Nucleic acid transfer carrier, nucleic acid transfer carrier set, nucleic acid transfer composition, and nucleic acid transfer method

Abstract

To provide carriers, carrier sets, compositions and methods for introducing nucleic acids that can introduce target nucleic acids into cellular genomes more efficiently and more safely.SOLUTION: A carrier for introducing a nucleic acid according to an embodiment is used to introduce a first sequence into a cellular genome. The carrier comprises a donor DNA containing the first sequence, an RNA agent containing at least an RNA encoding a protein participating to the introduction of the first sequence into the genome, and a lipid particle encapsulating the donor DNA and the RNA agent.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] Embodiments of the present invention relate to a nucleic acid transfer carrier, a nucleic acid transfer carrier set, a nucleic acid transfer composition, and a nucleic acid transfer method. [Background technology]

[0002] In recent years, many functional proteins useful in genetic engineering have been discovered, such as CRISPR-associated protein 9 (Cas9), which cuts DNA site-specifically, and transposase, which cuts out target DNA and inserts it into the genome of a cell. In order to utilize such functional proteins in genetic engineering, it is desirable to develop a technology that can introduce and express functional proteins more efficiently into cells.

[0003] For example, a method is used in which DNA (e.g., a vector) that codes for a functional protein is introduced into a cell to express the functional protein in the cell. However, it is difficult for DNA alone to pass through the cell membrane and enter the cell. One method for introducing DNA into a cell is to use lipofectamine. Lipofectamine binds to nucleic acids to form a complex, making it easier to introduce the nucleic acid into the cell. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Lingmin et al. NPG Asia Materials (2017) 9, e441 [Non-Patent Document 2] Zeming Chen et al. Adv. Funct. Mater. 2017, 27, 1703036 [Non-Patent Document 3] Shai Zhen et al. Oncotarget, 2017, Vol. 8, (No. 6), pp: 9375-9387 Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to provide a nucleic acid transfer carrier, a nucleic acid transfer carrier set, a nucleic acid transfer composition, and a nucleic acid transfer method that can transfer a target DNA sequence into the genome of a cell more efficiently and safely. [Means for solving the problem]

[0006] The nucleic acid transfer carrier according to the embodiment is used for transferring a first sequence into the genome of a cell. The nucleic acid transfer carrier includes a donor DNA including the first sequence, an RNA agent including at least an RNA encoding a protein involved in the transfer of the first sequence into the genome, and a lipid particle encapsulating the donor DNA and the RNA agent. [Brief description of the drawings]

[0007] [Figure 1] FIG. 1 is a cross-sectional view showing an example of a nucleic acid introduction carrier according to an embodiment. [Diagram 2] FIG. 2 is a flow chart showing an example of a nucleic acid introduction method according to an embodiment. [Diagram 3] FIG. 3 is a cross-sectional view showing an example of a nucleic acid introduction carrier according to an embodiment. [Figure 4] FIG. 4 is a cross-sectional view showing an example of a nucleic acid introduction carrier set according to an embodiment. [Diagram 5] FIG. 5 is a graph showing the experimental results of Example 1. [Figure 6] FIG. 6 is a photomicrograph showing the experimental results of Example 1. [Figure 7] FIG. 7 is a histogram showing the experimental results of Example 2. [Figure 8] FIG. 8 is a histogram showing the experimental results of Example 3. [Figure 9] FIG. 9 is a graph showing the experimental results of Example 4. [Figure 10] FIG. 10 is a histogram showing the experimental results of Example 4. [Figure 11]FIG. 11 is a graph showing the experimental results of Example 5. [Figure 12] FIG. 12 is an electrophoretic photograph showing the experimental results of Example 6. [Figure 13] FIG. 13 is a photomicrograph showing the experimental results of Example 6. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] Various embodiments will be described below with reference to the drawings. Each figure is a schematic diagram for facilitating understanding of the embodiment, and the shapes, dimensions, ratios, etc. may differ from the actual ones, but these can be appropriately modified in design by taking into account the following explanation and known techniques.

[0009] The nucleic acid transfer carrier according to the embodiment includes a donor DNA including a first sequence, an RNA agent including at least an RNA encoding a protein involved in the transfer of the first sequence into a genome, and a lipid particle encapsulating the donor DNA and the RNA agent. The nucleic acid transfer carrier is used to transfer the first sequence into the genome of a cell. In addition, according to the embodiment, a nucleic acid transfer carrier set in which the donor DNA and the RNA agent are encapsulated in separate lipid particles, a nucleic acid transfer composition including the nucleic acid transfer carrier or the nucleic acid transfer carrier set, and a nucleic acid transfer method using the nucleic acid transfer carrier or the nucleic acid transfer carrier set are also provided. The nucleic acid transfer carrier, the nucleic acid transfer carrier set, the nucleic acid transfer composition, and the nucleic acid transfer method are described in detail below.

[0010] (First embodiment) Nucleic acid delivery carrier FIG. 1 is a cross-sectional view showing an example of a nucleic acid transfer carrier of the first embodiment. The nucleic acid transfer carrier 1 includes a donor DNA 2, an RNA agent 3, and a lipid particle 4 encapsulating the donor DNA 2 and the RNA agent 3. The donor DNA 2 includes a first sequence 5 to be introduced into the genome of a cell. The RNA agent 3 includes an RNA 3a and a guide RNA 3b. The RNA 3a is an RNA that encodes a protein involved in the introduction of the first sequence 5 into the genome. The guide RNA 3b is an RNA that includes a sequence corresponding to a sequence on the genome into which the first sequence 5 is introduced (hereinafter referred to as the "second sequence"). The donor DNA 2 and the RNA agent 3 are encapsulated in a state condensed with a nucleic acid condensing peptide 6, for example. The lipid particle 4 is made of a lipid membrane formed by arranging a plurality of lipid molecules 4a with no gaps by non-covalent bonds, and is an approximately spherical hollow body, and the donor DNA 2 and the RNA agent 3 are encapsulated in the central lumen 4b.

[0011] Each component will be described in detail below.

[0012] The donor DNA 2 is, for example, a double-stranded linear DNA. The donor DNA 2 may be a single-stranded DNA or a circular DNA. The length of the donor DNA 2 is, for example, 3 to about 20,000 bases.

[0013] The first sequence 5 contained in the donor DNA 2 is a sequence to be introduced into the genome of a cell, and is, for example, a gene expression cassette including a promoter sequence, a specific gene, and a transcription termination sequence, a base sequence encoding a specific gene or a part of a gene, or a natural or non-natural base sequence that is not a gene. Alternatively, it may be a base sequence encoding one to several amino acids or a sequence consisting of three to several tens of nucleotides. The length of the first sequence is, for example, 3 to about 20,000 bases.

[0014] The donor DNA 2 may contain, for example, a sequence other than the first sequence 5. Such a sequence is a recognition sequence for a protein encoded by the RNA 3a, or a recognition sequence for the guide RNA 3b.

[0015] The configuration of the donor DNA 2, i.e., the type of the first sequence 5, the types and base lengths of other sequences, etc., will be described in detail later, and are selected depending on the application of the nucleic acid transfer carrier 1. It is preferable that the nucleic acid transfer carrier 1 contains 1 to 100 molecules of the donor DNA 2.

[0016] RNA3a is RNA encoding a protein involved in the introduction of the first sequence 5 into the genome. The protein is, for example, an enzyme that has an activity of cutting, binding, inserting and / or repairing DNA, and is involved in the introduction of a DNA sequence into the genome by this activity. Hereinafter, such a protein is also simply referred to as "enzyme". The enzyme is preferably, for example, an enzyme having endonuclease activity, transposase, reverse transcriptase and intecrase (enzymes for retroviral retrotransposons), reverse transcriptase and endonuclease (enzymes for non-retroviral retrotransposons), etc.

[0017] Examples of enzymes having endonuclease activity include CRISPR-Associated Protein 9 (Cas9), zinc finger nucleases (ZFNs), transcription activation effector nucleases (TALENs), meganucleases, etc. As will be described in detail later, endonucleases are involved in the introduction of the first sequence 5 into the genome by cleaving a phosphodiester bond at the position on the genome where the first sequence 5 is to be introduced.

[0018] Examples of transposase include PiggyBac, SleepingBeauty, Frog Prince, Hsma, Minos, Tol1, Tol2, Passport, hAT, Ac / Ds, PIF, Harbinger, Harbinger3-DR, Himar1, Hermes, Tc3, and Mos1. Transposase has an activity of excising a sequence including the first sequence 5 from the donor DNA 2 and introducing it into the genome, thereby participating in the introduction of the first sequence 5 into the genome.

[0019] RNA3a may be, for example, mRNA of the gene of the enzyme. RNA3a may have an additional sequence in addition to the sequence encoding the gene of the enzyme. The additional sequence may be, for example, a 5'-end leader sequence, an IRES (Internal Ribosome Entry Site), a transcription termination sequence, or a Poly(A) sequence. RNA3a may have a CAP structure.

[0020] The length of the RNA 3a is, for example, about 20 to about 5000 bases. It is preferable that 1 to about 1000 molecules of the RNA 3a are contained in the nucleic acid introduction carrier 1. The RNA 3a may contain a plurality of RNAs each encoding a plurality of types of enzymes.

[0021] Guide RNA 3b is RNA having a base sequence corresponding to the second sequence or its complementary sequence. The second sequence is, for example, a 15-25 base sequence around the position where the first sequence 5 is introduced on the genome of the cell. Since the second sequence is DNA and guide RNA 3b is RNA, the "corresponding base sequence" refers to a homologous base sequence or its complementary sequence, except that T (thymine) of the second sequence is U (uridine) in guide RNA 3b.

[0022] Guide RNA3b is preferably used when RNA3a is an RNA encoding Cas9. In that case, guide RNA3b may be a guide RNA in a CRISPR-Cas9 system that can be designed by a person skilled in the art based on the second sequence according to common knowledge. In this case, guide RNA3b may be an RNA in which a 3'-end crRNA containing a PAM sequence is linked to the 3'-end of the second sequence, or an RNA (sgRNA) in which a 3'-end crRNA containing a PAM sequence and a sequence consisting of a part of tracrRNA are linked to the 3'-end of the second sequence. The length of such guide RNA3b is, for example, about 40 to about 150 bases.

[0023] Guide RNA3b forms a complex with the endonuclease expressed from RNA3a and serves to guide the endonuclease to the second sequence. Therefore, the first sequence 5 can be introduced site-specifically by using guide RNA3b. When it is not necessary to introduce the first sequence 5 site-specifically or when Cas9 is not used as the enzyme, guide RNA3b does not need to be used.

[0024] The nucleic acid transfer carrier 1 preferably contains 1 to about 1000 molecules of guide RNA 3b.

[0025] The RNA agent 3 may contain further RNA. The further RNA is, for example, an RNA having a function of modifying DNA, such as methylating, demethylating, repairing and / or binding to DNA. For example, these RNAs may be RNAs encoding proteins having the above-mentioned modification activity. By including these RNAs, it is possible to add the above-mentioned modification to the first sequence 5 introduced into the genome and its surrounding sequences. This allows, for example, further functional modification to be added to the cell.

[0026] The RNA contained in the RNA agent 3 is preferably modified to be resistant to degradation. For example, the modification may be any known modification that prevents the RNA from being degraded by RNase present inside or outside the cell. Such modifications include, for example, the use / introduction of naturally occurring modified nucleotides or non-naturally occurring nucleotides into the RNA, the use / addition of non-naturally occurring sequences, or the addition of natural / non-naturally occurring CAP structures.

[0027] Examples of natural modified nucleotides include pseudouridine, 5-methylcytidine, 1-methyladenosine, etc. Examples of non-natural nucleotides include bridged nucleotides (BNAs), locked nucleotides (LNAs), and peptide nucleotides (PNAs).

[0028] The non-natural sequence is, for example, an artificially created base sequence that does not exist in nature, such as a random base sequence, or a hybrid sequence of natural / non-natural amino acids and nucleic acids, etc. The non-natural sequence is preferably added, for example, to the end of RNA.

[0029] Examples of natural CAP structures include CAP0 (m7GpppN), CAP1 (m7GpppNm), etc. Examples of non-natural CAP structures include ARCA (Anti-Reverse Cap Analog) or LNA-guanosine, etc. It is preferable that the non-natural CAP structure is added to the 5' end of RNA, for example.

[0030] By using RNA modified as described above, degradation of RNA by RNase present inside and outside the cell can be prevented, and as a result, the efficiency of introduction of the first sequence 5 can be further increased.

[0031] The nucleic acid condensing peptide 6 is a peptide for condensing a larger amount of nucleic acid into a smaller amount, and efficiently encapsulating a large amount of nucleic acid within the lipid particle 4. As such a peptide, for example, a peptide having cationic properties is preferably used. For example, a cationic peptide can condense nucleic acid by entering into gaps in the helical shape of anionic nucleic acid and narrowing the gaps.

[0032] A preferred nucleic acid-condensing peptide 6 is, for example, a peptide containing 45% or more cationic amino acids in total. A more preferred nucleic acid-condensing peptide 6 has RRRRRR (first amino acid sequence) at one end and the sequence RQRQR (second amino acid sequence) at the other end. Between both amino acid sequences, there is contained 0 or 1 or more intermediate sequences consisting of RRRRRR or RQRQR. In addition, there are contained 2 or more neutral amino acids between two adjacent sequences of the first amino acid sequence, the second amino acid sequence, and the intermediate sequence. The neutral amino acid is, for example, G or Y.

[0033] The nucleic acid-condensing peptide 6 preferably has the following amino acid sequence: RQRQRYYRQRQRGGRRRRRR (SEQ ID NO:1) RQRQRGGRRRRRR (sequence number 2).

[0034] Such a nucleic acid-condensing peptide can efficiently condense nucleic acids due to the cationic property of R and can weaken the anionic property of the nucleic acid, making it possible to efficiently encapsulate nucleic acids within lipid particles 4. Furthermore, since the nucleic acid-condensing peptide efficiently dissociates nucleic acids within cells, it is possible to efficiently express nucleic acids introduced into cells within the cells.

[0035] Alternatively, the nucleic acid-condensing peptide 6 has RRRRRR (third amino acid sequence) at one end and RRRRRR (fourth amino acid sequence) at the other end. Between the two amino acid sequences, there is contained zero or one or more intermediate sequences consisting of RRRRRR or RQRQR. In addition, there are contained two or more neutral amino acids between two adjacent sequences of the third amino acid sequence, the fourth amino acid sequence, and the intermediate sequence. The neutral amino acid is, for example, G or Y.

[0036] Such a nucleic acid-condensing peptide 6 preferably has the following amino acid sequence: RRRRRRYYRQRQRGGRRRRRR (sequence number 3).

[0037] Such a nucleic acid-condensing peptide 6 has strong cationicity at both ends and has high binding affinity with nucleic acids. Therefore, it is possible to more efficiently condense nucleic acids and encapsulate more nucleic acids within the lipid particles 4. As a result, the amount of nucleic acid remaining outside the lipid particles 4 is reduced, which prevents aggregation of nucleic acid delivery carriers, making it easier for the nucleic acid delivery carriers to be taken up into cells.

[0038] Furthermore, nucleic acid-condensing peptide 6 having the following amino acid sequence can also be used in combination with any of the nucleic acid-condensing peptides described above. GNQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (M9) (sequence number 4).

[0039] This peptide can further condense the nucleic acid aggregates condensed by the nucleic acid-condensing peptide 6. Therefore, it is possible to obtain a nucleic acid delivery carrier with a smaller particle size. Such a nucleic acid delivery carrier is easily taken up into cells, making it possible to more efficiently deliver nucleic acid to the genome of cells.

[0040] For example, donor DNA 2 and RNA agent 3 can be condensed by stirring and mixing them with nucleic acid-condensing peptide 6 prior to encapsulation in lipid particle 4. Donor DNA 2 and RNA agent 3 may be condensed together or separately.

[0041] Use of the nucleic acid-condensing peptide 6 is preferable because it provides the effects described above; however, depending on the types of donor DNA 2 and RNA agent 3 used, or the type of cell to be transfected, the nucleic acid-condensing peptide 6 may not be used.

[0042] The lipid particle 4 may be a lipid monolayer or a lipid bilayer. In addition, the lipid particle 4 may be made of a single membrane or a multilayer membrane.

[0043] As the material for the lipid particles 4, for example, lipids, which are the main components of biological membranes, can be used. Examples of such lipids include diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebroside. Among these, diacylphosphatidylcholine and diacylphosphatidylethanolamine are preferred because they allow easy control of the structure and particle size of the lipid particles 4 and can also be imparted with membrane fusion ability. The length of the hydrocarbon chain of the acyl group contained in the lipid is C 10 ~C 20 The hydrocarbon chain may be a saturated or unsaturated hydrocarbon group.

[0044] For example, lipids include 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-stearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1,2-di-O-octadecyl-3-trimethylammonium propane (DOTMA), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), 1,2-Dimyristoyl-3-dimethylammonium propane (14:0 DAP), 1,2-dipalmitoyl-3-dimethylammonium propane (16:0 DAP), 1,2-distearoyl-3-dimethylammonium propane (18:0 DAP), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propane (DOBAQ), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), 1,2-dioleoyl-sn-glycero-3-phosphochorin (DOPC), 1,2-dilinoleoyl-sn-glycero-3-phosphochorin (DLPC), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), or cholesterol It is preferable to use the above. These have the function of forming the lipid particles 4, and are also highly effective in fusing with the cell membrane when the nucleic acid transfer carrier is transferred into the cell.

[0045] The lipid particles 4 may be composed of a single lipid, but are preferably a lipid mixture composed of multiple types of lipids. The type of lipid used in the lipid particles 4 is appropriately selected in consideration of the size of the lipid particles 4, the type of encapsulation, the stability in the cells to be introduced, and the like.

[0046] In addition to the above lipids, the lipid particles 4 preferably further contain a first biodegradable lipid compound. The first biodegradable lipid compound can be represented by the formula Q-CHR2. (In the formula, Q is a nitrogen-containing aliphatic group containing two or more tertiary nitrogen atoms and no oxygen; R is independently C 12 ~C 24 is an aliphatic group of At least one R contains in its main chain or in a side chain a linking group L-R selected from the group consisting of -C(=O)-O-, -OC(=O)-, -OC(=O)-O-, -SC(=O)-, -C(=O)-S-, -C(=O)-NH-, and -NHC(=O)-.

[0047] When the lipid particle 4 contains the first biodegradable lipid compound, the surface of the lipid particle 4 becomes non-cationic, which reduces the hindrance to cell introduction and increases the rate of introduction of the nucleic acid, thereby enabling the first sequence 5 to be introduced into the genome of the cell more efficiently.

[0048] For example, it is preferable to use a lipid having a structure represented by the following formula as the first biodegradable lipid compound, since this leads to a superior amount of nucleic acid encapsulated therein and a superior efficiency of nucleic acid transfer. [ka] [ka] [ka]

[0049] For example, the lipid particle 4 preferably further contains a second biodegradable lipid compound. The second biodegradable lipid compound can be represented by the formula P-[XWY-W'-Z]2. (In the formula, P is an alkyleneoxy containing one or more ether bonds in the main chain; Each X is independently a divalent linking group containing a tertiary amine structure, Each W is independently a C1-C6 alkylene; each Y is independently a divalent linking group selected from the group consisting of a single bond, an ether bond, a carboxylate bond, a thiocarboxylate bond, a thioester bond, an amide bond, a carbamate bond, and a urea bond; Each W' is independently a single bond or a C1-C6 alkylene; Z is independently a fat-soluble vitamin residue, a sterol residue, or C 12 ~C 22 (an aliphatic hydrocarbon group).

[0050] When the second biodegradable lipid compound is contained, the oxygen constituting the ether bond contained in P forms a hydrogen bond with the encapsulated nucleic acid, resulting in a larger amount of nucleic acid or the like being encapsulated.

[0051] For example, the second biodegradable lipid compound having the following structure is preferably used because it has a superior nucleic acid encapsulation amount and nucleic acid transfer efficiency. [ka] [ka] [ka]

[0052] When the lipid particles 4 containing the first and second biodegradable lipid compounds described above are used, the rate of nucleic acid introduction is high and cell death of the introduced cells can be reduced. When both the first and second biodegradable lipid compounds are contained, the application to gene therapy, nucleic acid medicine, genome diagnosis, etc. is facilitated. In particular, it is preferable to use the compound of formula (1-01) or formula (1-02) and the compound of formula (2-01) since the amount of nucleic acid encapsulated and the efficiency of nucleic acid introduction are particularly excellent.

[0053] The lipid particles 4 may further contain other lipids. Such other lipids may be arbitrarily selected from those commonly used in lipid particles 4. Examples of other substances include lipids that reduce aggregation of lipid particles 4, such as polyethylene glycol (PEG)-modified lipids, particularly polyethylene glycol (PEG) dimyristoyl glycerol (DMG-PEG), polyamide oligomers derived from omega-amino (oligoethylene glycol) alkanoic acid monomers (U.S. Pat. No. 6,320,017), and monosialogangliosides; lipids with relatively low toxicity for adjusting toxicity; lipids having functional groups for binding ligands to lipid particles 4; lipids for suppressing leakage of encapsulated substances such as sterols, for example, cholesterol, and the like.

[0054] For example, the lipid particle 4 preferably contains a compound of formula (1-01) or (1-02), a compound of formula (2-01), DOPE and / or DOTAP, cholesterol, and DMG-PEG, because the amount of nucleic acid encapsulated and the efficiency of nucleic acid introduction are particularly excellent. For example, it is preferable that the lipid particle 4 contains these components in any one of compositions 1 to 6 shown in Table 1 below. [Table 1]

[0055] The lipid particle 4 may encapsulate additional compounds in addition to the donor DNA 2 and the RNA agent 3. Such compounds include, for example, compounds that regulate the expression of nucleic acids in cells, such as retinoic acid, cyclic adenosine monophosphate (cAMP) or ascorbic acid; therapeutic agents such as peptides, polypeptides, cytokines, growth factors, apoptotic factors, differentiation inducers, cell surface receptors and their ligands, anti-inflammatory compounds, antidepressants, stimulants, analgesics, antibiotics, contraceptives, antipyretics, vasodilators, angiogenesis inhibitors, vascular agonists, signal transduction inhibitors, cardiovascular drugs, oncology drugs, hormones and steroids.

[0056] The nucleic acid transfer carrier 1 can be prepared by using, for example, a known method used for encapsulating small molecules in lipid particles, such as the Bangham method, organic solvent extraction method, surfactant removal method, freeze-thaw method, etc. For example, the material of the lipid particle 4 is contained in an organic solvent such as alcohol, and an aqueous buffer containing the donor DNA 2 and the RNA agent 3 is added to the mixture obtained, and the mixture is stirred to suspend the material, thereby preparing the nucleic acid transfer carrier 1. The ratio of the amount of the donor DNA 2 and the RNA agent 3 encapsulated in the lipid particle 4 can be easily adjusted by changing the ratio of the two in the aqueous buffer.

[0057] The amount of DNA and RNA contained therein can be confirmed, for example, using a commercially available DNA and RNA quantification kit.

[0058] The average particle size of the nucleic acid introduction carrier 1 is about 50 nm to about 300 nm, preferably about 50 nm to about 200 nm. When the nucleic acid introduction carrier 1 is intended to be used for medical purposes, the nucleic acid introduction carrier 1 is preferably made into particles of nano-order size. For example, the particle size can be reduced by ultrasonic treatment. It is also possible to adjust the size of the nucleic acid introduction carrier 1 by passing it through a polycarbonate membrane or a ceramic membrane. The average particle size of the nucleic acid introduction carrier 1 can be measured, for example, by a Zetasizer using dynamic light scattering.

[0059] ·Nucleic acid introduction method A method for introducing nucleic acid using the above-mentioned nucleic acid introduction carrier will be described below. The method for introducing nucleic acid is a method for introducing a first sequence into the genome of a cell, and includes contacting the nucleic acid introduction carrier with the cell.

[0060] 2 is a schematic flow chart showing an example of a method for introducing nucleic acid, which includes, for example, the following steps: (S1) contacting a nucleic acid transfer carrier with a cell; (S2) expressing a protein in the cell from the RNA contained in the RNA agent by said contacting; and (S3) Introducing the first sequence into the genome of the cell through the activity of the protein.

[0061] In the nucleic acid transfer method, when the person carrying out the method performs step (S1), steps (S2) and (S3) can occur naturally due to the activity of the molecules contained in the nucleic acid transfer carrier and mechanisms that are inherently present in the cell.

[0062] The cells may be, for example, of human, animal or plant origin, or of microbial origin, such as bacteria or fungi. The cells are preferably animal cells, more preferably mammalian cells, and most preferably human cells. The cells are preferably, for example, blood and immune system cells, mesenchymal cells, epithelial cells, endothelial cells, or tissue stem cells or pluripotent stem cells.

[0063] The cells may be cells removed from a living body, for example, cells isolated from a body fluid such as blood, a tissue, or a biopsy. The cells may be, for example, isolated or established cells. Alternatively, the cells may be cells in vivo.

[0064] When the cells are cells or microorganisms removed from a living body, the nucleic acid transfer carrier 1 can be brought into contact with the cells 7 by, for example, adding a composition containing the nucleic acid transfer carrier 1 onto the cultured cells or microorganisms. For example, after the addition, the cells are preferably cultured for 30 to 48 hours under conditions suitable for cell survival.

[0065] When the cell is a cell in a living animal, the contact is performed, for example, by administering to the living body a composition containing the nucleic acid transfer carrier 1. The administration can be performed, for example, by parenteral routes, such as subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intraocular, intrahepatic, intralesional, and intracranial injection or infusion.

[0066] When the cells are plant cells, the contact can be achieved by immersing the plant in a composition containing the nucleic acid transfer carrier 1, or by injecting the composition into the plant using a syringe or the like.

[0067] The enzymes used in the nucleic acid introduction method of the embodiment are not limited to Cas9 and transposase, and the first sequence 5 can be introduced in a similar manner using other enzymes involved in nucleic acid introduction.

[0068] According to the nucleic acid introduction method of the embodiment, the enzyme is introduced in the form of RNA, which makes it possible to omit the transcription step compared to the case of introducing the enzyme in the form of DNA, and therefore the enzyme is expressed more rapidly and efficiently, and therefore the first sequence 5 can be introduced more efficiently.

[0069] Furthermore, when an enzyme is introduced in the form of a protein, it is necessary to adjust the size and composition of the lipid particle 4 according to the size and properties of the encapsulated protein. On the other hand, when the enzyme is introduced in the form of RNA, as in the nucleic acid introduction method of the embodiment, there are relatively no limitations on the composition of the lipid particle 4, and therefore it is possible to reduce the effort and cost required for producing a nucleic acid introduction carrier compared to when the enzyme is introduced in the form of a protein.

[0070] Furthermore, when an enzyme is introduced in the form of DNA, the gene for the enzyme may be incorporated into the genome of the cell, which may have adverse effects on the cell or the living body containing the cell. In contrast, according to the nucleic acid introduction method of the embodiment, the enzyme is introduced in the form of RNA, and is not incorporated into the genome of the cell, which can prevent adverse effects.

[0071] In addition, when nucleic acids are introduced into cells by binding them to lipids such as lipofectamine, the nucleic acids may be decomposed or aggregate with unnecessary molecules. In addition, it is difficult to adjust the abundance ratio of the nucleic acids to be introduced. In contrast, according to the method of the embodiment, the donor DNA 2 and the RNA agent 3 are encapsulated in the lumen 4b of the lipid particle 4, so that the donor DNA 2 and the RNA agent 3 can be protected from decomposition and aggregation. In addition, it is possible to easily adjust the quantitative ratio of the donor DNA 2 and the RNA agent 3 to be introduced. Therefore, the donor DNA 2 and the RNA agent 3 can be efficiently introduced into cells, the RNA can be expressed, and the first sequence 5 can be introduced.

[0072] Furthermore, by using the nucleic acid-condensing peptide 6 as described above, imparting resistance to degradation to the RNA, and including a biodegradable lipid compound in the lipid particle 4, it is possible to further increase the transfer efficiency.

[0073] The nucleic acid introduction method can be used, for example, for the introduction of DNA in genome editing or gene recombination. For example, when the first sequence 5 includes a gene expression cassette, a gene, or a part of a gene, the gene can be introduced into the genome of a cell by the above-mentioned nucleic acid introduction method, and the cell can obtain a new function due to the gene. For example, it is possible to impart the normal function of the gene to a cell in which the gene is deleted, a cell with a small amount of the gene, a cell with an abnormality in the gene, or a cell with a partial abnormality.

[0074] Alternatively, the introduction of the first sequence 5 can knock out a gene in the genome of the cell. For example, it is possible to provide normal function to the cell by knocking out a gene that expresses a product that has a detrimental effect on the cell or an overexpressed gene. It is also possible to create a gene knockout model organism.

[0075] Although not limited thereto, the nucleic acid introduction method can be used in various fields such as gene therapy, production of model animals, gene function analysis, drug discovery, gene drive, or production of genetically modified crops. According to the method of the embodiment, the gene can be introduced more efficiently, so that the gene therapy effect, the production efficiency of model animals, the gene function analysis effect, the drug discovery efficiency, the gene drive effect or efficiency, or the production effect or efficiency of genetically modified organisms can be further improved.

[0076] ·Composition According to an embodiment, there is also provided a composition comprising the nucleic acid transfer carrier 1 and a carrier.

[0077] The carrier may be, for example, water, saline such as physiological saline, aqueous glycine or a buffer solution.

[0078] The composition of the embodiment may contain additional components in addition to the nucleic acid transfer carrier and the carrier. The additional components include, but are not limited to, agents for improving the stability of glycoproteins such as albumin, lipoproteins, apolipoproteins, globulins, etc.; for pharmaceutical use, for example, pH adjusters, buffering agents, tonicity adjusters, etc., which are pharma- ceutically acceptable and bring the pharmaceutical composition closer to physiological conditions, such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc.; and compounds such as lipid protectants for improving storage stability, such as lipophilic free radical quenchers such as α-tocopherol, which inhibit damage caused by free radicals, and water-soluble chelators such as ferrioxamine, which inhibit peroxidative damage to lipids. The carrier and other components are preferably added after the formation of the nucleic acid transfer carrier.

[0079] The composition may be, for example, a pharmaceutical composition comprising a pharma- ceutical administrable composition. The composition of the embodiment may be sterilized by conventional, well-known methods.

[0080] The compositions may be provided as liquids or as dry powders, for example, ready for use by dissolving in a suitable liquid.

[0081] The concentration of the nucleic acid transfer carrier contained in the composition of the embodiment is not limited, but is preferably 0.01 to 30% by mass, and more preferably 0.05 to 10% by mass. The concentration is appropriately selected depending on the purpose.

[0082] ·kit According to the embodiment, a kit including a nucleic acid transfer carrier is provided. The kit includes, for example, the above-mentioned composition including the nucleic acid transfer carrier and a reagent for transferring the nucleic acid transfer carrier into a cell. Alternatively, a kit in which a dispersion of the material of the lipid particle 4 dispersed in a carrier, the donor DNA 2, and the RNA agent 3 are contained in separate containers, or a kit in which the dried lipid particle 4, the donor DNA 2, the RNA agent 3, and the carrier are contained in separate containers, etc. are provided. Furthermore, the dried lipid particle 4 or the dispersion of the material of the lipid particle 4, the donor DNA 2, and the RNA agent 3 can be made into separate products, so that the user can select each product according to the purpose.

[0083] The kit may contain additional agents in separate containers that can be included in the composition.

[0084] Second embodiment In the second embodiment, there is provided a nucleic acid delivery carrier in which the donor DNA 2 and the RNA agent 3 have a core-shell structure. A cross-sectional view of the nucleic acid delivery carrier of the second embodiment is shown in FIG.

[0085] 3(a) has a core-shell structure including a donor DNA core 15 containing a donor DNA 2, and an RNA agent shell 16 containing an RNA agent 3 coating the donor DNA core 15. The core-shell structure is encapsulated in a lipid particle 4.

[0086] The nucleic acid transfer carrier 100 can be manufactured, for example, as follows. First, the donor DNA 2 is condensed with a nucleic acid condensing peptide to prepare a donor DNA core 15. Next, the RNA agent 3 is brought into contact with the donor DNA core 15, so that the RNA contained in the RNA agent 3 is electrostatically attached to the periphery of the donor DNA core 15, and an RNA agent shell 16 is formed. The RNA agent 3 may be condensed with a nucleic acid condensing peptide. As a result, a core-shell structure is formed. Next, the core-shell structure is added to a solvent containing the material of the lipid particle 4, and the mixture is stirred to encapsulate the core-shell structure in the lipid particle 4, and the nucleic acid transfer carrier 100 is manufactured.

[0087] Such a structure allows for time-delayed introduction of donor DNA 2 and RNA agent 3. For example, when nucleic acid introduction carrier 100 is introduced into a cell, RNA agent 3, which is the shell, is released prior to donor DNA 2, which is the core, and an enzyme generated from RNA contained in RNA agent 3 reaches the nucleus prior to donor DNA 2. Therefore, introduction of first sequence 5 begins immediately after donor DNA 2 reaches the nucleus, and introduction efficiency can be increased.

[0088] 3(b) comprises a core-shell structure including an RNA agent core 17 containing an RNA agent 3, and a donor DNA shell 18 containing donor DNA 2 coating the RNA agent core 17. The core-shell structure is encapsulated in a lipid particle 4.

[0089] Nucleic acid delivery carrier 101 is prepared, for example, by condensing RNA agent 3 with a nucleic acid-condensing peptide to produce RNA agent core 17, which is then contacted with donor DNA 2 to form donor DNA shell 18. Donor DNA 2 may be condensed with a nucleic acid-condensing peptide. The resulting core-shell structure is then added to a solvent containing the material for lipid particle 4 and stirred to produce nucleic acid delivery carrier 101.

[0090] With this configuration, for example, when nucleic acid delivery carrier 101 is delivered to a cell, donor DNA 2, which is the shell, is released prior to RNA agent 3, which is the core, and RNA agent 3 is gradually released. Therefore, even if the enzyme produced from the RNA agent is decomposed within the cell, RNA is released again from RNA agent core 17 to supply the enzyme, so that the delivery effect of first sequence 5 can be sustained for a long period of time.

[0091] The configuration of the nucleic acid transfer carrier is selected depending on the type of cells used. For example, in cells where there is no problem even if the enzyme generated from the RNA agent is transferred into the nucleus first, such as cells with slow protein degradation, the transfer efficiency can be increased by using the nucleic acid transfer carrier 100 shown in (a) of Figure 3. Alternatively, in cells where the enzyme is easily decomposed or consumed, such as cells with a fast division cycle or cells with fast protein degradation, the transfer efficiency can be increased by using the nucleic acid transfer carrier 101 shown in (b) of Figure 3.

[0092] The release rate and release duration of the nucleic acid contained in the core can be adjusted by the composition or amount of the nucleic acid-condensing peptide, or the amount of the nucleic acid.

[0093] The nucleic acid transfer carriers 100 and 101 can be used in a nucleic acid transfer method in the same manner as the nucleic acid transfer carrier of the first embodiment. In addition, they may be provided as a composition or kit similar to the first embodiment.

[0094] (Third embodiment) According to the third embodiment, a nucleic acid transfer carrier set is provided in which donor DNA 2 and RNA agent 3 are separately encapsulated in lipid particles 4. An example of a nucleic acid transfer carrier set is shown in FIG. 4. Nucleic acid transfer carrier set 200 comprises a first carrier 201 and a second carrier 202. First carrier 201 comprises donor DNA 2 and a first lipid particle 41 encapsulating donor DNA 2. Second carrier 202 comprises RNA agent 3 and a second lipid particle 42 encapsulating RNA agent 3. Donor DNA 2 and RNA agent 3 may each be encapsulated in a state condensed with a nucleic acid-condensing peptide.

[0095] First carrier 201 and second carrier 202 can be manufactured separately. For example, first carrier 201 and second carrier 202 can be obtained by condensing donor DNA 2 and RNA agent 3 with a nucleic acid condensing peptide, respectively, and mixing and stirring them in separate solutions containing lipid particle materials.

[0096] The nucleic acid introduction carrier set 200 may be provided as a composition or kit similar to that of the first embodiment. For example, the first carrier 201 and the second carrier 202 are provided as compositions contained in separate containers or compositions contained in the same container.

[0097] The nucleic acid introduction carrier set 200 can be used in the nucleic acid introduction method in the same manner as the nucleic acid introduction carrier of the first embodiment. According to such a nucleic acid introduction carrier set 200, either the first carrier 201 or the second carrier 202 can be contacted with the cells first in the nucleic acid introduction method.

[0098] For example, when using cells in which it is preferable to first transfer the RNA agent 3 into the nucleus as described above, it is preferable to contact the second carrier 202 with the cells before the first carrier 201. For example, it is preferable to contact the first carrier 201 30 minutes to 48 hours after contacting the second carrier 202.

[0099] Alternatively, when using cells in which it is preferable to first transfer donor DNA 2 into the nucleus and then slowly release RNA agent 3 as described above, it is preferable to contact first carrier 201 with the cells prior to contacting second carrier 202. For example, it is preferable to contact second carrier 202 30 minutes to 48 hours after contacting first carrier 201.

[0100] Alternatively, both may be contacted with the cells at the same time.

[0101] By using the nucleic acid delivery carrier set 300, when the donor DNA 2 and the RNA agent 3 are delivered with a time lag, the difference in delivery time can be easily adjusted.

[0102] [example] An example of the production and use of the nucleic acid transfer carrier of the embodiment will be described below.

[0103] Example 1. Evaluation of the transfection and expression efficiency of the NanoLuc gene using a carrier encapsulating DNA Preparation of DNA-encapsulated carriers The DNA used was a plasmid DNA in which the NanoLuc gene was linked downstream of the cytomegalovirus promoter. A cationic peptide was added to the DNA solution containing this DNA to form a DNA-peptide condensate. This was then added to an ethanol-dissolved lipid solution (FFT10 (biodegradable lipid compound of the above formula (1-01)) / DOTAP / cholesterol / PEG-DMG=73 / 44 / 59 / 4 mol), and 10 mM HEPES (pH 7.3) was gently added, followed by washing and concentration by centrifugal ultrafiltration to obtain a DNA-encapsulated carrier. The amount of DNA encapsulated in the carrier was measured using the Quant-iT (registered trademark) PicoGreen dsDNA Assay Kit (manufactured by Thermo Fisher Scientific), and it was confirmed that a sufficient amount of DNA was encapsulated.

[0104] Preparation of Jurkat and delivery of nucleic acid using DNA-encapsulated carriers Human T-cell leukemia cells (Jurkat, ATCC) were cultured in TexMACS medium (Miltenyi Biotec), and the cells were harvested by centrifugation. 7 The cells were suspended in fresh TexMACS to give 1.0 × 10 cells in a 48-well culture plate. 6 150 μL each of the cell suspension and TexMACS were added to give cells / well.

[0105] Thereafter, the DNA-encapsulating carriers were added to the wells so that the DNA was 0.5 μg / well, and the cells were cultured at 37° C. in a 5% CO 2 atmosphere.

[0106] -Transfer of DNA into cells using Lipofectamine 3000 For comparison, the above plasmid DNA was introduced into Jurkat using Lipofectamine 3000 Reagent (Invitrogen). The introduction was performed according to the manual attached to the reagent. The plasmid DNA was added to Jurkat at 0.5 μg / well and cultured at 37° C. in a 5% CO2 atmosphere.

[0107] Measurement of NanoLuc expression level (NanoLuc luminescence assay) 48 hours after addition of the plasmid DNA mixed with the DNA-encapsulating carrier or Lipofectamine 3000, the culture plate was removed from the incubator, and the luminescence intensity of NanoLuc was measured using a luminometer (Infinite (registered trademark) F200 PRO, Tecan) with the Nano-Glo Luciferase Assay System (Promega). The measurement was performed according to the manuals attached to the kit and the device.

[0108] NanoLuc luminescence assay results Figure 5 shows the results of measuring the luminescence intensity of NanoLuc. The luminescence intensity was higher when DNA was introduced using a DNA-encapsulating carrier than when it was introduced using Lipofectamine 3000. From this result, it is clear that DNA was well introduced and the NanoLuc gene was well expressed in cells into which DNA was introduced using a DNA-encapsulating carrier. This shows that the method of introducing DNA by encapsulating it in a carrier has higher DNA introduction efficiency and gene expression efficiency than the method using a complex of Lipofectamine and DNA.

[0109] -Detection of luminescent cells by microscope Next, the above cells to which DNA had been added using a carrier or Lipofectamine 3000 were used to detect luminescent cells using a luminescence microscope system (LV200, Olympus). 24 hours after addition, 100 μL of cell culture solution was transferred to a 4-well culture dish, and NanoLuc substrate (LiveCellLuciferaseAssayKit, Promega) was added. After setting the culture dish in a designated position on the luminescence microscope system (LV200, Olympus), bright field images and luminescent images of the cells were taken.

[0110] Microscopic observation results Figure 6 shows a photograph of the luminescent cells (a superimposed image of a bright-field image and a luminescent image taken by Matamorph software). The white dots indicated by the arrows in the figure are luminescent cells. Figure 6(a) shows a microscopic image of the cells using the DNA-encapsulated carrier, and Figure 6(b) shows a microscopic image of the cells using Lipofectamine 3000. Comparing the two, it is clear that the number of luminescent cells is much greater when the DNA-encapsulated carrier is used than when Lipofectamine 3000 is used. This result, like the NanoLuc luminescence assay, indicates that the DNA introduction efficiency and gene expression efficiency are higher when the DNA-encapsulated carrier is used than when the complex of Lipofectamine and DNA is used.

[0111] Example 2. Evaluation of the transfection and expression efficiency of the green fluorescent protein (GFP) gene using a carrier encapsulating mRNA Preparation of RNA-encapsulating carriers As messenger RNA (mRNA), green fluorescent protein (GFP) mRNA (Oz Biosciences) was used as a reporter gene. The RNA solution containing this mRNA was added to an ethanol-dissolved lipid solution (FFT10 / DOPE / cholesterol / PEG-DMG=73 / 44 / 59 / 4 mol), suspended by pipetting, and then 10 mM HEPES (pH 7.3) was gently added. The solution was washed and concentrated by centrifugal ultrafiltration to obtain an RNA-encapsulated carrier. The amount of RNA encapsulated in the carrier was measured using QuantiFluor (registered trademark) RNA System (Promega), and it was confirmed that a sufficient amount of mRNA was encapsulated.

[0112] Preparation of Jurkat and delivery of nucleic acid using RNA-encapsulated carrier Jurkat cells were cultured in TexMACS medium, harvested by centrifugation, and then diluted with 0.65 × 10 7 The cells were suspended in fresh TexMACS to give 1.0 × 10 cells in a 48-well culture plate. 6 150 μL each of the cell suspension and TexMACS were added to give cells / well.

[0113] Thereafter, the RNA-encapsulating carrier was added to the wells so that the mRNA was 0.5 μg / well, and the cells were cultured at 37° C. in a 5% CO 2 atmosphere.

[0114] -Transfection of mRNA into cells using Lipofectamine 3000 For comparison, the above mRNA was introduced into Jurkat using Lipofectamine 3000 reagent. The introduction was performed according to the manual attached to the reagent. The mRNA was added to Jurkat at 0.5 μg / well and cultured at 37° C. in a 5% CO2 atmosphere.

[0115] GFP expression detection 24 hours after addition of mRNA mixed with RNA-encapsulating carriers or Lipofectamine 3000, the culture plate was removed from the incubator, and the cells were harvested by centrifugation and then suspended in phosphate buffered saline (PBS) containing 1% BSA (Gibco, Thermo Fisher Scientific). The green fluorescence of GFP was detected using a fluorescence-activated cell sorter (FACS; FACSVerse®, BD Biosciences).

[0116] ·result The detection results are shown in Figure 7. Figure 7(a) shows the results for the RNA-encapsulating carrier, and Figure 7(b) shows the results for Lipofectamine 3000. The graph shows a histogram with the vertical axis representing the cell number (%) and the horizontal axis representing the GFP expression intensity. The solid line histogram shows the distribution in cells into which RNA was introduced, and the dashed line histogram shows the distribution in cells into which RNA was not introduced (control).

[0117] As shown in Figure 7(a), when RNA was introduced using the carrier, the distribution of the fluorescence intensity of the cells was significantly shifted to the right compared to the control, indicating that GFP was well expressed in the cells. This demonstrates that GFP mRNA was well introduced and that GFP was well expressed.

[0118] In contrast, as shown in Figure 7(b), when RNA was introduced using Lipofectamine reagent, the distribution of fluorescence intensity was almost the same as that of the control, suggesting that the introduction or expression of GFP mRNA was weak.

[0119] Therefore, the method of introducing mRNA by encapsulating it in a carrier shows higher efficiency of mRNA introduction and gene expression than the method using a complex of Lipofectamine and mRNA.

[0120] Example 3. Evaluation of the introduction efficiency and expression efficiency of the GFP gene in the introduction in the form of mRNA (RNA-containing carrier) and in the form of DNA (DNA-containing carrier) Preparation of RNA-encapsulating carriers As the mRNA, the GFP mRNA described in Example 2 was used. The RNA solution containing the GFP mRNA was added to an ethanol-dissolved lipid solution (FFT10 / DOPE / cholesterol / PEG-DMG=73 / 44 / 59 / 4 mol), suspended by pipetting, and then 10 mM HEPES (pH 7.3) was gently added. The solution was washed and concentrated by centrifugal ultrafiltration to obtain an RNA-encapsulating carrier. The amount of RNA encapsulated in the carrier was measured using the QuantiFluor (registered trademark) RNA System, and it was confirmed that a sufficient amount of mRNA was encapsulated.

[0121] Preparation of DNA-encapsulated carriers The DNA used was a plasmid DNA in which the GFP gene was linked downstream of the cytomegalovirus promoter. A cationic peptide was added to the DNA solution containing this DNA to condense the DNA, and then the solution was added to an ethanol-dissolved lipid solution (FFT10 / DOTAP / cholesterol / PEG-DMG=73 / 44 / 59 / 4 mol), and 10 mM HEPES (pH 7.3) was gently added. The solution was then washed and concentrated by centrifugal ultrafiltration to obtain a DNA-encapsulated carrier. The amount of DNA encapsulated in the carrier was measured using the Quant-iT (registered trademark) PicoGreen dsDNA Assay Kit, and it was confirmed that a sufficient amount of DNA was encapsulated.

[0122] Preparation of Jurkat and delivery of nucleic acid using carrier Jurkat cells were cultured in TexMACS medium, harvested by centrifugation, and then diluted with 0.65 × 10 7 The cells were suspended in fresh TexMACS to give 1.0 × 10 cells in a 48-well culture plate. 6 150 μL each of the cell suspension and TexMACS were added to give cells / well.

[0123] The RNA-encapsulated carriers and the DNA-encapsulated carriers were added to the wells of separate well culture plates at 1.0 μg / well, and cultured at 37° C. in a 5% CO 2 atmosphere.

[0124] GFP expression detection 24 hours after adding the carrier, the culture plate was removed from the incubator, and the cells were collected by centrifugation and suspended in phosphate buffered saline (PBS) containing 1% BSA (Gibco, Thermo Fisher Scientific). Green fluorescence (GFP) of the cells was detected by FACS.

[0125] ·result The detection results are shown in Figure 8. Figure 8(a) shows the results for RNA-encapsulated carriers, and Figure 8(b) shows the results for DNA-encapsulated carriers. The graph shows a histogram with the vertical axis representing the cell count (%) and the horizontal axis representing the GFP expression intensity. The solid histogram shows the distribution in cells into which RNA or DNA was introduced using a carrier, and the dashed histogram shows the distribution in cells into which RNA or DNA was not introduced (control).

[0126] As shown in Figure 8(a), when the GFP gene was introduced in the form of mRNA, the distribution of fluorescence intensity was significantly shifted to the right compared to the control, indicating that GFP was well expressed in the cells. This demonstrates that GFP mRNA was well introduced and that GFP was well expressed.

[0127] In contrast, as shown in Figure 8(b), when the GFP gene was introduced in the form of DNA, the distribution of fluorescence intensity was almost the same as that of the control, suggesting that the introduction of DNA or the expression of GFP was weak.

[0128] Therefore, it is shown that introduction of GFP in the form of mRNA results in higher nucleic acid introduction efficiency and gene expression efficiency than introduction in the form of DNA.

[0129] Example 4 Evaluation of the transfer efficiency and expression efficiency of the GFP gene using DNA / RNA co-encapsulating carriers Preparation of RNA / DNA co-encapsulating carriers and their introduction into Jurkat mice A mixed solution containing the plasmid DNA containing the NanoLuc gene described in Example 1 and the mRNA of the GFP gene described in Example 2 was added to an ethanol-dissolved lipid solution (FFT10 / DOPE / cholesterol / PEG-DMG=73 / 44 / 59 / 4 mol), and 10 mM HEPES (pH 7.3) was gently added, followed by washing and concentration by centrifugal ultrafiltration to obtain an RNA / DNA-encapsulated carrier. The amount of RNA encapsulated in the carrier was measured using the QuantiFluor (registered trademark) RNA System, and the amount of DNA encapsulated was measured using the Quant-iT (registered trademark) PicoGreen dsDNA Assay Kit, and it was confirmed that sufficient amounts of mRNA and DNA were contained.

[0130] Jurkat cells were cultured in TexMACS medium, harvested by centrifugation, and then diluted with 0.65 × 10 7 The cells were suspended in fresh TexMACS to give 1.0 × 10 cells in a 48-well culture plate. 6 150 μL each of the cell suspension and TexMACS were added to the wells so that there were cells / well. Then, the DNA / RNA-encapsulating carriers were added to the wells so that the mRNA and DNA were each 0.5 μg / well, and the wells were cultured at 37° C. in a 5% CO2 atmosphere.

[0131] - Lipofectamine 3000 for delivery of mRNA and DNA into cells For comparison, mRNA and plasmid DNA were introduced into Jurkat cells using Lipofectamine 3000 reagent. The introduction was performed according to the manual attached to the reagent. The mRNA and plasmid DNA were added to Jurkat cells at 0.5 μg / well and cultured at 37° C. in a 5% CO2 atmosphere.

[0132] -Expression detection of NanoLuc and GFP Expression of NanoLuc from NanoLucDNA was detected by the Nano-Glo Luciferase Assay System, and expression of GFP from GFPmRNA was detected by FACS, as described in Examples 1 and 2, respectively.

[0133] ·result The results of detecting the expression of NanoLuc are shown in Figure 9. The graph in Figure 9 reveals that the relative luminescence intensity was much higher when DNA and RNA were introduced using a carrier than when Lipofectamine 3000 was used. This result indicates that the use of a carrier results in higher DNA introduction efficiency and higher gene expression efficiency from DNA.

[0134] Figure 10 shows the results of detecting GFP expression. Figure 10(a) shows the results for the carrier, and Figure 10(b) shows the results for Lipofectamine 3000. These histograms reveal that the GFP fluorescence intensity is higher when the carrier is used than when Lipofectamine 3000 is used. This result indicates that the use of the carrier results in higher efficiency of mRNA introduction and gene expression from mRNA.

[0135] From the above results, it was demonstrated that the DNA / RNA co-encapsulating carrier enables simultaneous introduction of both DNA and RNA with high introduction and expression efficiency.

[0136] Example 5. Evaluation of time-delayed delivery of DNA and RNA using DNA-encapsulated carriers and RNA-encapsulated carriers Preparation of DNA-encapsulated carriers The DNA used was a plasmid DNA incorporating a NanoLuc gene expression cassette in which the cytomegalovirus promoter and the NanoLuc gene were linked. The DNA-encapsulating carrier was prepared by the method described in Example 1.

[0137] Preparation of RNA-encapsulating carriers The RNA used was transposase RNA. The RNA-encapsulating carrier was prepared by the method described in Example 2.

[0138] Preparation of cells and delivery of nucleic acids using carriers Frozen commercially available human peripheral blood mononuclear cells (PBMC, Lonza) were thawed in a 37°C incubator, and then the cells were collected by centrifugation. The cells were suspended in TexMACS containing two types of cytokines (10 ng / mL IL-7, 5 ng / mL IL-15 (Miltenyi)), then seeded on a 6 cm culture dish, and cultured in an incubator at 37°C and 5% CO2. After overnight culture, the culture dish was removed from the incubator, and the cells were collected by centrifugation, suspended in TexMACS (containing 10 ng / mL IL-7, 5 ng / mL IL-15), and then cultured overnight at 37°C and 5% CO2 in a 48-well culture plate coated with CD3 antibody (Miltenyi) and CD28 antibody (Miltenyi).

[0139] Transposase RNA-encapsulated carriers (4 μg) were added to the cell culture medium and cultured in a 5% CO2 atmosphere. After 2 hours, NanoLuc DNA-encapsulated carriers (4 μg) were added and culture was continued.

[0140] For comparison, a transposase RNA-encapsulated carrier (4 μg) and a NanoLuc DNA-encapsulated carrier (4 μg) were simultaneously added to the same cell culture medium and cultured in a 5% CO2 atmosphere.

[0141] NanoLuc luminescence detection 48 hours after the initial carrier addition, the culture plates were removed from the incubator and the luminescence intensity of NanoLuc was measured using the Nano-Glo Luciferase Assay System (Promega) and a luminometer (Infinite (registered trademark) F200 PRO, Tecan). Luminescence measurement was performed according to the manuals attached to the kit and the device.

[0142] ·result FIG. 11 shows the results of measuring the NanoLuc luminescence intensity. The luminescence intensity of NanoLuc was higher when the DNA-containing carrier was added 2 hours after the addition of the RNA-containing carrier than when the DNA-containing carrier and the RNA-containing carrier were added simultaneously. This result revealed that the time-delayed introduction of transposase mRNA, which assists the introduction of DNA, and DNA containing the sequence to be introduced is effective in increasing the amount of protein expressed from DNA. In addition, it was revealed that the method of the embodiment allows efficient introduction and expression of DNA even in PBMC, which is generally considered to have low nucleic acid introduction efficiency.

[0143] Example 6. Evaluation of nucleic acid transfer efficiency using a carrier with a DNA / RNA core-shell structure Preparation of DNA / RNA core-shell encapsulated carriers As DNA, a plasmid DNA incorporating a CAR gene expression cassette in which a cytomegalovirus promoter and a CAR gene were linked was used, and as RNA, the GFP mRNA described in Example 2 was used. A cationic peptide was added to a DNA solution containing the DNA to form a DNA core, and then the RNA was added to prepare a solution containing a DNA / RNA core shell in which the DNA core was enveloped with RNA as a shell. This was added to an ethanol-dissolved lipid solution (FFT10 / DOTAP / cholesterol / PEG-DMG=73 / 44 / 59 / 4 mol), and 10 mM HEPES (pH 7.3) was gently added, followed by washing and concentration by centrifugal ultrafiltration to obtain a carrier having a DNA / RNA core shell structure.

[0144] Preparation of DNA / RNA mixed encapsulation carrier For comparison, a cationic peptide was added to the mixed solution containing the above DNA and RNA to prepare a solution containing a DNA / RNA mixed core, which was then added to an ethanol-dissolved lipid solution (FFT10 / DOTAP / cholesterol / PEG-DMG=73 / 44 / 59 / 4 mol), and 10 mM HEPES (pH 7.3) was gently added. The solution was then washed and concentrated by centrifugal ultrafiltration to obtain a DNA / RNA mixed encapsulating carrier.

[0145] Confirmation of DNA / RNA inclusion within the carrier Next, to confirm whether DNA and RNA were encapsulated in both of the obtained carriers, the carriers were cleaved (by adding a surfactant: sodium dodecyl sulfate) and the core structure was destroyed (by adding polyglutamic acid), and the released DNA / RNA were detected by agarose electrophoresis.

[0146] The results are shown in Figure 12. In both the DNA / RNA core-shell encapsulated carrier and the DNA / RNA mixed core encapsulated carrier, DNA and RNA signals (arrows in the figure) were detected only under the condition where both carrier cleavage and core structure destruction were performed simultaneously. From this result, it was confirmed that both the DNA / RNA core-shell encapsulated carrier and the DNA / RNA mixed core encapsulated carrier contained DNA and RNA aggregates.

[0147] Fluorescence microscopy of cells 20 hours after the DNA / RNA core-shell encapsulating carrier and the DNA / RNA mixed core encapsulating carrier were introduced into Jurkat, cells with green fluorescence (GFP-expressing cells) were detected by a fluorescence microscope. FIG. 13 shows micrographs showing the results. In the DNA / RNA mixed core encapsulating carrier, GFP-expressing cells were not detected 20 hours after the introduction of the carrier (FIG. 13(b)), but GFP-expressing cells were detected 4 days later (arrow in FIG. 13(d)). In contrast, in the DNA / RNA core-shell encapsulating carrier, GFP-expressing cells were detected 20 hours after the addition of the carrier (arrow in FIG. 13(a)). This result suggests that the expression of RNA in the DNA / RNA core-shell is faster than that in the DNA / RNA mixed core. Therefore, it was revealed that in the DNA / RNA core-shell encapsulating carrier, the DNA / RNA core is decomposed in the cell with a time difference, and proteins can be expressed in the order of the RNA shell and the core DNA. The invention as originally claimed in the present application is set forth below. [1] a donor DNA comprising a first sequence; an RNA agent comprising at least an RNA encoding a protein involved in the introduction of the first sequence into a genome; a lipid particle encapsulating the donor DNA and the RNA agent; Equipped with A nucleic acid transfer carrier used for transferring said first sequence into the genome of a cell. [2] The nucleic acid transfer carrier according to [1], wherein the donor DNA and the RNA agent have a core-shell structure in which the donor DNA is the core and the RNA agent is the shell. [3] The nucleic acid transfer carrier described in [1], wherein the donor DNA and the RNA agent have a core-shell structure in which the RNA agent is the core and the donor DNA is the shell. [4] The nucleic acid transfer carrier according to any one of [1] to [3], wherein the protein is an enzyme having endonuclease activity. [5] The nucleic acid transfer carrier described in [4], wherein the protein is CRISPR-associated protein 9 (Cas9). [6] The nucleic acid transfer carrier described in [5], wherein the RNA agent further contains a guide RNA including a sequence corresponding to a sequence on the genome for transferring the first sequence. [7] The nucleic acid transfer carrier according to any one of [1] to [3], wherein the protein is a transposase. [8] The nucleic acid transfer carrier described in [7], wherein the protein is PiggyBac, SleepingBeauty, Frog Prince, Hsma, Minos, Tol1, Tol2, Passport, hAT, Ac / Ds, PIF, Harbinger, Harbinger3-DR, Himar1, Hermes, Tc3 or Mos1. [9] The nucleic acid transfer carrier according to any one of [1] to [8], wherein the RNA agent further comprises RNA encoding a protein that methylates DNA, RNA encoding a protein that demethylates DNA, RNA encoding a protein that repairs DNA, and / or RNA encoding a protein that binds DNA.

[10] The nucleic acid transfer carrier according to any one of [1] to [9], wherein the RNA contained in the RNA agent is modified so as to be resistant to degradation.

[11] The nucleic acid transfer carrier according to any one of [1] to

[10] , wherein the donor DNA and / or the RNA agent is condensed with a nucleic acid-condensing peptide.

[12] The lipid particles have the formula Q-CHR 2 (In the formula, Q is a nitrogen-containing aliphatic group containing two or more tertiary nitrogen atoms and no oxygen; R is independently C 12 ~C 24 is an aliphatic group of The nucleic acid transfer carrier according to any one of [1] to

[11] , wherein at least one R further comprises a first biodegradable lipid represented by the formula (LR) containing a linking group selected from the group consisting of -C(=O)-O-, -OC(=O)-, -OC(=O)-O-, -SC(=O)-, -C(=O)-S-, -C(=O)-NH-, and -NHC(=O)- in its main chain or side chain.

[13] The lipid particles have the formula P-[XWY-W'-Z] 2 (In the formula, P is an alkyleneoxy containing one or more ether bonds in the main chain; Each X is independently a divalent linking group containing a tertiary amine structure, W is independently C 1 ~C 6 is alkylene, each Y is independently a divalent linking group selected from the group consisting of a single bond, an ether bond, a carboxylate bond, a thiocarboxylate bond, a thioester bond, an amide bond, a carbamate bond, and a urea bond; Each W' is independently a single bond or C 1 ~C 6 is alkylene, Z is independently a fat-soluble vitamin residue, a sterol residue, or C 12 ~C 22 (It is an aliphatic hydrocarbon group) The nucleic acid transfer carrier according to any one of [1] to

[12] , further comprising a second biodegradable lipid represented by the following formula:

[14] a first carrier comprising a donor DNA containing a first sequence and a first lipid particle encapsulating the donor DNA; and A second carrier comprising an RNA agent comprising at least an RNA encoding a protein involved in the introduction of said first sequence into the genome, and a second lipid particle encapsulating said RNA agent. Including, A nucleic acid transfer carrier set used for transferring the first sequence into the genome of a cell.

[15] A nucleic acid transfer carrier according to any one of [1] to

[13] or a nucleic acid transfer carrier set according to

[14] , Carrier and A nucleic acid transfer composition comprising:

[16] A method for introducing a first sequence into the genome of a cell using a nucleic acid introduction carrier comprising donor DNA containing a first sequence, an RNA agent containing at least RNA encoding a protein involved in the introduction of the first sequence into the genome, and a lipid particle encapsulating the donor DNA and the RNA, the method comprising contacting the nucleic acid introduction carrier with the cell.

[17] expressing the protein from the RNA in the cell by contacting the nucleic acid transfer carrier with the cell; and introducing said first sequence into said genome of said cell through the activity of said protein. The method according to

[16] , further comprising:

[18] The method according to

[16] or

[17] , wherein the donor DNA and the RNA agent of the nucleic acid transfer carrier have a core-shell structure in which the donor DNA is the core and the RNA agent is the shell.

[19] The method of claim 18, wherein the protein expressed from the RNA reaches the nucleus of the cell faster than the donor DNA.

[20] The method according to

[16] or

[17] , wherein the donor DNA and the RNA agent of the nucleic acid transfer carrier have a core-shell structure in which the RNA agent is the core and the donor DNA is the shell.

[21] The method according to claim 20, wherein the RNA is slowly released within the cell.

[22] A first carrier comprising a donor DNA containing a first sequence and a lipid particle encapsulating the donor DNA; and A second carrier comprising an RNA agent comprising at least an RNA encoding a protein involved in the introduction of said first sequence into a genome, and a lipid particle encapsulating said RNA agent. 1. A method for introducing the first sequence into a genome of a cell using A method for introducing nucleic acid, comprising contacting the first carrier and the second carrier with the cell.

[23] The method according to

[22] , wherein the first carrier is contacted with the cells, and then the second carrier is contacted with the cells.

[24] The method according to

[22] , further comprising contacting the second carrier with the cells and then contacting the second carrier with the cells.

[25] The method according to

[23] , wherein the first carrier and the second carrier are contacted with the cells simultaneously.

[26] The method according to any one of

[16] to

[25] , wherein the protein is an enzyme having endonuclease activity.

[27] The method according to claim 26, wherein the protein is CRISPR-associated protein 9 (Cas9).

[28] The method according to claim 27, wherein the RNA agent further comprises a guide RNA comprising a sequence corresponding to a sequence on the genome for introducing the first sequence.

[29] The method according to any one of

[16] to

[25] , wherein the protein is a transposase.

[30] The method according to claim 29, wherein the protein is PiggyBac, SleepingBeauty, Frog Prince, Hsma, Minos, Tol1, Tol2, Passport, hAT, Ac / Ds, PIF, Harbinger, Harbinger3-DR, Himar1, Hermes, Tc3 or Mos1. [Explanation of symbols]

[0148] 1, 100, 101...Nucleic acid delivery carrier 2. Donor DNA 3. RNA agents 3a…RNA 3b…Guide RNA 4...Lipid particles 5...First sequence 6. Nucleic acid condensing peptide 200...Nucleic acid delivery carrier set 201…First career 202…Second Career

Claims

1. a donor DNA comprising a first sequence; RNA comprising at least an RNA sequence encoding a protein involved in the introduction of said first sequence into the genome; A lipid particle encapsulating the donor DNA and the RNA; Equipped with The donor DNA and the RNA encapsulated in the lipid particles form a two-layer core-shell structure composed of a donor DNA core layer having the donor DNA, and an RNA shell layer coating the donor DNA core layer and having the RNA in contact with the donor DNA of the donor DNA core layer. A nucleic acid transfer carrier used for transferring said first sequence into the genome of a cell.

2. a donor DNA comprising a first sequence; RNA comprising at least an RNA sequence encoding a protein involved in the introduction of said first sequence into the genome; A lipid particle encapsulating the donor DNA and the RNA; Equipped with The donor DNA and the RNA encapsulated in the lipid particles form a two-layer core-shell structure composed of an RNA core layer comprising the RNA, and a donor DNA shell layer coating the RNA core layer and comprising the donor DNA in contact with the RNA of the RNA core layer. A nucleic acid transfer carrier used for transferring said first sequence into the genome of a cell.

3. 3. The nucleic acid transfer carrier according to claim 1, wherein the protein is an enzyme having an endonuclease activity.

4. The nucleic acid transfer carrier according to claim 3, wherein the protein is CRISPR-associated protein 9 (Cas9).

5. The nucleic acid transfer carrier according to claim 4, wherein the RNA comprises a guide RNA comprising a sequence corresponding to a sequence on the genome for transferring the first sequence.

6. The nucleic acid transfer carrier according to claim 1 or 2, wherein the protein is a transposase.

7. The nucleic acid transfer carrier according to claim 6, wherein the protein is PiggyBac, Sleeping Beauty, Frog Prince, Hsma, Minos, Tol1, Tol2, Passport, hAT, Ac / Ds, PIF, Harbinger, Harbinger3-DR, Himar1, Hermes, Tc3 or Mos1.

8. The nucleic acid transfer carrier according to any one of claims 1 to 7, wherein the RNA comprises an RNA sequence encoding a protein that methylates DNA, an RNA sequence encoding a protein that demethylates DNA, an RNA sequence encoding a protein that repairs DNA, and / or an RNA sequence encoding a protein that binds DNA.

9. 9. The nucleic acid transfer carrier according to claim 1, wherein the RNA is modified so as to be resistant to degradation.

10. The nucleic acid transfer carrier according to any one of claims 1 to 9, wherein the donor DNA and / or the RNA is condensed with a nucleic acid-condensing peptide.

11. The lipid particles have the formula Q-CHR ２ (In the formula, Q is a nitrogen-containing aliphatic group containing two or more tertiary nitrogen atoms and no oxygen; Each R is independently C １２ ~C ２４ is an aliphatic group of The nucleic acid transfer carrier according to any one of claims 1 to 10, further comprising a first biodegradable lipid represented by the formula (LR) selected from the group consisting of -C(=O)-O-, -O-C(=O)-, -O-C(=O)-O-, -S-C(=O)-, -C(=O)-S-, -C(=O)-NH-, and -NHC(=O)- in its main chain or side chain.

12. The lipid particles have the formula P-[X-W-Y-W'-Z] ２ (In the formula, P is an alkyleneoxy containing one or more ether bonds in the main chain; Each X is independently a divalent linking group containing a tertiary amine structure, Each W is independently C １ ~C ６ is alkylene, Each Y is independently a divalent linking group selected from the group consisting of a single bond, an ether bond, a carboxylate bond, a thiocarboxylate bond, a thioester bond, an amide bond, a carbamate bond, and a urea bond; Each W' is independently a single bond or C １ ~C ６ is alkylene, Z is independently a fat-soluble vitamin residue, a sterol residue, or C １２ ~C ２２ aliphatic hydrocarbon group) The nucleic acid transfer carrier according to any one of claims 1 to 10, further comprising a second biodegradable lipid represented by:

13. A nucleic acid transfer carrier according to any one of claims 1 to 12, Carrier and A nucleic acid transfer composition comprising:

14. A method for introducing the first sequence into the genome of a cell existing outside of a living body using a nucleic acid introduction carrier described in any one of claims 1 to 13, the method comprising contacting the nucleic acid introduction carrier with the cell.

15. expressing the protein from the RNA in the cell by contacting the nucleic acid transfer carrier with the cell; and 15. The method of claim 14, further comprising introducing the first sequence into the genome of the cell by the activity of the protein.

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