Method for producing protein / nucleic acid complex, population of protein / nucleic acid complex, and protein / nucleic acid complex

Abstract

Provided is a method for producing a protein / nucleic acid complex which comprises a capsid protein derived from an adeno-associated virus and a genomic nucleic acid having at least one inverted terminal repeat sequence and has infectiousness to mammals. The genomic nucleic acid further includes a target nucleic acid that encodes a target protein. The method comprises: (a-1) a step for providing an isolated first nucleic acid that comprises a linear single-stranded nucleic acid, a linear double-stranded nucleic acid, or a circular single-stranded nucleic acid and includes at least one inverted terminal repeat sequence and a target nucleic acid that encodes a target protein; (a-2) a step for providing an isolated first nucleic acid that includes at least one inverted terminal repeat sequence and a target nucleic acid that encodes a target protein; and (b) a step for bringing a first protein into contact with the first nucleic acid in a solvent to form the protein / nucleic acid complex.

Description

Method for producing protein / nucleic acid complexes, population of protein / nucleic acid complexes, and protein / nucleic acid complexes

[0001] This disclosure relates to a method for producing protein / nucleic acid complexes containing a capsid protein and genomic nucleic acid derived from adeno-associated virus, a population of protein / nucleic acid complexes, and protein / nucleic acid complexes. The protein / nucleic acid complexes of this disclosure are useful as vectors for gene therapy.

[0002] Various gene transfer technologies have been developed to introduce and express target genes in mammalian cells or tissues, or in mammalian individuals. Among these, viral vectors are promising in the field of gene therapy because they can efficiently deliver the target gene, which is DNA, into the nucleus. Various viral vectors derived from lentiviruses, adenoviruses, adeno-associated viruses, herpes simplex viruses, and others are known.

[0003] Adeno-associated virus (AAV) vectors have recently become a promising gene delivery vector for gene therapy. AAV is a non-enveloped virus belonging to the parvovirus family. AAV is non-pathogenic and lacks self-renewal ability, so it cannot autonomously replicate and requires co-infection with adenoviruses or other viruses for replication, resulting in extremely low transmissibility. Furthermore, genes introduced by AAV vectors do not become inserted into the genome of target cells but function as extragenomic genes for several years. AAV vectors also have low immunogenicity in humans. Due to these characteristics, AAV vectors can be considered gene therapy vectors that combine safety and effectiveness. In addition, there are more than a dozen serotypes of AAV, each with different tissue targeting properties. For this reason, AAV vectors are expected to be applicable to gene therapy for a wide range of diseases.

[0004] Gene therapy drugs containing AAV vectors are already on the market. Onasemnogene abeparvovec, a treatment for spinal muscular atrophy (SMA), is a gene therapy drug containing an AAV vector. However, this drug is very expensive. One of the reasons for this is the high manufacturing cost, i.e., the low production efficiency of AAV vectors.

[0005] Generally, AAV vectors are produced by cell culture. Specifically, they are produced by introducing three plasmids into human-derived HEK293 cells: a plasmid encoding the capsid protein and Rep protein, a plasmid encoding the genomic nucleic acid, and a plasmid expressing a supplementary factor derived from the helper virus adenovirus, and then culturing these recombinant cells. However, this method has low productivity for AAV vectors, and it is estimated that a culture scale of 1000-2000 L is required for a single dose of gene therapy. Furthermore, the population of AAV vectors obtained by this method may contain unintended viral particles, such as hollow particles without genomic DNA, viral particles with genomic DNA that is less than the full length (designed length), and viral particles with DNA derived from plasmids or host cells.

[0006] One approach to overcome these challenges is to enhance the expression of the Rep protein in HEK293 cells. The Rep protein is a factor involved in the replication and packaging of genomic DNA. However, this approach is said to worsen host cell proliferation due to the enhanced expression of the Rep protein. Thus, there are difficulties in achieving mass production of AAV vectors through cell culture methods.

[0007] Several methods for producing AAV vectors other than cell culture have been proposed. Patent Document 1 describes an idea in which hollow AAV particles are denatured and dissociated by urea treatment, heating, pH adjustment, etc., and genomic DNA or RNA is embedded in vitro. However, this method may reduce the infectivity to humans because the denatured hollow particles are reconstituted. Patent Document 2 describes the introduction of small molecular weight compounds and small double-stranded RNA (siRNA: small interfering RNA) of about 20 bp into hollow AAV particles treated with a surfactant. However, no experimental examples are shown for the introduction of long nucleic acids exceeding 1 kb, such as viral genomes. Patent Document 3 discloses matters relating to an in vitro synthesis method of single-stranded AAV genome using the RCA (rolling circle amplification) method.

[0008] Non-Patent Document 1 describes the in vitro production of complete AAV vector particles by mixing an extract of adenovirus-infected HEK293 cells that produce Rep protein and capsid (VP) protein, a plasmid containing the AAV genome, and dNTPs (deoxyribonucleotides). Patent Document 4 further improves upon the technique of Non-Patent Document 1 by supplementing the HEK293 cell extract expressing adenovirus factors, Rep protein, and VP protein with separately prepared VP protein, etc. However, these systems that rely on HEK293 cell extracts involve the replication reaction of the AAV genome, which involves multiple types of adenovirus factors and host cell factors. Furthermore, it is generally difficult to prepare and store large quantities of cell extracts with consistent quality. For these reasons, these systems cannot be directly applied to mass production.

[0009] Furthermore, Patent Document 4 states that, separate from the technique using the HEK cell extract described above, an AAV vector can be produced by a solution reaction with a circular double-stranded plasmid containing the AAV genome, Rep protein, VP protein, ATP, and dNTPs as its basic components. However, Patent Document 4 does not provide evidence that the reaction product infects human cells or that it has the function of a vector, such as expressing the target gene. In addition, since this technique uses plasmids produced from E. coli as raw materials for the AAV genome, it is difficult to completely avoid contamination of the AAV vector with unintended genes.

[0010] Non-patent document 2 describes that capsid hollow particles produced and reconstituted in E. coli could be internalized upon contact with human cells.

[0011] Japanese Patent Publication No. 10-502526, International Publication No. 2012 / 144446, International Publication No. 2021 / 048366, International Publication No. 2025 / 983192

[0012] Zhou X. et al., "In Vitro Packaging of Adeno-Associated Virus DNA", J. Viol. 1998, 72 (4), 3241-3247Le, DT et al., "Adeno-associated virus capsid protein expression in Escherichia coli and chemically defined capsid assembly", Sci. Rep. 2019, 9(1), 18631

[0013] As described above, technologies for in vitro production of AAV vectors containing long-chain genomic nucleic acids, which can be used for mass production of AAV vectors, have not been sufficiently explored. Therefore, this disclosure aims to provide a series of technologies for in vitro production of AAV vectors containing long-chain genomic nucleic acids.

[0014] Furthermore, this disclosure aims to provide a protein / nucleic acid complex that can be used as an AAV vector with fewer side effects.

[0015] One aspect of the present disclosure is a method for producing a protein / nucleic acid complex that is infectious to mammals, comprising a capsid protein derived from adeno-associated virus and a genomic nucleic acid having at least one reverse-terminal repeat sequence, wherein the genomic nucleic acid further comprises a target nucleic acid encoding a target protein, and the method comprises: (a-1) providing an isolated first protein consisting of a capsid protein derived from adeno-associated virus; (a-2) providing an isolated first nucleic acid consisting of a linear single-stranded nucleic acid, a linear double-stranded nucleic acid, or a cyclic single-stranded nucleic acid, comprising at least one reverse-terminal repeat sequence and a target nucleic acid encoding a target protein; and (b) contacting the first protein and the first nucleic acid in a solvent to form the protein / nucleic acid complex.

[0016] Preferably, the process further comprises (a-3) providing an isolated secondary protein comprising a Rep protein, wherein in step (b), the first protein, the first nucleic acid, and the secondary protein are brought into contact in a solvent containing nucleotides.

[0017] Preferably, the first nucleic acid is linear single-stranded DNA or linear double-stranded DNA, and the at least one reverse-end repeat sequence includes a first reverse-end repeat sequence and a second reverse-end repeat sequence, wherein the first reverse-end repeat sequence is located at the 3' end of the first nucleic acid and the second reverse-end repeat sequence is located at the 5' end of the first nucleic acid.

[0018] Preferably, the first nucleic acid is a linear double-stranded DNA or a circular single-stranded DNA, and the at least one reverse-end repeat sequence includes a first reverse-end repeat sequence and a second reverse-end repeat sequence.

[0019] Preferably, the first protein comprises at least VP3.

[0020] Preferably, the second protein comprises at least one selected from the group consisting of Rep40, Rep52, Rep68, and Rep78.

[0021] Preferably, the second protein comprises at least one selected from the group consisting of Rep68 and Rep78.

[0022] Preferably, the first nucleic acid is ssAVg, dsAVg, or scAVg.

[0023] Preferably, the first nucleic acid is scAVg.

[0024] Preferably, the first nucleic acid is neAVg.

[0025] Preferably, the first nucleic acid is synthesized using the PCR method and / or the RCA method.

[0026] Preferably, the first nucleic acid is denatured.

[0027] Preferably, the first protein does not contain a sugar chain.

[0028] Preferably, the first protein is denatured.

[0029] Preferably, the solvent contains a water-soluble polymer.

[0030] Preferably, it further includes the step of providing an isolated third protein consisting of a single-stranded DNA-binding protein, and in the step (b), the third protein is further contacted.

[0031] One aspect of the present disclosure is a population of protein / nucleic acid complexes comprising a capsid protein derived from adeno-associated virus and a genomic nucleic acid having at least one inverted terminal repeat sequence, and having infectivity to mammals, wherein the genomic nucleic acid further comprises a target nucleic acid encoding a target protein, the plurality of protein / nucleic acid complexes include a first protein / nucleic acid complex having scAVg as the genomic nucleic acid, and the ratio of the number of molecules of the first protein / nucleic acid complex to the total number of molecules of the plurality of protein / nucleic acid complexes is 50% or more.

[0032] One aspect of the present disclosure is a group of protein / nucleic acid complexes comprising a plurality of protein / nucleic acid complexes that are infectious to mammals, each comprising a capsid protein derived from an adeno-associated virus and a genomic nucleic acid having at least one reverse-terminal repeat sequence, wherein the genomic nucleic acid further comprises a target nucleic acid encoding a target protein, and the plurality of protein / nucleic acid complexes each comprises a second protein / nucleic acid complex having ssAVg or dsAVg as the genomic nucleic acid, and the ratio of the number of molecules of the second protein / nucleic acid complex to the total number of molecules of the plurality of protein / nucleic acid complexes is 50% or more.

[0033] One aspect of the present disclosure is a group of protein / nucleic acid complexes comprising a plurality of protein / nucleic acid complexes that are infectious to mammals, each comprising a capsid protein derived from an adeno-associated virus and a genomic nucleic acid having at least one reverse-terminal repeat sequence, wherein the genomic nucleic acid further comprises a target nucleic acid encoding a target protein, and the plurality of protein / nucleic acid complexes each comprises a third protein / nucleic acid complex having ssAVg, dsAVg, or scAVg as the genomic nucleic acid, and the ratio of the number of molecules of the third protein / nucleic acid complex to the total number of molecules of the plurality of protein / nucleic acid complexes is 90% or more.

[0034] One aspect of the present disclosure is a group of protein / nucleic acid complexes comprising a plurality of protein / nucleic acid complexes that are infectious to mammals, each comprising a capsid protein derived from an adeno-associated virus and a genomic nucleic acid having at least one reverse-terminal repeat sequence, wherein the genomic nucleic acid further comprises a target nucleic acid encoding a target protein, and the ratio of the number of molecules of protein / nucleic acid complexes in which the genomic nucleic acid contains nucleic acids other than the target nucleic acid to the total number of molecules of the plurality of protein / nucleic acid complexes is 1% or less.

[0035] Preferably, the capsid protein does not contain sugar chains.

[0036] One aspect of the present disclosure is a protein / nucleic acid complex that is infectious to mammals, comprising an adeno-associated virus-derived capsid protein and a genomic nucleic acid having at least one reverse-terminal repeat sequence, wherein the capsid protein is sugar-free.

[0037] Preferably, the genomic nucleic acid includes a target nucleic acid that encodes a target protein.

[0038] Preferably, the mammal is a human.

[0039] Preferably, the capsid protein is produced by a prokaryote into which the nucleic acid encoding the capsid protein has been introduced.

[0040] Preferably, the prokaryote is a bacterium.

[0041] Preferably, the prokaryote is Escherichia coli.

[0042] Preferably, the genomic nucleic acid is a linear single-stranded DNA, and the at least one reverse-end repeat sequence includes a first reverse-end repeat sequence and a second reverse-end repeat sequence, wherein the first reverse-end repeat sequence is located at the 3' end of the single-stranded DNA and the second reverse-end repeat sequence is located at the 5' end of the single-stranded DNA.

[0043] Preferably, the genomic nucleic acid is a single-stranded DNA of the scAVg type.

[0044] Preferably, the genomic nucleic acid is ssAVg type single-stranded DNA.

[0045] According to this disclosure, AAV vectors containing long-chain genomic nucleic acids can be mass-produced in vitro. Furthermore, a population of high-purity AAV vectors containing a large amount of the target genomic nucleic acid can be obtained.

[0046] This disclosure provides a protein / nucleic acid complex having a capsid that does not contain glycans. This protein / nucleic acid complex is useful as a vector for gene therapy.

[0047] This is an explanatory diagram showing the structure of reverse terminal repeat sequences (ITRs). This is an explanatory diagram showing the structures of ssAVg, dsAVg, scAVg, and neAVg. This is an explanatory diagram showing the structures of VP1, VP2, and VP3. This is an explanatory diagram showing the structures of Rep78, Rep68, Rep52, and Rep40. This is a photograph showing the results of agarose gel electrophoresis performed in Experimental Example 1. This is a photograph showing the results of SDS-PAGE performed in Experimental Example 2. This is a photograph showing the results of Western blotting performed in Experimental Example 2. This is a graph showing the results of Experimental Example 3. This is a micrograph showing the results of Experimental Example 3. This is a graph showing the results of Experimental Example 4. This is a graph showing the results of Experimental Example 5. This is a micrograph showing the results of Experimental Example 5. This is a photograph showing the results of SDS-PAGE performed in Experimental Example 6. This is a chromatogram showing the results of size exclusion chromatography performed in Experimental Example 7, showing the results using VP1, VP2, and VP3. This is a chromatogram showing the results of size exclusion chromatography performed in Experimental Example 7, showing the results using only VP3. This is a chromatogram showing the results of size exclusion chromatography performed in Experimental Example 7, showing the results using only VP1. This is a chromatogram showing the results of size exclusion chromatography performed in Experimental Example 7, showing the results using only VP2. This is a micrograph showing the results of Experimental Example 7, showing the results using VP1, VP2, and VP3. This is a micrograph showing the results of Experimental Example 7, showing the results using only VP3. This is a micrograph showing the results of Experimental Example 7, where C indicates the results using HEK293 cell-produced hollow particles. This is a graph showing the results of Experimental Example 8. This is an explanatory diagram showing an overview of the experiments performed in Experimental Examples 3, 5, and 8. This is an explanatory diagram showing an overview of the synthesis method performed in Experimental Example 9. This is a photograph showing the results of agarose gel electrophoresis performed in Experimental Example 9. This is a graph showing the results of Experimental Example 10, showing the results using PEG20000. This is a graph showing the results of Experimental Example 10, showing the results using PEG200. This graph shows the results of Experimental Example 10, specifically the results using PEG2000. This graph shows the results of Experimental Example 10, specifically the results using PEG8000. This graph shows the results of Experimental Example 10, specifically the results using PEG20000. This graph shows the results of Experimental Example 10, specifically the results using dextran 40000.This graph shows the results of Experiment Example 10, specifically the results using dextran 150000. This graph shows the results of Experiment Example 10, specifically the results using Ficol 70. This graph shows the results of Experiment Example 10, specifically the results using Ficol 400. This graph shows the results of Experiment Example 10, specifically the results using polyvinylpyrrolidone K90. This is a micrograph showing the results of Experiment Example 10. This graph shows a comparison of the inclusion rates of Experiment Example 10 and Experiment Example 11. This graph shows a comparison of the ratio of plasmid-derived sequences (AmpR) to ITR sequences in the inclusion of nucleic acids in Experiment Example 10 and Experiment Example 11. This is a photograph showing the results of agarose gel electrophoresis performed in Experiment Example 11.

[0048] The protein / nucleic acid complex of this disclosure basically comprises an AAV-derived capsid protein and a genomic nucleic acid containing the target nucleic acid, and is infectious to mammals. Furthermore, the method for producing the protein / nucleic acid complex of this disclosure basically includes a step of contacting an isolated capsid protein (first protein) and an isolated genomic nucleic acid (first nucleic acid) in a solvent.

[0049] <Protein / Nucleic Acid Complexes> In this disclosure, a protein / nucleic acid complex refers to a structure formed by the assembly and integration of a protein and a nucleic acid. A viral particle having a capsid and genomic nucleic acid is an example of a protein / nucleic acid complex.

[0050] In this disclosure, AAV complete particles refer to AAV particles containing full-length genome nucleic acids. Typically, AAV complete particles function as AAV vectors. AAV complete particles are contained within a protein / nucleic acid complex. Full-length genome nucleic acids, in other words, are genome nucleic acids that have the designed length.

[0051] In this disclosure, AAV hollow particles refer to structures consisting solely of AAV capsids and lacking genomic nucleic acids. AAV hollow particles are not included in protein / nucleic acid complexes. Hollow particles include virus-like particles (VLPs). AAV complete particles and AAV hollow particles can be separated by density gradient ultracentrifugation, ion exchange chromatography, etc.

[0052] In this disclosure, AAV incomplete particles refer to AAV particles containing genomic nucleic acids of incomplete length. AAV incomplete particles typically do not function as AAV vectors, or if they do, only to a limited extent. AAV incomplete particles are basically contained in protein / nucleic acid complexes, but there may be embodiments in which they are not. In general, it is difficult to separate AAV complete particles from AAV incomplete particles.

[0053] The protein / nucleic acid complexes of this disclosure are infectious to mammals. "Infectious to mammals" can be rephrased as "biologically active." For example, even AAV-like particles containing genomic nucleic acid and capsid protein that are biologically inactive and not infectious to mammals are not included in the protein / nucleic acid complexes of this disclosure. The mammals referred to are typically humans. Other mammals include mice, rabbits, and monkeys. Because the protein / nucleic acid complexes of this disclosure are infectious to mammals such as humans, they can function, for example, as vectors for gene therapy.

[0054] "Infection" includes, at a minimum, the entry of the complex into a mammalian cell; further, the translocation of the complex to the nucleus; and further, the expression of the target gene.

[0055] <Capsid Proteins> Generally, the AAV capsid is a structure composed of three types of capsid proteins: VP1, VP2, and VP3. The ratio of the number of molecules of VP1, VP2, and VP3 in the AAV capsid is said to be 1:1:10. The molecular weight of VP1 (SEQ ID NO: 3) is approximately 82k, the molecular weight of VP2 (SEQ ID NO: 4) is approximately 67k, and the molecular weight of VP3 (SEQ ID NO: 5) is approximately 60k. The sequence of VP3 is a common sequence of the three types of capsid proteins. VP1 and VP2 have an extra sequence at the N-terminus. This N-terminal extra sequence contains multiple sequences consisting of basic amino acids, which are said to be nuclear localization signals (NLS). VP3 also contains one NLS sequence at its N-terminus (Sonntag, F. et al., J. Virol. 2006, 80(22), 11040). In addition, VP1 has a unique region called the phospholipase A2 domain. This motif is thought to be necessary for AAV to migrate outside the endosome after infection (Zadori, Z. et al., Develop. Cell, 2001, 1, 291).

[0056] The "AAV-derived capsid protein," a component of the protein / nucleic acid complex of this disclosure, includes VP1, VP2, or VP3. In this disclosure, VP1, VP2, and VP3 may be collectively referred to as VP protein or VP.

[0057] The manufacturing method of the present disclosure includes the step of providing an isolated first protein consisting of a capsid protein derived from AAV. The first protein is not particularly limited as long as it can ultimately form the protein / nucleic acid complex described above. In one embodiment, the first protein comprises VP1, VP2, and VP3. In another embodiment, the first protein comprises at least VP3 and optionally VP1 and VP2. The ratio of the number of VP1, VP2, and VP3 molecules in the first protein may be, for example, 1:1:10, the same as that of the AAV capsid, but is not limited thereto. This ratio may be changed depending on the purpose. For example, the first protein may contain an excess of any of the VP proteins. It has been reported that gene transfer ability can be improved by changing the ratio of the number of VP1, VP2, and VP3 molecules (Onishi, T. et al., Molecular Therapy - Methods & Clinical Development, 31, 101142 (2023)).

[0058] <Capsid Protein Serotypes> To date, more than 100 variants of AAV have been isolated. Of these, more than a dozen serotypes are available for use in human therapy (Yoon DS et al, Int. J. Med. Sci. 2021, 18, 3353). Since tissue targeting differs depending on the serotype, it is possible to select a serotype according to the therapeutic objective. Furthermore, it is possible to obtain modified capsid proteins using rational or evolutionary engineering methods to improve tissue delivery, infectivity, immunogenicity, blood stability, solubility, etc. (Wang, D. et al., Nat. Rev. Drug Discov. 2019, 18, 358). For example, AAV particles have numerous loop structures on their surface (Gurda, BL et al., J. Viol. 2013, 87(16), 9111) (located within the VP3 sequence), but it is also possible to improve the tissue delivery and infectivity of AAV vectors by introducing arbitrary sequences into this region.

[0059] In this disclosure, a VP protein corresponding to the target serotype can be selected as the primary protein. Alternatively, a modified VP protein can be selected as the primary protein depending on the purpose.

[0060] <Capsid Protein Glycans> Treatment involving the systemic administration of large amounts of AAV vectors is considered to carry the risk of serious side effects such as inflammation that cannot be avoided with conventional therapeutic drugs (Hamilton, BA et al., Front. Immunol. 2021, 12, article 675897). In recent years, the relationship between inflammation and glycosylation abnormalities of glycoproteins has attracted attention. Here, it is possible that VP proteins produced by HEK293 cells are conjugated with glycans specific to immortalized cells. As a means of avoiding this, it is suggested to use VP proteins that do not have glycans or VP proteins whose glycan structure has been improved by enzymatic treatment, etc.

[0061] Capsid hollow particles produced and reconstituted in Escherichia coli have been shown to be internalized upon contact with human cells (Le, DT et al., Sci. Rep. 2019, 9, 18631). Therefore, it is clear that AAV vectors constructed using VP proteins without glycosylation exhibit infectivity and expression of target genes. The primary structure of the VP protein, as well as modified versions including glycosylation, are applicable to the protein / nucleic acid complexes of this disclosure.

[0062] The VP protein included in the first protein of this disclosure may be either containing or not containing a sugar chain.

[0063] <Obtaining Capsid Proteins> One method for obtaining isolated VP proteins that constitute the primary protein is to obtain them from cultures of recombinant organisms that produce VP proteins. Suitable hosts include individual animals, individual plants, animal cells, insect cells, yeast, plant cells, bacteria, etc. An optimal production system can be constructed, specifically for the production of three types of VP proteins. One expression method is the co-expression of the three types of VP proteins (VP1, VP2, VP3). This system yields a mixture of the three types of VP proteins. Alternatively, a production system can be constructed for each VP protein to obtain isolated VP1, VP2, and VP3 individually, and then these can be mixed.

[0064] Based on our experience in producing AAV vectors for gene therapy, when using animal cells as a host for VP protein production, HEK293 cells are preferred. When using insect cells as a host, sf9 cells are preferred. Yeast can also be used as a host, but in that case, high-mannose type glycans are added to the VP protein, requiring enzymatic in vitro glycosylation. Humanized yeast that can be conjugated with human-type glycans (Hamilton, SR et al., Science 2006, 313, 1441) may also be used. VP proteins produced in eukaryotes can be subjected to glycosylation as needed.

[0065] On the other hand, if glycosylation of the VP protein is to be avoided, bacterial hosts such as Escherichia coli, Bacillus subtilis, and Corynebacterium can be used. When producing using Gram-positive bacteria, contamination with pyrogenic substances can be easily avoided. When producing VP protein in Escherichia coli, it may be expressed as inclusion bodies in the cytoplasm. In this case, the inclusion bodies can be reconstituted into virus-like particles (VLPs) by dissolving them in high concentrations of urea or guanidine hydrochloride and then reducing the denaturant concentration by dialysis (Le, DT et al., Sci. Rep. 2019, 9(1), 18631). When producing VP protein as a soluble protein, secretory expression is preferred. When secretory expression is used, a secretory signal suitable for each host cell and VP protein can be newly added to the VP protein sequence. Co-expression of Assembly-Activating Protein (AAP) and VP in E. coli is also effective (Fu, C., Gobbooru, S., Martino, AT & Low, W.-K. Protein Expression and Purification 220, 106502 (2024)). AAP has been shown to improve VP folding in vitro (Le DT et al, Sci Rep. 2019, 9(1), 18631).

[0066] Furthermore, in the manufacturing method disclosed herein, it is not essential that the first protein itself forms an aggregate, for example, a hollow particle. As will be described later, a VP protein denatured with urea or the like may be used as the first protein. Denatured VP proteins are thought to be dissociated from each other and not form aggregates. In the manufacturing method disclosed herein, regardless of the structure of the first protein, a protein / nucleic acid complex containing genomic nucleic acid and capable of infecting mammals such as humans can ultimately be produced.

[0067] <Rep Proteins> Generally, four types of Rep proteins (Figure 4), called Rep40, Rep52, Rep68, and Rep78, are involved in the replication and genome packaging of the AAV genome. Rep proteins are classified into Small-Rep and Large-Rep based on their molecular weight. Small-Rep includes Rep40 (molecular weight approximately 35k, SEQ ID NO: 6) and Rep52 (molecular weight approximately 45k, SEQ ID NO: 7). Large-Rep includes Rep68 (molecular weight approximately 61k, SEQ ID NO: 8) and Rep78 (molecular weight approximately 71k, SEQ ID NO: 9).

[0068] These Rep proteins share a common ATP-dependent helicase domain and a nuclear localization signal (NLS). Rep40 essentially consists of the aforementioned helicase domain and nuclear localization signal. Rep68 and Rep78 have an ITR binding site at their N-terminus. Rep68 and Rep78 also possess nicking activity against TRS (Figure 1), which is present in the double-stranded genome intermediate formed during the replication process of the AAV genome. Rep78 and Rep52 have a zinc finger domain at their C-terminus and exhibit DNA binding ability. On the other hand, the function of the C-terminal surplus sequences of Rep40 and Rep68 is unknown, but it is expected to be involved in binding to other Rep proteins, capsid proteins, or host factors.

[0069] In a preferred embodiment, the manufacturing method of the present disclosure further includes the step of providing an isolated secondary protein comprising a Rep protein. The secondary protein is then brought into contact with the primary nucleic acid and the primary protein in a solvent. The secondary protein may contain all four Rep40, Rep52, Rep68, and Rep78, or at least one of them. In a preferred embodiment, the secondary protein contains at least one of Rep68 and Rep78, more preferably both Rep68 and Rep78. Even more preferably, the secondary protein contains all four Rep40, Rep52, Rep68, and Rep78. The secondary protein may contain only Rep40.

[0070] The Rep protein used as the secondary protein may be the wild type or a modified version with some amino acids substituted. Modified Rep proteins include those with improved helicase activity, ITR binding ability, and nicking activity. On the other hand, using Rep68 or Rep78, which lack nicking activity but retain ITR binding ability, makes it possible to efficiently synthesize scAVg in vitro.

[0071] Furthermore, AAV has multiple serotypes, and tissue targeting is determined by the primary structure of the capsid protein. However, in this embodiment, a Rep protein of a serotype different from that of the capsid may be used. When using multiple types of Rep proteins, Rep proteins of different serotypes may be mixed together.

[0072] There are no particular limitations on the method for obtaining isolated Rep proteins, but for example, they can be obtained from cultures of recombinant organisms that produce Rep proteins. Suitable hosts include individual animals, individual plants, animal cells, insect cells, yeast, plant cells, and bacteria. For example, an optimal production system can be constructed that is specifically designed for the production of the four types of Rep proteins. Since Rep proteins do not originally possess sugar chains, using bacteria as a host is preferable in terms of productivity.

[0073] Because Rep proteins have a tendency to aggregate, fusion proteins with molecular chaperones or co-expression systems with molecular chaperones may be used. When using fusion proteins of Rep proteins and molecular chaperones, they may be used as is, or the fusion protein may be cleaved with a sequence-specific protease to recover the Rep protein, which may then be used. Examples of molecular chaperones include MBP (malotose binding protein), FKBP (FK506 binding protein), chaperonin, TrxA (thioredoxin), NusA (N-utilization substance protein A), and SUMO (small ubiquitin-related modifier) ​​(Lebendiker, M. et al., FEBS Lett. 2014, 588, 236).

[0074] <Genomic Nucleic Acid> The genomic nucleic acid contained in the protein / nucleic acid complex of this disclosure comprises at least one reverse terminal repeat (ITR) and a target nucleic acid. The target nucleic acid encodes a target protein. In a preferred embodiment, the genomic nucleic acid has ITRs at both ends and the target nucleic acid between the ITRs.

[0075] The genomic nucleic acid is typically single-stranded DNA. This single-stranded DNA may be either a positive-positive or negative-positive strand. Regardless of whether it is a positive-positive or negative-positive strand, the presence of an ITR (internal tracer) in this single-stranded DNA allows for the formation of a folded structure at the end. Using this as a scaffold, complementary strand synthesis by the host's DNA polymerase becomes possible, and double-stranded DNA is formed. Transcription then begins from the formed double-stranded DNA. Furthermore, the single-stranded DNA may be either linear or circular.

[0076] The manufacturing method of the present disclosure includes the step of providing a first nucleic acid. The first nucleic acid consists of a linear single-stranded nucleic acid, a linear double-stranded nucleic acid, or a circular single-stranded nucleic acid, and includes at least one ITR and a target nucleic acid encoding a target protein. In a preferred embodiment, the first nucleic acid is a linear single-stranded or double-stranded DNA having ITRs at both ends and the target nucleic acid (DNA) between the ITRs. The following description will focus on embodiments in which the first nucleic acid is a linear DNA having ITRs at both ends.

[0077] <Reverse End Repeat Sequences> Generally, the AAV genome consists of approximately 4.7 kb of single-stranded DNA and has reverse end repeat sequences (ITRs) at both ends. The length of the ITR is usually around 130 to 145 b. The ITR contains a terminal resolution site (TRS) and two rep binding elements (RBEs) (Figure 1). An example of the sequence of the 5' end ITR is shown in Sequence ID 1. An example of the sequence of the 3' end ITR is shown in Sequence ID 2.

[0078] It has been suggested that the 3' terminal ITR is involved in the initiation of AAV genome packaging (King, JA et al., EMBO J. 2001,20(12),3282). In this disclosure, the primary nucleic acid may have at least one ITR, preferably one at the 3' terminal. More preferably, the primary nucleic acid (DNA) has one ITR each at the 3' and 5' terminals. In this disclosure, the 3' terminal ITR may be referred to as the 3' ITR (or primary ITR), and the 5' terminal ITR may be referred to as the 5' ITR (or secondary ITR).

[0079] The ITR sequence may be the natural ITR sequence (SEQ ID NOs: 1 and 2), or a modified sequence, as long as it retains its function as an ITR. For example, it may be a partial sequence of the natural ITR, or a modified sequence in which 1 to 10 nucleotides have been substituted, deleted, or inserted. Development is underway to modify ITRs by nucleotide substitution, etc. (Shitik, EM et.al., Front. Med. 2023, 10, 1106085). Furthermore, it is preferable that the modified ITR maintains its ability to bind to Rep68 or Rep78.

[0080] An extra sequence of about 20 bases may be added to the 5' (upstream) and 3' (downstream) ends of the ITR, regardless of whether double-strand formation occurs. Additionally, an intervening sequence of about 20 bases may be added within each ITR. For example, if the 3' end sequence of the ITR can be used as a primer for DNA polymerase extension, then substitution with an artificial sequence or the addition of an artificial sequence is possible. Furthermore, if the 3' end sequence of the ITR can be acted upon by a DNA polymerase with a 3'→5' exonuclease proofreading function, then a sequence of 20 bases or less without complementary strand formation may be present on the 3' end.

[0081] <Morphology of the first nucleic acid> There are four types of morphologies for the first nucleic acid: single-stranded DNA (ssAVg), double-stranded DNA (dsAVg), self-complementary DNA (scAVg), and no-end DNA (neAVg) (Figure 2).

[0082] ssAVg consists of a single-stranded DNA molecule with positive or negative strands, each having an ITR (Integrated Transistor) at both ends. When the primary nucleic acid is ssAVg, it can contain a target nucleic acid (DNA) of up to approximately 5 kb. ssAVg is an example of a "linear single-stranded nucleic acid."

[0083] dsAVg consists of a positive-straight ssAVg and a complementary negative-straight ssAVg, which form a hybrid. When the primary nucleic acid is dsAVg, it can contain a target nucleic acid (DNA) of up to approximately 5 kb. dsAVg is an example of a "linear double-stranded nucleic acid."

[0084] scAVg consists of a single-stranded DNA molecule in which the 3' ITR, positive strand, intervening sequence, negative strand, and 5' ITR are linked in that order. It then bends at the intervening sequence, and the positive and negative strands self-complementarily form a hybrid. When the primary nucleic acid is scAVg, it can contain a target nucleic acid (DNA) of up to approximately 2.5 kb. The intervening sequence may be the same sequence as the ITR. scAVg is an example of a "linear single-stranded nucleic acid."

[0085] neAVg has a structure in which the single-stranded region of scAVg forms a double helix and the ends are closed. When the first nucleic acid is neAVg, it can contain a target nucleic acid (DNA) of up to approximately 2.5 kb. neAVg is an example of a "linear double-stranded nucleic acid." From another perspective, neAVg is also an example of a "cyclic single-stranded nucleic acid." That is, neAVg can take on a double-stranded linear structure under undenatured conditions and a single-stranded cyclic structure under denatured conditions.

[0086] In this disclosure, closed linear DNA, such as neAVg, that is, linear DNA whose ends are covalently closed and which does not have free 5' phosphate groups and 3' hydroxyl groups at the ends, is included in "linear double-stranded nucleic acids." In this disclosure, circular double-stranded nucleic acids, such as E. coli plasmids, are not included in the first nucleic acid.

[0087] When ssAVg or dsAVg is used as the primary nucleic acid, a protein / nucleic acid complex containing ssAVg-type single-stranded DNA as the genomic nucleic acid is obtained. When scAVg is used as the primary nucleic acid, a protein / nucleic acid complex containing scAVg-type single-stranded DNA as the genomic nucleic acid is obtained. When neAVg is used as the primary nucleic acid, a protein / nucleic acid complex containing neAVg-type DNA as the genomic nucleic acid is obtained.

[0088] Furthermore, when using scAVg or neAVg as the primary nucleic acid, in reaction systems where Large-Rep is present, there is a possibility of nicks being introduced into the TRS. If nicks are introduced into the TRS, using scAVg as the primary nucleic acid may result in a protein / nucleic acid complex containing single-stranded DNA with ITR at one or both ends as the genomic nucleic acid. Similarly, if nicks are introduced into the TRS, using neAVg as the primary nucleic acid may result in a protein / nucleic acid complex containing single-stranded DNA with ITR at one end as the genomic nucleic acid. In these embodiments, the primary nucleic acid can include target nucleic acid (DNA) up to approximately 5 kb.

[0089] The first nucleic acid includes at least one ITR and the target nucleic acid (target gene), but may also include other elements. Other elements include promoters, enhancers, transcriptional regulatory elements, poly-A addition signals, terminators, packaging signals, etc. Specific examples of enhancers / promoters include the CMV (cytomegalovirus) promoter, CAG (CMV-chicken β-actin hybrid) promoter, TBG (thyroxine-binding globulin) promoter, and hAAT (human α-1 antitrypsin) promoter. Specific examples of transcriptional regulatory elements include the WPRE (woodchuck hepatitis virus posttranscriptional regulatory element).

[0090] <Preparation of Primary Nucleic Acid> Since the manufacturing method disclosed herein does not amplify genomic nucleic acids within cells, it is desirable to secure a substantial amount of primary nucleic acid. There are no particular limitations on the method for preparing primary nucleic acid, but using a polymerase replication reaction is preferable for mass production. Examples of polymerase replication reactions include PCR (polymerase chain reaction) and RCA (rolling circle amplification). The RCA method is an isothermal reaction using a DNA polymerase (such as Phi29 DNA polymerase) that has both strand substitution activity and 3'→5' exonuclease activity.

[0091] When using polymerase-mediated replication reactions, it is crucial to secure high-purity DNA resources with mutations minimized as template DNA. One method for obtaining large quantities of template DNA fragments is to have E. coli produce plasmids containing the cloned DNA fragments. When using this method, it is preferable to use strains such as Stbl3 (Thermo Fisher Scientific) or JW0387 (KEIO Collection) of E. coli to avoid AAV genome mutations during plasmid amplification as much as possible. Methods independent of E. coli include chemical synthesis of long-chain DNA on nano-processed solid-phase surfaces (Hoose, A. et al., Nature Rev. Chem. 2023, 7, 144) and long-chain DNA synthesis systems using Bacillus subtilis, an organism with a high GC content (Tsuge, K. et al., Sci. Rep. 2015, 5, 10655). High-purity DNA resources obtained by these methods can be used as templates.

[0092] As an example of a method for preparing dsAVg, first, a plasmid containing the target AAV genome (first nucleic acid) is obtained, cloned from E. coli. Then, a DNA fragment containing the AAV genome is obtained from this plasmid by restriction enzyme digestion or PCR. This DNA fragment is circularized using T4 DNA ligase to obtain circular double-stranded DNA. This circular double-stranded DNA is used as a template for RCA to obtain an amplification product. This amplification product is a polymer in which fragments containing the AAV genome are linked head-to-tale. By digesting this polymer with restriction enzymes, dsAVg monomers (first nucleic acid) can be obtained. Note that when preparing dsAVg, it is not necessary to use specific primers in the RCA method; random hexamer oligonucleotides may be used.

[0093] ssAVg can be prepared by asymmetric PCR (Wooddell, CI et al., Genome Res. 1996, 6(9), 886) or by RCA (Ali, MM et al., Chem. Soci. Rev. 2014, 43(10), 3324). The method for synthesizing ssAVg by RCA is described in International Publication No. 2021 / 048366 (Patent Document 3). When preparing ssAVg by RCA, a primer specific to one strand is generally used to promote elongation of only the target strand, but elongation reactions often occur in both strands. In this case, by introducing a Nicking enzyme recognition site inside a circular template DNA (double-stranded), single-stranded circular DNA can be prepared by Nicking enzyme treatment. Using this as a template makes it possible to elongate only the target strand. Furthermore, the molar ratio of specific primers to the single-stranded circular DNA template is also important for the preparation of ssAVg.

[0094] The above RCA method uses DNA polymerases that possess strand-displacement activity. Typical examples include Phi29 DNA polymerase (which has 3'→5' exonuclease activity) and Bst DNA polymerase (which does not have 3'→5' exonuclease activity) that can withstand high-temperature reactions above 65°C, but this is not limited to these.

[0095] In the RCA synthesis process for dsAVg and ssAVg, when using sequence-specific primers, it is also effective to react BstDNA polymerase at a high temperature of 60°C or higher to promote a precise extension reaction.

[0096] scAVg can be produced by an extension reaction using ssAVg or dsAVg as a template. For example, ssAVg or dsAVg that has been denatured with alkali beforehand is prepared as a template. Meanwhile, a DNA polymerase having strand substitution activity, such as Phi29 DNA polymerase or Bst DNA polymerase, is prepared. The DNA polymerase and dNTPs are added to the template, and the complementary strand extension reaction is carried out by incubation at an appropriate temperature. Alternatively, a similar reaction can be carried out using a combination of Rep protein and the DNA polymerase. By these methods, a single-stranded DNA, i.e., scAVg, can be produced in which the positive and negative strands are linked in tandem via ITRs (intervening sequences), and further having ITRs at the 5' and 3' ends. The production of scAVg can be confirmed by alkali gel electrophoresis.

[0097] neAVg can be produced by an extension reaction using ssAVg or dsAVg as a template. For example, ssAVg or dsAVg that has been denatured with alkali beforehand is prepared as a template. Meanwhile, a mixed solution of a DNA polymerase that does not have strand substitution activity, such as T4 DNA polymerase, and T4 DNA ligase is prepared. By incubating these in the presence of dNTPs at an appropriate temperature, double-strand formation between ITRs and end ligation proceed. DNA with unreacted ends can be removed by degrading it from the 3' end using exonuclease III. On the other hand, a method that does not involve denaturing the template DNA involves partially degrading dsAVg from the 3' end with exonuclease III, leaving the complementary strand region intact, then inactivating the exonuclease by heat treatment, etc., and finally reacting it with DNA polymerase and T4 DNA ligase (Snyder RO et al, Cell 1990, 60, 105). In this case as well, unreacted products can be removed by reacting with exonuclease III after the reaction.

[0098] The Terminal Resolution Site (TRS) of scAVg, dsAVg, and neAVg may be modified. For example, mutations may be introduced into the TRS, or the TRS itself may be deleted. This makes it possible to avoid nickeling of the TRS by Rep68 or Rep78 during the formation process of the protein / nucleic acid complex of this disclosure. As a result, it becomes possible to more reliably package full-length scAVg and dsAVg.

[0099] ssAVg, dsAVg, scAVg, and neAVg may have their CpG sequences methylated by methyltransferase. Methylation is expected to evade recognition by TLR9 and suppress the induction of immunity. The presence or absence of methylation can be confirmed by the state of cleavage when a restriction enzyme that is affected by methylation of the CpG sequence is applied.

[0100] The ssAVg, dsAVg, scAVg, and neAVg prepared by the above method are of extremely high purity. In other words, in the above method, ssAVg and dsAVg are produced by amplification by RCA using double-stranded DNA or single-stranded circular DNA containing a full-length genome as a template, and by cleavage with restriction enzymes. Therefore, the ssAVg, dsAVg, scAVg, and neAVg finally obtained are, in principle, all full-length genomes. Furthermore, in principle, no contamination of foreign genes occurs. By using such high-purity primary nucleic acids, the genome purity contained in the produced protein / nucleic acid complex is also extremely high.

[0101] <Single-stranded DNA-binding protein> In one embodiment, when the first protein and the first nucleic acid are brought into contact, a third protein consisting of a single-stranded DNA-binding protein is further brought into contact. In this embodiment, a second protein (Rep protein) may also be used.

[0102] <Solvent> In the manufacturing method of this disclosure, the first protein and the first nucleic acid are brought into contact in a solvent (step (b)). The solvent is not particularly limited as long as it is capable of forming the desired protein / nucleic acid complex. For example, a buffer solution with a pH of about 6 to 9 can be used as a base, with other components added as needed. Other components include adenosine triphosphate (ATP), dithiothreitol (DTT), water-soluble polymers, etc.

[0103] In the manufacturing method of this disclosure, when a secondary protein (Rep protein) is used, it is preferable to include nucleotides. Examples of nucleotides include adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), uridine triphosphate (UTP), thymidine triphosphate (TTP), deoxyadenosine triphosphate (dATP), deoxythymidine triphosphate (dTTP), deoxyguanosine triphosphate (dGTP), and deoxycytidine triphosphate (dCTP). It is preferable to use a mixture of dATP, dTTP, dGTP, and dCTP (dNTPs).

[0104] By including water-soluble polymers in the solvent, crowding effects and other phenomena can be exhibited, improving reaction efficiency. Examples of water-soluble polymers include synthetic substances such as polyethylene glycol (PEG), polyvinylpyrrolidone, Lipidure®, and glycerin; polysaccharides such as dextran, Ficol®, chitosan, heparin, and pullulan; and proteins or polypeptides such as bovine serum albumin and polylysine. The molecular structure of water-soluble polymers is not particularly limited, and examples include linear and dendritic structures.

[0105] <Contact Conditions> In step (b), there are no particular limitations on the conditions under which the primary protein and the primary nucleic acid are brought into contact in the solvent. For example, contact can be carried out at 15°C to 37°C for several minutes to several days, preferably 1 hour to 24 hours.

[0106] There are no particular limitations on the ratio of primary protein to primary nucleic acid used in step (b), however, it is preferable to use an excess of primary nucleic acid to suppress the formation of hollow particles. Note that the protein / nucleic acid complex (complete particle) and the unreacted primary nucleic acid can be easily separated.

[0107] <Denaturation of the First Protein> Generally, there are two main models for the mechanism of viral genome packaging (Bingham RJ et al., "Genome Packaging", 2021, P.488, Elsevier Ltd.). One is called the energy-independent model. In this model, the assembly of capsid proteins and genome packaging occur simultaneously. More specifically, secondary structures called packaging signals present in the genome interact with capsid proteins, causing them to spontaneously assemble and ultimately form the complete viral structure. Examples of energy-independent models include STNV (Satellite tobacoo necrosis virus) (Patel, N. et al., PNAS 2017, 114, 12255), bacteriophage M13 (Peeters, BP et al., DNA 1987, 6(2), 139), and BFDV (Beak and feather disease virus) (Sarker, S. et al., Nat. Commu. 2016, 7, 13014). These are all viruses with single-stranded DNA or RNA genomes of approximately 6 kb or less. In the case of AAV, the ITR acts as a packaging signal, and interaction with the capsid may lead to the formation of a complete particle according to this model. Furthermore, it is expected that the Rep protein plays an important role in maintaining a genome conformation suitable for packaging.

[0108] Another model is the energy-dependent model. In this model, genomic nucleic acid is introduced into a hollow particle formed by the association of capsid proteins, and genome packaging takes place. More specifically, a single-stranded genome is inserted through the gap in the formed viral hollow particle by the energy-dependent drive of a motor protein that binds to it. Examples of the energy-dependent model include bacteriophage T4 and bacteriophage φ29 (Rao, VB et al., Annu. Rev. Viol. 2015, 2, 351), VEEV (Venezuelan equine encephalitis virus) (Mendes, A. et al., Viruses 2018, 10, 138), and HSV (Herpes simplex virus) (Deiss, LP et al., J. Viol. 1986, 59(3), 605). These are viruses that have a genome of single-stranded or double-stranded DNA or RNA of 6 kb or more.

[0109] In AAV genome packaging, there is a hypothesis that the single-stranded genome generated during the replication process is inserted from outside a pre-formed hollow particle by an ATP-driven motor in which Rep proteins work in coordination (i.e., an energy-dependent model), but there is no conclusive evidence.

[0110] In the formation of the protein / nucleic acid complex in this disclosure, the first protein that serves as the raw material for the capsid may be either undenatured (potentially forming hollow particles) or denatured (presumably not forming hollow particles).

[0111] In one embodiment, an undenatured VP protein is used as the first protein and is brought into contact with a first nucleic acid (ssAVg, dsAVg, scAVg, or neAVg). If necessary, a second protein (Rep protein) is also brought into contact. Note that nucleotides such as ATP are required for the helicase activity of the Rep protein to be expressed.

[0112] In another embodiment, a VP protein denatured by urea treatment or the like is used as the first protein, and this is brought into contact with a first nucleic acid (ssAVg, dsAVg, scAVg, or neAVg). Subsequently, operations such as dialysis and dilution are performed to promote particle formation and genome packaging. In this embodiment as well, it is preferable to have a second protein (Rep protein) in the presence of a second protein to control the conformation of the genome suitable for packaging. The denaturation treatment of the first protein can be carried out by, for example, treatment with a denaturing agent such as urea or guanidine hydrochloride at a concentration of about 0.5 to 8 M; treatment with a surfactant such as Triton X-100, NP-40, Tween 20, octyl-β-glucoside, or CHAPS at a concentration of about 0.1 to 20%; PEG treatment; high salt concentration treatment with NaCl, KCl, etc. at a concentration of about 1 to 3 M; or pH change. Alternatively, these treatments may be combined with heat treatment.

[0113] The genome packaging efficiency of protein / nucleic acid complexes formed by the method disclosed herein can be evaluated by qPCR. Infectivity can be evaluated by in vitro infection experiments in human cells, etc. For example, in infection experiments with AAV vectors in HEK293 cells, the multiplicity of infection (MOI) is typically 10. ５ ~10 ６ That is the case.

[0114] <Population of Protein / Nucleic Acid Complexes> This disclosure includes a population of protein / nucleic acid complexes, which includes multiple of the protein / nucleic acid complexes described above.

[0115] Analysis of genomes extracted from AAV vector particles produced by conventional cell culture methods using a Sequel II long-read sequencer revealed that approximately 12% of the AAV vector genome reads were incomplete in length, and approximately 2% of the reads were from sources other than the AAV vector genome (Takara Bio website, URL: https: / / catalog.takara-bio.co.jp / com / tech_info_detail.php?mode=3&masterid=M100007672&unitid=U100004297). Here, reads other than the AAV vector genome include plasmid-derived sequences used when introducing the AAV vector into HEK293 cells, HEK293 cell-derived sequences, etc.

[0116] In contrast, the manufacturing method of this disclosure uses isolated and purified primary nucleic acids and does not use cells, so the purity of the primary nucleic acid is directly reflected in the purity of the protein / nucleic acid complex. Therefore, as long as a primary nucleic acid containing the full-length genome is used, the formation of unintended complexes will not occur in principle. According to this disclosure, it is possible to obtain a population of high-purity protein / nucleic acid complexes with an extremely high content of protein / nucleic acid complexes containing the full-length genome.

[0117] Furthermore, in this disclosure, as long as a high-purity primary nucleic acid free from nucleic acids other than the target nucleic acid (such as heterologous genes) is used, the contamination rate of sequences not derived from the AAV vector genome is, in principle, significantly reduced compared to conventional techniques. According to this disclosure, it is possible to obtain a population of high-purity protein / nucleic acid complexes with an extremely low content (contamination rate) of protein / nucleic acid complexes containing genomic nucleic acids other than the target nucleic acid. The aforementioned contamination rate (number of molecules) is usually 1% or less, preferably 0.1% or less, more preferably 0.01% or less, and even more preferably 0.001% or less. The aforementioned contamination rate can be calculated using next-generation sequencing technology.

[0118] Furthermore, the prevalence of scAVg by the conventional AAV vector genome analysis method described above was approximately 36% of all reads (according to the Takara Bio website). Thus, with conventional AAV vector production using cells, it is not possible to control the proportion of scAVg-type AAV vectors in the AAV vector population.

[0119] In contrast, the manufacturing method of this disclosure uses isolated and purified primary nucleic acids and does not use cells. Therefore, as long as scAVg primary nucleic acids are used, the possibility of generating protein / nucleic acid complexes having genomic nucleic acids other than the scAVg type is extremely low. Similarly, as long as ssAVg or dsAVg primary nucleic acids are used, the possibility of generating protein / nucleic acid complexes having genomic nucleic acids other than the ssAVg type is extremely low. According to this disclosure, it is possible to obtain a population of high-purity protein / nucleic acid complexes with an extremely high content of protein / nucleic acid complexes containing genomic nucleic acids having the desired structure. The "desired structure" includes the ssAVg type and the scAVg type.

[0120] In this disclosure, a protein / nucleic acid complex having scAVg-type single-stranded DNA as the genomic nucleic acid is referred to as the first protein / nucleic acid complex. The first protein / nucleic acid complex is obtained by using scAVg as the first nucleic acid. Here, scAVg-type single-stranded DNA refers to full-length DNA. The ratio of the number of molecules of the first protein / nucleic acid complex to the total number of protein / nucleic acid complex molecules included in the population is usually 50% or more, preferably 60% or more, more preferably 70% or more, even more preferably 80% or more, and particularly preferably 90% or more.

[0121] In this disclosure, a protein / nucleic acid complex having ssAVg-type single-stranded DNA as the genomic nucleic acid is referred to as the second protein / nucleic acid complex. The second protein / nucleic acid complex is obtained by using ssAVg or dsAVg as the first nucleic acid. Here, ssAVg-type single-stranded DNA refers to full-length DNA. The ratio of the number of molecules of the second protein / nucleic acid complex to the total number of molecules of protein / nucleic acid complexes included in the population is usually 80% or more, preferably 90% or more, and more preferably 95% or more.

[0122] In this disclosure, a protein / nucleic acid complex having full-length single-stranded DNA (regardless of type) as a genomic nucleic acid is referred to as a third protein / nucleic acid complex. The ratio of the number of molecules of the third protein / nucleic acid complex to the total number of molecules of protein / nucleic acid complexes included in the population is usually 90% or more, preferably 95% or more.

[0123] The proportion of the aforementioned molecules can be calculated using next-generation sequencing technology.

[0124] <Sugar-Free Protein / Nucleic Acid Complexes> This disclosure includes a protein / nucleic acid complex that is infectious to mammals, comprising an AAV-derived VP protein and a genomic nucleic acid having at least one reverse terminal repeat sequence (ITR), wherein the capsid protein is sugar-free. The genomic nucleic acid may also contain a target nucleic acid encoding a target protein. All of the above embodiments are possible with respect to the sugar-free protein / nucleic acid complexes of this disclosure.

[0125] This disclosure includes the following items: [Item 1] A method for producing a protein / nucleic acid complex that is infectious to mammals, comprising a capsid protein derived from adeno-associated virus and a genomic nucleic acid having at least one reverse-terminal repeat sequence, wherein the genomic nucleic acid further comprises a target nucleic acid encoding a target protein, and the method comprises: (a-1) providing an isolated first protein consisting of a capsid protein derived from adeno-associated virus; (a-2) providing an isolated first nucleic acid consisting of a linear single-stranded nucleic acid, a linear double-stranded nucleic acid, or a circular single-stranded nucleic acid, comprising at least one reverse-terminal repeat sequence and a target nucleic acid encoding a target protein; and (b) contacting the first protein and the first nucleic acid in a solvent to form the protein / nucleic acid complex.

[0126] [Item 2] A method for producing a protein / nucleic acid complex according to Item 1, further comprising the step of (a-3) providing an isolated second protein consisting of a Rep protein, wherein in step (b), the first protein, the first nucleic acid, and the second protein are brought into contact in a solvent containing nucleotides.

[0127] [Item 3] A method for producing a protein / nucleic acid complex according to Item 1 or 2, wherein the first nucleic acid is linear single-stranded DNA or linear double-stranded DNA, the at least one reverse-end repeat sequence comprises a first reverse-end repeat sequence and a second reverse-end repeat sequence, the first reverse-end repeat sequence is located at the 3' end of the first nucleic acid, and the second reverse-end repeat sequence is located at the 5' end of the first nucleic acid.

[0128] [Item 4] A method for producing a protein / nucleic acid complex according to Item 1 or 2, wherein the first nucleic acid is a linear double-stranded DNA or a circular single-stranded DNA, and the at least one reverse-end repeat sequence comprises a first reverse-end repeat sequence and a second reverse-end repeat sequence.

[0129] [Item 5] A method for producing a protein / nucleic acid complex according to any one of Items 1 to 4, wherein the first protein comprises at least VP3.

[0130] [Item 6] The method for producing a protein / nucleic acid complex according to Item 2, wherein the second protein comprises at least one selected from the group consisting of Rep40, Rep52, Rep68, and Rep78.

[0131] [Item 7] The method for producing a protein / nucleic acid complex according to Item 2, wherein the second protein comprises at least one selected from the group consisting of Rep68 and Rep78.

[0132] [Item 8] The method for producing a protein / nucleic acid complex according to Item 3, wherein the first nucleic acid is ssAVg, dsAVg, or scAVg.

[0133] [Item 9] The method for producing the protein / nucleic acid complex described in Item 3, wherein the first nucleic acid is scAVg.

[0134] [Item 10] The method for producing the protein / nucleic acid complex described in Item 4, wherein the first nucleic acid is neAVg.

[0135] [Item 11] A method for producing a protein / nucleic acid complex according to any one of items 8 to 10, wherein the first nucleic acid is synthesized using the PCR method and / or the RCA method.

[0136] [Item 12] A method for producing a protein / nucleic acid complex according to any one of items 1 to 11, wherein the first nucleic acid is denatured.

[0137] [Item 13] A method for producing a protein / nucleic acid complex according to any one of items 1 to 12, wherein the first protein does not contain sugar chains.

[0138] [Item 14] A method for producing a protein / nucleic acid complex according to any one of items 1 to 13, wherein the first protein is denatured.

[0139] [Item 15] A method for producing a protein / nucleic acid complex according to any one of Items 1 to 14, wherein the solvent contains a water-soluble polymer.

[0140] [Item 16] A method for producing a protein / nucleic acid complex according to any one of Items 1 to 15, further comprising the step of (a-4) providing an isolated third protein comprising a single-stranded DNA-binding protein, wherein in step (b), the third protein is further contacted.

[0141] [Item 17] A protein / nucleic acid complex that is infectious to mammals, comprising a capsid protein derived from adeno-associated virus and a genomic nucleic acid having at least one reverse terminal repeat sequence, wherein the capsid protein does not contain glycans.

[0142] [Item 18] The genomic nucleic acid is the protein / nucleic acid complex described in Item 17, comprising the target nucleic acid encoding the target protein.

[0143] [Item 19] The protein / nucleic acid complex described in item 17 or 18, wherein the mammal is a human.

[0144] [Item 20] The protein / nucleic acid complex according to any one of items 17 to 19, wherein the capsid protein is produced by a prokaryote into which the nucleic acid encoding the capsid protein has been introduced.

[0145] [Item 21] The prokaryote is a bacterium, the protein / nucleic acid complex described in Item 20.

[0146] [Item 22] The prokaryote is Escherichia coli, the protein / nucleic acid complex described in Item 20.

[0147] [Item 23] The protein / nucleic acid complex according to any one of items 17 to 22, wherein the genomic nucleic acid is linear single-stranded DNA, the at least one reverse-end repeat sequence comprises a first reverse-end repeat sequence and a second reverse-end repeat sequence, the first reverse-end repeat sequence is located at the 3' end of the single-stranded DNA, and the second reverse-end repeat sequence is located at the 5' end of the single-stranded DNA.

[0148] [Item 24] The genomic nucleic acid is a single-stranded DNA of the scAVg type, as described in Item 23, and is a protein / nucleic acid complex.

[0149] [Item 25] The genomic nucleic acid is a single-stranded DNA of the ssAVg type, as described in Item 23, and is a protein / nucleic acid complex.

[0150] [Experimental Example 1] Synthesis of dsAVg-type genomic DNA

[0151] Plasmid pAAV2 (SEQ ID NO: 10), which encodes the AAV vector genome, was constructed (contract synthesis by Vector Builder). Plasmid pAAV2 encodes the AAV genome containing the EGFP gene as the target gene. PCR was performed on pAAV2 using primers PD1-F4 (SEQ ID NO: 11) and PD1-R4 (SEQ ID NO: 12) with PrimeSTAR Max DNA polymerase (Takara Bio). The resulting approximately 3.5 kb PCR fragment was cleaved with restriction enzyme NotI to obtain a DNA fragment containing approximately 2.5 kb of AAV double-stranded genome. This DNA fragment was circularized using T4 DNA ligase (Nippon Gene Co., Ltd.).

[0152] To perform the RCA reaction, circularized template DNA and random hexamer primers (5'-nnnnnn-3') were incubated at 98°C for 15 minutes, then left at room temperature for primer annealing. An RCA reaction solution (400 μL) was prepared containing template circular DNA (400 ng), primers (40 pmole), 20 μL of dNTPs (25 mM each), 8 μL of BSA (2 mg / mL), 40 μL of 10x buffer, and 8 μL of Phi29 DNA polymerase (NEB), and incubated at 30°C for 40 hours. After the reaction was complete, DNA extraction was performed using phenol / chloroform / isoamyl alcohol (25:24:1) and chloroform. The RCA product was recovered by ethanol precipitation. The recovered RCA product was cleaved with restriction enzyme PvuII and separated by 0.8% agarose gel electrophoresis. A band equivalent to 2.5 kb was excised from the gel, and after gel thawing with heat-resistant β-agarase (Nippon Gene Co., Ltd.), DNA fragments (dsAVg) were recovered using phenol / chloroform / isoamyl alcohol (25:24:1) and chloroform.

[0153] Figure 5 shows the results of agarose gel electrophoresis of the recovered dsAVg (2.5 kb). Lane 1 is dsAVg (double-stranded) dissolved in water, and lane 2 is a sample immediately after neutralization with 10x TAE (Tris-acetate EDTA buffer) of dsAVg denatured in 50 mM NaOH. The shift of the band in lane 2 to the lower molecular weight side indicates that dsAVg in lane 2 dissociated into positive and negative ssAVg strands upon NaOH treatment.

[0154] [Experimental Example 2] Production and Purification of Rep Protein in E. coli

[0155] Genes encoding Rep40 (SEQ ID NO: 13), Rep52 (SEQ ID NO: 14), Rep68 (SEQ ID NO: 15), and Rep78 (SEQ ID NO: 16), each having a His-tag sequence at the C-terminus, were synthesized (synthesis was outsourced to GenScript). The synthesized Rep40, Rep52, and Rep68 genes were introduced into the NdeI-SacI region of pET21a, respectively. The synthesized Rep78 gene was introduced into the NdeI-XhoI region of pET21a. Each of the resulting Rep expression vectors was introduced into Escherichia coli BL21 star (DE3) (Invitorgen) to obtain recombinants expressing Rep proteins.

[0156] The resulting recombinant cells were cultured, and Rep protein expression was induced by adding 0.4 mM IPTG. After culturing, the cells were collected and suspended in Rep buffer (25 mM Tris-HCl / 450 mM NaCl / 20% glycerol / 10 mM imidazole / 0.1% Tween 20 / 0.5 mM AEBSF: aminoethyl benzylsulfonyl fluoride, pH 7.9). The cells were lysed by sonication, and the supernatant obtained by centrifugation was passed through a 0.22 μm filter. This cell extract was applied to a HisTrap-HP column (1 mL) pre-equalized with Rep buffer, and then washed with 10 mL or more of Rep buffer. Each Rep protein retained in the column was eluted with Rep buffer containing 400 mM imidazole. The eluted fractions were analyzed by SDS-PAGE (Figure 6) and Western blotting using an anti-Rep monoclonal antibody (Figure 7). As a result, bands for Rep40 (lane 1), Rep52 (lane 2), Rep68 (lane 3), and Rep78 (lane 4) were identified in each fraction.

[0157] Each eluted fraction containing the Rep protein was treated with 1 mM EDTA and incubated for at least 30 minutes, after which it was dialyzed against 1xPBS containing 10% glycerol and 1 mM DTT.

[0158] [Experimental Example 3] Reconstitution using HEK293 cell-produced AAV hollow particles and dsAVg <1>

[0159] (1) Titer evaluation by AAV reconstruction and qPCR. The dsAVg prepared in Experiment Example 1 was heat-treated in a 50 mM NaOH solution at 90°C for 15 minutes to denature it, and then left at room temperature. This denatured dsAVg (as ssAVg) was 1.4 × 10⁶ １１ In 10 μL of alkaline solution containing molecules, various combinations of Rep proteins (each Rep molecule numbering 1.4 × 10) are added. １２ Reconstituted buffer (5 mM MgCl) containing molecules ２ 90 μL of (5 mM ATP / 1 mM DTT / 50 mM NaCl / 25 mM Hepes-KOH, pH 7.4) was added and mixed. The following combinations of Rep proteins were set. For the four-component mixture, a system without ATP was also set. ・Rep40 / Rep52 / Rep68 / Rep78 (four-component mixture) ・Rep40 / Rep52 / Rep68 (three-component mixture) ・Rep40 / Rep52 / Rep78 (three-component mixture) ・Rep52 / Rep68 / Rep78 (three-component mixture) ・Rep40 / Rep68 / Rep78 (three-component mixture)

[0160] To these solutions, add 1 μL of HEK293 cell-produced AAV hollow particles (Vector Builder) (0.8 × 10⁶ particles). １１ The particles were added and incubated at 30°C for 24 hours. After incubation, the titer of complete particles in each solution was measured by qPCR using the AAVpro Titration kit (for real time PCR) Ver.2 (Takara Bio). In this method, DNaseI treatment was performed for more than 2 hours.

[0161] The results are shown in Figure 8. First, when AAV hollow particles and dsAVg were brought into contact without using the Rep protein, the titer was zero, and no reconstitution occurred at all (Figure 8a).

[0162] When using the four types of Rep proteins in the presence of ATP, reconstitution was promoted (Figure 8b). On the other hand, in the absence of ATP (Figure 8g), the reconstitution rate decreased significantly. This indicates that the function of Rep proteins in the presence of ATP (helicase activity) plays a major role in reconstitution.

[0163] Among Large-Rep, when Rep78 was removed from the system, the recombination rate decreased significantly (c in Fig. 8). However, even when Rep68 was removed from the system, no decrease in the recombination rate was observed (d in Fig. 8). Also, among Small-Rep, no significant decrease in the recombination rate was observed when either Rep40 (e in Fig. 8) or Rep52 (f in Fig. 8) was removed from the system.

[0164] From the above, it was shown that the function of Rep protein is essential for the recombination of AAV vector from AAV empty particles and dsAVg.

[0165] (2) Evaluation of Infectivity Using AAV Reconstitution and HEK293 Cells Alkaline-denatured dsAVg (2.6×10 １１ molecules as ssAVg) and multiple types of Rep proteins (26×10 １１ molecules for each Rep protein) were brought into contact in a reconstitution buffer (5 mM MgCl ２ / 5 mM ATP / 1 mM DTT / 50 mM NaCl / 25 mM Hepes-KOH, pH 7.4). Then, 1 / 50 volume of an undenatured AAV empty particle solution (containing 1.6×10 １１ particles) was added, and the mixture was incubated at 30°C for one day and night. The volume of each reaction solution was set to 100 μL. As combinations of Rep proteins, the following were set. For the 4-component mixture, a system without ATP was also set. ・ Rep40 / Rep52 / Rep68 / Rep78 (4-component mixture) ・ Rep40 / Rep52 / Rep78 (3-component mixture)

[0166] After incubation, 0.001% Pluronic-F68 was added to each sample. 20 μL of the reconstitution reaction product (per well of a 96-well culture plate) was used to infect HEK293 cells in 200 μL of culture medium at a multiplicity of infection (MOI) of 10 ５ to 10 ６ . Five days after infection, the emission of EGFP was observed using a fluorescence microscope. As a control, AAV complete particles at the same concentration as AAV empty particles (produced using the AAV genome-containing plasmid VB010000-9394npt and commissioned to Vector Builder) were used at 1.7×10 １０ per well.We also conducted experiments to infect the organism using particles.

[0167] The results are shown in Figure 9. First, when the Rep protein was not used, no EGFP luminescence was observed at all (Figure 9a).

[0168] When the Rep4 protein was acted upon in the presence of ATP (Figure 9b), luminescence was observed, similar to the control (Figure 9e). On the other hand, when the Rep protein was acted upon in the absence of ATP (Figure 9d), no luminescence was observed at all.

[0169] When Rep68, one of the four Rep proteins, was removed from the system, luminescence was still observed, but a significant decrease was seen (Figure 9c).

[0170] In summary, it has been shown that by applying the Rep protein to AAV hollow particles and dsAVg, an AAV vector with the ability to infect cells can be reconstructed.

[0171] [Experimental Example 4] Reconstruction using urea-denatured AAV perfect particles

[0172] AAV perfect particle (Experimental Example 3) 1.3 × 10⁻⁶ ９ The particles were treated with 1-8 M urea for at least 16 hours at room temperature. After treatment, samples treated with 8 M urea, 6 M urea, or 5 M urea underwent three-stage dialysis with 4 M urea, 2 M urea, and 0 M urea. Samples treated with 4 M urea underwent two-stage dialysis with 2 M urea and 0 M urea. Samples treated with 2 M urea or 1 M urea underwent dialysis with 0 M urea. Each urea solution was 25 mM NaCl / 5 mM MgCl ２ 5 mM ATP / 1 mM DTT / 25 mM Hepes-KOH (pH 7.4) was used as the base, and urea was added to prepare each concentration. For the urea-free solution (0 M urea), DTT was not included. After dialysis, the residual titer of complete particles in each solution was measured by qPCR using the AAVpro Titration kit (for real time PCR) Ver.2 (Takara Bio Inc.). In this method, DNaseI treatment was performed for more than 2 hours.

[0173] The results are shown in Figure 10. No significant decrease in titer was observed with urea treatment at 5M or lower concentrations, but the titer was almost completely lost when treated with 6M and 8M urea. This indicates that AAV complete particles are almost completely disintegrated by urea treatment at 6M or higher concentrations and cannot be spontaneously reconstructed.

[0174] [Experimental Example 5] Reconstitution using HEK293 cell-produced AAV hollow particles and dsAVg <2>

[0175] (1) Titer evaluation by AAV reconstruction and qPCR method: Similar to Experiment Example 2, alkali-denatured dsAVg (as ssAVg, 1.4 × 10) １１ Various combinations of Rep proteins (1.4 × 10⁶ Rep molecules) １２ 90 μL of reconstituted buffer containing the molecule was added and mixed, and incubated at 30°C for 1 hour. The following combinations of Rep proteins were set: • Rep40 / Rep52 (2-combination) • Rep68 / Rep78 (2-combination) • Rep40 / Rep52 / Rep68 / Rep78 (4-combination)

[0176] 90 μL of this reaction solution is mixed with 10 μL of AAV hollow particles (Experimental Example 3) denatured with 6 M urea (1.6 × 10⁶). １１ The reconstitution reaction was carried out by adding the substance (containing particles) to the reconstitution buffer and performing dialysis at 30°C. Dialysis was performed using a 500 μL capacity dialysis machine, Slide-A-Lyzer. TM The procedure was performed overnight using MINI Dialysis Devices, 10K MWCO (Thermo Scientific). As a control, 1.6 × 10⁶ AAV perfect particles (Experimental Example 3) were used. １１ 100 μL of reconstituted buffer containing particles was maintained in the same dialysis machine. After dialysis, the titer of complete particles in each solution was measured by qPCR using the AAVpro Titration kit (for real time PCR) Ver.2 (Takara Bio Inc.). In this method, DNaseI treatment was performed for more than 2 hours.

[0177] The results are shown in Figure 11. When Large-Rep was applied (Figures 11c and d), the titer indicating complete particle formation was lower compared to the control (Figure 11e), but it was clearly higher than in the absence of Rep (Figure 11a) and when only Small-Rep was applied (Figure 11b). This indicates that Large-Rep plays an important role in genome packaging from the dissociated state of hollow particles.

[0178] (2) Evaluation of infectivity using AAV rearrangement and HEK293 cells 20 μL of the above dialysis fluid (3.2 × 10 as hollow particles) per well of a 96-well culture plate １０ (including particles), multiple infection degree (MOI) 10 ５ ~10 ６ HEK293 cells were then infected. Culturing was performed in a total of 200 μL (per well) of culture medium. EGFP luminescence was observed using a fluorescence microscope 5 days after infection.

[0179] The results are shown in Figure 12. When the four types of Rep proteins were applied in the presence of ATP (Figure 12d), EGFP luminescence was observed, similar to the control (Figure 12e). When only Large-Rep was applied (Figure 12c), slight luminescence was observed. On the other hand, in the absence of Rep proteins (Figure 12a) and when only Small-Rep was applied (Figure 12b), no luminescence was observed at all. From the above, it was shown that AAV vectors with the ability to infect cells can be reconstructed by applying Rep proteins to hollow particles and dsAVg denatured and dissociated with 6M urea.

[0180] [Experimental Example 6] Production of capsid protein in E. coli

[0181] A recombinant organism producing VP1 was created by introducing the VP1 gene (SEQ ID NO: 17) into the NdeI-BamHI region of pET11a (Novagen). A recombinant organism producing VP3 was created by introducing the VP3 gene (SEQ ID NO: 19) into the NdeI-BamHI region of pET11a. A recombinant organism producing VP2 was created by introducing the VP2 gene (SEQ ID NO: 18) into the NdeI-BamHI region of pET21a (Novagen). The synthesis of each VP gene was outsourced to GenScript.

[0182] The resulting recombinant cells were cultured, and VP protein expression was induced by adding 0.54 mM IPTG. After culturing, the cells were collected and suspended in buffer A (50 mM Tris-HCl / 0.6 M sugar / 1 mM EDTA / 10 mM NaCl, pH 8.0), and left on ice for 1 hour. The cells were collected by centrifugation and suspended in buffer B (50 mM Tris-HCl / 1 mM EDTA / 50 mM NaCl / 0.1% Triton X-100, pH 8.0), and left on ice for 1 hour. The cells were disrupted by sonication, and the supernatant and precipitate were separated by centrifugation. When the expression of each VP was confirmed by SDS-PAGE, all three VPs were detected in the precipitate fraction.

[0183] The precipitate fraction was washed twice with 1x PBS containing 0.1% Triton X-100, and the precipitate was dissolved in buffer C (50 mM Tris-HCl, 300 mM NaCl, 5 mM imidazole, 8 M urea, pH 8.0). This VP solution was centrifuged at 18,000 g for 30 minutes, and the supernatant was filtered through a 0.22 μm filter. Each of the obtained VP solutions was applied to His-Trap HP (1 ml) (Cytiva) pre-equilibrated with buffer C, and then washed with 10 mL of buffer C. After washing, the VP protein was eluted with buffer C containing 400 mM imidazole.

[0184] Figure 13 shows the results of SDS-PAGE analysis of the eluates. Bands for VP1 (lane 1), VP2 (lane 2), and VP3 (lane 3) were confirmed in each eluate. Thus, VP1, VP2, and VP3 could be recovered from the culture of recombinant E. coli in the form of inclusion bodies.

[0185] [Experimental Example 7] Formation of hollow particles from inclusion bodies

[0186] (1) Formation of hollow particles and analysis by size exclusion chromatography. Each precipitate fraction containing VP1, VP2, or VP3 obtained in Experimental Example 6 was dissolved in buffer C. Each VP solution was mixed so that the molar ratio of VP1, VP2, and VP3 was 1:1:10. The final protein concentration was approximately 600 μg / mL. Dialysis was performed using a 500 μL capacity Slide-A-Lyzer. TM Dialysis was performed at room temperature using MINI Dialysis Devices, 10K MWCO (Thermo Scientific). The first dialysis session consisted of 4M urea / 1mM DTT / 1mM MgCl. ２ For 200 mL of PBS-II (pH 7.4), the second solution was 2 M urea / 1 mM DTT / 1 mM MgCl. ２ For the third test, 1 mM MgCl was added to 200 mL of PBS-II (pH 7.4). ２ Each test was performed for at least 6 hours in 200 mL of PBS-II (pH 7.4). PBS-II was prepared by supplementing 1x D-PBS solution with 200 mM NaCl. After dialysis, 0.001% Pluronic F-68 was added to the recovered dialysate, and the system was centrifuged at 20,000 g for 10 minutes. The resulting supernatant was filtered through a 0.22 μm filter, and 100 μL of it was subjected to size exclusion chromatography using a Superose 6 increase 10 / 300 (Cytiva) pre-equilibriumized with PBS-II containing 0.001% Pluronic F-68.

[0187] The same procedure was performed using VP1 only, VP2 only, and VP3 only, instead of a mixture of VP1 / VP2 / VP3.

[0188] The results are shown in Figures 14A to 14D. As shown in Figures 14A and 14B, the reconstitutes of the three VP mixture and the reconstitute of VP3 alone showed similar main peaks immediately after the exclusion limit (>4,000 kDa) elution point. This strongly suggested that hollow AAV particles (theoretical molecular weight: 3,840 kDa) were reconstituted in both cases. Furthermore, VP proteins were detected in the main peak fractions by Western blotting with an anti-VP antibody. As a result, three bands corresponding to VP1 / VP2 / VP3 were detected in the VP mixture (data not shown).

[0189] On the other hand, as shown in Figure 14C, in the case of VP1 alone, no peak immediately after the exclusion limit was found. As shown in Figure 14D, in the case of VP2 alone, the peak immediately after the exclusion limit accounted for about 50% of the total peaks, and the remaining peaks could be determined to be incomplete VP2 oligomers.

[0190] (2) STEM analysis by transmission electron microscope The main peak of the three VP mixture (Figure 14A) (sample concentration: 9 μg / mL) and the main peak of VP3 alone (Figure 14B) (sample concentration: 15 μg / mL) were isolated and subjected to STEM analysis by transmission electron microscope. Negative staining with 0.5% phosphotungstic acid solution was performed. As a control, HEK cell-produced hollow particles (sample concentration: 100 μg / mL) were observed in the same manner.

[0191] The results are shown in Figures 15A to 15C. Hollow particle images with an average particle size of approximately 23 nm were observed in both the three-part VP mixture (Figure 15A) and the VP3-only sample (Figure 15B). In the HEK cell-produced hollow particle sample (Figure 15C), hollow particle images with an average particle size of approximately 22 nm were observed.

[0192] From the above, it was found that VP protein produced as an inclusion body in E. coli can form hollow particles similar to those produced by VP protein in HEK cells.

[0193] [Experimental Example 8] Reconstitution of protein / nucleic acid complexes using AAV hollow particles without sugar chains

[0194] (1) The dsAVg prepared in Reconstitution Experiment Example 1 using urea-free AAV hollow particles was heat-treated in a 50 mM NaOH solution at 95°C for 15 minutes to denature it, and then left at room temperature. This denatured dsAVg (as ssAVg) was 1.4 × 10⁶ １１ Add 10 μL of Rep40 / Rep52 / Rep68 / Rep78 mixed Rep proteins (each Rep molecule numbering 1.4 × 10) to 10 μL of alkaline solution containing Rep40 / Rep52 / Rep68 / Rep78 molecules. １２ 90 μL of reconstituted buffer containing the molecule was added and mixed. Separately, a system without ATP was also established.

[0195] To these solutions, add the AAV hollow particles (0.8 × 10) prepared using VP1 / VP2 / VP3 in Experimental Example 7. １１ The solution was added to the particles and incubated at 30°C for 24 hours (total reaction solution volume: 100 μL). After incubation, the titer of the complete particles in each solution was measured by qPCR using the AAVpro Titration kit (for real time PCR) Ver.2 (Takara Bio). In this method, DNaseI treatment was performed for more than 2 hours. Separately, a system using hollow particles (containing glycans) produced with HEK293 cells was also established.

[0196] (2) Reconstitution experiment using urea-modified AAV hollow particles: 1.5 × 10⁶ urea solutions of VP1, VP2, and VP3 obtained in Experiment Example 6 are mixed in a ratio of 1:1:10 to form 1.5 × 10⁶ hollow particles. １１ (Solution A) was mixed to the extent corresponding to (per 10 μL). On the other hand, the modified dsAVg (as ssAVg, 1.4 × 10) prepared in (1) was used. １１ Add 10 μL of Rep40 / Rep52 / Rep68 / Rep78 mixed Rep proteins (each Rep molecule numbering 1.4 × 10) to 10 μL of alkaline solution containing Rep40 / Rep52 / Rep68 / Rep78 molecules. １２ 90 μL of reconstitution buffer containing the molecule was added and mixed, and incubated at 30°C for 2 hours (Solution B). Immediately after adding the entire volume of Solution B to Solution A, a 500 μL capacity dialysis machine, Slide-A-Lyzer, was used. TMThe reconstitution reaction was carried out by dialysis at 30°C using MINI Dialysis Devices, 10K MWCO (Thermo Scientific). The reaction was carried out overnight. After the reaction was complete, titer measurement was performed by qPCR as in (1).

[0197] (3) The titer measurement results are shown in Figure 16. In Figure 16, "ecVPu" represents hollow particles produced by E. coli (with urea denaturation), "ecVP" represents hollow particles produced by E. coli (without urea denaturation), "hkVP" represents hollow particles produced by HEK293 cells (without urea denaturation), and "Rep" represents a mixture of Rep40 / 52 / 68 / 78.

[0198] It was shown that genome packaging is possible using VP protein without sugar chains (hollow particles) and Rep protein (Figures 16b, c, e, and f). However, the titer was lower compared to when hollow particles with sugar chains were used (Figure 16g). Surprisingly, no ATP dependence was observed with urea-denatured VP (ecVPu) (Figures 16b and c), while ATP dependence was observed with untreated VP (ecVP), similar to the results of Experimental Example 3 (Figures 16e and f). From this, it is thought that the function of Rep differs depending on whether urea-denatured VP is used (genome contacts dissociated VP) or undenatured VP is used (genome contacts VP after hollow particles are formed). At least in the case of the two, the need for ATP-dependent helicase activity of Rep differs.

[0199] Experimental Examples 3 and 5 demonstrate Rep-dependent genome packaging from a capsid protein composed of dsAVg and VP1 / 2 / 3, based on two different mechanisms, regardless of the presence or absence of glycans in the VP protein. Initial infection of human cells with particles consisting solely of VP3 produced and reconstituted in E. coli has been shown (Le, DT et al., Sci. Rep. 2019, 9, 18631). The glycan-free AAV vectors produced in this example also possess phospholipase A2 domains derived from VP1 and VP2, as well as a nuclear localization signal (NLS, Figure 3), clearly demonstrating that infection and nuclear localization can achieve expression of target genes.

[0200] Figure 17 shows an overview of the experiments conducted in Experimental Examples 3, 5, and 8.

[0201] [Experimental Example 9] Using the dsAVg prepared in Experimental Example 1 for in vitro synthesis of scAVg, a dsAVg solution (50 ng / μL) was prepared by denaturing with 50 mM NaOH. The extension reaction was initiated by diluting 1 μL of this solution with 19 μL of a solution containing DNA polymerase and dNTPs. Phi29 DNA polymerase or Bst DNA polymerase was used as the DNA polymerase. The reaction temperature was 30°C for the former and 65°C for the latter. The reaction time was 2 hours for both. An overview of this in vitro synthesis is shown in Figure 18.

[0202] After the reaction was complete, the formation of scAVg in each reaction solution was analyzed by 0.8% alkaline agarose (50 mM NaOH / 1 mM EDTA) electrophoresis. The results are shown in Figure 19. In Figure 19, lane 1 represents the reaction product with alkali-denatured dsAVg, lane 2 represents the reaction product with phi29 DNA polymerase, and lane 3 represents the reaction product with Bst DNA polymerase. In both cases, a band of approximately 5.0 kb corresponding to scAVg (genome dimer) was confirmed (lanes 2 and 3) using phi29 DNA polymerase (reaction temperature 30°C) and Bst DNA polymerase (reaction temperature 65°C). In the 65°C reaction, the formation of a genome tetramer was observed due to a further extension reaction from the generated scAVg (genome dimer) (lane 3). From these results, it was shown that scAVg can be synthesized from dsAVg in vitro.

[0203] Although unreacted materials (monomers) and by-products (tetramers) are present, these impurities can be easily removed by extraction from the agarose gel or by anion exchange chromatography. This makes it possible to separate high-purity scAVg. Furthermore, since scAVg can be synthesized from dsAVg, it is clear that scAVg can also be synthesized from ssAVg.

[0204] [Experimental Example 10] Reconstitution using HEK293 cell-produced AAV hollow particles and dsAVg in the presence of water-soluble polymers

[0205] (1) AAV reconstitution using water-soluble polymer and titer evaluation by qPCR experiment Example 1: The dsAVg prepared in the experiment is set to ssAVg and 1 × 10 １１ The molecules, HEK293 cell-produced AAV hollow particles (Vector Builder), are measured in 5 × 10⁶ particles. １０ The particles and Rep40 / Rep68 (a mixture of Rep40 and Rep68) are each represented by 2 × 10 molecules. １２ Molecules were prepared. To these mixtures, polyethylene glycol (PEG) 20000 was added as a water-soluble polymer to a final concentration of 20%, and further, a final concentration of 1 mM MgCl was added. ２ A buffer solution was added to achieve the following composition: 5 mM ATP / 1 mM DTT / 10 mM NaCl / 140 mM KCl / 25 mM Tris-HCl, pH 7.5. As controls, solutions with the same composition but without ATP and PEG20000, and solutions with the same composition but without ATP were also prepared. 10 μL of each solution was incubated at 37°C for 24 hours. After incubation, the titer of complete particles in each solution was measured by qPCR. The percentage of reconstructed AVg, where the amount of AAV hollow particles added to the reaction system was set to 100%, was calculated as the encapsulation rate (%).

[0206] The results are shown in Figure 20. First, similar to the results of Experimental Example 8, the encapsulation rate was very low in the absence of ATP (Figure 20a), but the encapsulation rate improved with the addition of ATP (Figure 20b). In contrast, when PEG20000 was further added as a water-soluble polymer, the encapsulation rate improved by about 100 times (Figure 20c).

[0207] (2) Screening of water-soluble polymers The same procedure as in (1) above was performed using various water-soluble polymers, and the encapsulation rate (%) was calculated. The water-soluble polymers selected were PEG200, PEG2000, PEG8000, PEG20000, dextran 40000, dextran 150000, ficol 70, ficol 400, and polyvinylpyrrolidone (PVP) K90. Multiple final concentrations of the water-soluble polymers were set in the range of 5 to 30%.

[0208] The results are shown in Figures 21A to 21I. In other words, the encapsulation rate improved in all cases where a water-soluble polymer was added. Of these, the encapsulation rate was particularly high when PEG20000 was used (Figure 21D).

[0209] (3) AAV reconstitution using water-soluble polymers and evaluation of infectivity using HEK293 cells A solution containing PEG20000 was prepared by the same procedure as in (1) above. 10 μL of this solution was incubated at 37°C for 24 hours. After incubation, this reaction solution was diluted 10-fold with DMEM medium containing 10% FBS and added to HeLa cells seeded in 96 wells. EGFP expression was observed 3 days after addition.

[0210] The results are shown in Figure 22. Specifically, compared to the case using a reaction solution without PEG20000 (PEG(-)), a significantly larger number of EGFP-positive cells were observed when using a reaction solution containing PEG20000 (PEG(+)).

[0211] [Experimental Example 11] In vitro packaging using E. coli plasmids

[0212] (1) Titer evaluation by AAV rearrangement and qPCR method Based on the description in Patent Document 4 (International Publication No. 2025 / 983192), a plasmid containing EGFP as GOI (VB010000-9394npt, Vector Builder) was measured at 109 ng / μL, and HEK293 cell-produced AAV hollow particles (Vector Builder) were measured as particles of 4.8 × 10⁶. １０ Particles / μL, Rep40 / Rep78 5 μM each, ATP 2.5 mM, GTP 0.1 mM, CTP 0.1 mM, UTP 0.1 mM, Creatine kinase 200 μg / mL, MgCl ２A reconstituted solution was prepared with the following concentrations: 3.5 mM AAV, 2.5 mM DTT, 50 μM dNTPs, 50 mM creatine phosphate, and 15 mM HEPES (pH 6.0). 20 μL of this solution was incubated at 37°C for 24 hours. After incubation, the titer of complete particles in the solution was measured by qPCR. The percentage of reconstituted AVg, relative to the amount of AAV hollow particles added to the reaction system (100%), was calculated as the inclusion rate (%). This inclusion rate was compared with that of Experimental Example 10 (using dsAVg).

[0213] The results are shown in Figure 23A. Inclusion was also confirmed in Experimental Example 11, which used an E. coli plasmid. However, the inclusion rate in Experimental Example 11 was significantly lower compared to Experimental Example 10, which used dsAVg.

[0214] Furthermore, the ratio of plasmid-derived sequences (AmpRs) to ITR sequences in the encapsulated nucleic acid was examined. The results are shown in Figure 23B. In other words, a very large number of AmpRs were detected in Experimental Example 11. Thus, in Experimental Example 11, only a very low encapsulation rate was obtained, and a large number of plasmid-derived sequences, i.e., unintended sequences, were detected in the encapsulated nucleic acid.

[0215] (2) Genome excision from circular double-stranded DNA by Rep: Reconstruction of a chemically defined AAV vector in Patent Document 4 requires excision of the AAV genome from a plasmid by Rep as a prerequisite. This was verified in this experimental example.

[0216] AAV vector plasmid (VB10000-9394npt) 1.5 μg (5 kbp: 5.4 × 10⁻¹⁴) １１ For mole, each Rep is approximately 1.2 × 10 １３ Mole was added. The reaction was 1 mM DTT / 5 mM MgCl ２The reaction was carried out at 37°C for 2 hours in a solution of 5 mM ATP / 50 mM NaCl / BSA (100 μg / mL) / 50 mM Hepes-KOH (pH 7.4). In addition to Rep, the restriction enzyme AclI / BspHI was also used in each of the above reactions to confirm the generation of dsAVg (2.5 kbp). These restriction enzymes cleave the non-AAV genome region on the plasmid. As control reactions to confirm dsAVg and the fragment containing it (3.5 kbp), reactions using the restriction enzymes PvuII / AclI / BspHI and AclI / BspHI alone were also performed. After the above reactions were completed, the AAV genome (2.5 kb) was examined by electrophoresis on a 0.8% agarose gel, either directly or after denaturation with 50 mM NaOH.

[0217] The results are shown in Figure 24. In the reactions treated with Rep68 (lane 4) or Rep78 (lane 5), no band corresponding to dsAVg (2.5 kbp) was generated. The same was true when Rep40 / 52 / 68 / 78 was treated (lane 7). On the other hand, in the alkali-denatured samples (lanes 8-14), an overall improvement in mobility due to single-stranding was observed. Although the generation of low-molecular-weight bands, presumably due to the nicking effect of Rep68 and Rep78, was observed, these bands were longer than the denatured AAV genome (denatured-dsAVg) (lane 8) (lanes 11, 12, 14). These fragments are thought to contain non-AAV sequences derived from plasmids. From the above, it was concluded that it is difficult to encapsulate only the AAV genome using the technology described in Patent Document 4.

[0218] [SEQ ID NO: 1] 5'-ttggccactccctctctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcct-3'

[0219] [SEQ ID NO: 2] 5'-aggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagagagggagtggccaa-3'

[0220] [Sequence ID 3] MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPA DVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTN VDIEKVMITDEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL

[0221] [Sequence ID 4] MAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNP LIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQR GNRQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL

[0222] [Sequence ID 5] MATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGAS DIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGVL PGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL

[0223] [Sequence ID 6] MELVGWLVDKGITSEKQWIQEDQASYISFNAASNSRSQIKAALDNAGKIMSLTKTAPDYLVGQQPVEDISSNRIYKILELNGYDPQYAASVFLGWATKKFGKRNTIWLFGPATTGKTNIAEAIAHTVPFYGCVNWTNENFPFNDCVDKMVIWWEEGKMTAKVVESAKAILGGSKVRVDQKCKSSAQIDPTPVIVTSNTNMCAVIDGNSTTFEHQQPLQDRMFKFELTRRLDHDFGKVTKQEVKDFFRWAKDHVVEVEHEFYVKKGGAKKRPAPSDADISEPKRVRESVAQPSTSDAEASINYADRLARGHSL

[0224] [Sequence ID 7] MELVGWLVDKGITSEKQWIQEDQASYISFNAASNSRSQIKAALDNAGKIMSLTKTAPDYLVGQQPVEDISSNRIYKILELNGYDPQYAASVFLGWATKKFGKRNTIWLFGPATTGKTNIAEAIAHTVPFYGCVNWTNENFPFNDCVDKMVIWWEEGKMTAKVVESAKAILGGSKVRVDQKCKSSAQIDPTPVIVTSNTNMCAVIDGNSTTFEHQQPLQDRMFKFELTRRLDHDFGKVTKQEVKDFFRWAKDHVVEVEHEFYVKKGGAKKRPAPSDADISEPKRVRESVAQPSTSDAEASINYADRYQNKCSRHVGMNLMLFPCRQCERMNQNSNICFTHGQKDCLECFPVSESQPVSVVKKAYQKLCYIHHIMGKVPDACTACDLVNVDLDDCIFEQ

[0225] [Sequence ID 8] MPGFYEIVIKVPSDLDEHLPGISDSFVNWVAEKEWELPPDSDMDLNLIEQAPLTVAEKLQRDFLTEWRRVSKAPEALFFVQFEKGESYFHMHVLVETTGVKSMVLGRFLSQIREKLIQRIYRGIEPTLPNWFAVTKTRNGAGGGNKVVDECYIPNYLLPKTQPELQWAWTNMEQYLSACLNLTERKRLVAQHLTHVSQTQEQNKENQNPNSDAPVIRSKTSARYMELVGWLVDKGITSEKQWIQEDQASYISFNAASNSRSQIKAALD NAGKIMSLTKTAPDYLVGQQPVEDISSNRIYKILELNGYDPQYAASVFLGWATKKFGKRNTIWLFGPATTGKTNIAEAIAHTVPFYGCVNWTNENFPFNDCVDKMVIWWEEGKMTAKVVESAKAILGGSKVRVD QKCKSSAQIDPTPVIVTSNTNMCAVIDGNSTTFEHQQPLQDRMFKFELTRRLDHDFGKVTKQEVKDFFRWAKDHVVEVEHEFYVKKGGAKKRPAPSDADISEPKRVRESVAQPSTSDAEASINYADRLARGHSL

[0226] [Sequence ID 9] MPGFYEIVIKVPSDLDEHLPGISDSFVNWVAEKEWELPPDSDMDLNLIEQAPLTVAEKLQRDFLTEWRRVSKAPEALFFVQFEKGESYFHMHVLVETTGVKSMVLGRFLSQIREKLIQRIYRGIEPTLPNWFAVTKTRNGAGGGNKVVDECYIPNYLLPKTQPELQWAWTNMEQYLSACLNLTERKRLVAQHLTHVSQTQEQNKENQNPNSDAPVIRSKTSARYMELVGWLVDKGITSEKQWIQEDQASYISFNAASNSRSQIKAALDNAGKIMSLTKTAPDYLVGQQPVEDISSNRIYKILELNGYDPQ YAASVFLGWATKKFGKRNTIWLFGPATTGKTNIAEAIAHTVPFYGCVNWTNENFPFNDCVDKMVIWWEEGKMTAKVVESAKAILGGSKVRVDQKCKSSAQIDPTPVIVTSNTNMCAVIDGNSTTFEHQQPLQDRMFKFELTRRLDHDFGKVTKQE VKDFFRWAKDHVVEVEHEFYVKKGGAKKRPAPSDADISEPKRVRESVAQPSTSDAEASINYADRYQNKCSRHVGMNLMLFPCRQCERMNQNSNICFTHGQKDCLECFPVSESQPVSVVKKAYQKLCYIHHIMGKVPDACTACDLVNVDLDDCIFEQ

[0227]

[0228] [Sequence ID 11] 5'-agcagagcgcagataccaaatac-3'

[0229] [Sequence ID 12] 5'-gaccgaaatcggcaaaatccc-3'

[0230] 〔SEQ ID NO: 13〕 5'-catatggagctggtcgggtggctcgtggacaaggggattacctcggagaagcagtggatccaggaggaccaggcctcatacatctccttcaatgcggcctccaactcgcggtcccaaatcaaggctgccttggacaatgcgggaaagattatgagcctgactaaaaccgcccccgactacctggtgggccagcagcccgtggaggacatttccagcaatcggatttataaaattttggaactaaacgggtacgatccccaatatgcggcttccgtctttctgggatgggccacgaaaaagttcggcaagaggaacaccatctggctgtttgggcctgcaactaccgggaagaccaacatcgcggaggccatagcccacactgtgcccttctacgggtgcgtaaactggaccaatgagaactttcccttcaacgactgtgtcgacaagatggtgatctggtgggaggaggggaagatgaccgccaaggtcgtggagtcggccaaagccattctcggaggaagcaaggtgcgcgtggaccagaaatgcaagtcctcggcccagatagacccgactcccgtgatcgtcacctccaacaccaacatgtgcgccgtgattgacgggaactcaacgaccttcgaacaccagcagccgttgcaagaccggatgttcaaatttgaactcacccgccgtctggatcatgactttgggaaggtcaccaagcaggaagtcaaagactttttccggtgggcaaaggatcacgtggttgaggtggagcatgaattctacgtcaaaaagggtggagccaagaaaagacccgcccccagtgacgcagatataagtgagcccaaacgggtgcgcgagtcagttgcgcagccatcgacgtcagacgcggaagcttcgatcaactacgcagacagattggctcgaggacactctctcggttctcatcatcatcatcatcattgagagctc-3'

[0231]

[0232]

[0233]

[0234]

[0235]

[0236]

Claims

1. A method for producing a protein / nucleic acid complex that is infectious to mammals, comprising a capsid protein derived from adeno-associated virus and a genomic nucleic acid having at least one reverse-terminal repeat sequence, wherein the genomic nucleic acid further comprises a target nucleic acid encoding a target protein, and the method comprises: (a-1) providing an isolated primary protein consisting of a capsid protein derived from adeno-associated virus; (a-2) providing an isolated primary nucleic acid consisting of a linear single-stranded nucleic acid, a linear double-stranded nucleic acid, or a circular single-stranded nucleic acid, comprising at least one reverse-terminal repeat sequence and a target nucleic acid encoding a target protein; and (b) contacting the primary protein and the primary nucleic acid in a solvent to form the protein / nucleic acid complex.

2. A method for producing a protein / nucleic acid complex according to claim 1, further comprising the step of (a-3) providing an isolated second protein comprising a Rep protein, wherein in step (b), the first protein, the first nucleic acid, and the second protein are brought into contact in a solvent containing nucleotides.

3. The method for producing a protein / nucleic acid complex according to claim 1 or 2, wherein the first nucleic acid is linear single-stranded DNA or linear double-stranded DNA, the at least one reverse-end repeat sequence comprises a first reverse-end repeat sequence and a second reverse-end repeat sequence, the first reverse-end repeat sequence is located at the 3' end of the first nucleic acid, and the second reverse-end repeat sequence is located at the 5' end of the first nucleic acid.

4. The method for producing a protein / nucleic acid complex according to claim 1 or 2, wherein the first nucleic acid is a linear double-stranded DNA or a circular single-stranded DNA, and the at least one reverse-end repeat sequence comprises a first reverse-end repeat sequence and a second reverse-end repeat sequence.

5. The method for producing a protein / nucleic acid complex according to claim 1 or 2, wherein the first protein comprises at least VP3.

6. The method for producing a protein / nucleic acid complex according to claim 2, wherein the second protein comprises at least one selected from the group consisting of Rep40, Rep52, Rep68, and Rep78.

7. The method for producing a protein / nucleic acid complex according to claim 2, wherein the second protein comprises at least one selected from the group consisting of Rep68 and Rep78.

8. The method for producing a protein / nucleic acid complex according to claim 3, wherein the first nucleic acid is ssAVg, dsAVg, or scAVg.

9. The method for producing a protein / nucleic acid complex according to claim 3, wherein the first nucleic acid is scAVg.

10. The method for producing a protein / nucleic acid complex according to claim 4, wherein the first nucleic acid is neAVg.

11. The method for producing a protein / nucleic acid complex according to claim 8, wherein the first nucleic acid is synthesized using PCR and / or RCA.

12. The method for producing a protein / nucleic acid complex according to claim 1 or 2, wherein the first nucleic acid is denatured.

13. The method for producing a protein / nucleic acid complex according to claim 1 or 2, wherein the first protein does not contain a sugar chain.

14. The method for producing a protein / nucleic acid complex according to claim 1 or 2, wherein the first protein is denatured.

15. The method for producing a protein / nucleic acid complex according to claim 1 or 2, wherein the solvent contains a water-soluble polymer.

16. A method for producing a protein / nucleic acid complex according to claim 1 or 2, further comprising the step of (a-4) providing an isolated third protein comprising a single-stranded DNA-binding protein, wherein in step (b), the third protein is further contacted.

17. A group of protein / nucleic acid complexes comprising a plurality of protein / nucleic acid complexes that are infectious to mammals, each comprising a capsid protein derived from adeno-associated virus and a genomic nucleic acid having at least one reverse-terminal repeat sequence, wherein the genomic nucleic acid further comprises a target nucleic acid encoding a target protein, and the plurality of protein / nucleic acid complexes each include a first protein / nucleic acid complex having scAVg-type single-stranded DNA as the genomic nucleic acid, and the ratio of the number of molecules of the first protein / nucleic acid complex to the total number of molecules of the plurality of protein / nucleic acid complexes is 50% or more.

18. A group of protein / nucleic acid complexes comprising a plurality of protein / nucleic acid complexes that are infectious to mammals, each comprising a capsid protein derived from adeno-associated virus and a genomic nucleic acid having at least one reverse-terminal repeat sequence, wherein the genomic nucleic acid further comprises a target nucleic acid encoding a target protein, and each plurality of protein / nucleic acid complexes comprises a second protein / nucleic acid complex having ssAVg-type single-stranded DNA as the genomic nucleic acid, and the ratio of the number of molecules of the second protein / nucleic acid complex to the total number of molecules of the plurality of protein / nucleic acid complexes is 80% or more.

19. A group of protein / nucleic acid complexes comprising a plurality of protein / nucleic acid complexes that are infectious to mammals, each comprising a capsid protein derived from adeno-associated virus and a genomic nucleic acid having at least one reverse-terminal repeat sequence, wherein the genomic nucleic acid further comprises a target nucleic acid encoding a target protein, and each plurality of protein / nucleic acid complexes comprises a third protein / nucleic acid complex having full-length single-stranded DNA as the genomic nucleic acid, and the ratio of the number of molecules of the third protein / nucleic acid complex to the total number of molecules of the plurality of protein / nucleic acid complexes is 90% or more.

20. A group of protein / nucleic acid complexes comprising multiple protein / nucleic acid complexes that are infectious to mammals, each comprising a capsid protein derived from adeno-associated virus and a genomic nucleic acid having at least one reverse terminal repeat sequence, wherein the genomic nucleic acid further comprises a target nucleic acid encoding a target protein, and the ratio of the number of molecules of protein / nucleic acid complexes in which the genomic nucleic acid contains nucleic acids other than the target nucleic acid to the total number of molecules of the multiple protein / nucleic acid complexes is 1% or less.

21. The group of protein / nucleic acid complexes according to any one of claims 17 to 20, wherein the capsid protein is free of sugar chains.

22. A protein / nucleic acid complex that is infectious to mammals, comprising a capsid protein derived from adeno-associated virus and a genomic nucleic acid having at least one reverse-terminal repeat sequence, wherein the capsid protein does not contain glycans.

23. The protein / nucleic acid complex according to claim 22, wherein the genomic nucleic acid comprises a target nucleic acid encoding a target protein.

24. The protein / nucleic acid complex according to claim 22, wherein the mammal is a human.

25. The protein / nucleic acid complex according to claim 22, wherein the capsid protein is produced by a prokaryote into which the nucleic acid encoding the capsid protein has been introduced.

26. The protein / nucleic acid complex according to claim 25, wherein the prokaryote is a bacterium.

27. The protein / nucleic acid complex according to claim 25, wherein the prokaryote is Escherichia coli.

28. The protein / nucleic acid complex according to any one of claims 22 to 27, wherein the genomic nucleic acid is linear single-stranded DNA, the at least one reverse-end repeat sequence comprises a first reverse-end repeat sequence and a second reverse-end repeat sequence, the first reverse-end repeat sequence is located at the 3' end of the single-stranded DNA, and the second reverse-end repeat sequence is located at the 5' end of the single-stranded DNA.

29. The protein / nucleic acid complex according to claim 28, wherein the genomic nucleic acid is a single-stranded DNA of the scAVg type.

30. The protein / nucleic acid complex according to claim 28, wherein the genomic nucleic acid is ssAVg type single-stranded DNA.

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