JP7142815B2 - Adeno-associated virus virions for gene transfer into human liver - Google Patents

Description

The present invention relates to recombinant adeno-associated virus (rAAV) vectors that are less susceptible to neutralizing antibodies in serum. More specifically, the present invention relates to mutants that have reduced cross-reactivity with neutralizing antibodies in the serum of living organisms and that enable more efficient gene transfer into livers of living organisms (for example, humans). Regarding rAAV.

Adeno-associated virus (AAV)-based gene transfer vectors introduce genes into cells in the body, such as nerve cells, hepatocytes (liver parenchymal cells), retinal cells, muscle cells, cardiomyocytes, vascular endothelial cells, and adipocytes. and can be expressed for a long period of time (Patent Documents 1 to 3). Therefore, clinical application is progressing as a gene therapy vector for hemophilia, retinitis pigmentosa, Parkinson's disease, and the like (Non-Patent Documents 1 and 2). Furthermore, recently, it is frequently used as a vector for introducing genes of sgRNA and CAS9 protein in gene editing (Patent Document 3, Non-Patent Document 3). For hemophilia, promising results have been reported in gene therapy in which an AAV vector expressing factor VIII or factor IX is introduced into hepatocytes (Non-Patent Documents 4 and 5).

However, AAV3 and AAV8, which have high gene transfer efficiency into hepatocytes, cross-react with the neutralizing antibodies against AAV2 that more than 60% of adults have, so it has been reported that sufficient effects cannot be expected in many patients ( Non-Patent Documents 6, 7, 8). In addition, it is being promoted to perform gene therapy on patients who were not eligible for gene therapy because they have neutralizing antibodies, and to perform gene therapy again on patients who did not obtain sufficient effects from the first gene therapy. ing.

WO 2008/124724 WO2012/057363 International application JP2018/00150

Dunber CE, et al., Science 359: eaan4672, 2018. Hastie E, Samulski RJ, Hum Gene Ther 26: 257-265, 2015. Ohmori T, et al., Sci Rep 7: 4159, 2017. George LA, et al., N Engl J Med 377: 2215-2227, 2017. Rangarajan S, et al., N Engl J Med 377: 2519-2530, 2017. Mimuro J, et al., J Med Virol 86: 1990-1997, 2014. Ling C, et al., J Integr Med 13: 341-346, 2015. Meliani A, et al., Hum Gene Ther Methods 26: 45-53, 2015.

Therefore, there is a demand for AAV vectors, such as novel AAV vectors derived from AAV3 and AAV8, that have reduced cross-reactivity to serum neutralizing antibodies against AAV2 and the like and have high gene transfer efficiency into hepatocytes.

In order to solve the above-mentioned problems, the present inventors, after various trials and errors, modified some amino acids of the coat protein (capsid) of AAV, resulting in cross-reactivity to neutralizing antibodies in serum. We have created a novel modified AAV vector with reduced virality. Furthermore, when using this modified vector, it was found that gene introduction into cultured human liver-derived cells was possible with higher efficiency, and the present invention was completed.

That is, the present invention provides AAV vectors with reduced cross-reactivity to neutralizing antibodies in serum and high gene transfer efficiency into hepatocytes, such as recombinant adeno-associated viruses for gene therapy targeting hepatocytes. The inventions exemplified below are provided, including vectors and pharmaceutical compositions comprising the same.
[1] An amino acid sequence in which at least one of 472nd serine, 587th serine, and 706th asparagine in the amino acid sequence set forth in SEQ ID NO: 2 or 3 is replaced with another amino acid, An adeno-associated virus vector comprising a capsid protein comprising an amino acid sequence in which 1 to 6 amino acid residues are deleted, substituted, inserted or added at the residue positions of , and is effective against type 2 AAV present in serum an adeno-associated virus vector that does not cross-react with antibodies.
[2] A capsid having an amino acid sequence in which serine at position 472, serine at position 587 and asparagine at position 706 are each replaced with an amino acid selected from the group consisting of glycine, alanine, valine, leucine, threonine and isoleucine. The adeno-associated virus vector of [1] above, which contains a protein.
[3] The adeno-associated virus vector of [1] above, comprising a capsid protein having an amino acid sequence in which at least one of the serine at position 472, serine at position 587 and asparagine at position 706 is substituted with alanine.
[4] The adeno-associated virus vector of [1] above, wherein the capsid protein comprises a protein having the amino acid sequence of SEQ ID NO:4.
[5] The adeno-associated virus vector of [1] above, wherein the neutralizing antibody is an antibody against an adeno-associated virus of a type different from AAV3 or AAV8.
[6] The adeno-associated virus vector of [1] above, wherein the neutralizing antibody is an antibody against AAV2.
[7] The adeno-associated virus vector of [1] above, which has a viral genome containing a liver cell-specific promoter sequence.
[8] the liver cell-specific promoter sequence is ApoE promoter, antitrypsin promoter, cKit promoter, liver-specific transcription factors (HNF-1, HNF-2, HNF-3, HNF-6, C/ERP, DBP) promoter, an albumin promoter, a thyroxine-binding globulin (TBG) promoter, and a polynucleotide sequence having 90% or more homology to a polynucleotide sequence selected from the group consisting of the polynucleotide sequence of SEQ ID NO: 1; The adeno-associated virus vector of [1] above, which functions as a promoter.
[8a] The adeno-associated virus vector of [1] above, comprising a therapeutic gene operably linked to the liver cell-specific promoter sequence.
[8b] The above, wherein the therapeutic gene encodes blood coagulation factor VIII (FVIII), blood coagulation factor IX (FIX), hepatocyte growth factor (HGF), or hepatocyte growth factor receptor (c-Kit) The adeno-associated virus vector of [1].
[9] An amino acid sequence in which at least one of 472nd serine, 587th serine and 706th asparagine in the amino acid sequence set forth in SEQ ID NO: 2 or 3 is replaced with another amino acid, amino acid sequences with deletions, substitutions, insertions or additions of 1 to 6 amino acid residues at residue positions;
an amino acid sequence in which the 472nd serine, 587th serine and 706th asparagine are each substituted with an amino acid selected from the group consisting of glycine, alanine, valine, leucine, threonine and isoleucine;
an amino acid sequence in which at least one of the serine at position 472, serine at position 587 and asparagine at position 706 is substituted with alanine, or the amino acid sequence set forth in SEQ ID NO: 4; Polynucleotide.
[9a] The polynucleotide of [9] above, further comprising a nucleotide sequence encoding the amino acid sequence of any one of SEQ ID NOS:6-8.
[10] A pharmaceutical composition for gene transfer into the liver of a living organism, comprising the adeno-associated virus vector of any one of [1] to [8] above.
[11] The pharmaceutical composition according to [10] above, wherein the living body is a human.

INDUSTRIAL APPLICABILITY The present invention provides AAV vectors, such as AAV3- or AAV8-derived AAV vectors, that have reduced cross-reactivity to neutralizing antibodies against AAV2 and the like and have high gene transfer efficiency into hepatocytes. Furthermore, by using the AAV vector according to the present invention, genetic therapy for patients who originally had neutralizing antibodies and were not eligible for gene therapy, or the first gene therapy did not produce a sufficient effect. It becomes possible to perform gene therapy again on the patient. By using the AAV vector of the present invention, gene transfer into hepatocytes can be particularly improved.

FIG. 1A shows an alignment of the VP1 protein amino acids of AAV3A, AAV3B and AAVGT5. FIG. 1B shows a continuation of FIG. 1A. Figure 1C shows an alignment of the Rep protein amino acid sequences of AAV3A, AAV3B and AAVGT5 (denoted as ARep, BRep and baRep respectively). FIG. 1D shows a continuation of FIG. 1C. FIG. 2A shows the GFP expression level in AAVGT5 human liver-derived HepG2 cells. FIG. 2B shows the state of the transfected GFP-expressing cells in FIG. 2A. FIG. 3A shows the GFP expression level in AAVGT5 human liver-derived PXB cells. FIG. 3B shows the state of the transfected GFP-expressing cells in FIG. 3A. FIG. 4A shows a schematic diagram of antibody titer measurement. FIG. 4B shows the results of obtaining Serum 1 to Serum 4 antibody titers. The dashed line in the figure indicates 50% of the Serum(-) value. FIG. 5A shows four sera (Serum 1 to Serum 4) containing neutralizing antibodies against AAV2, reacted with AAV3-CMV-AcGFP or AAVGT5-CMV-AcGFP, and then infected HEK293 cells with each AAV vector. The results of comparing the expression of GFP are shown. FIG. 5B shows the state of the transfected GFP-expressing cells in FIG. 5A.

The present invention provides recombinant adeno-associated virus vectors that improve the efficiency of gene transfer into liver cells, pharmaceutical compositions containing the vectors, and the like.

1. Recombinant adeno-associated virus vector according to the present invention 1.1. About Adeno-Associated Viruses Adeno-associated viruses include viruses of many known blood groups. Adeno-associated viruses with tropism for hepatocytes (liver parenchymal cells) include, for example, type 2, type 3 (3A and 3B), and type 8 blood group viruses. In the present invention, various adeno-associated virus vectors described in Patent Document 1 (WO 2008/124724), for example, can be used as vectors for delivery to living hepatocytes.

Adeno-associated viruses in nature are non-pathogenic viruses. Utilizing this characteristic, various recombinant virus vectors carrying desired genes have been produced and used for gene therapy (for example, WO2003/018821, WO2003/053476, WO2007/001010, Pharmaceutical Journal 126(11) 1021-1028). The wild-type AAV genome is a single-stranded DNA molecule with a total nucleotide length of approximately 5 kb, either the sense strand or the antisense strand. AAV genomes generally have inverted terminal repeat (ITR) sequences approximately 145 nucleotides long at both the 5' and 3' ends of the genome. This ITR is known to have a variety of functions, such as a function as a replication origin of the AAV genome and a function as a packaging signal of this genome into virions (for example, the above-mentioned article, Pharmaceutical Journal 126 (11) 1021-1028, etc.). The internal region of the wild-type AAV genome flanked by ITRs (hereinafter, internal region) contains the AAV replication (rep) gene and the capsid (cap) gene. These rep and cap genes are respectively a protein involved in viral replication (Rep) and a capsid protein (for example, at least one of VP1, VP2 and VP3) that forms a virus particle that is an icosahedral shell. ). For further details see, for example, Human Gene Therapy, 13, pp.345-354, 2002; Neuronal Development 45, pp.92-103, 2001; Experimental Medicine 20, pp.1296-1300, 2002; ) 1021-1028, Hum Gene Ther, 16, 541-550, 2005, etc.

The rAAV vectors of the present invention include native adeno-associated viruses type 1 (AAV1), type 2 (AAV2), type 3 (AAV3A/AAV3B), type 4 (AAV4), type 5 (AAV5), type 6 (AAV6), Type 7 (AAV7), Type 8 (AAV8), Type 9 (AAV9), Type 10 (AAV10), Type RH10 (AVVrh10; Hu, C. et al., Molecular Therapy vol. 22 no. 10 oct. 2014, 1792-1802 ), but not limited to. The nucleotide sequences of these adeno-associated virus genomes are known and are GenBank accession numbers: AF063497.1 (AAV1), AF043303 (AAV2), NC_001729 (AAV3), NC_001829.1 (AAV4), NC_006152.1 (AAV5), respectively. , AF028704.1 (AAV6), NC_006260.1 (AAV7), NC_006261.1 (AAV8), AY530579 (AAV9), AY631965.1 (AAV10).
In the present invention, it is preferred to utilize capsid proteins (VP1, VP2, VP3, etc.) derived from AAV2, AAV3B (AF028705.1), AAV8, or AAV9, particularly for hepatocyte tropism. The amino acid sequences of each of these capsid proteins are known, and reference can be made, for example, to the sequences registered under the above GenBank accession numbers corresponding to each AAV.

1.2. Capsid Protein According to the Present Invention The rAAV vector used in the present invention contains a mutated capsid protein. Such mutant capsid proteins include at least one of serine 472, serine 587, and asparagine 706 in the amino acid sequence set forth in SEQ ID NO: 2 or 3, such as one, preferably Two, more preferably all three, have been substituted with other amino acids, and multiple amino acid residues have been deleted, substituted, inserted or added at residue positions other than positions 472, 587 and 706. It includes mutant proteins that contain an amino acid sequence and that can function as a capsid protein. Combinations of two or more of these deletions, substitutions, insertions and additions may be included at the same time.
In the rAAV vector used in the present invention, in the amino acid sequence shown in SEQ ID NO: 2 or 3, serine at position 472 is at least one of serine at position 587 and asparagine at position 706, for example, one, preferably An amino acid sequence in which two, more preferably all three, are substituted with amino acids selected from the group consisting of glycine, alanine, valine, leucine, threonine and isoleucine, preferably with alanine (e.g. SEQ ID NO: 4 ) and further includes amino acid sequences having deletions, substitutions, insertions or additions of multiple amino acid residues at residue positions other than 472nd, 587th and 706th.
The number of deletions, substitutions, insertions or additions of the above amino acid residues is, for example, 1 to 50, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20. , 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9 (1-several), 1-8, 1-7 1-6, 1-5, 1-4, 1-3, 1-2 or 1. Generally, the smaller the number of deletions, substitutions, insertions and/or additions of amino acid residues, the better.
The mutant capsid protein of the rAAV vector used in the present invention is preferably about 90% or more, 91% or more, 92% or more, 93% or more, 94% of the amino acid sequence of any of the above SEQ ID NOs: 2 to 4. 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, It may be a protein having an amino acid sequence with 99.8% or more, 99.9% or more identity and capable of functioning as a capsid protein.
In the present invention, functioning as a capsid protein refers to a protein capable of forming a viral vector capable of infecting target cells. Capsid proteins used in the present invention can form viral vectors alone or together with other capsid protein members (eg, VP2, VP3, etc.). Packaged within the viral vector is a polynucleotide containing a therapeutic gene of interest for delivery to target cells, such as liver cells.
Preferably, the viral vector containing the mutant capsid protein has the same infectivity as the viral vector containing the wild-type capsid protein, specifically, compared to the infectivity per viral genome weight of the wild-type. preferably 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more means greater than or equal to Methods known in the art, such as reporter assays, can be used to measure infectivity.

Examples of mutually substitutable amino acid residues in the protein (polypeptide) of the present invention are shown below. Amino acid residues included in the same group can be substituted for each other.
Group A: leucine, isoleucine, norleucine, valine, norvaline, alanine, 2-aminobutanoic acid, methionine, o-methylserine, t-butylglycine, t-butylalanine, cyclohexylalanine;
Group B: aspartic acid, glutamic acid, isoaspartic acid, isoglutamic acid, 2-aminoadipic acid, 2-aminosuberic acid;
Group C: Asparagine, Glutamine;
Group D: lysine, arginine, ornithine, 2,4-diaminobutanoic acid, 2,3-diaminopropionic acid;
Group E: proline, 3-hydroxyproline, 4-hydroxyproline;
Group F: Serine, Threonine, Homoserine;
Group G: phenylalanine, tyrosine.
Proteins containing desired amino acid residue substitutions can be produced according to techniques known to those skilled in the art, such as general genetic engineering techniques and chemical synthesis. Such genetic manipulation procedures are described, for example, in Molecular Cloning 4th Edition, J. Sambrook et al., Cold Spring Harbor Lab. Press. 2012, Current Protocols in Molecular Biology, John Wiley & Sons 1987-2018 (ISSN:1934- 3647, etc.).

The rAAV viral vectors used in the present invention may contain a gene encoding a Rep protein for replication in the genome of the host virus or in the genome of the helper virus. In order to replicate the rAAV of the present invention, such a Rep protein has the function of recognizing the ITR sequence and carrying out genome replication depending on the sequence, packaging the wild-type AAV genome (or rAAV genome) into a viral vector, As long as it has the function to sing and the function to form the rAAV vector of the present invention to the same extent as the wild type, it is preferably about 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more , 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, 99.9 They may have amino acid sequences with greater than % identity. Alternatively, 60 or less, 50 or less, 40 or less, 30 or less, 20 or less, 15 or less, 10 or less, or 5 or less amino acid deletions or substitutions, It may contain insertions and/or additions. Here, having the same level as the wild type means having a specific activity of 50%, 60%, 70%, 80%, 90% or more relative to the wild type. In the present invention, a known AAV3-derived Rep protein, such as AAV3A or AAV3B Rep protein, or a fusion protein thereof (amino acid sequence of SEQ ID NO: 8) is preferably used, but not limited thereto.

In one embodiment of the present invention, the capsid proteins VP1 and the like (VP1, VP2 and/or VP3) encoded by the internal regions of the wild-type AAV genome described above, and the Rep proteins, are isolated from the AAV helper by polynucleotides encoding these proteins. It is incorporated into a plasmid and used to obtain the rAAV of the present invention. Capsid proteins (VP1, VP2 and/or VP3) and Rep proteins used in the present invention may be incorporated into one, two, three or more plasmids as required. Optionally, one or more of these capsid proteins and Rep proteins may be included in the AAV genome. In the present invention, capsid proteins (VP1, VP2 and/or VP3) and Rep proteins are preferably all encoded in one polynucleotide and provided as an AAV helper plasmid.

1.3. About the viral genome (1) About the rAAV viral genome The polynucleotide (that is, the polynucleotide) packaged in the rAAV vector of the present invention is located between the ITRs located on the 5' and 3' sides of the wild-type genome. The polynucleotide of the internal region (i.e., one or both of the rep gene and the cap gene), the polynucleotide encoding the protein of interest (genome editing means and / or repair gene) and for transcribing this polynucleotide It can be produced by replacing it with a gene cassette containing a promoter sequence and the like. Preferably, the 5' and 3' located ITRs are located at the 5' and 3' ends of the AAV genome, respectively. Preferably, in the rAAV genome of the present invention, the ITRs located at the 5' and 3' ends are the 5' and 3' ITRs contained in the genome of AAV1, AAV2, AAV3, AAV6, AAV8, or AAV9. Including but not limited to these specific AAVs. In general, since the ITR portion easily adopts a sequence in which complementary sequences are exchanged (flip and flop structure), the ITR contained in the rAAV genome of the present invention may have the 5' and 3' directions reversed. . In the rAAV genome of the present invention, the length of the polynucleotide to be replaced with the internal region (ie, the genome editing means and/or the repair gene) is practically preferably the same as the length of the original polynucleotide. That is, the total length of the rAAV genome of the present invention is preferably about the same as the wild-type total length of 5 kb, for example, about 2-6 kb, preferably about 4-6 kb. The length of the therapeutic gene to be incorporated into the rAAV genome of the present invention is preferably It is about 0.01-3.7 kb in length, more preferably about 0.01-2.5 kb in length, more preferably about 0.01-2 kb in length, but is not limited thereto. Furthermore, using a known method such as intervening a known internal ribosome entry site (IRES) sequence, T2A sequence, etc., two or more therapeutic genes can be simultaneously administered as long as the total length of the rAAV genome is within the above range. can be incorporated. When the rAAV of the present invention expresses two or more proteins, the genes encoding each protein may be arranged in the same orientation or in different orientations.

In general, if the polynucleotide to be packaged in the recombinant adeno-associated virus vector is single-stranded, the gene of interest (such as a therapeutic gene) may take time (several days) to be expressed. . In this case, the target gene to be introduced can be designed to be self-complementary (sc), thereby shortening the time until expression. Specifically, for example, it is described in Foust K.D. et al. (Nat Biotechnol. 2009 Jan;27(1):59-65). Polynucleotides packaged in rAAV vectors of the invention may be of the non-sc or sc type. Preferably, the polynucleotides packaged in the rAAV vectors of the invention are non-sc.

Capsid proteins for use in the present invention can be encoded by polynucleotides that are appropriately modified based on, for example, codon preferences in the host cell. Polynucleotides encoding preferred capsid proteins used in the present invention include, for example, one or more (eg, 1 to 50, 1 to 40, 1 ~30, 1~25, 1~20, 1~15, 1~10, 1~9 (1~several), 1~8, 1~7, 1~6, 1-5, 1-4, 1-3, 1-2, 1, etc.) nucleotide deletions, substitutions, insertions and/or additions, wherein SEQ ID NOs:2-4 or a capsid protein containing an amino acid sequence in which one or more of the above amino acids are deleted, substituted, inserted and/or added in the amino acid sequences of SEQ ID NOS: 2 to 4 (capsomeres can be formed) is) comprising a polynucleotide encoding a protein. Combinations of two or more of these deletions, substitutions, insertions and additions may be included at the same time. Generally, the smaller the number of nucleotide deletions, substitutions, insertions and/or additions, the better. In addition, preferred polynucleotides in the present invention are, for example, polynucleotides encoding the amino acid sequences of SEQ ID NOs: 2 to 4, or polynucleotides that are capable of hybridizing under stringent hybridization conditions to their complementary sequences. A polynucleotide encoding a protein comprising an amino acid sequence of 2 to 4, or a capsid protein comprising an amino acid sequence in which one or more of the above amino acids are deleted, substituted, inserted and/or added in the amino acid sequences of SEQ ID NOs: 2 to 4 including.

Hybridization can be performed according to a known method or a method analogous thereto, for example, the method described in Molecular Cloning (4th Edition, J. Sambrook et al., Cold Spring Harbor Lab. Press. 2012). Alternatively, when using a commercially available library, it can be performed according to the method described in the instructions provided by the manufacturer. Here, "stringent conditions" may be any of low stringent conditions, medium stringent conditions and high stringent conditions. “Low stringent conditions” are, for example, 5×SSC, 5×Denhardt's solution, 0.5% SDS, 50% formamide, and 32°C. In addition, "moderately stringent conditions" are, for example, 5 x SSC, 5 x Denhardt's solution, 0.5% SDS, 50% formamide, and 42°C. "Highly stringent conditions" are, for example, 5 x SSC, 5 x Denhardt's solution, 0.5% SDS, 50% formamide, and 50°C. Under these conditions, it can be expected that the higher the temperature, the more efficiently DNAs with higher homology can be obtained. However, multiple factors such as temperature, probe concentration, probe length, ionic strength, time, and salt concentration can be considered as factors that affect the stringency of hybridization, and those skilled in the art can select these factors as appropriate. It is possible to achieve similar stringency in .

As a hybridizable polynucleotide, when compared with the control polynucleotide sequence, when calculated by homology search software such as FASTA and BLAST using default parameters, for example, 70% or more, 80% or more , 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3 % or greater, 99.4% or greater, 99.5% or greater, 99.6% or greater, 99.7% or greater, 99.8% or greater, 99.9% or greater identity. Generally, the higher the numerical value of homology, the better.

The identity or homology of amino acid sequences and polynucleotide sequences can be determined by algorithm BLAST by Carlin and Arthur (Proc. Natl. Acad. Sci. USA 872264-2268, 1990; Proc. Natl. Acad. Sci USA 90: 5873). , 1993). Programs called BLASTN and BLASTX based on the BLAST algorithm have been developed (Altschul SF, et al: J Mol Biol 215: 403, 1990). When analyzing a base sequence using BLASTN, the parameters are, for example, Expect threshold=100 and Word size=12. When analyzing an amino acid sequence using BLASTX, the parameters are, for example, Expect threshold=50 and Word size=3. When using BLAST and Gapped BLAST programs, the default parameters of each program are preferably used, but these parameters may be changed as appropriate within the range known to those skilled in the art.

2. Cross-Reactivity of Neutralizing Antibody The adeno-associated virus vector of the present invention does not cross-react with neutralizing antibodies against type 2 AAV (AAV2) or type 8 AAV (AAV8) present in serum.
In the present invention, a neutralizing antibody refers to an antibody that reduces or eliminates the activity of a certain antigen by binding to the antigen, when the antigen has activity such as toxicity or infectivity. In the present invention, "neutralizing" in the context of antibody activity means, for example, that an anti-adeno-associated virus neutralizing antibody (e.g., IgG, IgM, IgA) in serum binds to an antigen, an AAV vector, resulting in Refers to reducing or eliminating the ability of AAV vectors to introduce genes. This neutralization reaction includes a series of complement reactions such as binding of an antigen (for example, an AAV vector) to an antibody, binding of the Fc portion of an antibody to the first component of complement (C1), and the like, resulting in the AAV The vector reduces or loses its ability to infect (gene transfer) target cells. In the present invention, a neutralizing antibody may also include serum components, that is, components that bind to antibodies such as complement and are directly involved in neutralizing reactions such as antigen inactivation and removal. intended.

In the present invention, cross-reactivity of neutralizing antibodies refers to reactions in which neutralizing antibodies bind to a target substance (eg, AAV3) different from the original target antigen (eg, AAV2). In addition, in the present invention, "does not cross-react with neutralizing antibody" means that the neutralizing antibody does not sufficiently function in the binding reaction with a target substance that is not the original target antigen, preferably does not detectably bind. or, even if detectable, the amount of target substance bound (molar or mass ratio) is significantly reduced when compared to the original target antigen. The amount of the target substance to be reduced in this way is several percent (about 5%), about 10%, about 15%, about 20%, about 25%, about Binding amounts of 30%, about 35%, about 40%, about 45%, about 50%, about 60% are included. This ratio can be obtained using a known method such as a method for measuring the amount of protein bound to the antibody. Alternatively, cross-reactivity of neutralizing antibodies can be indirectly measured and compared using proteins encoded within the recombinant virus (such as reporter genes).

Neutralizing antibodies according to the present invention are preferably of mammalian origin, more preferably of primate origin, and most preferably of human origin. Neutralizing antibodies used in the present invention are neutralizing antibodies against AAV2 unless otherwise specified. Normally, humans are highly likely to have antibodies against AAV2, but most of them are not neutralizing antibodies, and it is reported that 18-32% have neutralizing ability (Chirmule N et al., Gene Ther 6: 1574-1583, 1999; Moskalenko M, et al., J Virol 74: 1761-1766, 2000). Furthermore, many of the human sera containing neutralizing antibodies against AAV2 are known to contain neutralizing antibodies against AAV3A and AAV3B (Fu H et al., Hum Gene Ther Clin Dev 2017; 28:187-196, Ling CJ et al. Integr Med 2015;13:341-346). This is also suggested by the relatively high amino acid identity (specifically about 87-88%) between the AAV2 VP1 protein (SEQ ID NO: 1) and the AAV3A or AAV3B VP1 protein (SEQ ID NO: 2 or 3). be done.
Furthermore, neutralizing antibodies for use in the present invention may comprise multiple isotypes (eg, IgG, IgM, IgA, etc.). Therefore, the neutralizing antibody against AAV2 according to the present invention needs to be verified in advance for performance such as antibody concentration or antibody titer. Such verification means are known, and for example, the means described in Ito et al. (Ito et al., Ann Clin Biochem 46: 508-510, 2009) can be used. Specifically, it is described that the titer of neutralizing antibodies (antibody titer) is analyzed by evaluating the inhibitory ability when an AAV2 vector containing a β-galactosidase reporter gene is introduced into HEK293 cells. (Ito et al., Methods).

The neutralizing antibody used in the present invention has an antibody titer of 4 to 640, preferably 20 to 640, 40 to 320, more preferably 40 to 160 (Ito et al., Ann Clin Biochem, 46: 508- 510, 2009). Specific methods for measuring the antibody titer in the present invention are described in "(e) Measurement of antibody titer of neutralizing AAV2 antibody in serum" and "(3) Measurement of antibody titer of neutralizing AAV2 antibody" in Examples. , and FIGS. 4A and 4B, etc. FIG.
In addition, Li C, et al., Gene Ther 19: 288-294, 2012; Methods described in literature such as Methos 26:45-53, 2015 can be used.

The adeno-associated virus vectors of the present invention are less susceptible to neutralizing antibodies in serum than wild-type vectors. Specifically, the viral vectors of the present invention (for example, those containing a capsid protein having the amino acid sequence of SEQ ID NO: 4) have at least the ability to transduce genes into target cells after being treated with neutralizing antibodies in serum. 60%, preferably 70% or more, more preferably 75% or more, still more preferably 80% or more, 85% or more, 90% or more, 95% or more. Here, known assays such as reporter assay, in situ hybridization, and radioisotope labeling can be used for comparison of gene introduction ability. On the other hand, wild-type viral vectors (for example, those containing a capsid protein having the amino acid sequence of SEQ ID NO: 2 or 3) lack the ability to transduce genes into target cells after similarly being treated with neutralizing antibodies in serum. It can be retained less than about 60%, less than about 50%, less than about 40%, less than about 35%, less than about 30%.
Alternatively, the viral vector of the present invention is 1.2-fold or more, preferably 1.5-fold or more, more preferably 1.75-fold more effective against target cells after being treated with neutralizing antibodies in serum compared to the wild-type viral vector. More than twice, preferably more than twice the amount of gene can be introduced.

In the present invention, AAV2-neutralizing antibodies may also have neutralizing activity against AAV3 due to cross-reactivity. Sera containing AAV2 neutralizing antibodies may also have cross-reactivity to AAV of other blood groups (Fu H et al., Hum Gene Ther Clin Dev 2017; 28:187-196, Ling CJ et al. Integr Med 2015;13:341-346, Mimuro J, et al., J Med Virol 86: 1990-1997, 2014.). Here, the amino acid sequence identity between the AAV2 capsid protein VP1 and AAV3A VP1 is calculated to be 87%, and the identity between AAV2 VP1 and AAV3B VP1 is 88% (these identities can be found on the Blastp website ( https://blast.ncbi.nlm.nih.gov/Blast.cgi)). Further studies on epitope mapping of anti-AAV2 neutralizing antibodies are described by Moskalenko, M. et al. (J Virol 74: 1761-1766, 2000).
In addition, the following extracellular receptors related to AAV cell infection are known (Summerfold et al., Mol Ther 24: 2016; Pillay et al., Nature 530:108-112, 2016).
primary receptor
AAV2, 3, 6: heparan sulfate proteoglycans
AAV9: N-terminal galactose
AAV1, 4, 5, 6: specific N-linked or O-linked sialic acid moieties Secondary receptors
AAV2: fibroblast growth factor receptor and integrin
AAV2, 3: hepatocyte growth factor receptor (c-Met)
AAV5: Platelet-derived growth factor (which can be modified by sialic acid)
Based on the disclosure of the present application and knowledge of the above receptors, designing and screening a recombinant vector that is less susceptible to inhibition by a living body's neutralizing antibody against AAV2, etc., based on the recombinant virus vector of the present invention. , may also be acquired.

3. Genes Contained in the Viral Vector of the Present Invention In one embodiment, the rAAV vector of the present invention preferably contains a polynucleotide containing a liver-specific promoter sequence and a gene of interest operably linked to the promoter sequence ( ie, such polynucleotides are packaged). As the promoter sequence used in the present invention, a promoter sequence specific to cells in the liver, for example, a promoter sequence specific to liver parenchymal cells, hepatic non-parenchymal cells (astrocytes, etc.), and the like can be used. Specific examples of such promoter sequences include ApoE promoter, antitrypsin promoter, cKit promoter, liver-specific transcription factors (HNF-1, HNF-2, HNF-3, HNF-6, C/ERP, DBP ) promoters, thyroxine-binding globulin (TBG) promoters (Ran FA, et al., Nature 520(7546):186-91, 2015), promoters of other liver-specific proteins (such as albumin), and combinations of these promoters synthetic promoters, etc., but are not limited to these. In addition, these promoters can be further combined with known enhancer sequences, preferably liver-specific enhancer sequences. Such enhancer sequences include the ApoE enhancer sequence and the like. These promoter sequences and enhancer sequences can be used singly or in combination. Furthermore, synthetic promoters using the above liver-specific promoters and enhancers can also be used. The rAAV vector of the present invention may preferably contain the sequence of liver-specific, more preferably hepatocyte (hepatocyte)-specific ApoE promoter, antitrypsin promoter, TBG promoter, or known HCRhAAT synthetic promoter.

In addition, the liver-specific promoter sequence used in the present invention has one or more (eg, 1 to 100, 1 to 50, 1 to 40, 1 to 30, 1 ~25, 1~20, 1~15, 1~10, 1~9 (1~several), 1~8, 1~7, 1~6, 1~5, 1-4, 1-3, 1-2, 1, etc.) nucleotide deletions, substitutions, insertions and/or additions that can function as liver-specific promoter sequences. You can also use things. In the present specification, the ability to function as a liver-specific promoter sequence means that, for example, when liver-derived cells and non-liver-derived cells are compared in a reporter assay, non-liver-derived cells have a detection threshold of about or express about 25% or less, about 20% or less, about 15% or less, about 10% or less, or less of the reporter compared to liver-derived cells. In addition, the polynucleotide having the above deletion, substitution, insertion and/or addition means that the specific activity relative to the original promoter sequence is 50%, 60%, 70%, 80%, 90% or more. do. The fewer the number of mutations mentioned above, the better.

Diseases to be treated using the AAV vector of the present invention include hemophilia associated with hepatocyte genomic disorder, acute intermittent porphyria, ornithine transcobalamin deficiency, Wilson's disease, phenylketonuria, and familial hypercholesterolemia. blood, etc. For example, hemophilia A is caused by deficiency or abnormality of blood coagulation factor VIII (also called FVIII), and hemophilia B is caused by deficiency or abnormality of blood coagulation factor IX (also called FIX). Therefore, gene therapy supplementation with blood clotting factor VIII or IX can be considered.
The recombinant viral vectors used in the present invention may contain various therapeutic genes to perform the above treatments. Examples of proteins encoded by such therapeutic genes include blood coagulation factor VIII (FVIII), blood coagulation factor IX (FIX), hepatocyte growth factor (HGF), and hepatocyte growth factor receptor (c-Kit). Examples include, but are not limited to, proteins such as Moreover, the therapeutic gene used in the present invention may be a gene (antisense nucleotide, CAS9, etc.) for inhibiting the target function. Examples of such genes include, but are not limited to, antisense nucleotides against genes encoding thrombin, protein C, or protein S. As used in the present invention, an antisense nucleotide is a polynucleotide for altering (e.g., disrupting, reducing) the function of a targeted endogenous gene, or altering (e.g., reducing) the expression level of an endogenous protein. Refers to a polynucleotide for. Examples of such polynucleotides include, but are not limited to, antisense molecules, ribozymes, interfering RNAs (iRNAs), microRNAs (miRNAs), sgRNAs. Methods for preparing and using double-stranded RNA (dsRNA, siRNA, shRNA or miRNA) are known from many documents (Japanese Patent Publication No. 2002-516062; US Publication No. 2002/086356A; Nature Genetics, 24(2), 180-183, 2000 Feb., etc.). Furthermore, the recombinant viral vectors used in the present invention may contain one or more therapeutic genes.

4. About Pharmaceutical Compositions In a further embodiment of the invention there is provided a pharmaceutical composition comprising the rAAV vector (rAAV virion) of the invention. By using a pharmaceutical composition containing the rAAV vector of the present invention (hereinafter referred to as the pharmaceutical composition of the present invention), a therapeutic gene can be introduced into the liver cells of a subject with high efficiency, and the introduced treatment Provided is a pharmaceutical composition capable of treating a disease of interest by expressing a gene for use. rAAV vectors containing such therapeutic genes are included in the pharmaceutical compositions of the invention. Examples of such therapeutic genes include, but are not limited to, genome editing means and genome repair means as described above.

In one embodiment, the rAAV vector of the invention preferably comprises a liver cell-specific promoter sequence and a gene operably linked to the promoter sequence. The rAAV vectors of the present invention can contain genes that are useful in the treatment of hemophilia and the like, and can incorporate those genes into liver cells, preferably liver parenchymal cells.

When the pharmaceutical composition of the present invention is used, it can be administered, for example, orally, parenterally (intravenously), intramuscularly, buccal mucosa, rectally, vaginally, transdermally, nasally, or by inhalation. It is preferred to administer Intravenous administration is even more preferred. The active ingredients of the pharmaceutical composition of the present invention may be formulated singly or in combination, and may also be provided in the form of preparations by blending them with pharmaceutically acceptable carriers or pharmaceutical additives. can. In this case, the active ingredient of the present invention can be contained, for example, in an amount of 0.1-99.9% by weight in the formulation.

The active ingredients of the pharmaceutical composition of the present invention may be formulated singly or in combination, and may also be provided in the form of preparations by blending them with pharmaceutically acceptable carriers or pharmaceutical additives. can. In this case, the active ingredient of the present invention has, for example, a potency of 10 ⁵ to 10 ¹⁶ vg/mL (900 fg/mL to 90 mg/mL) and a potency of 10 ⁶ to 10 ¹⁵ vg/mL ( 9.0 pg/mL to 9.0 mg/mL), 10 ⁷ to 10 ¹⁴ vg/mL (90 pg/mL to 900 μg/mL), 10 ⁸ to 10 ¹³ vg/mL (900 pg/mL to 90 pg/mL) μg/mL), titers 10 ⁹ - 10 ¹² vg/mL (9 ng/mL - 9 μg/mL), titers 10 ¹⁰ - 10 ¹¹ vg/mL (90 ng/mL - 900 ng/mL) can contain Examples of pharmaceutically acceptable carriers or additives include excipients, disintegrants, disintegration aids, binders, lubricants, coating agents, dyes, diluents, solubilizers, solubilizers, Tonicity agents, pH adjusters, stabilizers and the like can be used.

Formulations suitable for parenteral administration include, for example, injections and suppositories. For parenteral administration, a solution of the active ingredient of the present invention in either sesame oil or peanut oil, or in aqueous propylene glycol solution can be used. The aqueous solutions should be suitably buffered (preferably pH 8 or greater) if necessary and the liquid diluent first rendered isotonic. Physiological saline, for example, can be used as such a liquid diluent. The aqueous solutions prepared are suitable for intravenous injection, while the oily solutions are suitable for intra-articular, intramuscular and subcutaneous injection. The aseptic production of all these solutions is readily accomplished by standard pharmaceutical techniques well known to those skilled in the art. Furthermore, the active ingredient of the present invention can also be administered topically such as to the skin. Topical administration in the form of creams, jellies, pastes or ointments is desirable in this case according to standard pharmaceutical practice.

Examples of formulations suitable for oral administration include powders, tablets, capsules, fine granules, granules, liquids, syrups, and the like. For oral administration, various excipients such as microcrystalline cellulose, sodium citrate, calcium carbonate, dipotassium phosphate, glycine are added to starch, preferably corn, potato or tapioca starch, and alginic acid or seed. and granule-forming binders such as polyvinylpyrrolidone, sucrose, gelatin, gum arabic.

The dosage of the pharmaceutical composition of the present invention is not particularly limited, and an appropriate dosage is selected according to various conditions such as the type of disease, age and symptoms of the patient, administration route, purpose of treatment, and presence or absence of concomitant drugs. It is possible to The dosage of the pharmaceutical composition of the present invention is, for example, 1 to 5000 mg, preferably 10 to 1000 mg per day for an adult (eg, body weight 60 kg), but is not limited thereto. These daily doses may be administered in 2 to 4 divided doses. In addition, when vg (vector genome) is used as a dosage unit, for example, administration in the range of 10 ⁶ to 10 ¹⁴ vg, preferably 10 ⁸ to 10 ¹³ vg, more preferably 10 ⁹ to 10 ¹² vg per kg body weight. Amounts can be selected, but are not limited to these.

5. Administration of Viral Vectors of the Invention Viral vectors of the invention are preferably more safely and easily administered to a subject by peripheral administration. As used herein, the term "peripheral administration" refers to administration routes generally understood by those skilled in the art as peripheral administration, such as intravenous administration, intraarterial administration, intraperitoneal administration, intracardiac administration, and intramuscular administration.

The viral vector of the invention can be administered to a subject to infect cells of the liver and provide the above-described genome editing means delivered by the virus in the infected cells, resulting in genome editing. The viral vectors of the invention are preferably capable of infecting liver parenchymal cells and effecting genome editing. The viral vector of the present invention preferably has a ratio of cells undergoing genome editing among hepatocytes of the liver of the administered subject to preferably 70% or more, 80% or more, 85% or more, 90% or more, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, 99.1% or higher, 99.2% or higher, 99.3% or higher, 99.4% ≥99.5%, ≥99.6%, ≥99.7%, ≥99.8%, ≥99.9%, or 100%.

6. Kits for Preparing rAAV Vectors of the Present Invention In another embodiment, the present invention provides kits for producing the rAAV of the present invention. Such kits can include, for example, (a) a first polynucleotide for expressing capsid protein VP1 or the like, and (b) a second polynucleotide packaged within a rAAV vector. For example, the first polynucleotide comprises a polynucleotide encoding the amino acids of SEQ ID NO: For example, the second polynucleotide may or may not contain a therapeutic gene of interest, but preferably contains various restriction enzyme cleavage sites for incorporating such a therapeutic gene of interest. .

Kits for making rAAV virions of the invention can further comprise any of the components described herein (eg, AdV helpers, etc.). Kits of the invention can also further include instructions describing a protocol for generating rAAV virions using the kits of the invention.

7. Other terms used in this specification Each term used in this specification has the following meaning. Terms not specifically described herein are intended to have the meanings of those terms as commonly understood by those skilled in the art.

As used herein, unless otherwise stated, the terms "viral vector," "viral virion," and "viral particle" are used interchangeably. "Adeno-associated virus vector" also includes adeno-associated virus containing genetic recombination.

As used herein, the term "polynucleotide" is used interchangeably with "nucleic acid", "gene" or "nucleic acid molecule" and intends a polymer of nucleotides. As used herein, the term "nucleotide sequence" is used interchangeably with "nucleic acid sequence" or "base sequence" and is a sequence of deoxyribonucleotides (abbreviated as A, G, C and T) is shown as For example, a "polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 or a fragment thereof" is intended to be a polynucleotide comprising the sequence represented by each deoxynucleotide A, G, C and/or T of SEQ ID NO: 1, or a fragment portion thereof. be done.

Each of the "viral genome" and "polynucleotide" according to the present invention may exist in the form of DNA (eg, cDNA or genomic DNA), but may optionally be in the form of RNA (eg, mRNA). Each of the viral genome and polynucleotides used herein can be double-stranded or single-stranded DNA. For single-stranded DNA or RNA, it may be the coding strand (also known as the sense strand) or the non-coding strand (also known as the antisense strand).
Unless otherwise stated, in this specification, when describing the arrangement of genes such as promoters, target genes, and polyadenylation signals encoded by the rAAV genome, when the rAAV genome is the sense strand, the strand itself For the antisense strand, its complementary strand is described. Also, in this specification, "r" representing recombination may be omitted when it is clear from the context.

As used herein, "protein" and "polypeptide" are used interchangeably and are intended to be polymers of amino acids. For polypeptides used herein, the left end is the N-terminus (amino terminus) and the right end is the C-terminus (carboxyl terminus) according to the convention of peptide designation. The partial peptide of the polypeptide of the present invention (which may be abbreviated as the partial peptide of the present invention in this specification) is a partial peptide of the above-described polypeptide of the present invention, preferably the above-described polypeptide of the present invention. It has properties similar to those of peptides.

As used herein, the term "plasmid" refers to various known genetic elements such as plasmids, phages, transposons, cosmids, chromosomes, and the like. Plasmids are capable of replication in a particular host and transport gene sequences between cells. As used herein, plasmids contain various known nucleotides (DNA, RNA, PNA and mixtures thereof) and may be single-stranded or double-stranded, preferably double-stranded. . For example, as used herein, unless otherwise specified, the term "rAAV vector plasmid" is intended to include the duplex formed by the rAAV vector genome and its complement. Plasmids used in the present invention may be linear or circular.

As used herein, the term "packaging" refers to events including preparation of the single-stranded viral genome, assembly of the coat protein (capsid), and encapsidation of the viral genome. Say. When a suitable plasmid vector (usually multiple plasmids) is introduced into a packaging-capable cell line under appropriate conditions, recombinant viral particles (i.e., viral virions, viral vectors) are assembled and placed in culture. secreted.

Terms not specifically described herein are intended to have the meanings of those terms as commonly understood by those skilled in the art.

EXAMPLES The present invention will be described in more detail with reference to examples below, but the scope of the present invention is not limited to the following examples.

A. Materials and methods
(a) AAV vectors Four types of AAV vectors (AAV2, AAV3B, AAVGT5, AAV8) were used.
AAVGT5 has three amino acid substitutions in the coat protein VP1 of AAV3A and 3B: Serine (S) at position 472 to alanine (A), Serine (S) at position 587 to alanine (A), and Serine (S) at position 587 to alanine (A). contains an amino acid sequence in which asparagine (N) of is replaced with alanine (A).
In each AAV vector, an expression cassette consisting of the cytomegalovirus promoter (CMV) promoter, green fluorescent protein (AcGFP) cDNA, and SV40 poly(A) was inserted between the inverted terminal repeats (ITR) of AAV3A. In the AAV2 vector, instead of the green fluorescent protein (AcGFP) cDNA, an expression cassette containing a LacZ sequence encoding β-galactosidase was also used to construct AAV2-CMV-AcGFP and AAV2-CMV-LacZ vectors, respectively.
AAV2: GenBank Accession # NC_001401.2 (whole genome sequence)
(The amino acid sequence of AAV2 capsid protein VP1 is shown in SEQ ID NO: 1.)
AAV3A: GenBank Accession # U48704 (genome sequence)
(The amino acid sequence of AAV3A capsid protein VP1 is shown in SEQ ID NO: 2.)
AAV3B: GenBank Accession # AF028705.1 (genome sequence)
(The amino acid sequence of AAV3B capsid protein VP1 is shown in SEQ ID NO:3.)
AAVGT5: (produced in this application)
(The amino acid sequence of capsid protein VP1 is shown in SEQ ID NO: 4.)
AAV8: GenBank Accession # NC_006261 (genome sequence)
(The amino acid sequence of AAV8 capsid protein VP1 is shown in SEQ ID NO:5.)

(b) Cell culture 1) HEK 293 cells
HEK293 cells were plated at 5×10 ⁴ cells/well and cultured at 37° C. with 5% CO ₂ using 10% fetal calf serum (FCS)-DMEM/F12 medium (Thermo Fisher Scientific).
2) HepG2 cells
HepG2 cells were plated at 5×10 ⁴ cells/well and cultured at 37° C. in 5% CO ₂ using 10% FCS-DMEM Low glucose medium (Thermo Fisher Scientific).
3) PXB cells (Phoenix Bio)
Cells collected from PXB mouse liver, more than 90% human hepatocytes. Cells were seeded at 7×10 ⁴ cells/well and cultured at 37° C. in 5% CO ₂ using a dedicated dHCGM medium (Phoenix Bio).

The activity of neutralizing antibodies against AAV2 was measured by the method described in Ito T et al., Ann Clin Biochem. 46(Pt 6):508-510, 2009, and LacZ Four sera that completely inhibited expression were used.

(c) vector infection
Various AAV vectors expressing GFP (AAV2-CMV-AcGFP, AAV8-CMV-AcGFP, AAV3-CMV-AcGFP, or AAVGT5-CMV-AcGFP) or AAV vectors expressing β-Galactosidase described in (a) (AAV2-CMV-LacZ) was added at 1-5×10 ⁸ vg/well to each cultured cell and cultured for 2-7 days.

(d) Evaluation of GFP or β-Galactosidase Expression Using a plate reader (Biotech Japan), fluorescence intensity of GFP expressed by various AAV vectors or absorbance by β-galactosidase assay was measured and compared. In addition, an image of a representative field of view of the GFP-expressing cells was taken with a fluorescence microscope (Olympus IX83).

(e) Measurement of antibody titer of AAV2 neutralizing antibody in serum
Antibody titer of neutralizing antibody against AAV2 was measured in advance by the method described in Ito T et al., Ann Clin Biochem. 46(Pt 6):508-510, 2009, ), which completely inhibited the expression of LacZ by the AAV2 vector (Serum 1-4).
HEK293 cells 5×10 ⁴ cells/well were seeded in a 96-well plate (Thermo Fisher Scientific) and cultured in a 37° C., 5% CO ₂ incubator for 1 day. Test sera (Serums 1 to 4) were serially diluted 2-fold up to 128-fold from the undiluted solution. Fetal calf serum (FCS) was used for dilution.
AAV2-CMV-LacZ was diluted to 1×10 ⁸ vg/μL with a 50 mM Hepes, 150 mM NaCl(HN) buffer solution and mixed with the aforementioned diluted serum at a ratio of 1:2 (AAV: serum volume/volume). As a control (Sample (-)), a mixture of FCS containing no test serum and AAV2-CMV-LacZ was used. Specifically, 5 μL of 2×10 ⁷ vg/μL AAV and 10 μL of diluted serum were reacted per well at room temperature for 1 hour, and then 15 μL/well was added to cell wells containing 85 μL of medium. Serum dilution in the medium at this point ranges from 10-fold to 1280-fold (Figs. 4A, 4B).
After 2 days of culture in a 37°C, 5% CO ₂ incubator, β-galactosidase expression was evaluated by measuring absorbance with a plate reader using β-Gal assay Kit (Thermo Fisher Scientific).
The antibody titer was defined as the highest dilution at which the sample (-) well exhibited less than 50% of the absorbance, and was expressed as the serum dilution ratio in the medium.

(f) Verification of cross-reactivity of neutralizing antibodies
To compare the cross-reactivity of AAV3 or AAV GT5 to AAV2 neutralizing antibodies, these AAV3 vectors or AAV GT5 vectors were pre-reacted with serum containing neutralizing antibodies, and then the transduction ability of these vectors was tested in HEK293 cells. were compared by GFP expression in HEK293 cells were seeded at 5×10 ⁴ cells/well in a 96-well optical bottom plate and used the next day.
As the neutralizing antibody-positive sera, four types of human sera that completely inhibited AAV2 expression from the results of the measurement in (e) above were used. AAV3-CMV-AcGFP and AAVGT5-CMV-AcGFP were mixed with these sera at 1:2 (volume/volume), reacted at room temperature for 1 hour, and then administered to the cells. Specifically, 5 μL of 2×10 ⁷ vg/μL AAV and 10 μL of diluted serum were mixed and reacted per well and administered to cell wells containing 85 μL of medium. FCS alone and AAV were mixed and reacted to serve as a control (No Serum). After culturing in a 37°C, 5% CO ₂ incubator for 3 days, the fluorescence intensity of GFP was measured using a plate reader (Biotech Japan) and compared relative to the control value.

B. Results
(1) Preparation of Mutant AAVGT5 DNA containing the Rep sequence fused between adeno-associated virus AAV3A and AAV3B and the VP sequence of AAV3B was artificially synthesized. At that time, the adeno-associated virus mutant AAVGT5 was obtained by genetically manipulating the VP1 of AAV3B to have mutations S472A, S587A, and N706A.
Alignments of the VP1 protein amino acid sequences (SEQ ID NOs:2-4) and Rep protein amino acid sequences (SEQ ID NOs:6-8) of AAV3A, AAV3B and AAVGT5, respectively, are shown in FIGS. 1A-1D.

(2) Confirmation of ability of AAVGT5 to infect human liver-derived cells HepG2 and PXB The ability of AAVGT5 prepared above to infect human liver-derived cells was confirmed.
AAV8-CMV-AcGFP, AAV2-CMV-AcGFP, AAV3-CMV-AcGFP and AAVGT5-CMV-AcGFP the day after seeding HepG2 cells, a human liver-derived cell line, at 5×10 ⁴ cells/well in a 96-well optical bottom plate. 5×10 ⁸ vg/well was administered. After culturing in a 37°C, 5% CO ₂ incubator for 7 days, the fluorescence intensity of GFP was measured with a plate reader and compared (Fig. 2A). The state of the cells at this time is shown in Fig. 2B.
FIG. 3 shows the results of gene transfer into PXB cells, which were collected from the PXB mouse liver in the same manner and in which human hepatocytes account for 90% or more. PXB cells (Phoenix Bio Inc.) were seeded at 7×10 ⁴ cells/well in a 96-well plate, and after 6 days, AAV8-CMV-AcGFP, AAV2-CMV-AcGFP, AAV3-CMV-AcGFP, and AAVGT5-CMV-AcGFP 5×10 ⁸ Administered vg/well. After culturing in a 37°C, 5% CO ₂ incubator for 7 days, the fluorescence intensity of GFP was measured with a plate reader and compared (Fig. 3A). The state of the cells at this time is shown in FIG. 3B.
The GFP expression level of AAVGT5-CMV-AcGFP was approximately 1.1 times that of AAV3 in both HepG2 cells and PXB cells. AAVGT5 is considered to be capable of gene transfer equivalent to or greater than that of AAV3 into these human liver-derived cells. On the other hand, AAV8 and AAV2 had lower gene transfer efficiencies into human hepatocytes than AAV3 and AAVGT5 (FIGS. 2 and 3).

(3) Measurement of Antibody Titer of AAV2 Neutralizing Antibody Antibody titers of AAV2 antibodies in test sera (Serums 1 to 4) were measured according to the above procedure (e) (FIG. 4A).
As a result, the antibody titers of Serum 1-4 were 40, 160, 80 and 160, respectively (Fig. 4B, Table 3 below). These Serums 1-4 were used for cross-reactivity experiments.

(4) Comparison of AAV expression reacted with neutralizing antibody-positive serum against AAV2
After reacting 4 types of sera containing neutralizing antibodies that completely block AAV2 expression (Serum 1 to Serum 4) with AAV3-CMV-AcGFP or AAVGT5-CMV-AcGFP, each AAV vector was added to HEK293 cells. and compared the expression of GFP (Fig. 5A).
HEK293 cells 5×10 ⁴ cells/well were seeded in a 96-well Optical bottom plate and used for measurement of GFP activity on the next day. Comparison was made relative to the control (No Serum).
Reaction with sera containing high titers of neutralizing antibodies (Serum 1–Serum 4) reduced AAV3 GFP expression to 53–28% of control, whereas AAVGT5 expression decreased 85–75%. maintained at high levels (Fig. 5A). The appearance of the cells at this time is shown in FIG. 5B.
Thus, the AAV3 vector, mutated to the AAV GT5 vector, was made more resistant (i.e., less cross-reactive) to neutralizing antibodies in all four sera, resulting in improved gene transfer to cells. I was able to

The recombinant adeno-associated virus vectors of the present invention can reduce attack from neutralizing antibodies of living organisms and, for example, allow more efficient gene transfer to target cells. Thus, the recombinant adeno-associated virus vector of the present invention can be used, for example, in liver-specific promoters or genetic diseases associated with genomic disorders of hepatocytes, such as hemophilia, thrombosis, thrombocytopenia, hereditary hemorrhagic capillary. More efficient if you have therapeutic genes for ectasia, hereditary liver metabolic disorders, hepatocellular carcinoma, etc. (e.g. factor VIII or factor IX gene therapy for hemophilia, cholesterol metabolizing enzymes for hyperlipidemia) gene therapy becomes possible.

SEQ ID NO: 1: Amino acid sequence of AAV2 capsid protein SEQ ID NO: 2: Amino acid sequence of AAV3A capsid protein SEQ ID NO: 3: Amino acid sequence of AAV3B capsid protein SEQ ID NO: 4: Amino acid sequence of AAVGT5 mutant capsid protein (S472A, S587A, N706A) SEQ ID NO: 5: amino acid sequence of AAV8 capsid protein SEQ ID NO: 6: amino acid sequence of AAV3A Rep protein (ARep) SEQ ID NO: 7: amino acid sequence of AAV3B Rep protein (BRep) SEQ ID NO: 8: amino acid sequence of AAVGT5 Rep protein (baRep)

Claims (6)

deletion of 1 to 3 amino acid residues at the amino acid sequence set forth in SEQ ID NO: 4 or other residue positions at positions 472 , 587 and 706 in the amino acid sequence set forth in SEQ ID NO: 4; An adeno-associated virus vector comprising a capsid protein comprising a substituted, inserted or added amino acid sequence, which does not cross-react with neutralizing antibodies against type 2 AAV present in serum. Claim 1, wherein the amino acid sequence is substituted with 1 to 3 amino acid residues at other residue positions of the 472nd, 587th and 706th amino acid residues in the amino acid sequence set forth in SEQ ID NO:4. The adeno-associated virus vector described in . 3. The amino acid sequence of claim 1 or 2, wherein one amino acid residue is substituted at other residue positions of the 472nd, 587th and 706th amino acid residues in the amino acid sequence set forth in SEQ ID NO:4. The adeno-associated virus vector described in . The adeno-associated virus vector according to any one of claims 1 to 3 , which has a viral genome containing a liver cell-specific promoter sequence. the liver cell-specific promoter sequence is ApoE promoter, antitrypsin promoter, cKit promoter, liver-specific transcription factor (HNF-1, HNF-2, HNF-3, HNF-6, C/EBP , DBP) promoter; 5. The adeno-associated virus vector of claim 4 , which is selected from the group consisting of albumin, thyroxine-binding globulin (TBG) promoters, and HCRhAAT synthetic promoters . An amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, inserted or added at residue positions other than the 472nd, 587th and 706th amino acid residues in the amino acid sequence shown in SEQ ID NO: 4 ,
An amino acid sequence in which 1 to 3 amino acid residues are substituted at other residue positions of the 472nd, 587th and 706th amino acid residues in the amino acid sequence set forth in SEQ ID NO: 4,
472nd, 587th and 706th amino acid residues in the amino acid sequence set forth in SEQ ID NO: 4 in which one amino acid residue is substituted at other residue positions, or the amino acid set forth in SEQ ID NO: 4 array
wherein an adeno-associated viral vector comprising a capsid protein comprising said amino acid sequence does not cross-react with neutralizing antibodies against type 2 AAV present in serum .

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