Gene vector based on metal nanoparticle-nucleic acid conjugate

Abstract

The present invention relates to a gene vector, a use thereof, a gene expression method using the same, and a preparation method therefor, the gene vector comprising a nucleic acid molecule comprising a gene of interest bound to the surface of a metal nanoparticle, which is delivered into a cell and expressed.

Description

Technical Field

[0001] The present invention relates to a gene carrier, a gene expression method using the same and a preparation method thereof. The gene carrier comprises metal nanoparticles and double-helix nucleic acid molecules combined with the surface of the nanoparticles. Background Art

[0002] Nanoparticles are nanometer-sized particles that have various physical and chemical properties due to their small size and large surface area. Gold nanoparticles, the most widely used nanoparticles, generate surface electromagnetic resonance (SPR) caused by light absorption and scattering in the visible light range according to their size and shape, so they are used in detection and imaging through fluorescent labeling, etc., and are easy to introduce surface functional groups and have high biocompatibility and stability, so they can also be used in the delivery of biomaterials such as DNA, RNA, protein, antibodies, and various drugs.

[0003] Research is ongoing on technologies that deliver genetic material into cells to produce antigens or proteins for treatment or prevention, such as cell therapy agents. In particular, vaccine development technology has significantly advanced due to the pandemics in recent years, leading to the development of various gene vaccines such as DNA vaccines, mRNA vaccines, and viral vector vaccines.

[0004] DNA is the simplest genetic material and is easy to be genetically modified, so the development cycle for therapeutic agents such as vaccines can be shortened. Moreover, compared with viruses, proteins, and RNA as other vaccine candidate substances, it is very economical in terms of production facility construction and production costs, and has excellent stability, so it has the advantages of easy storage and transportation. However, DNA vaccines need to be delivered to the nucleus of the cell and make the process of directly producing mRNA in the nucleus. The DNA injected into the nucleus can continuously produce mRNA and continuously produce antigen proteins, but due to the injection of other genotypes into the nucleus of the cell in the human body, there is a risk of side effects such as innate immune response. Moreover, when delivered by plasmid, DNA also delivers bacterial-derived genes in addition to antigens, which may cause side effects such as mutations in the body. Therefore, it is necessary to study the carriers and delivery technologies for safely delivering DNA. Summary of the invention

[0005] Technical issues

[0006] Therefore, in order to effectively deliver nucleic acid molecules that can be delivered into cells and expressed alone, especially double-stranded DNA (dsDNA), into the cell nucleus, the inventors have made intensive research and efforts to develop nucleic acid delivery technology. As a result, nucleic acid molecules containing genes of interest are directly bonded to the surface of metal nanoparticles through covalent bonds, thereby completing the delivery system that can be delivered into cells, the gene expression method based on this, and the preparation method of the delivery system.

[0007] Therefore, the object of the present invention is to provide a gene vector comprising: metal nanoparticles; and nucleic acid molecules, which are combined with the metal nanoparticles and contain one or more genes of interest that are delivered into cells and expressed.

[0008] Furthermore, another object of the present invention is to provide a pharmaceutical composition comprising the gene vector.

[0009] Furthermore, another object of the present invention is to provide a composition for detecting a target substance, which comprises the gene vector.

[0010] Furthermore, another object of the present invention is to provide a composition for delivering a gene of interest into a cell, which comprises the gene vector.

[0011] Furthermore, another object of the present invention is to provide a method for preparing a gene carrier, the method comprising the following steps: modifying the surface of the metal nanoparticles by treating the metal nanoparticles with an acidic solution; and combining the surface of the metal nanoparticles with a nucleic acid molecule containing one or more genes of interest that are delivered to and expressed in cells.

[0012] Furthermore, another object of the present invention is to provide a method for expressing a gene of interest through a nucleic acid molecule that is bound to the surface of metal nanoparticles and expressed alone in cells.

[0013] Technical Solution

[0014] In order to achieve the above-mentioned purpose, the present invention provides a gene carrier, which comprises: metal nanoparticles; and nucleic acid molecules, which are combined with the metal nanoparticles and contain one or more genes of interest that are delivered into cells and expressed.

[0015] Furthermore, in order to achieve another object of the present invention, the present invention provides a pharmaceutical composition comprising the gene vector.

[0016] Furthermore, in order to achieve another object of the present invention, the present invention provides a composition for detecting a target substance, which comprises the gene vector.

[0017] Furthermore, in order to achieve another object of the present invention, the present invention provides a composition for delivering a gene of interest into a cell, comprising the gene vector.

[0018] Furthermore, in order to achieve another object of the present invention, the present invention provides a method for preparing a gene carrier, the method comprising the following steps: modifying the surface of the metal nanoparticles by treating the metal nanoparticles with an acidic solution; and combining the surface of the metal nanoparticles with a nucleic acid molecule containing one or more genes of interest that are delivered to and expressed in cells.

[0019] Furthermore, in order to achieve another object of the present invention, the present invention provides a method for expressing a gene of interest through a nucleic acid molecule that is bound to the surface of metal nanoparticles and expressed alone in cells.

[0020] Hereinafter, the present invention will be described in detail.

[0021] As one embodiment, the present invention relates to a gene vector, comprising: metal nanoparticles; and nucleic acid molecules, which are combined with the metal nanoparticles and contain one or more genes of interest that are delivered into cells and expressed.

[0022] The gene carrier of the present invention comprises metal nanoparticles. The metal nanoparticles have a nanometer-scale diameter, and there is no limit to the size thereof, but preferably, the diameter is 5nm to 500nm, and more preferably, the diameter is 10nm to 200nm. Nanoparticles of this size are easily prepared in the form of stable particles, and the size is easily adjusted in the preparation process. Moreover, as described in the present invention, in the case of metal nanoparticles used as gene carriers, when the diameter of the metal nanoparticles increases to more than 500nm, not only the characteristics as nanoparticles disappear, but also the combination of the metal surface and the functional group becomes weak, so there is a disadvantage that it is difficult to prepare a carrier using nanoparticles. Moreover, preferably, the metal nanoparticles can be gold nanoparticles. Unlike heavy metals such as manganese, aluminum, cadmium, lead, mercury, cobalt, nickel, and beryllium, the gold nanoparticles are harmless to the human body and therefore have high biocompatibility.

[0023] As an example, for example, the gold nanoparticles used in the present invention can be prepared as follows: HAuCl4 is used as a gold source, and sodium citrate is used as a reducing agent to prepare the gold nanoparticles 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.

[0024] The surface of the metal nanoparticles of the present invention is bound to a nucleic acid molecule containing a gene of interest. The nucleic acid molecule is bound to the surface of the metal nanoparticles for the purpose of delivering the gene of interest for introduction into cells and individual expression.

[0025] There is no restriction on the type of nucleic acid molecule. In the present invention, nucleic acid molecule refers to a compound having a structure in which a base, a sugar and a phosphoric acid are connected by a phosphodiester bond, including naturally occurring oligonucleotides such as 2'-deoxyribonucleic acid (hereinafter referred to as "DNA") and ribonucleic acid (hereinafter referred to as "RNA") and nucleic acids containing modified sugar residues, modified phosphate residues or modified necleobases. Modification of sugar residues includes substitution of ribose rings with hexose, cyclopentyl or cyclohexyl rings. On the other hand, the D-ribose ring of a naturally occurring nucleic acid can be substituted with an L-ribose ring, or the β-anomer of a naturally occurring nucleic acid can be substituted with an α-anomer. Nucleic acid molecules can also include one or more abasic residues (abasic moieties). Modified phosphate residues can also include thiophosphates (phosphorothioates), dithiophosphates (phosphorodithioates), methylphosphonates (methylphosphonates) and methyl phosphate (methyl phosphate). Such nucleic acid analogs (analogs) are well known in the art. For example, nucleic acid molecules containing two or more mixtures in the mixture can be produced by a mixture of deoxyribose- or ribonucleosides, especially by a mixture of deoxyribonucleosides such as 2'-O-methylribonucleosides (2'-O-methylribonucleoside) or 2'-O-methoxyethylribonucleoside and 2'-O-substituted ribonucleosides.

[0026] More specifically, the nucleic acid molecule can be selected from DNA, RNA or DNA / RNA molecules, and more specifically, can be double-stranded DNA. The double-stranded DNA can include cDNA, gDNA, plasmid DNA and PCR DNA that can be expressed alone. As an embodiment, the double-stranded DNA is combined with gold nanoparticles in a double-stranded state, and is used to deliver the double-stranded DNA into cells, which is different from the situation in which single-stranded DNA is combined with gold nanoparticles, introduced into cells and hybridized with complementary strands.

[0027] And, described nucleic acid molecule can comprise more than one gene of interest and be introduced into cell and express separately.In the present invention, term " gene of interest " comprises any nucleic acid or the nucleic acid of coding interested functional peptide or polypeptide (protein) (native state or modified peptide / protein) with treatment, diagnosis and / or preventive effect and / or induction required biology and / or pharmacological effect.

[0028] In the present invention, the "single expression" refers to the process in which the gene of interest contained in the nucleic acid molecule is transcribed and / or translated separately without being integrated into the genome of the injected cell. As an example, although not limited to this, for single expression, the nucleic acid molecule may include more than one promoter, open reading frame or terminator, and more preferably, may include more than one promoter operably connected to the gene of interest. The promoter sequence is usually derived from a virus or a cut eukaryotic promoter, and therefore, the promoter may be a pro-opiomelanocortin promoter (POMC), an adenovirus promoter, a baculovirus promoter, a CMV promoter, a parvovirus promoter, a herpes virus promoter, a poxvirus promoter, an adeno-associated virus promoter, a Semliki Forest virus promoter, an SV40 promoter, a vaccinia virus promoter or a retrovirus promoter. Examples of promoters include human herpes simplex virus thymidine kinase (HSV TK or mini-TK) promoter, cauliflower mosaic virus (CaMV) 35S promoter, human cytomegalovirus CMV promoter (mini-CMV), CMV53 (minCMV with upstream GC box added), simian virus 40 promoter (minSV40), MLP (-38 to +6 region of adenovirus major late promoter), minP (synthetic promoter consisting of TATA box and transcription start site-Promega product), pJB42CAT5 (promoter from human junB gene), YB_TATA and super core promoter 1 (SCP1) promoter. Several promoters (sometimes referred to as "core promoters") are described in the literature (Ede et al., ACS Synth Biol. 2016 May 20; 5(5): 395-404).

[0029] The terms "operably configured", "operably associated" and "operably linked" mean that a promoter (and / or enhancer) is in the correct functional site and orientation relative to a nucleic acid sequence in order to control transcription initiation and expression of its nucleic acid. An enhancer is "operably linked" to a promoter when it is in the correct functional site and orientation for increasing the transcriptional activity of the promoter.

[0030] In one example, the nucleic acid molecule further comprises a polyadenylation (poly A) sequence. The poly A sequence causes appropriate polyadenylation of the nucleic acid (transcriptome) of interest. Examples of representative poly A sequences include SV40 poly A and / or bovine growth hormone poly A, which are known to be convenient and / or well-functioning in various target cells.

[0031] In one example, the nucleic acid molecule further comprises a transcription termination sequence. A "termination signal" or "termination factor" consists of a DNA sequence that participates in the specific termination of an RNA transcriptome by an RNA polymerase.

[0032] Furthermore, although not limited thereto, the nucleic acid molecule may produce a transcription and / or translation product through a transcription and / or translation expression process. The transcription and / or translation product is not limited thereto, but may include, for example, mRNA, non-coding RNA, protein, antigen or antibody, etc.

[0033] The gene carrier of the present invention is a surface binding of a nucleic acid molecule to a gold nanoparticle. In order to bind to the surface of the gold nanoparticle, the nucleic acid molecule contains one or more functionalities. The type of the functionality is not limited, and can be a thiol group or an amine group, which can be contained in one or more residues of the nucleic acid molecule as the delivery object. In one embodiment of the present invention, the nucleic acid molecule can contain one or more thiolated residues, thereby directly binding to the surface of the gold nanoparticle. The binding does not contain an additional spacer or linker. The thiolated residue can be one or more bases contained in the 3' end, the 5' end or the base sequence of the nucleic acid molecule.

[0034] Furthermore, one or more of the nucleic acid molecules are bound to the surface of the gold nanoparticles, although not limited thereto, for expression, 1 to 20 nucleic acid molecules can be bound to the surface of the gold nanoparticles. Furthermore, the length of the bound nucleic acid molecules can be 100 bp, 200 bp or 300 bp or more, and there is no limit to the length. Furthermore, in some cases, the nucleic acid molecules are 30 or more nucleotides. In another example, the nucleic acid molecules are 35 or more nucleotides. In another example, the length is at least 40 nucleotides. In another example, the length is at least 45 nucleotides. In another example, the length is at least 55 nucleotides. In another example, the length is at least 50 nucleotides. In another example, the length is at least 60 nucleotides. In another example, the length is at least 80 nucleotides. In another example, the length is at least 90 nucleotides. In another example, the length is at least 100 nucleotides. In another example, the length is at least 120 nucleotides. In another example, the length is at least 140 nucleotides. In another example, the length is at least 160 nucleotides. In another example, the length is at least 180 nucleotides. In another example, the length is at least 200 nucleotides. In another example, the length is at least 250 nucleotides. In another example, the length is at least 300 nucleotides. In another example, the length is at least 350 nucleotides. In another example, the length is at least 400 nucleotides. In another example, the length is at least 450 nucleotides. In another example, the length is at least 500 nucleotides. In another example, the length is at least 600 nucleotides. In another example, the length is at least 700 nucleotides. In another example, the length is at least 800 nucleotides. In another example, the length is at least 900 nucleotides. In another example, the length is at least 1000 nucleotides. In another example, the length is at least 1100 nucleotides. In another example, the length is at least 1200 nucleotides. In another example, the length is at least 1300 nucleotides. In another example, the length is at least 1400 nucleotides. In another example, the length is at least 1500 nucleotides. In another example, the length is at least 1600 nucleotides. In another example, the length is at least 1800 nucleotides. In another example, the length is at least 2000 nucleotides. In another example, the length is at least 2500 nucleotides. In another example, the length is at least 3000 nucleotides. In another example, the length is at least 4000 nucleotides. In another example, the length is at least 5000 nucleotides or more.

[0035] As another embodiment of the present invention, the present invention relates to a pharmaceutical composition comprising a gene vector, wherein the gene vector comprises the above-mentioned gold nanoparticles and double-helix DNA. The pharmaceutical composition is used for preventing, improving or treating diseases, and its type is not limited, and its use can be changed according to the type of double-helix DNA bound to the gold nanoparticles and the expression product.

[0036] The pharmaceutical composition may be a cell gene therapy agent comprising a cell therapy agent, a gene-modified cell therapy agent, a gene therapy agent, or an RNA therapy agent, and the cell gene therapy agent may comprise a vaccine, an antibiotic, and an anticancer agent.

[0037] The cell therapy agent refers to a drug used for therapeutic, diagnostic and preventive purposes by proliferating living autologous, allogenic or xenogeneic cells in vitro, screening or changing the biological characteristics of cells through other methods in order to restore cell organization and function. When the genes in the cells are modified, it is also classified as a gene-modified cell therapy agent.

[0038] Furthermore, the gene therapy agent is a drug prepared by introducing normal genes and therapeutic genes into the patient's cells by genetic engineering such as gene recombination to modify defective genes or to treat or prevent gene defects by giving cells new functions.

[0039] Furthermore, the RNA therapeutic agent inhibits the protein production process that induces the disease from the target gene and exhibits the efficacy of a drug, and may include mRNA, RNAi, ASO (Antisense oligonucleotide), RNA aptamer, and the like.

[0040] The pharmaceutical composition of the present invention may also contain suitable carriers, excipients and diluents commonly used in the preparation of pharmaceutical compositions. The excipients may be, for example, one or more selected from the group consisting of diluents, binders, disintegrants, lubricants, adsorbents, humectants, film coating materials and controlled release additives.

[0041] The pharmaceutical composition of the present invention can be prepared according to conventional methods into powders, granules, sustained-release granules, enteric-coated granules, liquids, eye drops, elixirs, emulsions, suspensions, alcohol, lozenges, aromatic waters, lemonades, tablets, sustained-release tablets, enteric-coated tablets, sublingual tablets, hard capsules, soft capsules, sustained-release capsules, enteric-coated capsules, pills, tinctures, soft extracts, dry extracts, fluid extracts, injections, capsules, perfusion solutions, plasters, lotions, pastes, sprays, inhalants, patches, sterile injection solutions or aerosols and other external preparations for use, and the external preparations can have dosage forms such as creams, gels, patches, sprays, ointments, plasters, lotions, liniments, pastes or mud compresses.

[0042] The carriers, excipients and diluents contained in the pharmaceutical composition of the present invention may include lactose, glucose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.

[0043] When formulating, a commonly used filler, extender, binder, wetting agent, disintegrant, surfactant and other diluents or excipients can be used for preparation.

[0044] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" refers to an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level may depend on factors including the type of the patient's disease, severity, activity of the drug, sensitivity to the drug, administration time, route of administration, and excretion rate, treatment period, drugs used simultaneously, and other factors known in the medical field. The pharmaceutical composition of the present invention can be administered as a single therapeutic agent or in combination with other therapeutic agents, can be administered sequentially or simultaneously with existing therapeutic agents, and can be administered in a single or multiple doses. Taking all of the above factors into account, it is important to administer the drug in a minimum amount to obtain the maximum effect without side effects, which can be easily determined by those skilled in the art.

[0045] The pharmaceutical composition of the present invention can be administered to an individual by various routes. All modes of administration can be envisioned, for example, administration can be carried out by oral administration, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, injection into the periarticular space (intradura), sublingual administration, oral mucosal administration, rectal insertion, vaginal insertion, ocular administration, ear administration, nasal administration, inhalation, spraying through the mouth or nose, dermal administration, and transdermal administration.

[0046] The pharmaceutical composition of the present invention depends on various relevant factors such as the disease to be treated, the route of administration, the age, sex, weight of the patient, and the severity of the disease, as well as the type of drug as the active ingredient.

[0047] In the present invention, "individual" refers to a subject for whom disease treatment is required, and more specifically refers to a human or non-human primate, mouse, rat, dog, cat, horse, cow or other mammal.

[0048] In the present invention, "administering" means providing a prescribed composition of the present invention to an individual by any appropriate method.

[0049] In the present invention, "prevention" refers to all actions that inhibit or delay the onset of the target disease, "treatment" refers to all actions that improve or beneficially change the symptoms of the target disease and its metabolic abnormalities by administering the pharmaceutical composition of the present invention, and "improvement" refers to all actions that reduce parameters related to the target disease, such as the severity of symptoms, by administering the composition of the present invention.

[0050] As one embodiment of the present invention, the present invention provides a vaccine composition comprising a gene vector, wherein the gene vector comprises a nucleic acid molecule comprising the metal nanoparticles of the present invention and a gene of interest. The term "vaccine" is a biological preparation comprising an antigen that provides immunity to an individual, and refers to an immunogenic or antigenic substance that produces immunity by injection or oral administration to humans or animals in order to prevent disease.

[0051] The vaccine may be a DNA vaccine. The "DNA vaccine" refers to a vaccine that induces an immune response by artificially replicating part of the genes of pathogens or viruses and then administering the vaccine. The vaccine composition may form immunity against various infectious diseases, genetic diseases, other diseases or cancers.

[0052] The vaccine composition can be injected into an individual in various forms. The "injection" can be performed by any method selected from the group consisting of subcutaneous injection, intramuscular injection, subcutaneous injection, intraperitoneal injection, nasal administration, oral administration, transdermal administration and oral administration, and more preferably, it can be administered by any route suitable for administration of DNA vaccines, such as subcutaneous injection, intramuscular injection, intraperitoneal injection or intravenous injection.

[0053] The vaccine composition may include one or more adjuvants to improve or enhance the immune response. Suitable adjuvants may include a composition consisting of peptides, aluminum hydroxide, aluminum phosphate, aluminum oxide, mineral oils such as Marcol 52 or vegetable oils, and one or more emulsifiers, or surface active substances such as lysolecithin, polycations, polyanions, etc.

[0054] As another embodiment of the present invention, the present invention relates to a composition for diagnosing or detecting a target substance, comprising a gene vector, wherein the gene vector comprises a nucleic acid molecule containing the above-mentioned metal nanoparticles and a gene of interest.

[0055] As another embodiment of the present invention, the present invention relates to a composition for delivering a gene of interest into a cell, which comprises the metal nanoparticles and a nucleic acid molecule of the gene of interest.

[0056] Furthermore, as another embodiment of the present invention, the present invention relates to a method for preparing a gene carrier, the method comprising: modifying the surface of the metal nanoparticles by treating the metal nanoparticles with an acidic solution; combining the surface of the metal nanoparticles with a nucleic acid molecule containing one or more genes of interest that are delivered to and expressed in cells.

[0057] Furthermore, as another embodiment of the present invention, the present invention relates to a method for expressing a gene of interest through a nucleic acid molecule that is bound to the surface of a metal nanoparticle and expressed alone in a cell.

[0058] Effects of the Invention

[0059] The present invention relates to a gene carrier, a preparation method and use thereof. The gene carrier comprises metal nanoparticles and nucleic acid molecules containing a gene of interest bound to the surface of the metal nanoparticles, the nucleic acid molecules containing more than one gene, especially nucleic acid molecules in the form of double-helix DNA, which are directly bound to the surface of gold nanoparticles through covalent bonds and delivered to cells for expression, thereby achieving stable delivery and expression, and having the effect of being used as a gene therapy agent, a vaccine and a composition for diagnosing diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 Schematic diagram showing the structure of a nucleic acid molecule containing a gene of interest bound to gold nanoparticles in one embodiment of the present invention.

[0061] Figure 2 The results show the binding efficiency of nucleic acid molecules bound to the gold nanoparticles of the present invention via thiolated residues.

[0062] Figure 3 The results are shown for confirming whether or not a gene is delivered into cells based on the thiolated chains ((+), (-)) in the double-helix DNA bound to the gold nanoparticles of the present invention.

[0063] Figure 4 The following is an example of preparing AuNP-dsDNA in which thiolated double-stranded DNAs of different lengths are bound to gold nanoparticles and delivered to cells for expression.

[0064] Figure 5 Results showing the functionalization of thiolated double-helix DNA and its binding to the surface of gold nanoparticles are shown.

[0065] Figure 6 The graph shows the results of confirming gene delivery and expression in a small animal model using gold nanoparticle carriers loaded with double-stranded DNA of various lengths of SEQ ID NOs: 1 to 4.

[0066] Figure 7 Shown are the results of functionalizing the luciferase (SEQ ID NO: 5) gene onto the surface of gold nanoparticles.

[0067] Figure 8 The results of confirming whether the luciferase gene was delivered into cells via the gold nanoparticle carrier and expressed are shown. DETAILED DESCRIPTION

[0068] 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.

[0069] Example 1. Preparation of gold nanoparticles bound to double-stranded DNA (GFP) (AuNP-dsDNA)

[0070] The AuNP-dsDNA that can be delivered into cells and expressed by combining thiolated double-helix DNA with gold nanoparticles for intracellular expression was prepared by the following steps, and a simplified schematic diagram thereof is shown in Figure 1 middle.

[0071] 1-1. Preparation of dsDNA capable of expressing antigens in cells

[0072] The gene to be delivered was cloned between the CMV promoter and bGH terminator of the plasmid pcDNA3.1. The thiolated residues of the gene were thiolated using PCR primers to synthesize double-helix DNA with thiolation at the 5' end, 3' end or internal residues.

[0073] As the delivery target gene, the eGFP gene of SEQ ID NO: 1 was cloned by the method, and the 5' terminal thiolation primer sequence shown in Table 1 below was used to synthesize thiolated duplex DNA of eGFP in order to thiolate the 5' terminal, 3' terminal or internal residues.

[0074] Table 1

[0075]

[0076]

[0077] Add μl of 1N dithiothreitol (DTT) and react at room temperature for 60 minutes. To remove DTT containing unwanted thiol molecules, add 200 μl of ethyl acetate and mix, then remove the supernatant by centrifugation, and repeat this process 3 times. Then, precipitate the thiolated double-helix DNA using the EtOH precipitating method.

[0078] 1-3. Preparation of dsDNA-functionalized gold nanoparticles (AuNP-dsDNA)

[0079] The thiolated double helix DNA precipitated by pretreatment in the 1-2 process was dissolved in water, then added to the gold nanoparticles, and then combined by the salt aging method. Specifically, the thiolated double helix DNA (AuNP: thiolated double helix DNA = 1:40) was added to the 7nm gold nanoparticles and fully mixed, and then NaCl was added to a concentration of 0.1M and mixed for 4 hours. After 4 hours, NaCl was added to a concentration of 0.2M and mixed for 4 hours. After 4 hours, NaCl was added to a concentration of 0.3M and mixed for 12 hours.

[0080] After 12 hours, the mixture was centrifuged at 10,000 x g for 20 minutes to collect the thiolated duplex DNA and gold mixture, and then the unreacted duplex DNA in the supernatant was removed. This process was repeated 3 times.

[0081] The final AuNP-thiolated double-stranded DNA conjugate (AuNP-thiolated dsDNA) was dispersed in 10 mM sodium phosphate buffer (pH 7.4) containing 0.1 M NaCl. The prepared AuNP-thiolated double-stranded DNA conjugate was analyzed by electrophoresis on a 10% acrylamide 8M urea gel, and it was confirmed that one gold nanoparticle was bound to 1.98 to 9.27 thiolated double-stranded DNAs ( Figure 2 ).

[0082] Example 2. Confirmation of gene delivery and expression via AuNP-dsDNA (GFP) in a small animal model

[0083] For the convenience of the experiment, a xenograft tumor model was used. HeLa cells were injected into 6-week-old immunodeficient BALB / c-nu / nu mice (Central Lab Animal Inc, Korea) to induce cervical cancer, and AuNP-thiolated dsDNA was injected into the xenograft tumor. Then, 36 hours later, the xenograft tumor was removed and the expression of the injected GFP was observed using a Fluorescence In Vivo Imaging System (FOBI). As a result, it was confirmed that the injected tumor expressed the (+) chain or (-) chain or the 5' end of the (+) and (-) chains was thiolated or had a thiol group inside. Figure 3 ).

[0084] Example 3. Preparation of gold nanoparticles bound to dsDNA of different lengths

[0085] AuNP-dsDNA that can be delivered into cells and expressed was prepared by combining thiolated double-stranded DNA with different lengths with gold nanoparticles, and a simple schematic diagram is shown in Figure 4 middle.

[0086] 3-1. Preparation of double-stranded DNA that can be expressed in cells

[0087] The eGFP (SEQ ID NO: 1, SEQ ID NO: 2), A3APO-FLAG (SEQ ID NO: 3) and pFOX-FLAG (SEQ ID NO: 4) genes contained between the CMV promoter and the bGH terminator of the plasmid pcDNA3.1 were cloned respectively. The thiolated residues of the genes were synthesized by PCR using 5'-thiolated primers to synthesize double-helix DNAs with thiolated residues at the 5' end, 3' end or internal residues.

[0088] The synthesized thiolated double-stranded DNA was pretreated in the same manner as in Experimental Example 1-2, and the thiolated double-stranded DNA was precipitated using the EtOH precipitating method.

[0089] 3-2. Preparation of dsDNA-functionalized gold nanoparticles (AuNP-dsDNA)

[0090] The thiolated double-stranded DNA synthesized and precipitated by pretreatment in the above 3-1 process was functionalized onto the surface of the gold nanoparticles by the same process as in 1-3. The prepared AuNP-thiolated dsDNA conjugates were analyzed by electrophoresis on a 10% acrylamide 8M urea gel, and it was confirmed that one gold nanoparticle was bound to 2.38 to 5.67 thiolated double-stranded DNAs ( Figure 5 ).

[0091] Example 4. Confirmation of gene delivery and expression of gold nanoparticles loaded with dsDNA of different lengths in small animal models

[0092] For the convenience of the experiment, a xenograft tumor model was used. HeLa cells were injected subcutaneously into 6-week-old immunodeficient BALB / c-nu / nu mice (Central Lab Animal Inc, Korea) to induce tumors, and AuNP-thiolated dsDNA bound to double-helix DNA of SEQ ID NO: 1 to 4 (6kbp, 1.7kbp or 0.5kbp) was injected around the tumor. 36 hours after injection, the xenograft tumor or gastrocnemius muscle was removed and sections were generated by frozen sectioning, and the expression of proteins caused by the injected dsDNA was observed by immunofluorescence staining. As a result, protein expression of 6kbp, 1.7kbp or 0.5kbp dsDNA of SEQ ID NO: 1 to 4 was confirmed in the mouse tumor or gastrocnemius muscle, respectively. ( Figure 6 )

[0093] Example 5. Preparation of gold nanoparticles expressing luciferase

[0094] In order to confirm whether the gold nanoparticle carrier is well expressed in mouse tissues in vivo, AuNP-dsDNA capable of being delivered into cells and expressed was prepared and confirmed by combining thiolated double-stranded DNA expressing luciferase with gold nanoparticles.

[0095] 5-1. Preparation of double-stranded DNA that can be expressed in cells

[0096] The luciferase gene (SEQ ID NO: 5) contained between the CMV promoter and the bGH terminator of the plasmid pcDNA3.1 was cloned. The thiolated residues of the gene were synthesized by PCR using 5'-thiolated primers to synthesize double-helix DNA with thiolated residues at the 5' end, 3' end or internal residues.

[0097] The synthesized thiolated double-stranded DNA was pretreated in the same manner as in Experimental Example 1-2, and the thiolated double-stranded DNA was precipitated using the EtOH precipitating method.

[0098] 5-2. Preparation of dsDNA-functionalized gold nanoparticles (AuNP-dsDNA)

[0099] The thiolated double-stranded DNA synthesized and precipitated by pretreatment in the above 5-1 process was functionalized onto the surface of the gold nanoparticles in the same process as in 1-3. The prepared AuNP-thiolated dsDNA conjugate was subjected to electrophoresis analysis on a 10% acrylamide 8M urea gel, confirming that one gold nanoparticle was bound to 2.4 thiolated double-stranded DNAs. ( Figure 7 )

[0100] Example 6. Confirmation of gene delivery and expression via AuNP-dsDNA (luciferase) in a small animal model

[0101] Physiological saline or AuNP-thiolated dsDNA was injected into the gastrocnemius muscle of 7-week-old Balb / c mice on both sides. As a control group, the corresponding amount of AuNP or luciferase dsDNA was injected. After injection, the mice were anesthetized with isoflurane and D-luciferin was injected intraperitoneally every 12 hours or 24 hours to measure the activity of luciferase. The results of imaging using the Luciferase In Vivo Imaging System (LUCI) showed that the enzyme activity of luciferase was confirmed in the gastrocnemius muscle injected with luciferase dsDNA bound to AuNP. ( Figure 8 )

[0102] 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.

[0103] Embodiments of the present invention

[0104] As one embodiment of the present invention, the present invention relates to a gene carrier, which comprises: metal nanoparticles; and nucleic acid molecules, which are combined with the metal nanoparticles and contain one or more genes of interest that are delivered into cells and expressed.

[0105] The nucleic acid molecule comprises a promoter operably linked to a gene of interest.

[0106] The gene of interest is not replicated within the cell.

[0107] The gene of interest is not integrated into the genome of the injected cells.

[0108] The gene of interest produces a product in the cell through transcription and / or translation.

[0109] The product produced by the transcription and / or translation may be selected from the group consisting of mRNA, non-coding RNA, protein, antigen or antibody.

[0110] The nucleic acid molecules are expressed individually or independently in the cell.

[0111] The nucleic acid molecule is bound to the surface of the metal nanoparticle via one or more thiolated residues.

[0112] One or more thiolated residues may be contained at the 3' end, the 5' end or the base sequence of the nucleic acid molecule.

[0113] The size of the metal nanoparticles is 5 nm to 500 nm.

[0114] The metal nanoparticles may be gold nanoparticles.

[0115] The nucleic acid molecule may be selected from DNA, RNA or DNA / RNA molecules.

[0116] The DNA is double-helix DNA.

[0117] The nucleic acid molecule may have a length of 100 bp, 200 bp or more than 300 bp.

[0118] As another embodiment of the present invention, the present invention relates to a pharmaceutical composition comprising the above gene vector.

[0119] As another embodiment of the present invention, the present invention relates to a composition for detecting a target substance, which comprises the above-mentioned gene vector.

[0120] As another embodiment of the present invention, the present invention relates to a composition for delivering genes of interest into cells, comprising: metal nanoparticles; and nucleic acid molecules, which are combined with the metal nanoparticles and contain one or more genes of interest that are delivered into cells and expressed.

[0121] As another embodiment of the present invention, the present invention relates to a method for preparing a gene carrier, which comprises the following steps: modifying the surface of the metal nanoparticles by treating the metal nanoparticles with an acidic solution; and combining the surface of the metal nanoparticles with a nucleic acid molecule containing one or more genes of interest that are delivered to and expressed in cells.

[0122] As another embodiment of the present invention, the present invention relates to a method for expressing a gene of interest via a nucleic acid molecule that is bound to the surface of a metal nanoparticle and expressed alone in a cell.

Claims

1. A gene vector, characterized in that: Include: Metal nanoparticles; and A nucleic acid molecule is bound to the metal nanoparticle and comprises one or more genes of interest that are delivered into cells and expressed.

2. The gene carrier according to claim 1, characterized in that The nucleic acid molecule comprises a promoter operably linked to a gene of interest.

3. The gene carrier according to claim 1, characterized in that The gene of interest is not replicated within the cell.

4. The gene carrier according to claim 1, characterized in that The gene of interest is not integrated into the genome of the injected cells.

5. The gene carrier according to claim 1, characterized in that The gene of interest produces a product in the cell through transcription and / or translation.

6. The gene carrier according to claim 5, characterized in that The product produced by the transcription and / or translation is one or more selected from the group consisting of mRNA, non-coding RNA, protein, antigen or antibody.

7. The gene carrier according to claim 1, characterized in that The nucleic acid molecules are expressed individually or independently in the cell.

8. The gene carrier according to claim 1, characterized in that The nucleic acid molecule is bound to the surface of the metal nanoparticle via one or more thiolated residues.

9. The gene carrier according to claim 8, characterized in that One or more thiolated residues are contained at the 3' end, the 5' end or the base sequence of the nucleic acid molecule.

10. The gene carrier according to claim 1, characterized in that The size of the metal nanoparticles is 5 nm to 500 nm.

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

12. The gene carrier according to claim 1, characterized in that The nucleic acid molecule is selected from DNA, RNA or DNA / RNA molecules.

13. The gene carrier according to claim 12, characterized in that The DNA is double-helix DNA.

14. The gene carrier according to claim 1, characterized in that The nucleic acid molecule has a length of 100 bp, 200 bp or more than 300 bp.

15. A pharmaceutical composition, characterized in that Comprising the gene vector according to claim 1.

16. A composition for detecting a target substance, characterized in that: Comprising the gene vector according to claim 1.

17. A composition for delivering a gene of interest into a cell, characterized in that: Include: Metal nanoparticles; and A nucleic acid molecule is bound to the metal nanoparticle and comprises one or more genes of interest that are delivered into cells and expressed.

18. A method for preparing a gene vector, characterized in that: The steps include: modifying the surface of the metal nanoparticles by treating the metal nanoparticles with an acidic solution; and The surface of the metal nanoparticle is bound to a nucleic acid molecule comprising one or more genes of interest that are delivered into cells and expressed.

19. A method for expressing a gene of interest, characterized in that: Expression is via nucleic acid molecules that are bound to the surface of metal nanoparticles and expressed individually within cells.

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