JP7173490B2 - Nucleic Acid Encapsulating AAV Hollow Particles - Google Patents

Description

The present invention relates to a method for producing AAV hollow particles encapsulating nucleic acids.

Various gene transfer techniques have been developed to introduce and express a gene of interest into cells, tissues or individuals of mammals including humans. Viral vectors are one of them, and various vectors derived from lentiviruses, oncoretroviruses, adenoviruses, adeno-associated viruses and the like are known.

Among them, adeno-associated virus (AAV) vectors have recently been expected as useful gene transfer vectors in gene therapy. AAV is a non-pathogenic virus belonging to the parvovirus family. It lacks self-replicating ability and cannot propagate autonomously. Because it requires co-infection with adenovirus or herpes virus for propagation, its transmissibility is low. It is said that It also has properties of low immunogenicity in the host. Due to such characteristics, it has the advantage of being highly safe as a gene transfer vector. In addition, since it has a wide host range, it is possible to infect various cells, and vectors derived from each serotype AAV (for example, AAV1 to AAV9) have been developed. By applying their specificity, they are used to introduce genes into specific cells, tissues and organs such as nerve cells, muscle cells and hepatocytes. However, conventional viral vectors including AAV have various problems such as acquisition of self-replicating ability and contamination with wild-type virus.

In order to solve the above problems, Samulski et al. proposed preparing a capsid that constitutes the coat protein of AAV and using it as a drug delivery carrier (Patent Document 1). AAV hollow particles formed by capsids and containing no AAV viral genome inside have specific recognition of target cells, adsorption and entry into cells, threshing and other early viral infection activities, but the virus is necessary for self-replication. Since it does not have the originating gene, it has no viral growth activity. Therefore, hollow particles are capable of specifically delivering drugs such as proteins and nucleic acids to target cells, and are ideal drug delivery system (DDS) carriers that are safe for the individual being administered. is.

Samulski et al. have proposed an introduction method in which hollow particles are temporarily denatured by urea, heat, and pH conditions, and then reconstituted after the drug is incorporated inside (Patent Document 1). However, when a denaturing agent such as urea acts on the hollow particles, there is a problem that the capsid's initial infection activity is reduced, impairing its function as a delivery carrier. In addition, Okada et al. disclose a method of introducing protein or nucleic acid into hollow particles using a surfactant (Patent Document 2).

It is known that the DNA genome can take the form of circular double-stranded DNA (cAAV) during the replication process of AAV. This double-stranded DNA has one inverted terminal repeat (ITR) sequence. Musatov et al. reported that the replication and encapsulation of AAV genomic DNA requires a sequence consisting of the A region sequence and the D' region sequence of the ITR (Non-Patent Document 1). Musatov et al. disclose the production of AAV hollow particles encapsulating the DNA by introducing circular double-stranded DNA containing the A region-D' region sequence into cells.

U.S. Pat. No. 5,863,541 International Publication No. 2012/144446 pamphlet

Musatov, et al. , 2002, vol. 76, No. 24, p. 12792-12802

An object of the present invention is to develop and provide a simpler and more efficient method for encapsulating nucleic acids into hollow particles.

As a result of intensive research, the present inventors have succeeded in producing hollow particles encapsulating nucleic acid while maintaining the initial virus infection activity by a simpler and more efficient method. It was also confirmed that the nucleic acid encapsulated in the hollow particles can be effectively introduced into the target cells by mixing the nucleic acid-encapsulated hollow particles with the target cells. The present invention is based on these findings and results, and provides the following.

That is, the present invention
[1] A method for producing an adeno-associated virus (AAV) nucleic acid-encapsulating hollow particle comprising the following steps;
(1) (i) the sequence of the A region of the AAV inverted terminal repeat (ITR);
(ii) preparing a linear nucleic acid fragment comprising an AAV ITR A' region sequence and (iii) an AAV ITR D region sequence and/or D' region sequence;
(2) the step of introducing the nucleic acid fragment of (1) into cells that produce AAV hollow particles; and (3) the step of culturing the cells of (2).
[2] The method of [1], wherein the nucleic acid fragment comprises an A' region sequence, an A region sequence and a D' region sequence from the 5' end to the 3' end.
[3] The method of [1], wherein the nucleic acid fragment comprises a D region sequence, an A' region sequence and an A region sequence from the 5' end to the 3' end.
[4] The method of [1], wherein the nucleic acid fragment comprises, from the 5' end to the 3' end, a D region sequence, an A' region sequence, an A region sequence and a D' region sequence.
[5] The method of [1], wherein the step of preparing the nucleic acid fragment includes amplification of the nucleic acid fragment by a nucleic acid amplification reaction.
[6] The method of [1], wherein the hollow particle-producing cells are cells into which AAV Cap and Rep genes and AAV helper functions have been introduced.
[7] A method for producing AAV nucleic acid-encapsulating hollow particles, comprising the following steps;
(a) (i) at least three AAV ITR A-D' region sequences, or (ii) a sequence complementary to the A-D'region;
preparing a nucleic acid comprising
(b) introducing the nucleic acid of (a) into cells that produce AAV hollow particles;
(c) culturing the cells of (b);
[8] The method of [7], wherein the nucleic acid is a plasmid.
[9] The method of [7], wherein the hollow particle-producing cells are cells into which AAV Cap and Rep genes and AAV helper functions have been introduced.

INDUSTRIAL APPLICABILITY The present invention provides a simpler and more efficient method for producing nucleic acid-encapsulated hollow particles. The "AAV hollow particles encapsulating nucleic acid" in the present invention are AAV-like particles that do not retain the complete AAV genome, and are different from ordinary AAV.

According to the method for producing hollow particles of the present invention, it is possible to easily and efficiently enclose the nucleic acid of interest in hollow particles while reducing the number of virus-derived sequences. Thereby, hollow particles having high safety and target cell specificity can be provided.

FIG. 3 shows the structure of a plasmid containing the A region-D' region sequence. FIG. 10 is a diagram showing fluorescence observation of CHO-K1 cells contacted with AAV hollow particles prepared using a plasmid containing the A region-D′ region sequence. FIG. 3 shows the structure of a linear nucleic acid fragment containing the A region-D' region sequence. FIG. 10 is a diagram showing the titer of AAV hollow particles prepared using a linear nucleic acid fragment containing the A region-D′ region sequence. FIG. 2 shows regions of AAV ITRs.

As used herein, the term "inverted terminal repeat (ITR)" refers to the cis elements present at both ends of the AAV genomic DNA. ITRs are essential for replication, amplification and packaging of AAV genomic DNA. The ITR contains a Rep binding site (RBS, also described as RBE) and a terminal separation site (TRS) as well as a palindromic sequence that allows hairpin formation. The ITR includes A region, B region, B' region, C region, C' region and A' region in order from the end. The A region and the A' region, the B region and the B' region, and the C region and the C' region are complementary sequences in opposite directions. to form At the end of the A' region, the D region exists on the opposite side (3' terminal side or 5' terminal side) of the C' region.

As used herein, the term "3' terminal side" means located in the 3' terminal direction of the 3' terminal portion of a certain sequence (region). Similarly, "5' terminal side" means located in the direction of the 5' terminal portion of a certain sequence (region) and further toward the 5' terminal. Therefore, when the term "3' terminal side" or "5' terminal side" is used herein, the desired sequence or the like is placed in contact with the 3' terminal portion or 5' terminal portion of a certain sequence (region). and the target sequence etc. are arranged without contacting the 3′ terminal portion or 5′ terminal portion of a certain sequence (region) (that is, the 3′ terminal portion or 5′ terminal portion of a certain sequence (region) and any sequence intervening between the target sequence, etc.).

In the present invention, "capsid" is one of the elements that constitute a virus particle (virion), and is composed of a plurality of unit proteins (capsomeres) surrounding genomic DNA or a core. ). Particles composed only of capsid proteins without viral nucleic acids, cores or other material inside the capsid are referred to herein as "empty particles" and "empty capsid particles". )” is sometimes called.

In the present invention, "gene of interest" means any gene desired to be encapsulated in hollow particles and introduced into target cells. Genes of interest include structural genes (e.g., functional protein genes such as enzymes, transcription factors, reporter molecules, growth factors) and regulatory genes (e.g., antisense RNA, siRNA, miRNA, encode functional nucleic acids such as ribozymes). gene). Furthermore, the gene of interest may contain regulatory elements that control transcription and translation, such as promoter sequences, enhancer sequences, poly-A addition signal sequences, terminator sequences and the like.

The present invention will be described in detail below.

In one aspect, the present invention is a method for producing AAV nucleic acid-encapsulating hollow particles, comprising the following steps;
(1) (i) the sequence of the A region of the AAV ITR;
(ii) preparing a linear nucleic acid fragment comprising an AAV ITR A' region sequence and (iii) an AAV ITR D region sequence and/or D' region sequence;
(2) the step of introducing the nucleic acid fragment of (1) into cells that produce AAV hollow particles; and (3) the step of culturing the cells of (2).

(A) Step of preparing a nucleic acid The linear nucleic acid fragment in the step (1) has (i) the sequence of the A region of the ITR, (ii) the sequence of the A' region of the ITR, and (iii) the sequence of the D region. and/or the sequence of the D' region. As these sequences, sequences of genomic DNA of known natural AAV serotypes (eg, serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11) can be used. Regions A, B, B', C, C', and A' are specified in the ITR of each serotype. The D region exists at the end of the ITR A' region opposite to the C' region (3' end side or 5' end side). The sequence of the D region of each serotype and the sequence of the D' region, which is complementary and inverse to that D region sequence, are also specified. The A region, A' region, D region and D' region may be derived from different serotypes. Furthermore, a mutant of the natural A region sequence can also be used within the range of retaining Rep binding ability and hairpin forming ability, in which case the A′ region sequence is complementary to the mutant A region sequence. is. The invention can also use variants of the native D region sequence, in which case the D' region sequence is complementary to the variant D region sequence.

In one aspect of the present invention, the linear nucleic acid fragment of the step (1) comprises, from the 5' end to the 3' end, the sequence of the A' region, the sequence of the A region, and the sequence of the D' region. In another embodiment, the linear nucleic acid fragment comprises a D region sequence, an A' region sequence and an A region sequence from the 5' end to the 3' end. Furthermore, the linear nucleic acid fragment of another embodiment comprises, from the 5' end to the 3' end, a D region sequence, an A' region sequence, an A region sequence and a D' region sequence.

The linear nucleic acid fragment in step (1) may contain a loop sequence (spacer sequence) between the A′ region sequence and the A region sequence. The loop sequence may be a sequence capable of forming a stem-loop structure (hairpin structure) together with the sequences of the A' region and the A region. The chain length of the loop sequence is 3-500 bp, preferably 5-200 bp, more preferably 7-100 bp.

The linear nucleic acid fragment in step (1) can further contain a gene of interest. The gene of interest can be exogenous to AAV. The gene of interest can be placed at the 5′ end of the A′ region sequence, between the A′ region sequence and the A region sequence, or at the 3′ end of the A region sequence. Or it can be arranged in a plurality of locations.

The linear nucleic acid fragment in step (1) includes at least one pair of sequences (i), (ii) and (iii). The present invention can use nucleic acid fragments containing one set, two sets, three sets, four sets, or five or more sets of the above sequences.

The linear nucleic acid fragment in step (1) is DNA or RNA, preferably DNA. Furthermore, the nucleic acid fragment may be a double-stranded nucleic acid in which two complementary nucleic acid molecules are annealed, or a single single-stranded nucleic acid molecule. The linear nucleic acid means that both ends of the nucleic acid are not covalently bound.

The linear nucleic acid fragment in step (1) can be prepared by a known nucleic acid preparation method. The nucleic acid fragment can be prepared in vitro by nucleic acid amplification reaction such as PCR or chemical synthesis. In addition, the nucleic acid can be prepared by extracting nucleic acids produced in vivo, such as in eukaryotic cells or prokaryotic cells, by polymerase reaction, replication and transcription from cells.

In one aspect, the present invention is a method for producing AAV nucleic acid-encapsulating hollow particles, comprising the following steps;
(a) (i) at least three AAV ITR A region-D' region sequences, or (ii) a sequence complementary to the A region-D' region (D region-A' region sequence),
preparing a nucleic acid comprising
(b) introducing the nucleic acid of (a) into cells that produce AAV hollow particles;
(c) culturing the cells of (b);

The nucleic acid of step (a) includes (i) at least three sequences of the A region-D' region of the AAV ITR, or (ii) a sequence complementary to the A region-D' region. Here, the “A region-D′ region sequence” means a continuous sequence from the A region to the D′ region of the AAV ITR or a sequence including the A region and the D′ region intermittently. Similarly, the "D region-A' region sequence" means a continuous sequence from the D region to the A' region of the AAV ITR or a sequence including the D region and the A' region intermittently. The A region-D' region sequence and the complementary sequence to this region (D region-A' region sequence) are known sequences of genomic DNA of natural AAV serotypes (e.g., serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11) can be used. The A region, A' region, D region and D' region may be derived from different serotypes. In addition, variants of the native A region sequence can also be used as long as they retain Rep binding activity and hairpin formation, where the A' region sequence is complementary to the variant A region sequence. The invention can also use variants of the native D region sequence, in which case the D' region sequence is complementary to the variant D region sequence.

A nucleic acid containing at least three A region-D' region sequences (hereinafter also referred to as "AD' sequences") in the nucleic acid of the step (a) is flanked by one AD' sequence and another AD' sequence. or may include a spacer sequence between one AD' sequence and another AD' sequence. The spacer sequence is 3-500 bp, preferably 5-200 bp, more preferably 7-100 bp base length.

The nucleic acid in step (a) can further contain a gene of interest. The gene of interest can be exogenous to AAV. The gene of interest can be placed at one or more sites on either the 5'-end side or the 3'-end side of the A region-D' region sequence.

The nucleic acid in step (a) is DNA or RNA, preferably DNA. It may be a double-stranded nucleic acid in which two complementary nucleic acid molecules are annealed, or one single-stranded nucleic acid molecule. Furthermore, it may be a circular nucleic acid or a linear nucleic acid. As one aspect of the present invention, the nucleic acid in step (a) is a circular double-stranded DNA, preferably a plasmid.

If the nucleic acid of step (a) is desired to replicate in a cell, the nucleic acid may further comprise an origin of replication that is functional in the host cell. Examples include prokaryotic origins of replication (ColE1 ori, f1 ori, etc.) and/or eukaryotic replication origins (SV40 ori, EBV ori, etc.). A nucleic acid having said origin of replication can be maintained episomally in a host cell.

As one aspect of the present invention, the nucleic acid fragment in step (1) and the nucleic acid in step (a) are sequences derived from AAV2, and the A' region sequence is SEQ ID NO: 8 and the A region sequence is SEQ ID NO: 9. Furthermore, the sequence of the D region is shown in SEQ ID NO: 7, and the sequence of the D' region is shown in SEQ ID NO: 10.

The nucleic acid fragment in step (1) and the nucleic acid in step (a) include natural nucleic acids, chemically modified nucleic acids, artificial nucleic acids, nucleic acid analogues, and combinations thereof. Natural nucleic acids are DNA and RNA in which only naturally occurring nucleotides are linked. Chemically modified nucleic acids are artificially chemically modified nucleic acids, such as methylphosphonate DNA/RNA, phosphorothioate DNA/RNA, phosphoramidate DNA/RNA, 2′-O-methyl DNA/ RNA and the like are exemplified. An artificial nucleic acid is a nucleic acid containing a non-natural nucleotide or a nucleic acid in which only a non-natural nucleotide is linked to a part of a natural nucleic acid. As used herein, the term “non-natural nucleotide” refers to an artificially constructed or artificially chemically modified nucleotide that does not exist in nature and has properties and/or structures similar to those of the natural nucleotide. Nucleic acid analogs are artificially constructed polymeric compounds that have similar structures and/or properties to naturally occurring nucleic acids. For example, peptide nucleic acid (PNA: Peptide Nucleic Acid), peptide nucleic acid having a phosphate group (PHONA), crosslinked nucleic acid (BNA/LNA: Bridged Nucleic Acid/Locked Nucleic Acid), morpholino nucleic acid (including morpholino oligo), etc. is exemplified.

The nucleic acid may be labeled with a phosphate group, sugar and/or base, if desired. A labeling substance known in the art can be used for labeling. For example, radioisotopes (e.g., 32P, 3H, 14C), DIG, biotin, fluorescent dyes (e.g., FITC, Texas, cy3, cy5, cy7, FAM, HEX, VIC, JOE, Rox, TET, Bodipy493, NBD, TAMRA), or luminescent materials (eg, acridinium esters).

As the nucleic acid fragment in step (1) and the nucleic acid in step (a), nucleic acids purified and extracted using known purification methods or commercially available products can be used. Examples of known purification methods include extraction with a phenol/chloroform mixed solution, alcohol precipitation, column purification, filter filtration, and agarose gel electrophoresis.

The size of the nucleic acid fragment and nucleic acid is not limited as long as it is a size that can be included in the AAV hollow particle, but is usually 5 kb or less. In addition, the nucleic acid fragment in the step (1) and the nucleic acid in the step (a) do not contain the sequence of the natural complete AAV genome, and lack the sequence of the AAV Rep gene and/or the AAV Cap gene. Preferably, the nucleic acid fragment and nucleic acid do not retain the sequence of the natural complete ITR, and the A region, A' region, and B region within the ranges described in steps (1) and (a) above. , B' region, C region, C' region, and D region. More preferably, the nucleic acid fragments and nucleic acids lack sequences in the B, B', C and C' regions.

(B) Step of introducing nucleic acid into cells The nucleic acid fragment of step (1) or the nucleic acid of step (a) is introduced into a cell that produces AAV hollow particles. Various eukaryotic cells such as insect cells and mammalian cells including mouse cells and primate cells (eg, human cells) can be used as cells into which nucleic acid fragments or nucleic acids are introduced. In the present specification, cells that produce AAV or nucleic acid-encapsulating hollow particles are sometimes referred to as packaging cells or producer cells. Suitable mammalian cells include, but are not limited to primary cells and cell lines, suitable cell lines include HEK293 cells, 293EB cells, COS cells, HeLa cells, Vero cells, 3T3 mouse fibroblasts, Examples include C3H10T1/2 fibroblasts, CHO cells, and cells derived from these cells. Suitable insect cells include, but are not limited to primary cells and cell lines, suitable cell lines include Sf9 cells and cells derived from these cells.

Cells that express the AAV rep and cap gene products are used as hollow particle-producing cells. These gene products may be encoded by AAV rep and cap genes stably integrated into the genome of the cell and introduced into the cell before, simultaneously with, or after introduction of said nucleic acid fragment or nucleic acid. may be encoded by a vector that When the rep gene and the cap gene are encoded by a vector, the rep gene and the cap gene may be encoded by the same vector, or may be encoded by separate vectors. The rep and cap genes can be sequences of AAV of any serotype (eg, serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11). Furthermore, the rep gene and the cap gene may be sequences derived from the same serotype or may be sequences derived from different serotypes.

As vectors encoding the rep gene and cap gene, expression vectors suitable for the cells to be introduced can be used as long as they can produce hollow particles, and examples thereof include viral vectors, plasmid vectors, cosmid vectors, and artificial chromosomes. . A plasmid vector that can be maintained episomally can be used as needed. Furthermore, the vector may be placed under the control of a promoter and terminator in a suitable expression system so that the rep and cap genes can be expressed within the cell. A nucleic acid expression system is a system that has at least one set of expression control elements required for expression of a gene in an operable state. Expression control elements include promoters and terminators, and optionally enhancers and poly-A addition signals.

Further helper functions can be introduced into the cells that produce the hollow particles. Helper functions are also called helper virus functions and accessory functions. Adenoviruses are commonly used to introduce helper functions, but viruses such as herpes simplex virus type 1 or type 2, vaccinia virus, etc. can also be used. When using a virus, the cells are infected with the virus as a helper virus. For example, an adenovirus that does not exhibit late gene expression may be used, since only expression of the adenoviral early genes is required for AAV particle packaging. Adenovirus mutants deficient in late gene expression (eg, ts100K or ts149 adenovirus mutants) can be used. Alternatively, the nucleic acid necessary for helper virus function isolated from the helper virus can be used to generate and introduce into cells a nucleic acid construct that provides the helper virus function. Constructs providing helper virus functions comprise one or more nucleotide sequences providing helper virus functions, including the E1 gene region, the E2A gene region, the E4orf6 gene and the gene encoding the VA RNA, and may be plasmids, phages, transposons. , cosmid or other viral form to the host cell.

Methods for introducing the nucleic acid fragment of step (1) and the nucleic acid of step (a) into cells include, for example, the calcium phosphate method, the lipofection method, the DEAE dextran method, the polyethyleneimine method, the electroporation method, and the direct microinjection method. , high-velocity microparticle bombardment, etc. can be used. In addition, commercially available reagents such as TransIT (registered trademark)-293 Reagent, TransIT (registered trademark)-2020 (manufactured by Mirus), Lipofectamine 2000 Reagent, Lipofectamine 2000CD Reagent (manufactured by Life Technologies), FuGene (registered trademark) ) Transfection Reagent (manufactured by Promega), PEI max (manufactured by Cosmo Bio) and the like may be used.

1 ng to 10 μg, preferably 5 ng to 1 μg, more preferably 10 ng to 500 ng of the nucleic acid fragment in step (1) is introduced per 10 ⁶ cells. 100 ng to 1000 μg, preferably 1 μg to 100 μg, more preferably 5 μg to 50 μg of the nucleic acid in step (a) is introduced per 10 ⁶ cells.

(C) Step of Culturing Cells Cells into which the nucleic acid fragment or nucleic acid has been introduced in (B) above can be cultured under known culture conditions depending on the cell. For example, culture at a temperature of 30-37° C., a humidity of 95% and a CO ₂ concentration of 5-10% is exemplified, but the present invention is not limited to such conditions. Temperatures, humidity, and _CO2 concentrations outside the above ranges may be used as long as desired cell growth and production of nucleic acid-encapsulating hollow particles can be achieved.

Known media can be used as the culture medium, such as DMEM, IMDM, Ham F12, RPMI-1640, etc., which are commercially available from Lonza, Life Technologies, Sigma-Aldrich, etc. can be obtained as The medium may be a serum-free medium or a medium supplemented with fetal bovine serum (FBS), human serum-derived albumin, or the like.

As culture equipment, cell culture equipment (vessels) such as petri dishes, flasks, bags, large-sized culture tanks and bioreactors can be used. As the bag, a CO ₂ gas permeable bag for cell culture is suitable. A large culture vessel may be used when a large amount of cells is required.

The culture period is not particularly limited, and is preferably 12 hours to 10 days, preferably 24 hours to 7 days. Nucleic acid-encapsulating hollow particles, ie, AAV-like particles that retain nucleic acid inside, are produced during culture in the cells and/or in the culture supernatant.

Furthermore, in the present invention, the step of obtaining nucleic acid-encapsulated hollow particles from the centrifugal supernatant of cell culture supernatant or collected cells resuspended in an appropriate buffer and disrupted (cell lysate). can be implemented. In the present invention, the culture supernatant and cell lysate supernatant thus obtained are used as they are, or further, for example, concentration and purification of hollow particles by known methods such as filter filtration, CsCl density gradient centrifugation, and commercially available products. After practice, it can be stored in a suitable manner, eg, frozen, until used for the desired application.

(D) Hollow Particle Encapsulating Nucleic Acid and Composition The present invention provides hollow particles encapsulating the nucleic acid fragment of the step (1) or the nucleic acid of the step (a). The hollow particles of the present invention do not carry the native complete AAV genome and do not carry nucleic acid containing the sequences of the AAV Rep gene and/or the AAV Cap gene. Preferably, the hollow particles of the present invention furthermore do not retain the natural complete ITRs, and the group consisting of A region, A' region, B region, B' region, C region, C' region, D region. It does not retain the sequence of at least one region more selected. Furthermore, the present invention provides a composition comprising the nucleic acid-encapsulating hollow particle. The composition of the present invention is characterized by containing at least one nucleic acid-encapsulating hollow particle obtained by the method of the present invention.

The composition of the present invention may contain nucleic acid-encapsulating hollow particles as an active ingredient, as well as carriers and/or other agents. Two or more different nucleic acid-encapsulating hollow particles may be included in the composition. In this case, two or more different nucleic acid-encapsulating hollow particles may be derived from viruses with different capsids, etc., and/or may be of different types in the encapsulated nucleic acids. For example, it may contain a plurality of nucleic acid-encapsulating hollow particles with different target cells. The carrier is a substance that is added to the extent that the formulation of the composition and application to the living body is facilitated and the action thereof is not inhibited or inhibited. Flow additive modifiers or lubricants include, but are not limited to. Preferably, a pharmaceutically acceptable carrier is used in the compositions of the invention.

The content of nucleic acid-encapsulating hollow particles in the composition of the present invention is not particularly limited. Type and/or effective amount of nucleic acid contained in the particles, applicable cells or individuals, method/route of application, purpose of application, form of composition (including form and size), and carrier It is determined as appropriate in consideration of the type of

Genes can be introduced into cells and animal individuals by using the nucleic acid-encapsulated hollow particles of the present invention and compositions containing the hollow particles. Such a gene introduction method is also an aspect of the present invention. Gene transfer into cells can be performed in vitro by contacting the hollow particles with the cells. Gene transfer into individual animals including humans can be achieved by administering the nucleic acid-encapsulated hollow particles of the present invention, preferably an appropriate composition containing the hollow particles, by intratissue (e.g., intramuscular), intravenous, subcutaneous, or intraperitoneal administration. It is carried out by administering by the route of

EXAMPLES The present invention will be specifically described below with reference to Examples, but the scope of the present invention is not limited to these Examples.

Example 1 Packaging of Plasmid DNA for AAV Hollow Particles (1) Construction of AD'-Loading Plasmid The AD' sequence present in the ITR of AAV2 genomic DNA (61 bp spanning A region-D' region: SEQ ID NO: 1), A double-stranded DNA having recognition sequences for the restriction enzyme AflIII at both ends was prepared by annealing both chemically synthesized strands. This double-stranded DNA is pAcGFP1-N1 Vector (manufactured by Clontech, Takara Bio USA) at the AflIII site upstream of the CMV promoter, and a plasmid AD-F in which the AD' sequence is inserted in the 5' to 3' direction, A reverse-inserted plasmid AD-R was prepared. In addition, a plasmid AD-3F was prepared in which the three AD' sequences were inserted in the order. As a negative control, a plasmid AD(-) containing no artificial gene was prepared. Furthermore, as positive controls, plasmids ss-AAV and plasmid ds-AAV that generate AAV genomes carrying the AcGFP gene were prepared. The plasmid ss-AAV has the AcGFP gene inserted into the multicloning site of the pAAV-MSC vector of AAV Helper-Free System (manufactured by Agilent Technologies). Plasmid ds-AAV replaces the eGFP gene of plasmid pdsAAV-CB-eGFP provided by Dr. Arun Srivastava with AcGFP. A schematic diagram of each plasmid is shown in FIG. In FIG. 1, the AD' sequence is described as "AD".

(2) Plasmid introduction into 293EB cells Each plasmid obtained in Example 1-(1) was adjusted to 1 μg/μL, and pAAV2/9 Vector (SEQ ID NO: 11) and pAd5N expressing AAV9 Cap and AAV2 Rep (Agilent Technologies) and Polyethyleneimine "Max" (manufactured by Cosmo Bio) were used to transfect 293EB cells (International Publication No. 2012/144446 pamphlet). After transfection, 293EB cells were cultured in DMEM medium containing 1/100 volume of GlutaMax (Gibco) at 37°C, 5% _CO2 for 3 days.

(3) Collection of Nucleic Acid-Encapsulating AAV Hollow Particles Nucleic acid-encapsulating AAV hollow particles (AAV-like particles) were purified from the culture supernatant of the 293EB cells cultured in Example 1-(2) by cesium chloride density gradient centrifugation.

2 μL of the purified AAV-like particle solution thus obtained, 116 μL of dH ₂ O, 15 μL of 50 mM MgCl ₂ , 15 μL of 0.1% Triton X-100 and 2 μL of 250 U/μL Benzonase were mixed and incubated at 37° C. for 30 minutes to obtain free Genomic DNA and plasmid DNA were degraded. Next, 100 µL of AL buffer (manufactured by Qiagen) was added to dissolve the AAV capsid, and the DNA packaged in the hollow particles was extracted. Using this DNA as a template, AcGFP-f primer (SEQ ID NO: 2) and AcGFP-r primer (SEQ ID NO: 3), and SYBR (registered trademark) Premix DimerEraser (trademark) (Perfect Real Time) (manufactured by Takara Bio Inc.) were used. Then, according to the instructions attached to the kit, the vector copy number (vector genome: vg/μL) contained in the AAV hollow particles was measured.

(4) Introduction into target cells CHO-K1 cells were infected with each AAV-like particle obtained from the culture supernatant in Example 1-(3) as follows. On the day before infection, 2.8×10 ⁴ cells of CHO-K1 cells were seeded on a 48-well plate. On the next day, each AAV-like particle obtained in Example 1-(3) was added to 4.0×10 ⁵ v. g. /cell. After infection, CHO-K1 cells were incubated at 37° C., 5% CO ₂ for 3 days, and then AcGFP expression was observed under a fluorescence microscope (FIG. 2). As shown in FIG. 2, the plasmid loaded with the reversed AD' sequences and the plasmid loaded with the three AD' sequences in the forward direction were more effectively hollowed out than the plasmid loaded with the forward AD' sequences. Incorporation into particles and expression of AcGFP in infected cells was confirmed. It was shown that AAV-like particles containing the AcGFP gene were produced from cells transfected with a plasmid containing the AD' sequence.

Example 2 Packaging of AAV Genome by Introduction of DNA Fragment (1) Preparation of DNA Fragment Containing AD Sequence Oligonucleotide Full described in SEQ ID NO: 4, Oligonucleotide Plus described in SEQ ID NO: 5, and SEQ ID NO: 6 A double-stranded DNA was prepared by chemically synthesizing an oligonucleotide Minus, chemically synthesizing each complementary strand, and annealing each of them. FIG. 3 shows the structure of each double-stranded DNA. AAV ITR A region sequence, D' region sequence, A' region sequence (complementary sequence to A region), and D region sequence (complementary sequence to D' region) are A and D, respectively. They are referred to as 'arrangement, A' arrangement and D arrangement, and are indicated as A, D(+), A' and D(-) in FIG. 3, respectively. As shown in FIG. 3, the oligonucleotide Full consists of a D sequence, an A' sequence, a loop sequence, an A sequence, and a D' sequence in order from the 5' end. Oligonucleotide Plus consists of an A' sequence, a loop sequence, an A sequence and a D' sequence in order from the 5' end. Oligonucleotide Minus consists of D sequence, A' sequence, loop sequence and A sequence in order from the 5' end. The D sequence is shown in SEQ ID NO: 7, the A' sequence in SEQ ID NO: 8, the A sequence in SEQ ID NO: 9, and the D' sequence in SEQ ID NO: 10, respectively.

Each of the DNA fragments was ligated to the pCR-Blunt vector of the Zero Blunt Cloning Kit (manufactured by ThermoFisher), and plasmid DNA was prepared according to the instructions. Using these plasmid DNAs as templates, PCR was performed using M13 Forward (-20) primer and M13 Reverse primer (manufactured by ThermoFisher) to amplify DNA fragment Full, DNA fragment Plus and DNA fragment Minus. As a negative control, PCR was performed using a pCR-Blunt vector containing an intervening sequence as a template to amplify a DNA fragment Mock.

The PCR reaction solution was subjected to 1.5% agarose gel electrophoresis, and the DNA fragment was extracted from the gel after electrophoresis and purified. Two bands were generated from the DNA fragment Full and extracted separately as DNA fragment Full-H and DNA fragment Full-M.

(2) Introduction into 293EB cells 75 ng of each DNA fragment obtained in Example 2-(1) was transfected into 293EB cells together with pAAV2/9 Vector and pAd5N (pHhelper Vector) using Polyethyleneimine "Max". Two days after transfection, 293EB cells were changed to DMEM/F12 (ThermoFisher) medium containing glucose, sodium bicarbonate and 1/100 volume of GlutaMax. It was then cultured at 37°C, 5% _CO2 for 5 days.

(3) Collection of AAV-like Particles After collecting the culture supernatant of the 293EB cells cultured in Example 2-(2), DNase I was added to the supernatant to degrade free DNA. After digestion, AL buffer (manufactured by Qiagen) was added in the same manner as above to dissolve the AAV capsid, and the packaged DNA was extracted. The DNA fragment copy number contained in the AAV-like particles was analyzed by quantitative PCR using a 3'sense primer (SEQ ID NO: 12) and a 3'antisense primer (SEQ ID NO: 13) (Fig. 4). As shown in FIG. 4, it was shown that the introduction of a DNA fragment containing an AAV ITR-derived sequence resulted in packaging of the DNA fragment into AAV hollow particles. Such DNA fragments can be prepared in large amounts by PCR, and AAV-like particles can be prepared more conveniently.

The present invention provides a simpler and more efficient method of encapsulating nucleic acids into hollow particles. Nucleic acid-encapsulating hollow particles produced by the method of the present invention and compositions containing the nucleic acid-encapsulating hollow particles as active ingredients are useful as gene transfer methods in the fields of gene therapy research and clinical practice.

SEQ ID NO: 1: AD' region sequence
SEQ ID NO: 2: AcGFP-f
SEQ ID NO: 3: AcGFP-r
SEQ ID NO: 4: Oligonucleotide Full
SEQ ID NO: 5: Oligonucleotide Plus
SEQ ID NO: 6: Oligonucleotide Minus
SEQ ID NO: 7: D region sequence
SEQ ID NO: 8: A' region sequence
SEQ ID NO: 9: A region sequence
SEQ ID NO: 10: D' region sequence
SEQ ID NO: 11: pAAV2/9 Vector
SEQ ID NO: 12: 3' sense primer
SEQ ID NO: 13: 3'antisense primer

Claims (5)

A method for producing an adeno-associated virus (AAV) nucleic acid-encapsulating hollow particle comprising the following steps;
(1) (i) the sequence of the A region of the AAV inverted terminal repeat (ITR);
(ii) a sequence of the A' region of the AAV ITR and (iii) a sequence of the D region and/or the sequence of the D' region of the AAV ITR. The nucleic acid fragment lacks the sequences of the B region, B' region, C region and C' region of the AAV ITR, and from the 5' end to the 3' end, the sequence of the A' region, the sequence of the A region and the sequence of the D' region ,
(2) the step of introducing the nucleic acid fragment of (1) into cells that produce AAV hollow particles; and (3) the step of culturing the cells of (2). A method for producing an adeno-associated virus (AAV) nucleic acid-encapsulating hollow particle comprising the following steps;
(1) (i) the sequence of the A region of the AAV inverted terminal repeat (ITR);
(ii) the sequence of the A' region of the AAV ITR, and
(iii) preparing a linear nucleic acid fragment comprising an AAV ITR D region sequence and/or a D′ region sequence, wherein the linear nucleic acid fragment comprises the AAV ITR B region, B′ lacking the sequence of the region, the C region and the C' region, and comprising the sequence of the D region, the sequence of the A' region and the sequence of the A region from the 5' end to the 3' end,
(2) introducing the nucleic acid fragment of (1) into a cell that produces AAV hollow particles, and
(3) A step of culturing the cells of (2) . A method for producing an adeno-associated virus (AAV) nucleic acid-encapsulating hollow particle comprising the following steps;
(1) (i) the sequence of the A region of the AAV inverted terminal repeat (ITR);
(ii) the sequence of the A' region of the AAV ITR, and
(iii) preparing a linear nucleic acid fragment comprising an AAV ITR D region sequence and/or a D′ region sequence, wherein the linear nucleic acid fragment comprises the AAV ITR B region, B′ region, C region and C' region sequences are deleted, and contains the D region sequence, the A' region sequence, the A region sequence and the D' region sequence from the 5' end to the 3' end ,
(2) introducing the nucleic acid fragment of (1) into a cell that produces AAV hollow particles, and
(3) A step of culturing the cells of (2) . 4. The method according to any one of claims 1 to 3, wherein the step of preparing the nucleic acid fragment comprises amplification of the nucleic acid fragment by a nucleic acid amplification reaction. The method according to any one of claims 1 to 4, wherein the hollow particle-producing cells are cells into which the AAV Cap gene, Rep gene and AAV helper function have been introduced.

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