical Sciences International, Ltd., 9, 2005). Further, “substitutions, deletions, insertions, and additions of one or several amino acid residues” in (xiv) and (xvii) described above also include naturally occurring mutations due to the difference in the origin of AAV-binding protein, the difference in species, and the like (mutants or variants).
The homology of the amino acid sequences in (xv) and (xviii) described above may be 70% or more, and the homology may be more than that (e.g., 80% or more, 85% or more, 90% or more, or 95% or more). The term “homology” used herein may refer to similarity or identity and may particularly refer to identity. The “amino acid sequence homology” means homology to the entire amino acid sequence. The “identity” between amino acid sequences means the proportion of amino acid residues of the same type in those amino acid sequences (Experimental Medicine, February 2013, Vol. 31, No. 3, YODOSHA CO., LTD.). The “similarity” between amino acid sequences means the sum of the proportion of amino acid residues of the same type in those amino acid sequences and the proportion of amino acid residues having similar side chain properties (Experimental Medicine, February 2013, Vol. 31, No. 3, YODOSHA CO., LTD.). The homology of the amino acid sequence can be determined by using an alignment program such as BLAST (Basic Local Alignment Search Tool) or FASTA.
In the present invention, the insoluble carrier for use in the immobilization of the AAV-binding protein is not particularly limited as long as it is insoluble in AAV-containing samples and solutions used for the analysis or purification (such as eluate, equilibration solution, and washing solution). Examples thereof include carriers made with polysaccharides such as agarose, alginate (alginic acid salt), carrageenan, chitin, cellulose, dextrin, dextran, and starch, carriers made with synthetic polymers such as polyvinyl alcohol, polymethacrylate, poly(2-hydroxyethyl methacrylate), and polyurethane, and carriers made with ceramics such as silica. Of these, carriers made with polysaccharides and carriers made with synthetic polymers are preferable as insoluble carriers. Examples of the preferable carrier include hydroxylated polymethacrylate gel such as TOYOPEARL (manufactured by Tosoh Corporation), agarose gel such as Sepharose (manufactured by Cytiva), and cellulose gel such as Cellufine (manufactured by JNC). The shape of insoluble carrier is not particularly limited, but a shape that can be filled in a column is preferred. The insoluble carrier may be, for example, a granular substance, a monolith-like substance, a film-like substance, a fibrous substance, or the like. Further, the insoluble carrier may be, for example, porous or non-porous.
The immobilization of the AAV-binding protein on the insoluble carrier can be carried out by immobilization, for example, via a covalent bond. Specifically, the AAV adsorbent for use in the present invention can be produced by immobilization of the AAV-binding protein on the insoluble carrier by covalently binding therebetween, for example, via an active group of the insoluble carrier. Examples of an active group include an N-hydroxy succinimide (NHS)-activated ester group, an epoxy group, a carboxy group, a maleimide group, a haloacetyl group, a tresyl group, a formyl group, and a haloacetamide group. As the insoluble carrier having an active group, for example, a commercially available insoluble carrier having an active group may be used as it is, or an active group may be introduced into the insoluble carrier for use. Examples of a commercially available carrier having an active group may include TOYOPEARL AF-Epoxy-650M, TOYOPEARL AF-Tresyl-650M (each manufactured by Tosoh Corporation), HiTrap NHS-activated HP Columns, NHS-activated Sepharose 4 Fast Flow, Epoxy-activated Sepharose 6B (each manufactured by Cytiva), and SulfoLink Coupling Resin (manufactured by Thermo Fisher Scientific).
As a method for introducing an active group on the carrier surface, a method in which a compound having two or more active sites is allowed to react, at one of the active sites, with a hydroxy group, an epoxy group, a carboxy group, an amino group, or the like present on the carrier surface can be exemplified.
Examples of a compound for introducing an epoxy group to a hydroxy group or an amino group present on the carrier surface may include epichlorohydrin, ethanediol diglycidyl ether, butanediol diglycidyl ether, hexanediol diglycidyl ether, and polyethylene glycol diglycidyl ether. Specific examples of polyethylene glycol diglycidyl ether may 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).
Examples of a compound for introducing a carboxy group to an epoxy group present on the carrier surface may include 2-mercaptoacetic acid, 3-mercaptopropionic acid, 4-mercaptobutyric acid, 6-mercaptobutyric acid, glycine, 3-aminopropionic acid, 4-aminobutyric acid, and 6-aminohexanoic acid.
Examples of a compound for introducing a maleimide group to a hydroxy group, an epoxy group, a carboxy group, or an amino group present on the carrier surface may 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)phenyl isocyanate, 2-maleimidoacetic acid, 3-maleimidopropionic acid, 4-maleimidobutyric acid, 6-maleimidohexanoic acid, N-(α-maleimidoacetoxy)succinimide ester, (m-maleimidobenzoyl)N-hydroxy succinimide ester, succinimidyl-4-(maleimidomethyl)cyclohexane-1-carbonyl-(6-aminohexanoic acid), succinimidyl-4-(maleimidomethyl)cyclohexane-1-carboxylic acid, (p-maleimidobenzoyl)N-hydroxy succinimide ester, and (m-maleimidobenzoyl)N-hydroxy succinimide ester.
Examples of a compound for introducing a haloacetyl group to a hydroxy group or an amino group present on the carrier surface may 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.
As a method for introducing an active group on the carrier surface, a method in which ω-alkenyl alkane glycidyl ether is reacted with a hydroxy group or an amino group present on the carrier surface, and then, ω-alkenyl is halogenated with a halogenating agent for activation can also be exemplified. Examples of ω-alkenyl alkane glycidyl ether may include allyl glycidyl ether, 3-butenyl glycidyl ether, and 4-pentenyl glycidyl ether. Examples of a halogenating agent may include N-hlorosuccinimide, N-bromosuccinimide, and N-iodosuccinimide.
Another example of a method for introducing an active group on the carrier surface is a method for introducing an active group to a carboxy group present on the carrier surface using a condensing agent and an additive. Examples of a condensing agent may include 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), dicyclohexylcarbodiamide, and carbonyldiimidazole. Examples of an additive may include N-hydroxysuccinimide (NHS), 4-nitrophenol, and 1-hydroxybenzotriazole.
Immobilization of the AAV-binding protein on an insoluble carrier can be carried out, for example, in a buffer. Examples of a buffer may include acetic acid buffer, phosphoric acid buffer, MES (2-morpholinoethanesulfonic acid) buffer, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer, Tris (Tris(hydroxymethyl)aminomethane) buffer, and boric acid buffer. The reaction temperature for immobilization may be set as appropriate in consideration of reactivity of an active group and stability of an AAV-binding protein. The reaction temperature at the time of immobilization may be, for example, from 4° C. to 50° C., preferably from 10° C. to 35° C.
The present invention is also characterized in that AAV contained in a sample is analyzed based on the strength of the infectivity, by a method comprising a step of allowing the AAV contained in the sample by an AAV adsorbent produced by the above-mentioned method (hereinafter, also simply referred to as “adsorption step”), and a step of eluting the AAV adsorbed by said adsorbent using an eluate (hereinafter, also simply referred to as “elution step”). It is preferable that the AAV adsorbent for use in the adsorption step and the elution step is filled in a column (hereinafter, also referred to as “AVR column”), since these steps can be easily performed. A detailed explanation of aspects using an AVR column will be given below as an example.
The sample containing AAV can be added (applied) to an AVR column using, for example, a liquid transfer means such as a pump. Adding a liquid to a column described herein is also referred to as “transferring the liquid to the column.” The sample containing AAV may be treated by solvent substitution with an appropriate buffer before being added to the AVR column. Also, the AVR column may be equilibrated with an appropriate buffer (equilibrating solution) before adding the sample containing AAV to the AVR column. It is expected that the equilibration will allow, for example, purification of AAV to a higher degree of purity. Examples of the buffer used for solvent substitution and equilibration may include a phosphate buffer, an acetate buffer, a succinate buffer, a citrate buffer, a Tris buffer, a HEPES buffer and an MES buffer which have a buffering capacity in the neutral region (herein refers to the pH 4.0 to 9.0 region). An inorganic salt such as 10 mM to 600 mM sodium chloride and calcium chloride may be further added to such buffer, for example. The buffer used for solvent substitution and the equilibrating solution may be the same or different. In addition, if components other than AAV such as contaminants remain in the AVR column after passing the sample containing AAV through the AVR column, such components may be removed (washed) from the AVR column before the AAV adsorbed by the AAV adsorbent is eluted with an eluate (i.e., before the elution step). Components other than AAV can be removed from the AVR column by using, for example, a suitable buffer as a washing solution. Regarding the washing solution, for example, the descriptions regarding the buffer solutions used for solvent replacement and equilibration can be applied mutatis mutandis.
In the elution step, in order to elute AAV based on the strength of the infectivity, elution is performed using a gradient. The gradient may be changed in two or more steps (stepwise) (stepwise gradient), or may be changed with a linear gradient (linear gradient). Examples of gradient elution include a gradient in which the pH is lowered from the neutral region to an acidic region (a region more acidic than the neutral region). The pH of the eluate at the beginning of the gradient may be in the neutral range, that is, pH 4.0 to 9.0 region, preferably pH 4.5 to 6.0. The pH of the eluate after gradient, which is an acidic region, may be 3.5 or lower, preferably 2.0 to 2.5. The lower limit of the flow rate is not particularly limited, and the upper limit depends on the back pressure of the insoluble carrier used in the AAV adsorbent. Typically, the flow rate is preferably 0.5 mL/min to 2.0 mL/min. The gradient time is sufficient as long as differences in the strength of the infectivity can be identified as a difference in the elution time. Specifically, the gradient time is preferably 5 minutes or longer, more preferably 20 minutes or longer, and even more preferably 45 minutes or longer.
The AAV that can be analyzed by the method of the invention may be a naturally occurring AAV or an artificially produced AAV. Examples thereof include AAVs of respective serotypes (such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and AAV13), AAV with amino acid substitution(s), AAV fused with peptide or protein, and AAV modified with polymer(s).
With use of the method of the invention, for example, it is possible to easily screen for AAV amino acid substitution product (AAV mutants) having desired infectivity. Specifically, screening for AAV mutants having desired infectivity can be performed by creating a mutant library of plasmids encoding AAV capsid using genetic modification such as error-prone PCR, then transforming the host with other plasmids required for AAV formation, and thereafter applying the AAV mutants expressed from the transformant to an AVR column.
According to the present invention, AAV contained in a sample can be analyzed depending on the strength of the infectivity. As a result, for example, AAV having high infectivity can be purified and used as a highly efficient gene therapy drug. Furthermore, if AAVs having different infectivity are mixed, the ratio thereof can be checked. Furthermore, it may also be used for quality control in the AAV production process to check for differences between lots and uniformity.
The adeno-associated virus (AAV)-binding protein of the invention is a protein in which amino acid residue(s) at specific positions within domains corresponding to the extracellular domain 1 (PKD1) and domain 2 (PKD2) of KIAA0319L (UniProt No. Q8IZA0) is(are) substituted with different amino acid residue(s). The AAV-binding protein of the invention has enhanced binding activity to AAV than conventional AAV-binding proteins. Furthermore, when the AAV-binding protein of the invention is produced by genetic engineering using recombinant E. coli, the production of decomposition products derived from the protein is suppressed compared to conventional AAV-binding proteins. Therefore, the AAV-binding protein can be efficiently produced, which is useful for industrial production of the protein. In addition, the present invention is characterized by that an adeno-associated virus (AAV) contained in a sample is analyzed based on the strength of the infectivity by a method comprising a step of allowing the AAV to be adsorbed by an adsorbent comprising an insoluble carrier and a polypeptide which is immobilized on the carrier and which comprises at least an amino acid sequence corresponding to the extracellular domain 1 or domain 2 of KIAA0319L, and a step of eluting the AAV adsorbed by said adsorbent using an eluate. According to the present invention, analysis of AAV contained in a sample based on the strength of the infectivity can be quickly and easily carried out, for which conventional methods require a lot of time and effort using cultured cells.
The invention will now be explained in greater detail using Examples and Comparative Examples, with the understanding that the invention is not limited to Examples.
Serotype 5 (AAV5) was selected among AAVs to prepare a virus vector used for the following examples.
As a result of qPCR, the concentration of the AAV vector (AAV5-EGFP) contained in the solution was 2.5×1013 cp/mL (cp: number of AAV particles). Furthermore, as a result of SDS-PAGE (silver staining), only bands corresponding to the three types of capsid proteins (VP1, VP2, VP3) constituting the AAV vector were observed, confirming that there was no problem with the purity.
A saturation substitution product library for amino acid residues corresponding to alanine (Ala330) at position 330 of SEQ ID NO: 1 (position 43 of SEQ ID NO: 4), tyrosine (Tyr331) at position 331 (position 44 of SEQ ID NO: 4), valine (Val332) at position 332 (position 45 of SEQ ID NO: 4), and leucine (Leu333) at position 333 (position 46 of SEQ ID NO: 4) was created using the vector pET-AVR10s (WO2021/106882) capable of expressing a polypeptide (SEQ ID NO: 4) containing the AAV-binding protein AVR10s as a template. A PelB signal peptide ranges from methionine (Met) at position 1 to alanine (Ala) at position 22, an AAV-binding protein AVR10s ranges from serine (Ser) at position 25 to aspartic acid (Asp) at position 213, and a tag sequence ranges from histidine (His) at position 214 to histidine (His) at position 219 in the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 4. AVR10s is a polypeptide with the following 10 amino acid substitutions in amino acid residues from position 312 to position 500 of SEQ ID NO: 1 corresponding to extracellular region domain 1 (PKD1) and domain 2 (PKD2) of KIAA0319L (UniProt No. Q8IZA0):
(1) A PCR primer set for creating the saturation substitution library was designed. Specifically,
Table 3 shows a list of the amino acid substitution sites for AVR10s and the binding activities to AAV5 (ratio when the above-described binding activity for AVR10s is set to 1) of the AAV-binding proteins expressed by the transformants selected in (5) above.
It is understood that the AAV-binding proteins, in which at least any one of amino acid substitutions of Ala330Cys (this notation indicates a substitution of alanine at position 330 of SEQ ID NO: 1 (position 43 of SEQ ID NO: 4) with cysteine; the same applies hereinafter), Ala330Phe, Ala330Leu, Ala330Arg, Ala330Val, Ala330Trp, Ala330Tyr, Tyr331Cys, Val332Trp, Val332Tyr, and Leu333Cys is present at the amino acid residues from serine (Ser) at position 312 to aspartic acid (Asp) at position 500 in the amino acid sequence set forth in SEQ ID NO: 1, have enhanced binding activity to AAV5, compared with the AAV-binding protein (AVR10s) without such an amino acid substitution.
Among them, AAV-binding proteins with any of the amino acid substitutions of Ala330Cys, Ala330Val, Ala330Tyr, and Val332Trp were found to have particularly enhanced binding activity to AAV5.
Among AAV-binding proteins with enhanced binding activity to AAV5 obtained in this Example, an AAV-binding protein with an amino acid substitution of Ala330Cys in AVR10s is named “AVR10s_A330C (SEQ ID NO: 15),”
A PelB signal peptide ranges from methionine (Met) at position 1 to alanine (Ala) at position 22, an AAV-binding protein (AVR10s amino acid substitution product) ranges from serine (Ser) at position 25 to aspartic acid (Asp) at position 213, and a tag sequence ranges from histidine (His) at position 214 to histidine (His) at position 219 in the polypeptides consisting of the amino acid sequences set forth in SEQ ID NOS: 15 to 25.
Table 4 shows the results. The AAV-binding proteins, in which at least any one of amino acid substitutions of Ala330Cys, Ala330Phe, Ala330Leu, Ala330Arg, Ala330Val, Ala330Trp, Ala330Tyr, Tyr331Cys, Val332Trp, Val332Tyr, and Leu333Cys is present at the amino acid residues from serine (Ser) at position 312 to aspartic acid (Asp) at position 500 in the amino acid sequence set forth in SEQ ID NO: 1, have a decreased proportion of an AAV-binding protein-derived decomposition product, compared with the AAV-binding protein without the amino acid substitution (AVR10s with a decomposition rate of 30.0%), suggesting enhanced efficiency of producing an AAV-binding protein using recombinant E. coli.
Among them, the AAV-binding protein (AVR10s_A330V or AVR10s_Y331C), in which an amino acid substitution of Ala330Val or Tyr331Cys is present, has a significantly decreased proportion of an AAV-binding protein-derived decomposition product (AVR10s_A330V with a decomposition rate of 1.9%, AVR10s_Y331C with a decomposition rate of 2.0%), promising that the efficiency of producing an AAV-binding protein using recombinant E. coli will be remarkably enhanced.
A saturation substitution product library for amino acid residues corresponding to glutamine (Gln347) at position 347 (position 60 of SEQ ID NO: 4), leucine (Leu348) at position 348 (position 61 of SEQ ID NO: 4), and isoleucine (Ile349) at position 349 (position 62 of SEQ ID NO: 4) was created using the vector pET-AVR10s (WO2021/106882) capable of expressing a polypeptide (SEQ ID NO: 4) comprising the AAV-binding protein AVR10s as a template.
Table 5 shows a list of the amino acid substitution sites for AVR10s and the binding activities to AAV5 (ratio when the above-described binding activity for AVR10s is set to 1) of the AAV-binding proteins expressed by the transformants selected in (3) above.
It is understood that the AAV-binding proteins, in which at least any one of amino acid substitutions of Gln347Ser, Leu348Cys, and Ile349Asn is present at the amino acid residues from serine (Ser) at position 312 to aspartic acid (Asp) at position 500 in the amino acid sequence set forth in SEQ ID NO: 1, have enhanced binding activity to AAV5, compared with the AAV-binding protein without the amino acid substitution (AVR10s).
Among AAV-binding proteins with enhanced binding activity to AAV5 obtained in this Example,
A PelB signal peptide ranges from methionine (Met) at position 1 to alanine (Ala) at position 22, an AAV-binding protein (AVR10s amino acid substitution product) ranges from serine (Ser) at position 25 to aspartic acid (Asp) at position 213, and a tag sequence ranges from histidine (His) at position 214 to histidine (His) at position 219 in the polypeptides consisting of the amino acid sequences set forth in SEQ ID NOS: 32 to 34.
Table 6 shows the results. The AAV-binding proteins, in which at least any one of amino acid substitutions of Gln347Ser, Leu348Cys, and Ile349Asn is present at the amino acid residues from serine (Ser) at position 312 to aspartic acid (Asp) at position 500 in the amino acid sequence set forth in SEQ ID NO: 1, have a decreased proportion of an AAV-binding protein-derived decomposition product, compared with the AAV-binding protein without the amino acid substitution (AVR10s with a decomposition rate of 18.4%), suggesting enhanced efficiency of producing an AAV-binding protein using recombinant E. coli.
Among the AAV-binding proteins evaluated in Examples 2 and 3, a protein with an amino acid substitution of Ala330Val (SEQ ID NO: 19, hereinafter also referred to as “AVR11a”) was selected, and a mutation was randomly introduced into the polynucleotide portion encoding the protein using error-prone PCR.
Tables 8 and 9 show lists of the amino acid substitution sites and the residual activities (%) after alkali treatment of the AAV-binding proteins expressed by the transformants selected in (7) above to AVR11a.
It can be said that the AAV-binding proteins, in which at least any one of amino acid substitutions of Ser312Pro, Ala313Val, Glu315Gly, Glu315Val, Gln318Arg, Lys323Met, Asn324His, Val326Glu, Gln327Leu, Leu328Gln, Leu328Arg, Leu328Pro, Asn329Tyr, Val332Glu, Val332Ala, Leu333Pro, Gln334Leu, Gln334His, Glu335Val, Pro337Gln, Lys338Glu, Glu340Asp, Thr341Ala, Thr343Pro, Thr343Ala, Thr343Ser, Asp345Gly, Ile349Thr, Thr350Ala, Asp354Val, Tyr355His, Met359Ile, Met359Leu, Glu360Asp, Ile366Thr, Leu367Val, Lys368Asn, Leu369Gln, Leu376Pro, Phe379Tyr, Lys380Asn, Lys380Arg, Ile(Val)382Asp (this notation indicates that isoleucine at position 382 of SEQ ID NO: 1 has been replaced with valine and then with aspartic acid; the same applies hereinafter), Ile(Val)382Ala, Glu384Val, Ala388Thr, His389Asp, His389Arg, His389Asn, His389Leu, Gly(Ser)390Asn, Gly392Arg, Val394Ala, Val394Asp, Asn395Asp, Val396Gly, Val398Ala, Arg403Ser, Arg403His, Lys404Ala, Arg406Ser, Ile409Asn, Ile411Thr, Ile411Leu, Val412Ile, Phe416Ser, Ile419Phe, Ile419Val, Ser420Thr, Thr423Ala, Ser425Gly, Ile428Val, Gly430Ala, Thr434Ala, Asp437Asn, Val440Ala, Glu446Asp, Lys448Glu, Lys448Asn, Leu451Ile, Glu453Lys, Lys455Glu, Lys455Arg, Lys464Asn, Lys464Glu, Val469Glu, Gly471Cys, Asn472Tyr, Asn472Asp, Tyr473His, Ser(Arg)476Gly, Thr478Ala, Val479Ala, Val480Asp, Val480Ala, Gly484Ser, Thr486Ser, Ser488Thr, Thr489Ala, Thr489Ser, Asn496Tyr, and Asn496Asp is present at the amino acid residues from serine (Ser) at position 312 to aspartic acid (Asp) at position 500 in the amino acid sequence set forth in SEQ ID NO: 1, have enhanced stability to alkali compared with AVR11a.
It was attempted to aggregate amino acid substitutions, which were found in Example 9 to be involved in enhancing the alkali stability of AAV-binding proteins, in AVR11a (SEQ ID NO: 19), thereby further enhancing the alkali stability. Specifically, 11 types of AAV-binding proteins shown in (a) to (k) below were designed and prepared.
Hereinafter, a method of preparing the 11 types of AAV-binding proteins shown in (a) to (k) above will be explained.
This protein was prepared by selecting Asn324His from among the amino acid substitutions involved in alkali stability revealed in Example 9 and introducing the amino acid substitution into AVR11a (SEQ ID NO: 19).
The amino acid sequence of AVR12a with a signal sequence and a polyhistidine tag is set forth in SEQ ID NO: 54. A PelB signal peptide ranges from methionine (Met) at position 1 to alanine (Ala) at position 22, an AAV-binding protein AVR12a (corresponding to a region from position 312 to position 500 in SEQ ID NO: 1) ranges from serine (Ser) at position 25 to aspartic acid (Asp) at position 213, and a tag sequence ranges from histidine (His) at position 214 to histidine (His) at position 219 in SEQ ID NO: 54. In addition, aspartic acid of Val317Asp is present at position 30, histidine of Asn324His is present at position 37, valine of Ala330Val is present at position 43, serine of Tyr342Ser is present at position 55, glutamic acid of Lys362Glu is present at position 75, asparagine of Lys371Asn is present at position 84, alanine of Val381Ala is present at position 94, valine of Ile382Val is present at position 95, serine of Gly390Ser is present at position 103, glutamic acid of Lys399Glu is present at position 112, arginine of Ser476Arg is present at position 189, and aspartic acid of Asn487Asp is present at position 200 in SEQ ID NO: 54.
This protein was prepared by selecting Glu335Val from among the amino acid substitutions involved in enhancing alkali stability revealed in Example 9 and introducing the amino acid substitution into AVR11a (SEQ ID NO: 19).
The amino acid sequence of AVR12b with a signal sequence and a polyhistidine tag is set forth in SEQ ID NO: 55. A PelB signal peptide ranges from methionine (Met) at position 1 to alanine (Ala) at position 22, an AAV-binding protein AVR12b (corresponding to a region from position 312 to position 500 in SEQ ID NO: 1) ranges from serine (Ser) at position 25 to aspartic acid (Asp) at position 213, and a tag sequence ranges from histidine (His) at position 214 to histidine (His) at position 219 in SEQ ID NO: 55. In addition, aspartic acid of Val317Asp is present at position 30, valine of Ala330Val is present at position 43, valine of Glu335Val is present at position 48, serine of Tyr342Ser is present at position 55, glutamic acid of Lys362Glu is present at position 75, asparagine of Lys371Asn is present at position 84, alanine of Val381Ala is present at position 94, valine of Ile382Val is present at position 95, serine of Gly390Ser is present at position 103, glutamic acid of Lys399Glu is present at position 112, arginine of Ser476Arg is present at position 189, and aspartic acid of Asn487Asp is present at position 200 in SEQ ID NO: 55.
This protein was prepared by selecting Asn324His and Glu335Val from among the amino acid substitutions involved in enhancing alkali stability revealed in Example 9 and introducing the amino acid substitutions into AVR11a (SEQ ID NO: 19).
The amino acid sequence of AVR13 with a signal sequence and a polyhistidine tag is set forth in SEQ ID NO: 56. A PelB signal peptide ranges from methionine (Met) at position 1 to alanine (Ala) at position 22, an AAV-binding protein AVR13 (corresponding to a region from position 312 to position 500 in SEQ ID NO: 1) ranges from serine (Ser) at position 25 to aspartic acid (Asp) at position 213, and a tag sequence ranges from histidine (His) at position 214 to histidine (His) at position 219 in SEQ ID NO: 56. In addition, aspartic acid of Val317Asp is present at position 30, histidine of Asn324His is present at position 37, valine of Ala330Val is present at position 43, valine of Glu335Val is present at position 48, serine of Tyr342Ser is present at position 55, glutamic acid of Lys362Glu is present at position 75, asparagine of Lys371Asn is present at position 84, alanine of Val381Ala is present at position 94, valine of Ile382Val is present at position 95, serine of Gly390Ser is present at position 103, glutamic acid of Lys399Glu is present at position 112, arginine of Ser476Arg is present at position 189, and aspartic acid of Asn487Asp is present at position 200 in SEQ ID NO: 56.
This protein is a protein for which Asn324His, Glu335Val, and Thr341Ala were selected from among the amino acid substitutions involved in enhancing alkali stability revealed in Example 9, and the amino acid substitutions were aggregated into AVR11a (SEQ ID NO: 19). Specifically, AVR14a was prepared by introducing mutations resulting in amino acid substitutions of Asn324His and Glu335Val into a polynucleotide (SEQ ID NO: 38) encoding the protein obtained in Example 9, in which an amino substitution of Thr341Ala was introduced into AVR11a (named “AVR11a_T341A,” SEQ ID NO: 39).
The amino acid sequence of AVR14a with a signal sequence and a polyhistidine tag is set forth in SEQ ID NO: 57. A PelB signal peptide ranges from methionine (Met) at position 1 to alanine (Ala) at position 22, an AAV-binding protein AVR14a (corresponding to a region from position 312 to position 500 in SEQ ID NO: 1) ranges from serine (Ser) at position 25 to aspartic acid (Asp) at position 213, and a tag sequence ranges from histidine (His) at position 214 to histidine (His) at position 219 in SEQ ID NO: 57. In addition, aspartic acid of Val317Asp is present at position 30, histidine of Asn324His is present at position 37, valine of Ala330Val is present at position 43, valine of Glu335Val is present at position 48, alanine of Thr341Ala is present at position 54, serine of Tyr342Ser is present at position 55, glutamic acid of Lys362Glu is present at position 75, asparagine of Lys371Asn is present at position 84, alanine of Val381Ala is present at position 94, valine of Ile382Val is present at position 95, serine of Gly390Ser is present at position 103, glutamic acid of Lys399Glu is present at position 112, arginine of Ser476Arg is present at position 189, and aspartic acid of Asn487Asp is present at position 200 in SEQ ID NO: 57.
This protein is a protein for which Asn324His, Gln334Leu, and Glu335Val were selected from among the amino acid substitutions involved in enhancing alkali stability revealed in Example 9, and the amino acid substitutions were aggregated in AVR11a (SEQ ID NO: 19). Specifically, AVR14b was prepared by introducing mutations resulting in amino acid substitutions of Asn324His and Glu335Val into a polynucleotide (SEQ ID NO: 36) encoding the protein obtained in Example 9, in which an amino substitution of Gln334Leu was introduced into AVR11a (named “AVR11a_Q334L,” SEQ ID NO: 37).
The amino acid sequence of AVR14b with a signal sequence and a polyhistidine tag is set forth in SEQ ID NO: 58. A PelB signal peptide ranges from methionine (Met) at position 1 to alanine (Ala) at position 22, an AAV-binding protein AVR14b (corresponding to a region from position 312 to position 500 in SEQ ID NO: 1) ranges from serine (Ser) at position 25 to aspartic acid (Asp) at position 213, and a tag sequence ranges from histidine (His) at position 214 to histidine (His) at position 219 in SEQ ID NO: 58. In addition, aspartic acid of Val317Asp is present at position 30, histidine of Asn324His is present at position 37, valine of Ala330Val is present at position 43, leucine of Gln334Leu is present at position 47, valine of Glu335Val is present at position 48, serine of Tyr342Ser is present at position 55, glutamic acid of Lys362Glu is present at position 75, asparagine of Lys371Asn is present at position 84, alanine of Val381Ala is present at position 94, valine of Ile382Val is present at position 95, serine of Gly390Ser is present at position 103, glutamic acid of Lys399Glu is present at position 112, arginine of Ser476Arg is present at position 189, and aspartic acid of Asn487Asp is present at position 200 in SEQ ID NO: 58.
This protein is a protein for which Asn324His, Glu335Val, and Met359Ile were selected from among the amino acid substitutions involved in enhancing alkali stability revealed in Example 9, and the amino acid substitutions were aggregated in AVR11a (SEQ ID NO: 19). Specifically, AVR14c was prepared by introducing mutations resulting in amino acid substitutions of Asn324His and Glu335Val into a polynucleotide (SEQ ID NO: 40) encoding the protein obtained in Example 9, in which an amino substitution of Met359Ile was introduced into AVR11a (named “AVR11a_M359I,” SEQ ID NO: 41).
The amino acid sequence of AVR14c with a signal sequence and a polyhistidine tag is set forth in SEQ ID NO: 59. A PelB signal peptide ranges from methionine (Met) at position 1 to alanine (Ala) at position 22, an AAV-binding protein AVR14c (corresponding to a region from position 312 to position 500 in SEQ ID NO: 1) ranges from serine (Ser) at position 25 to aspartic acid (Asp) at position 213, and a tag sequence ranges from histidine (His) at position 214 to histidine (His) at position 219 in SEQ ID NO: 59. In addition, aspartic acid of Val317Asp is present at position 30, histidine of Asn324His is present at position 37, valine of Ala330Val is present at position 43, valine of Glu335Val is present at position 48, serine of Tyr342Ser is present at position 55, isoleucine of Met359Ile is present at position 72, glutamic acid of Lys362Glu is present at position 75, asparagine of Lys371Asn is present at position 84, alanine of Val381Ala is present at position 94, valine of Ile382Val is present at position 95, serine of Gly390Ser is present at position 103, glutamic acid of Lys399Glu is present at position 112, arginine of Ser476Arg is present at position 189, and aspartic acid of Asn487Asp is present at position 200 in SEQ ID NO: 59.
This protein is a protein for which Asn324His, Glu335Val, and Phe379Tyr were selected from among the amino acid substitutions involved in enhancing alkali stability revealed in Example 9, and the amino acid substitutions were aggregated in AVR11a (SEQ ID NO: 19). Specifically, AVR14d was prepared by introducing mutations resulting in amino acid substitutions of Asn324His and Glu335Val into a polynucleotide (SEQ ID NO: 42) encoding the protein obtained in Example 9, in which an amino substitution of Phe379Tyr was introduced into AVR11a (named “AVR11a_F379Y,” SEQ ID NO: 43).
The amino acid sequence of AVR14d with a signal sequence and a polyhistidine tag is set forth in SEQ ID NO: 60. A PelB signal peptide ranges from methionine (Met) at position 1 to alanine (Ala) at position 22, an AAV-binding protein AVR14d (corresponding to a region from position 312 to position 500 in SEQ ID NO: 1) ranges from serine (Ser) at position 25 to aspartic acid (Asp) at position 213, and a tag sequence ranges from histidine (His) at position 214 to histidine (His) at position 219 in SEQ ID NO: 60. In addition, aspartic acid of Val317Asp is present at position 30, histidine of Asn324His is present at position 37, valine of Ala330Val is present at position 43, valine of Glu335Val is present at position 48, serine of Tyr342Ser is present at position 55, glutamic acid of Lys362Glu is present at position 75, asparagine of Lys371Asn is present at position 84, tyrosine of Phe379Tyr is present at position 92, alanine of Val381Ala is present at position 94, valine of Ile382Val is present at position 95, serine of Gly390Ser is present at position 103, glutamic acid of Lys399Glu is present at position 112, arginine of Ser476Arg is present at position 189, and aspartic acid of Asn487Asp is present at position 200 in SEQ ID NO: 60.
This protein is a protein for which Asn324His, Gln334Leu, Glu335Val, and Thr341Ala were selected from among the amino acid substitutions involved in enhancing alkali stability revealed in Example 9, and the amino acid substitutions were aggregated in AVR11a (SEQ ID NO: 19). Specifically, AVR15a was prepared by introducing mutations resulting in amino acid substitutions of Asn324His, Gln334Leu, and Glu335Val into a polynucleotide (SEQ ID NO: 38) encoding AVR11a_T341A (SEQ ID NO: 39).
The amino acid sequence of AVR15a with a signal sequence and a polyhistidine tag is set forth in SEQ ID NO: 61. A PelB signal peptide ranges from methionine (Met) at position 1 to alanine (Ala) at position 22, an AAV-binding protein AVR15a (corresponding to a region from position 312 to position 500 in SEQ ID NO: 1) ranges from serine (Ser) at position 25 to aspartic acid (Asp) at position 213, and a tag sequence ranges from histidine (His) at position 214 to histidine (His) at position 219 in SEQ ID NO: 61. In addition, aspartic acid of Val317Asp is present at position 30, histidine of Asn324His is present at position 37, valine of Ala330Val is present at position 43, leucine of Gln334Leu is present at position 47, valine of Glu335Val is present at position 48, alanine of Thr341Ala is present at position 54, serine of Tyr342Ser is present at position 55, glutamic acid of Lys362Glu is present at position 75, asparagine of Lys371Asn is present at position 84, alanine of Val381Ala is present at position 94, valine of Ile382Val is present at position 95, serine of Gly390Ser is present at position 103, glutamic acid of Lys399Glu is present at position 112, arginine of Ser476Arg is present at position 189, and aspartic acid of Asn487Asp is present at position 200 in SEQ ID NO: 61.
This protein is a protein for which Asn324His, Gln334Leu, Glu335Val, and Met359Ile were selected from among the amino acid substitutions involved in enhancing alkali stability revealed in Example 9, and the amino acid substitutions were aggregated in AVR11a (SEQ ID NO: 19). Specifically, AVR15b was prepared by introducing mutations resulting in amino acid substitutions of Asn324His, Gln334Leu, and Glu335Val into a polynucleotide (SEQ ID NO: 40) encoding AVR11a_M359I (SEQ ID NO: 41).
The amino acid sequence of AVR15b with a signal sequence and a polyhistidine tag is set forth in SEQ ID NO: 62. A PelB signal peptide ranges from methionine (Met) at position 1 to alanine (Ala) at position 22, an AAV-binding protein AVR15b (corresponding to a region from position 312 to position 500 in SEQ ID NO: 1) ranges from serine (Ser) at position 25 to aspartic acid (Asp) at position 213, and a tag sequence ranges from histidine (His) at position 214 to histidine (His) at position 219 in SEQ ID NO: 62. In addition, aspartic acid of Val317Asp is present at position 30, histidine of Asn324His is present at position 37, valine of Ala330Val is present at position 43, leucine of Gln334Leu is present at position 47, valine of Glu335Val is present at position 48, serine of Tyr342Ser is present at position 55, isoleucine of Met359Ile is present at position 72, glutamic acid of Lys362Glu is present at position 75, asparagine of Lys371Asn is present at position 84, alanine of Val381Ala is present at position 94, valine of Ile382Val is present at position 95, serine of Gly390Ser is present at position 103, glutamic acid of Lys399Glu is present at position 112, arginine of Ser476Arg is present at position 189, and aspartic acid of Asn487Asp is present at position 200 in SEQ ID NO: 62.
This protein is a protein for which Asn324His, Gln334Leu, Glu335Val, and Phe379Tyr were selected from among the amino acid substitutions involved in enhancing alkali stability revealed in Example 9, and the amino acid substitutions were aggregated in AVR11a (SEQ ID NO: 19). Specifically, AVR15c was prepared by introducing mutations resulting in amino acid substitutions of Asn324His, Gln334Leu, and Glu335Val into a polynucleotide (SEQ ID NO: 42) encoding AVR11a_F379Y (SEQ ID NO: 43).
The amino acid sequence of AVR15c with a signal sequence and a polyhistidine tag is set forth in SEQ ID NO: 63. A PelB signal peptide ranges from methionine (Met) at position 1 to alanine (Ala) at position 22, an AAV-binding protein AVR15c (corresponding to a region from position 312 to position 500 in SEQ ID NO: 1) ranges from serine (Ser) at position 25 to aspartic acid (Asp) at position 213, and a tag sequence ranges from histidine (His) at position 214 to histidine (His) at position 219 in SEQ ID NO: 63. In addition, aspartic acid of Val317Asp is present at position 30, histidine of Asn324His is present at position 37, valine of Ala330Val is present at position 43, leucine of Gln334Leu is present at position 47, valine of Glu335Val is present at position 48, serine of Tyr342Ser is present at position 55, glutamic acid of Lys362Glu is present at position 75, asparagine of Lys371Asn is present at position 84, tyrosine of Phe379Tyr is present at position 92, alanine of Val381Ala is present at position 94, valine of Ile382Val is present at position 95, serine of Gly390Ser is present at position 103, glutamic acid of Lys399Glu is present at position 112, arginine of Ser476Arg is present at position 189, and aspartic acid of Asn487Asp is present at position 200 in SEQ ID NO: 63.
This protein is a protein for which Asn324His, Gln334Leu, Glu335Val, Thr341Ala, and Phe379Tyr were selected from among the amino acid substitutions involved in enhancing alkali stability revealed in Example 9, and the amino acid substitutions were aggregated in AVR11a (SEQ ID NO: 19). Specifically, AVR16 was prepared by introducing mutations resulting in an amino acid substitution of Thr341Ala into a polynucleotide (SEQ ID NO: 64) encoding AVR15c (SEQ ID NO: 63).
The amino acid sequence of AVR16 with a signal sequence and a polyhistidine tag is set forth in SEQ ID NO: 65. A PelB signal peptide ranges from methionine (Met) at position 1 to alanine (Ala) at position 22, an AAV-binding protein AVR16 (corresponding to a region from position 312 to position 500 in SEQ ID NO: 1) ranges from serine (Ser) at position 25 to aspartic acid (Asp) at position 213, and a tag sequence ranges from histidine (His) at position 214 to histidine (His) at position 219 in SEQ ID NO: 65. In addition, aspartic acid of Val317Asp is present at position 30, histidine of Asn324His is present at position 37, valine of Ala330Val is present at position 43, leucine of Gln334Leu is present at position 47, valine of Glu335Val is present at position 48, alanine of Thr341Ala is present at position 54, serine of Tyr342Ser is present at position 55, glutamic acid of Lys362Glu is present at position 75, asparagine of Lys371Asn is present at position 84, tyrosine of Phe379Tyr is present at position 92, alanine of Val381Ala is present at position 94, valine of Ile382Val is present at position 95, serine of Gly390Ser is present at position 103, glutamic acid of Lys399Glu is present at position 112, arginine of Ser476Arg is present at position 189, and aspartic acid of Asn487Asp is present at position 200 in SEQ ID NO: 65.
Table 10 shows the results. It is understood that each aggregate of amino acid substitutions in AVR11a obtained in Example 10 has higher residual activity and enhanced alkali stability compared with AVR11a (SEQ ID NO: 19).
Among the AAV-binding proteins evaluated in Example 11, AVR16 (SEQ ID NO: 65) was selected, and a mutation was randomly introduced into the polynucleotide (SEQ ID NO: 66) portion encoding the protein using error-prone PCR.
Tables 11 and 12 show lists of the amino acid substitution sites and the residual activities (%) after alkali treatment of the AAV-binding proteins expressed by the transformants selected in (6) above to AVR11a.
It can be said that the AAV-binding proteins, in which at least any one of amino acid substitutions of Val(Asp)317Asn, Ile319Val, Lys323Asn, Glu325Lys, Val326Ala, Asn329Ser, Leu333Met, Lys338Arg, Gly339Glu, Met359Val, Gly361Glu, Lys(Glu)362Asp, Ser366Pro, Lys368Arg, Lys368Glu, Leu369Pro, Lys380Arg, Val(Ala)381Thr, Ile(Val)382Asp, Lys(Glu)399Gly, Leu421Pro, Pro422Ser, Ser425Gly, His443Tyr, His443Arg, Glu454Gly, Lys455Arg, Lys467Glu, Lys467Gln, Ser(Arg)476Gly, Ser482Thr, Ser488Phe, Ala491Ser, Asn492Asp, Asn496Tyr, and Asp500Gly is present at the amino acid residues from serine (Ser) at position 312 to aspartic acid (Asp) at position 500 in the amino acid sequence set forth in SEQ ID NO: 1, have enhanced stability to alkali even compared with AVR16 (SEQ ID NO: 65) with more enhanced alkali stability than AVR11a (SEQ ID NO: 19).
It was attempted to aggregate amino acid substitutions, which were found in Example 12 to be involved in enhancing the alkali stability of AAV-binding proteins, in AVR16 (SEQ ID NO: 65), thereby further enhancing the alkali stability. Specifically, seven types of AAV-binding proteins shown in (a) to (g) below were designed and prepared.
Hereinafter, a method of preparing the eight types of AAV-binding proteins shown in (a) to (g) above will be explained.
This protein is a protein for which Val326Ala, Lys467Gln, and Ser482Thr were selected from among the amino acid substitutions involved in enhancing alkali stability revealed in Example 12, and the amino acid substitutions were aggregated in AVR16 (SEQ ID NO: 65). Specifically, AVR19c was prepared by introducing a mutation resulting in an amino acid substitution of Ser482Thr into a polynucleotide (SEQ ID NO: 69) encoding a protein (named “AVR16_K467Q,” SEQ ID NO: 70) with an amino acid substitution of Lys467Gln in AVR16 obtained in Example 12 (named “AVR18”), followed by introducing a mutation resulting in an amino acid substitution of Val326Ala into a polynucleotide encoding the AVR18.
The amino acid sequence of AVR19a with a signal sequence and a polyhistidine tag is set forth in SEQ ID NO: 83. A PelB signal peptide ranges from methionine (Met) at position 1 to alanine (Ala) at position 22, an AAV-binding protein AVR19a (corresponding to a region from position 312 to position 500 in SEQ ID NO: 1) ranges from serine (Ser) at position 25 to aspartic acid (Asp) at position 213, and a tag sequence ranges from histidine (His) at position 214 to histidine (His) at position 219 in SEQ ID NO: 83. In addition, aspartic acid of Val317Asp is present at position 30, histidine of Asn324His is present at position 37, alanine of Val326Ala is present at position 39, valine of Ala330Val is present at position 43, leucine of Gln334Leu is present at position 47, valine of Glu335Val is present at position 48, alanine of Thr341Ala is present at position 54, serine of Tyr342Ser is present at position 55, glutamic acid of Lys362Glu is present at position 75, asparagine of Lys371Asn is present at position 84, tyrosine of Phe379Tyr is present at position 92, alanine of Val381Ala is present at position 94, valine of Ile382Val is present at position 95, serine of Gly390Ser is present at position 103, glutamic acid of Lys399Glu is present at position 112, glutamine of Lys467Gln is present at position 180, arginine of Ser476Arg is present at position 189, threonine of Ser482Thr is present at position 195, and aspartic acid of Asn487Asp is present at position 200 in SEQ ID NO: 83.
This protein is a protein for which Lys467Gln, Ser482Thr, and Asn492Asp were selected from among the amino acid substitutions involved in enhancing alkali stability revealed in Example 12, and the amino acid substitutions were aggregated in AVR16 (SEQ ID NO: 65). Specifically, AVR19b was prepared by introducing mutations resulting in an amino acid substitution of Asn492Asp into a polynucleotide (SEQ ID NO: 82) encoding AVR18 (SEQ ID NO: 81).
The amino acid sequence of AVR19b with a signal sequence and a polyhistidine tag is set forth in SEQ ID NO: 85. A PelB signal peptide ranges from methionine (Met) at position 1 to alanine (Ala) at position 22, an AAV-binding protein AVR19b (corresponding to a region from position 312 to position 500 in SEQ ID NO: 1) ranges from serine (Ser) at position 25 to aspartic acid (Asp) at position 213, and a tag sequence ranges from histidine (His) at position 214 to histidine (His) at position 219 in SEQ ID NO: 85. In addition, aspartic acid of Val317Asp is present at position 30, histidine of Asn324His is present at position 37, valine of Ala330Val is present at position 43, leucine of Gln334Leu is present at position 47, valine of Glu335Val is present at position 48, alanine of Thr341Ala is present at position 54, serine of Tyr342Ser is present at position 55, glutamic acid of Lys362Glu is present at position 75, asparagine of Lys371Asn is present at position 84, tyrosine of Phe379Tyr is present at position 92, alanine of Val381Ala is present at position 94, valine of Ile382Val is present at position 95, serine of Gly390Ser is present at position 103, glutamic acid of Lys399Glu is present at position 112, glutamine of Lys467Gln is present at position 180, arginine of Ser476Arg is present at position 189, threonine of Ser482Thr is present at position 195, aspartic acid of Asn487Asp is present at position 200, and aspartic acid of Asn492Asp is present at position 205 in SEQ ID NO: 85.
This protein is a protein for which Lys380Arg, Lys467Gln, and Ser482Thr were selected from among the amino acid substitutions involved in enhancing alkali stability revealed in Example 12, and the amino acid substitutions were aggregated in AVR16 (SEQ ID NO: 65). Specifically, AVR19c was prepared by introducing mutations resulting in an amino acid substitution of Lys380Arg into a polynucleotide (SEQ ID NO: 82) encoding AVR18 (SEQ ID NO: 81).
(c-2) A plasmid (expression vector) pET-AVR19c comprising a polynucleotide encoding AVR19c with 19 amino acid substitutions in the wild-type AAV-binding protein was obtained from the amplified PCR product using the same method as in (a-2) to (a-3). The nucleotide sequence of the plasmid was then analyzed and confirmed using the same method as in Example 3 (7).
The amino acid sequence of AVR19c with a signal sequence and a polyhistidine tag is set forth in SEQ ID NO: 87. A PelB signal peptide ranges from methionine (Met) at position 1 to alanine (Ala) at position 22, an AAV-binding protein AVR19c (corresponding to a region from position 312 to position 500 in SEQ ID NO: 1) ranges from serine (Ser) at position 25 to aspartic acid (Asp) at position 213, and a tag sequence ranges from histidine (His) at position 214 to histidine (His) at position 219 in SEQ ID NO: 87. In addition, aspartic acid of Val317Asp is present at position 30, histidine of Asn324His is present at position 37, valine of Ala330Val is present at position 43, leucine of Gln334Leu is present at position 47, valine of Glu335Val is present at position 48, alanine of Thr341Ala is present at position 54, serine of Tyr342Ser is present at position 55, glutamic acid of Lys362Glu is present at position 75, asparagine of Lys371Asn is present at position 84, tyrosine of Phe379Tyr is present at position 92, arginine of Lys380Arg is present at position 93, alanine of Val381Ala is present at position 94, valine of Ile382Val is present at position 95, serine of Gly390Ser is present at position 103, glutamic acid of Lys399Glu is present at position 112, glutamine of Lys467Gln is present at position 180, arginine of Ser476Arg is present at position 189, threonine of Ser482Thr is present at position 195, and aspartic acid of Asn487Asp is present at position 200 in SEQ ID NO: 87.
This protein is a protein for which Val326Ala, Lys467Gln, Ser482Thr, and Asn492Asp were selected from among the amino acid substitutions involved in enhancing alkali stability revealed in Example 12, and the amino acid substitutions were aggregated in AVR16 (SEQ ID NO: 65). Specifically, AVR20a was prepared by introducing mutations resulting in an amino acid substitution of Asn492Asp into a polynucleotide (SEQ ID NO: 84) encoding AVR19a (SEQ ID NO: 83).
The amino acid sequence of AVR20a with a signal sequence and a polyhistidine tag is set forth in SEQ ID NO: 88. A PelB signal peptide ranges from methionine (Met) at position 1 to alanine (Ala) at position 22, an AAV-binding protein AVR20a (corresponding to a region from position 312 to position 500 in SEQ ID NO: 1) ranges from serine (Ser) at position 25 to aspartic acid (Asp) at position 213, and a tag sequence ranges from histidine (His) at position 214 to histidine (His) at position 219 in SEQ ID NO: 88. In addition, aspartic acid of Val317Asp is present at position 30, histidine of Asn324His is present at position 37, alanine of Val326Ala is present at position 39, valine of Ala330Val is present at position 43, leucine of Gln334Leu is present at position 47, valine of Glu335Val is present at position 48, alanine of Thr341Ala is present at position 54, serine of Tyr342Ser is present at position 55, glutamic acid of Lys362Glu is present at position 75, asparagine of Lys371Asn is present at position 84, tyrosine of Phe379Tyr is present at position 92, alanine of Val381Ala is present at position 94, valine of Ile382Val is present at position 95, serine of Gly390Ser is present at position 103, glutamic acid of Lys399Glu is present at position 112, glutamine of Lys467Gln is present at position 180, arginine of Ser476Arg is present at position 189, threonine of Ser482Thr is present at position 195, aspartic acid of Asn487Asp is present at position 200, and aspartic acid of Asn492Asp is present at position 205 in SEQ ID NO: 88.
This protein is a protein for which Val326Ala, Lys380Arg, Lys467Gln, and Ser482Thr were selected from among the amino acid substitutions involved in enhancing alkali stability revealed in Example 12, and the amino acid substitutions were aggregated in AVR16 (SEQ ID NO: 65). Specifically, AVR20b was prepared by introducing mutations resulting in an amino acid substitution of Lys380Arg into a polynucleotide (SEQ ID NO: 84) encoding AVR19a (SEQ ID NO: 83).
The amino acid sequence of AVR20b with a signal sequence and a polyhistidine tag is set forth in SEQ ID NO: 90. A PelB signal peptide ranges from methionine (Met) at position 1 to alanine (Ala) at position 22, an AAV-binding protein AVR20b (corresponding to a region from position 312 to position 500 in SEQ ID NO: 1) ranges from serine (Ser) at position 25 to aspartic acid (Asp) at position 213, and a tag sequence ranges from histidine (His) at position 214 to histidine (His) at position 219 in SEQ ID NO: 90. In addition, aspartic acid of Val317Asp is present at position 30, histidine of Asn324His is present at position 37, alanine of Val326Ala is present at position 39, valine of Ala330Val is present at position 43, leucine of Gln334Leu is present at position 47, valine of Glu335Val is present at position 48, alanine of Thr341Ala is present at position 54, serine of Tyr342Ser is present at position 55, glutamic acid of Lys362Glu is present at position 75, asparagine of Lys371Asn is present at position 84, tyrosine of Phe379Tyr is present at position 92, arginine of Lys380Arg is present at position 93, alanine of Val381Ala is present at position 94, valine of Ile382Val is present at position 95, serine of Gly390Ser is present at position 103, glutamic acid of Lys399Glu is present at position 112, glutamine of Lys467Gln is present at position 180, arginine of Ser476Arg is present at position 189, threonine of Ser482Thr is present at position 195, and aspartic acid of Asn487Asp is present at position 200 in SEQ ID NO: 90.
This protein is a protein for which Lys380Arg, Lys467Gln, Ser482Thr, and Asn492Asp were selected from among the amino acid substitutions involved in enhancing alkali stability revealed in Example 12, and the amino acid substitutions were aggregated in AVR16 (SEQ ID NO: 65). Specifically, AVR20c was prepared by introducing mutations resulting in an amino acid substitution of Lys380Arg into a polynucleotide (SEQ ID NO: 86) encoding AVR19b (SEQ ID NO: 85).
The amino acid sequence of AVR20c with a signal sequence and a polyhistidine tag is set forth in SEQ ID NO: 91. A PelB signal peptide ranges from methionine (Met) at position 1 to alanine (Ala) at position 22, an AAV-binding protein AVR20c (corresponding to a region from position 312 to position 500 in SEQ ID NO: 1) ranges from serine (Ser) at position 25 to aspartic acid (Asp) at position 213, and a tag sequence ranges from histidine (His) at position 214 to histidine (His) at position 219 in SEQ ID NO: 91. In addition, aspartic acid of Val317Asp is present at position 30, histidine of Asn324His is present at position 37, valine of Ala330Val is present at position 43, leucine of Gln334Leu is present at position 47, valine of Glu335Val is present at position 48, alanine of Thr341Ala is present at position 54, serine of Tyr342Ser is present at position 55, glutamic acid of Lys362Glu is present at position 75, asparagine of Lys371Asn is present at position 84, tyrosine of Phe379Tyr is present at position 92, arginine of Lys380Arg is present at position 93, alanine of Val381Ala is present at position 94, valine of Ile382Val is present at position 95, serine of Gly390Ser is present at position 103, glutamic acid of Lys399Glu is present at position 112, glutamine of Lys467Gln is present at position 180, arginine of Ser476Arg is present at position 189, threonine of Ser482Thr is present at position 195, aspartic acid of Asn487Asp is present at position 200, and aspartic acid of Asn492Asp is present at position 205 in SEQ ID NO: 91.
This protein is a protein for which Val326Ala, Lys380Arg, Lys467Gln, Ser482Thr, and Asn492Asp were selected from among the amino acid substitutions involved in enhancing alkali stability revealed in Example 12, and the amino acid substitutions were aggregated in AVR16 (SEQ ID NO: 65). Specifically, AVR21 was prepared by introducing mutations resulting in an amino acid substitution of Lys380Arg into a polynucleotide (SEQ ID NO: 89) encoding AVR20a (SEQ ID NO: 88).
The sequence of the polynucleotide encoding a polypeptide containing AVR21 analyzed in (g-2) is set forth in SEQ ID NO: 93. The amino acid sequence of AVR21 with a signal sequence and a polyhistidine tag is set forth in SEQ ID NO: 92. A PelB signal peptide ranges from methionine (Met) at position 1 to alanine (Ala) at position 22, an AAV-binding protein AVR21 (corresponding to a region from position 312 to position 500 in SEQ ID NO: 1) ranges from serine (Ser) at position 25 to aspartic acid (Asp) at position 213, and a tag sequence ranges from histidine (His) at position 214 to histidine (His) at position 219 in SEQ ID NO: 92. In addition, aspartic acid of Val317Asp is present at position 30, histidine of Asn324His is present at position 37, alanine of Val326Ala is present at position 39, valine of Ala330Val is present at position 43, leucine of Gln334Leu is present at position 47, valine of Glu335Val is present at position 48, alanine of Thr341Ala is present at position 54, serine of Tyr342Ser is present at position 55, glutamic acid of Lys362Glu is present at position 75, asparagine of Lys371Asn is present at position 84, tyrosine of Phe379Tyr is present at position 92, arginine of Lys380Arg is present at position 93, alanine of Val381Ala is present at position 94, valine of Ile382Val is present at position 95, serine of Gly390Ser is present at position 103, glutamic acid of Lys399Glu is present at position 112, glutamine of Lys467Gln is present at position 180, arginine of Ser476Arg is present at position 189, threonine of Ser482Thr is present at position 195, aspartic acid of Asn487Asp is present at position 200, and aspartic acid of Asn492Asp is present at position 205 in SEQ ID NO: 92.
Table 13 shows the results. It is understood that each aggregate of amino acid substitutions in AVR16 obtained in this Example has higher residual activity and enhanced alkali stability even compared with AVR16 (SEQ ID NO: 65) with more enhanced alkali stability than AVR11a (SEQ ID NO: 19).
The infectivity of the amino acid substitution products shown in (a) to (m) above relative to the wild type (SEQ ID NO: 99) is summarized in Table 14 (Source: Lochrie, M. A., et al., Journal of Virology, 80(2), 821).
As a result, all solutions contained AAV2 at 8.2×109 cp/mL (cp indicates the number of AAV particles) or more. A solution that is sufficient to confirm the peak when detected using HPLC M40A (manufactured by Shimadzu Corporation) with a fluorescence intensity of 350 nm against excitation light of 280 nm could be obtained.
Table 15 shows the elution time of each AAV2 using the AVR10s column. In addition,
From Table 15 and
AAV2 was analyzed in the same manner as in Example 18, except that 1.25 mL of a POROS Capture Select AAVX Affinity Resin (manufactured by Thermo Fisher Scientific), which is an AAV adsorbent different from the AVR10s immobilization gel, was used as the gel packed into the stainless steel columns in Example 18 (1).
Table 16 shows the elution time of each AAV2 using a stainless steel column (hereinafter also referred to as “AAVX column”) packed with the POROS Capture Select AAVX Affinity Resin. In addition,
It is understood from Table 16 and
AAV-binding proteins were prepared in the same manner as in Example 16, except that one of the following was used as a plasmid containing a polynucleotide encoding a polypeptide containing an AAV-binding protein:
A PelB signal peptide ranges from methionine (Met) at position 1 to alanine (Ala) at position 22, an AAV-binding protein (AVR11a or AVR21) ranges from serine (Ser) at position 25 to aspartic acid (Asp) at position 213, and a cysteine tag sequence serving as an immobilization tag ranges from cysteine (Cys) at position 220 to glycine (Gly) at position 226 in SEQ ID NOS: 100 and 102. AVR11a is a polypeptide with the following amino acid substitutions <I> to <XI> in the extracellular domain 1 (PKD1) and the domain 2 (PKD2) (amino acid residue from serine at position 312 to aspartic acid at position 500 of SEQ ID NO: 94) of AAV receptor KIAA0319L, and AVR21 is a polypeptide with the following amino acid substitutions <I> to <XXI> in PKD1 and PKD2 above:
An AAV adsorbent was prepared in the same manner as in Example 17, except that AAV11a or AAV21 prepared in Example 19 was used as the AAV-binding protein. The AAV adsorbent with immobilized AVR11a is named “AVR11a immobilization gel,” and the AAV adsorbent with immobilized AVR21 is named “AVR21 immobilization gel.”
Table 17 shows the elution time of each AAV2 using the AVR11a column and AAV21 column. In addition,
From Table 17 and
AAV2 was analyzed in the same manner as in Example 20, except that 1.25 mL of a POROS Capture Select AAVX Affinity Resin (manufactured by Thermo Fisher Scientific), which is an AAV adsorbent different from AVR11a immobilization gel and AVR21 immobilization gel, was used as the gel packed into the stainless steel columns in Example 21 (1).
Table 18 shows the elution time of each AAV2 using a stainless steel column (hereinafter also referred to as “AAVX column”) packed with the POROS Capture Select AAVX Affinity Resin. In addition,
It is understood from Table 18 and
| Number | Date | Country | Kind |
|---|---|---|---|
| 2022-007922 | Jan 2022 | JP | national |
| 2022-043222 | Mar 2022 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2023/000872 | 1/13/2023 | WO |
