MECHANISM RNA VECTOR BASED ON NANOPARTICLE-Oligo T CONJUGATES

Abstract

The present invention relates to a gene vector for delivering a polyadenylated nucleic acid molecule derived from a target gene, comprising nanoparticles and Oligo T bound to the surface of the nanoparticles, the gene vector having an effect of efficiently delivering the target gene into a cell without requiring an additional processing process of binding the target gene to the vector.

Description

Technical Field

[0001] The present invention relates to a gene carrier for delivering polyadenylated nucleic acid molecules derived from a target gene, comprising nanoparticles and Oligo T bound to the surface of the nanoparticles. Background Art

[0002] Genes are organic substances that all living organisms have, and contain all the information necessary for the structure and maintenance of organisms and the organic relationships between cells. Genes can be damaged by various factors, and gene damage may develop into various diseases.

[0003] In order to treat diseases caused by gene damage, gene therapy methods that introduce foreign genes (DNA or RNA) required by patients are being studied. All organisms have defense mechanisms against foreign genes. Therefore, for effective gene therapy, it is necessary to develop an effective gene delivery system. So far, gene therapy agents are mainly viral vector technologies, but there is a fatal disadvantage of producing a strong immune response, and the range of applicable genes is limited. In order to overcome the limitations of this viral vector technology, non-viral vector technologies such as cationic lipids, polymers, dendritic polymers and peptides are developed. Compared with viral vector technology, non-viral vector technology improves the stability problem, but the gene delivery efficiency is reduced.

[0004] Therefore, a new gene delivery technology that can satisfy both stability and efficiency is needed. In recent years, nanoparticle non-viral vector technologies including carbon nanotubes, iron oxide, silicon dioxide and gold are being studied as alternatives.

[0005] So far, more than half of gene therapy studies use viral vector technology. Viral vector technology is a gene vector that uses the inherent proliferation mechanism of viruses. Representative examples include adenovirus, retrovirus, adeno-associated virus (AAV), etc. Viral vector technology improves the efficiency of gene delivery, but because it is a pathogenic virus, there are safety issues such as inducing a strong immune response, and the size of the gene that can be inserted into the viral vector is limited, so the applicable genes are limited.

[0006] In contrast, representative examples of non-viral vector technology include plasmids and liposomes. The advantages of non-viral vectors are that they do not introduce viral genetic material into human cells and are easy to produce. The size of the gene inserted into the vector can be unlimited, and it has the advantage of having few side effects due to low immune response to the host. However, compared with viral vectors, they have the disadvantage of low intracellular delivery efficiency.

[0007] In order to make up for the shortcomings of existing gene carriers, recently, nanoparticles including carbon nanotubes, iron oxide, silicon dioxide and gold are being studied as alternatives to gene carrier technology. In particular, gold nanoparticles are bioinert and nontoxic, and are easy to synthesize and can be functionalized, so they are attractive scaffolds for the development of gene delivery systems. So far, several gene delivery systems have been developed, including gold nanoparticles protected by mixed monolayers, complexes of polymers and gold nanoparticles, gold nanoparticles functionalized with double-stranded DNA, and gold nanoparticles functionalized with single-stranded DNA.

[0008] Compared with cationic liposomes (lipofectamine) or cytofectin (cytofectin) widely used as gene delivery systems for research, these nanoparticles show low cytotoxicity to cells, high gene delivery efficiency, and improved resistance to nucleases. However, since this system can only deliver genes connected to nanoparticles by covalent bonds, there is the inconvenience of needing to synthesize nanoparticles separately for each target gene and requiring an additional process for synthesis.

[0009] In order to overcome this shortcoming, the inventor introduces universal binding partner into nano material surface and in conjunction with the modified gene by covalent bond so as to have the binding pairing partner combined with this binding partner to develop new gene delivery system (patent 10-2011-0050338). However, this system needs to modify the gene to be delivered so that the gene has the extra process of the base sequence complementary to the universal binding partner. However, the modification of the base sequence added to the gene may reduce the efficiency of translation or cause the translation product to change. Therefore, it is necessary to study the gene carrier that can be used more conveniently without the need for additional modified gene. Summary of the invention

[0010] Technical issues

[0011] Therefore, the present inventors developed a gene delivery system, which introduces an Oligo T sequence as a universal binding partner into the surface of a nanomaterial by a covalent bond and contains a polyadenylated gene, so that the gene can be bound without performing an additional processing on the gene to be delivered to have a base sequence complementary to the universal binding partner, thereby completing the present invention.

[0012] Therefore, an object of the present invention is to provide a gene carrier comprising nanoparticles, Oligo T linked to the surface of the nanoparticles, and polyadenylated nucleic acid molecules.

[0013] Technical Solution

[0014] In order to achieve the above-mentioned purpose, the present invention provides a gene carrier, which comprises nanoparticles, Oligo T connected to the surface of the nanoparticles and polyadenylated nucleic acid molecules.

[0015] Hereinafter, the present invention will be described in detail.

[0016] The present invention relates to a gene carrier for delivering genes such as mRNA to a target, comprising nanoparticles, an Oligo T sequence connected to the surface of the nanoparticles through a covalent bond, and a polyadenylated nucleic acid molecule bound to the Oligo T.

[0017] Diseases caused by gene defects or damage can be treated by gene therapy techniques that replace or modify defective / damaged genes by introducing foreign genes. However, since all organisms have defense mechanisms against the introduction of foreign genes, it is necessary to develop effective gene delivery systems to deliver foreign genes into organisms. The gene vector of the present invention has no toxicity and can effectively deliver foreign genes into cells, and is developed to regulate the expression of target genes without affecting normal cells.

[0018] That is, the gene vector of the present invention is developed to introduce Oligo T capable of binding to polyadenylated nucleic acid molecules such as mRNA on the surface of nanoparticles by covalent bonds, and to bind the nucleic acid molecules to be expressed to Oligo T for delivery to target cells. That is, the present invention is a technology that can deliver the gene to be introduced by using Oligo T targeting the poly A tail (poly Atail) naturally present in the nucleic acid molecule without additional modification or processing.

[0019] In the present invention, the "nanoparticle (NP)" refers to a substance preferably having a size of 1-100 nm. The form and shape of the nanoparticle used in the present invention are not particularly limited, as long as it is a substance with a nanometer size (such as a particle, a tube, a rod or a regular tetrahedron). According to a preferred embodiment of the present invention, the "nanoparticle" refers to particles of various substances with a diameter of nanometer level, preferably 8-100 nm, more preferably 10-50 nm, and most preferably 12-14 nm. The nanoparticle is not particularly limited, as long as it has a nanometer size.

[0020] According to the most preferred embodiment of the present invention, the nanoparticles used in the present invention refer to gold nanoparticles. Gold nanoparticles are not only easily prepared in the form of stable particles, but also their size can be varied between 0.8nm and 200nm according to the purpose of use. In addition, gold can change its structure by combining with various types of molecules such as peptides, proteins, nucleic acids, etc., and reflect light at various wavelengths, so that it can be used to easily confirm the position in the cell. In addition, unlike heavy metals such as manganese, aluminum, cadmium, lead, mercury, cobalt, nickel, beryllium, etc., gold nanoparticles are harmless to the human body and therefore have high biocompatibility.

[0021] When the diameter of the gold nanoparticles increases to more than 100 nm, not only the characteristics of the nanoparticles disappear, but also the binding of the gold surface without the characteristics of the nanoparticles to the functional groups such as thiol groups becomes weaker, so it is difficult to prepare particles bound to Oligo T using gold particles as a medium. Therefore, preferably, the diameter of the gold nanoparticles of the present invention can be 8-100 nm, more preferably, 10-50 nm, and most preferably, 12-14 nm, but not limited thereto.

[0022] For example, the gold nanoparticles used in the present invention can be prepared as follows: using HAuCl4 as a gold source and sodium citrate as a reducing agent, the gold nanoparticles are prepared by reducing HAuCl4. In this case, the size of the gold nanoparticles can be adjusted by changing the amount of citrate added. That is, since nucleation increases with the increase in the amount of citrate added, the size of the gold nanoparticles decreases.

[0023] In the present invention, the "Oligo T" used to bind to the polyadenylated nucleic acid molecule has additional functionality to covalently bond to the surface of the nanoparticle. The additional functionality is located at the end of the oligonucleotide. More preferably, the end of the Oligo T is bound to a thiol group or an amine group, and more preferably, the additional functionality is located at the 5'-end.

[0024] As described above, the nucleic acid molecule can be directly bound to the site that imparts functionality, but preferably, a "linker (or spacer)" can be inserted in the middle. The linker plays a role in allowing Oligo T to bind to the nanoparticle at a high density and more easily bind to the target gene (e.g., mRNA). The linker connects the Oligo T and the target gene (e.g., mRNA), and its type is not limited, but can be an oligonucleotide or a polyether, preferably, it can include one or more linkers of Oligo A nucleotides, Oligo T nucleotides, Oligo G nucleotides or Oligo C nucleotides.

[0025] According to a preferred example of the present invention, the Oligo T may have a "5'-SH (or NH2)-linker-Oligo T nucleotide-3'" structure.

[0026] In the present invention, the length of the Oligo T is not particularly limited, but preferably, it may contain 3 to 100 Ts, more preferably, it may be composed of 10 to 50 Ts, and more preferably, it may be composed of 15 to 30 Ts (thymine).

[0027] The Oligo T can be combined with a target gene for delivery to a cell, such as a polyadenylation (poly A) sequence of mRNA, and the number of the polyadenylation sequence is not particularly limited, but can contain as short as more than 20 and as long as several hundred A (adenine), preferably, can contain about 20 to 250 A. The polyadenylation is added in the post-transcription process of mRNA, and the gene vector of the present invention can particularly contain polyadenylated mRNA as a nucleic acid molecule for delivery, and has the characteristic of being able to deliver mRNA to target cells.

[0028] As an example, the nucleic acid molecule derived from the target gene can be an expression construct comprising a promoter for expressing the target gene, a coding sequence of the target gene operatively linked to the promoter, and a polyadenylation sequence. In this specification, the term "operably linked" refers to the functional combination between a nucleic acid expression regulatory sequence (e.g., a promoter, a signal sequence, or an array of transcriptional regulatory factor binding sites) and other nucleic acid sequences, whereby the regulatory sequence can regulate the expression of other nucleic acid sequences.

[0029] The target gene can be a gene to be expressed in a cell, in particular, it can be RNA or DNA, more preferably, it can be an mRNA molecule containing a polyadenylation sequence. This can be of human origin, or can include genes of external origin (such as viruses, etc.) or therapeutic transgenes such as tumor suppressor genes, antigen genes, cytotoxic genes, cell proliferation inhibitory genes, apoptosis genes, and neovascularization inhibitory genes. In one embodiment of the present invention, the target gene is an antigen gene, and the intracellular delivery is confirmed using mRNA derived from the RBD of SARS-CoV2.

[0030] In the present invention, the gene vector can effectively deliver foreign genes into cells, and can be used as a gene vector capable of inducing expression, and can be used as a drug vector in drugs or vaccines using mRNA molecules themselves.

[0031] That is, as another embodiment of the present invention, the present invention can provide a use of the gene carrier, which can be used as a component of a gene therapy agent, a cell therapy agent or a vaccine. The gene carrier of the present invention can be used as a pharmaceutical composition for the purpose of gene delivery.

[0032] Effects of the Invention

[0033] The present invention relates to a gene carrier for delivering a polyadenylated nucleic acid molecule derived from a target gene, comprising nanoparticles and Oligo T connected to the surface of the nanoparticles. The gene carrier of the present invention can be widely used without an additional processing step for combining a target gene and a carrier, and has the effect of effectively delivering a target gene into a cell. RNAI Compared with nucleic acid carriers, it has the effect of increasing not only the binding ability to the nucleic acid to be delivered but also the efficiency of intracellular delivery. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Schematic diagram of a gene delivery system based on gold nanoparticles functionalized with Oligo T that binds specifically to the polyadenylic acid base sequence of mRNA.

[0035] Figure 2 Transmission microscopy analysis image of the synthesized gold nanoparticles.

[0036] Figure 3 The AuNP prepared in the present invention is shown dT 8M urea (Urea) 10% polyacrylamid gel (polyacrylamid gel) electrophoresis analysis image.

[0037] Figure 4 To deliver the AuNPs prepared in the present invention dT -Fluorescence microscopy images of HeLa cells expressing oligoA.

[0038] Figure 5 The AuNP prepared in the present invention dT -GFP mRNA, AuNP RNAI -GFP mRNA (A), AuNP dT -SARS-CoV-2RBD mRNA, AuNP RNAI -8 M urea (Urea) 10% polyacrylamid gel electrophoresis analysis image of SARS-CoV-2RBD mRNA (B).

[0039] Figure 6To show that AuNPs were expressed in the mouse xenograft model, immunohistochemical staining was used to confirm that dT Graph showing the results of delivering GFP mRNA.

[0040] Figure 7 To show that the AuNPs in rats were confirmed by ELISA analysis dT A graph of the results of delivering SARS-CoV-2 RBD mRNA.

[0041] Embodiments of the present invention

[0042] Hereinafter, detailed description will be given by way of examples to specifically illustrate this specification. However, the embodiments of this specification may be modified into various other forms, and the scope of this specification should not be interpreted as being limited to the above-described embodiments. The embodiments of this specification are provided to more fully illustrate this specification to those of ordinary skill in the art.

[0043] Example 1. Preparation of gold nanoparticles (AuNPs) functionalized with Oligo T dT )

[0044] The gene vector is prepared by covalently bonding Oligo T for binding to the polyadenylic acid sequence (poly A) of messenger RNA to gold nanoparticles. The schematic diagram of the preparation process is shown in FIG. Figure 1 shown.

[0045] 1-1. Preparation of 13 nm gold nanoparticles

[0046] The gold nanoparticles used in the present invention are prepared by using HAuCl4 as a gold source and reducing HAuCl4 using sodium citrate as a reducing agent. More specifically, 545 ml of a 0.92 mM HAuCl4 solution and 5 ml of a 388 mM sodium citrate solution are mixed and then reacted at a temperature of 100° C. for 15 minutes. The reactants are analyzed by transmission electron microscopy to confirm whether the nanoparticles are synthesized and the size ( Figure 2 ).

[0047] 1-2. Oligo T pretreatment

[0048] In order to prepare the gene vector of the present invention, the polyadenylation sequence of the messenger RNA is detected and the Oligo T sequence that specifically binds thereto is used. More specifically, the Oligo T uses Oligo T ((SH) 5'-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-3') of SEQ ID NO: 1 as an Oligo nucleotide whose 5' end is modified by a thiol group. The dried Oligo T is dissolved in water to make a final concentration of 100 μM, and then 20 μl of 3M sodium acetate (sodium acetate) (pH 5.2) and 30 μl of 1N dithiothreitol (DTT) are added to 150 μl of Oligo, and reacted at room temperature for 60 minutes. In order to remove DTT containing unnecessary thiol molecules, 200 μl of ethyl acetate (Ethylacetate) is added and mixed, and then the supernatant is removed by centrifugation, and this process is repeated 3 times. Then, Oligo T is precipitated using the EtOH precipitating method.

[0049] 1-3. Preparation of Oligo T functionalized gold nanoparticles (AuNP dT )

[0050] Oligo T pretreated and precipitated in the same manner as in Example 1-2 was dissolved in water and then added to the gold nanoparticles synthesized in Example 1-1, followed by binding by a salt aging method.

[0051] Specifically, Oligo T (AuNP:Oligo T ratio = 1:100-300) was added to 5-50 nm gold nanoparticles and mixed thoroughly, and then NaCl was added to make the concentration 0.1-1.0 M and mixed for 4 hours. After 4 hours, NaCl was added to double the concentration and mixed for 4 hours. After 4 hours, NaCl was added to make the concentration 0.3 M and mixed for 12 hours. After 12 hours, the mixture of Oligo T and gold was collected by centrifugation at ~10000*g for 20 minutes, and then the unreacted Oligo T in the supernatant was removed, and this process was repeated 3 times. Finally, the gold nanoparticle-Oligo T complex was added and dispersed in a 10 mM sodium phosphate buffer (sodium phosphate buffer) (pH 7.4) containing 0.1 M NaCl. The results of electrophoresis analysis of the prepared gold nanoparticle-Oligo T complex using 10% acrylamide 8M urea gel confirmed that one gold nanoparticle was bound to 130 to 150 Oligo T ( Figure 3 ).

[0052] Example 2. In vitro confirmation of AuNPs dT Intracellular delivery of genes

[0053] 2-1. Preparation of AuNPs dT -oligo A conjugate

[0054] OligoA (5'-AAAAAAAAAAAAAAAAAAAAA, SEQ ID NO: 2) as the polyadenylation sequence of mRNA was labeled with Cy3 to confirm the in vitro expression of AuNPs. dT The intracellular delivery ability of the polyadenylated nucleic acid molecule.

[0055] To prevent the formation of secondary structures of nucleic acid molecules, first, Oligo A was pre-incubated at 80° C. for 5 minutes. Oligo A and AuNP were mixed in a 10 mM sodium phosphate buffer (pH 7.4) containing 0.1 M NaCl. dT , then react at 55°C for 10 minutes, and then react again at 4°C for about 30 minutes. The resulting conjugate was centrifuged at 15000*g for 10 minutes, and then the supernatant was removed and electrophoresed on 10% acrylamide 8M urea gel to confirm binding.

[0056] 2-2. Confirmation of AuNP dT -Intracellular delivery ability of oligoA

[0057] In order to confirm that the AuNPs prepared in 2-1 dT The intracellular delivery ability of -oligoA was tested on human cervical carcinoma (HeLa) cells. HeLa cells were cultured in a 37°C incubator with 5% CO2 and regulated humidity in Dulbecco's modified Eagle's medium (DMEM) containing 10% heat-inactivated fetal bovine serum (Caisson, USA) and 1% penicillin-streptomycin (Welgene, Korea). In order to obtain a confluence of 50% to 60%, about 4×10 4The cells were plated in a 24-well plate in a medium containing 10% FBS. The AuNPs prepared in 2-1 were dT -oligoA was added to the HeLa cell culture medium to a final concentration of 1 nM, and fluorescence released from the cells was measured using a fluorescence microscope 24 hours later. Figure 4 As shown, it was confirmed that when the prepared gold nanocarrier was used, the intracellular movement of the nucleic acid molecule containing Oligo A was further increased.

[0058] Example 3. Confirmation of AuNPs in a small animal model dT Gene delivery capacity

[0059] Animal experiments were conducted to confirm the target gene delivery ability of the gold nanoparticle gene carrier of the present invention. dT -GFP mRNA and AuNP dT -SARS-CoV-2RBD mRNA to confirm delivery ability. Animals were kept in an animal breeding room with a humidity of 30% to 40% and a temperature of 22±1°C. The indoor lighting was set to a 12-hour light / dark cycle. The animal experimental protocol was approved by the Animal Experiment Support Center of Central University, and the animals were treated as described in the experimental protocol.

[0060] 3-1. In vitro synthesis of mRNA

[0061] Use the MEGA shortscript according to the manufacturer's instructions TM Messenger RNA was synthesized from the DNA templates of SEQ ID NO: 3 and SEQ ID NO: 4 comprising a T7 promoter sequence and a subsequent gene sequence using the PCR kit (Ambion, USA), CleanCap AG (Trilink, USA) and N1-Methylpseudo-UTP (JenaBioscience, Germany).

[0062] The DNA template used to synthesize GFP mRNA used a template consisting of the base sequence of SEQ ID NO: 3 (including T7 promoter), and the DNA template used to synthesize SARS-CoV-2 RBD mRNA used a template consisting of the base sequence of SEQ ID NO: 4. The synthesized mRNA was purified by phenol extraction.

[0063] 3-2. Preparation of AuNPs dT-mRNA conjugates

[0064] The AuNPs were prepared in the same manner as in Example 2-1. dT and GFP mRNA and SARS-CoV-2RBD mRNA conjugates. dT The mRNA conjugates were analyzed by electrophoresis on 10% acrylamide 8M urea gel, and it was confirmed that in each conjugate, one gold nanoparticle was bound to 30 to 40 mRNAs ( Figure 5 ). It can be confirmed that the AuNP is similar to the technology of the existing document (Korean Patent No. 10-2011-0050338). RNAI In comparison, the AuNPs of the present invention dT The mRNA binding capacity was increased by more than 30%.

[0065] 3-3. Confirmation of AuNPs in mice dT -GFP mRNA in vivo delivery capability

[0066] For the convenience of the experiment, a xenograft tumor model was used. Six-week-old immunodeficient BALB / c-nu / nu mice (Central Lab Animal Inc, Korea) were injected with HeLa cells to induce cervical cancer, and AuNPs were injected into the xenograft tumors every 24 hours. dT -GFP mRNA for 3 times, and then the xenograft tumors were extracted and the expression of GFP mRNA was determined by immunohistochemical staining ( Figure 6 ). As a result, the injection of AuNP dT -GFP mRNA was found in the tumor, and the AuNP dT -GFP mRNA intracellular transport. In addition, it was confirmed that the AuNPs were similar to the technology of the prior art (Korean Patent No. 10-2011-0050338). RNAI In comparison, the AuNPs of the present invention dT It has better mRNA delivery capability.

[0067] 3-4. Confirmation of AuNPs in rats dT -In vivo delivery capability of SARS-CoV-2 RBD mRNA

[0068] Six-week-old Sprague-Dawley rats (Samtako, Korea) were intramuscularly injected with AuNPs every two weeks. dT-SARS-CoV-2RBD mRNA for a total of 3 times, and then the muscles at the administration site were extracted and the expression of SARS-CoV-2RBD mRNA was determined by immunohistochemical staining ( Figure 7 ), then the serum of the rats was extracted and the ELISA method was used to determine whether antibodies against the RBD protein were formed ( Figure 8 ). As a result, Figure 7 As shown, it was confirmed that the injected AuNP dT -SARS-CoV-2RBD mRNA expresses RBD protein in muscle tissue, which shows that the AuNP dT -SARS-CoV-2RBD mRNA moves into cells in vivo. And, in this regard, Figure 8 As shown, it was confirmed that the injected AuNP dT -SARS-CoV-2RBD mRNA formed RBD antibodies in the serum of rats, thereby confirming the expression of mRNA that moved into cells.

[0069] So far, the present invention is described with the preferred embodiment as the center. It will be appreciated by those skilled in the art that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a restrictive point of view. The scope of the present invention is shown in the scope of the claims rather than the above description, and all differences within the equivalent scope thereof should be interpreted as included in the present invention. DETAILED DESCRIPTION

[0070] As one embodiment of the present invention, the present invention relates to a gene carrier, which comprises: a nanoparticle; Oligo T, which is bound to the surface of the nanoparticle through a covalent bond; and a nucleic acid molecule, which comprises a polyadenylation sequence bound to the Oligo T.

[0071] In the present invention, the nanoparticles are characterized in that they may have a size of 8-100 nm.

[0072] In the present invention, the nanoparticles are gold nanoparticles (AuNPs).

[0073] In the present invention, the terminal of the Oligo T is bonded to a thiol group or an amine group.

[0074] In the present invention, the Oligo T consists of 3 to 100 thymines.

[0075] In the present invention, the nucleic acid molecule comprising the polyadenylation sequence may be RNA or DNA.

[0076] In the present invention, the nucleic acid molecule comprising the polyadenylation sequence is mRNA.

[0077] In the present invention, the nucleic acid molecule may be derived from one or more genes selected from the group consisting of tumor suppressor genes, antigen genes, cytotoxic genes, cell proliferation inhibitory genes, apoptosis genes and angiogenesis inhibitory genes.

[0078] In the present invention, the nucleic acid molecule may be an antigen gene derived from a virus.

Claims

1. A gene vector, characterized in that: Include: Nanoparticles; Oligo T, bound to the surface of the nanoparticle via a covalent bond; and A nucleic acid molecule comprising a polyadenylation sequence bound to the Oligo T.

2. The gene carrier according to claim 1, characterized in that The size of the nanoparticles is 8-100 nm.

3. The gene carrier according to claim 1, characterized in that The nanoparticles are gold nanoparticles.

4. The gene carrier according to claim 1, characterized in that The end of the Oligo T is bonded to a thiol group or an amine group.

5. The gene carrier according to claim 1, characterized in that The Oligo T consists of 3 to 100 thymines.

6. The gene carrier according to claim 1, characterized in that The nucleic acid molecule comprising the polyadenylation sequence is RNA or DNA.

7. The gene carrier according to claim 1, characterized in that The nucleic acid molecule comprising the polyadenylation sequence is mRNA.

8. The gene carrier according to claim 1, characterized in that The nucleic acid molecule is derived from one or more genes selected from the group consisting of tumor suppressor genes, antigen genes, cytotoxic genes, cell proliferation inhibitory genes, apoptosis genes and angiogenesis inhibitory genes.

9. The gene carrier according to claim 8, characterized in that The nucleic acid molecule is an antigen gene derived from a virus.

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