Vaccine compositions comprising double helix DNA-binding gold nanoparticle carriers

Abstract

The present invention relates to a vaccine composition comprising double helix DNA transported into cells through gold nanoparticles, and more specifically, to a vaccine composition characterized in that the double helix DNA is derived from a virus, bacterial or oncogene and expresses an antigen to induce an immune response.

Description

Technical Field

[0001] The present invention relates to use of a gene carrier comprising gold nanoparticles and double-helix DNA bound to the surface of the gold nanoparticles as a vaccine. Background Art

[0002] In recent years, especially due to the recent epidemic, the technology of delivering genetic material into cells to produce antigens or proteins for treatment or prevention has developed significantly. Therefore, in addition to antigen vaccines, various gene vaccines are also being developed, such as DNA vaccines, mRNA (messenger RNA) vaccines or viral vector vaccines.

[0003] DNA is the simplest genetic material and is easy to genetically modify, thus shortening the R&D cycle for therapeutic agents such as vaccines. Furthermore, compared to other vaccine candidates such as viruses, proteins, and RNA, it is very economical in terms of production equipment construction and production costs, and has the advantages of excellent stability and ease of storage and transportation. However, DNA vaccines require delivery to the cell nucleus and direct production of mRNA within the nucleus, primarily using vectors such as plasmids for delivery. DNA injected into the nucleus can continuously produce mRNA and antigenic proteins, but since other genetic traits are injected into the cell nucleus in our bodies for delivery, there is a risk of side effects such as innate immune responses. In other words, for plasmids primarily used to transport DNA, there is a risk of side effects due to the potential for delivering bacterial genes. In view of this, in order to deliver and express therapeutic substances, various studies are underway on carriers for safely delivering genes into cells, with a particular need to develop delivery materials that improve delivery efficiency and stably express therapeutic substances.

[0004] In particular, with the recent outbreaks of large-scale infectious diseases such as coronaviruses and influenza, the rapid development of vaccines to combat these diseases has become extremely important. In response to the recent COVID-19 pandemic, in addition to existing antigen vaccines, various other vaccine types have been developed, including DNA vaccines and mRNA vaccines, along with various vaccine delivery vehicles for efficient delivery. Specifically, DNA vaccines offer the advantage of ensuring safety compared to temperature-sensitive and structurally unstable mRNA vaccines, but since they must be delivered into the nucleus, an efficient delivery technology is required. Summary of the Invention

[0005] Technical issues

[0006] In view of this, in order to effectively deliver non-cytotoxic, intracellularly expressed double-stranded DNA (dsDNA) to the cell nucleus, the present inventors have conducted extensive research and efforts to develop double-stranded DNA delivery technology. As a result, they invented a delivery system that can covalently attach thiolated double-stranded DNA to the surface of gold nanoparticles (AuNPs) to deliver genes to the cell nucleus. In addition, the present invention completes a gene carrier that can be used as a vaccine by introducing the double-stranded DNA into cells to express antigens alone.

[0007] Therefore, the object of the present invention is to provide a vaccine composition comprising: gold nanoparticles; and double-helix DNA, which is bound to the surface of the gold nanoparticles via residues containing one or more functional groups and expresses antigens alone in cells.

[0008] Technical Solution

[0009] To achieve the above objectives, the present invention provides a vaccine composition comprising: gold nanoparticles; and double-helix DNA, which is bound to the surface of the gold nanoparticles via residues containing one or more functional groups and expresses antigens alone in cells.

[0010] Hereinafter, the present invention will be described in detail.

[0011] The present invention relates to a vaccine composition comprising: gold nanoparticles; and double-helix DNA, which is combined with the surface of the gold nanoparticles via one or more thiolated residues and expresses antigens independently in cells.

[0012] The double-helix DNA of the present invention is intended to be introduced into cells for expression. The type of the double-helix DNA is not limited, and specifically can include cDNA, gDNA, plasmid DNA, and PCR DNA.

[0013] The double-stranded DNA is combined with gold nanoparticles in a double-helix form, and is intended to be transported into cells. This is different from single-stranded DNA, which, after combining with gold nanoparticles, enters the cell and hybridizes with the complementary sequence of the single strand. Specifically, the double-helix DNA can be derived from a gene capable of expressing an antigen. The antigen-expressing gene can be derived from a virus, bacteria, or cancer cell.

[0014] In addition, the double-helical DNA contains one or more genes that can be expressed independently after being introduced into the cell. In the present invention, the "independent expression" means that the double-helical DNA is transcribed and / or translated independently without being integrated into the genome of the cell into which it is injected. In order to express independently, the double-helical DNA may contain one or more promoters, open reading frames (open reading frames) or terminators, but is not limited thereto. Furthermore, the double-helical DNA can be transcribed and / or translated and produce its results, but is not limited thereto.

[0015] The vaccine composition of the present invention comprises a gene delivery agent, wherein the double-helix DNA is bound to the surface of gold nanoparticles. The double-helix DNA comprises one or more functional groups for binding to the surface of gold nanoparticles. The functional groups can be thiol groups or amine groups and can be contained in one or more residues of the double-helix DNA. In one embodiment of the present invention, the double-helix DNA comprises thiolated residues, thereby directly binding to the surface of gold nanoparticles. The thiolated residues can be one or more bases at the 3' end, 5' end, or in the double-helix DNA sequence.

[0016] In addition, one or more of the double-stranded DNAs are bound to the surface of the gold nanoparticles, but the present invention is not limited thereto. From 1 to 20 identical or different double-stranded DNAs can be bound to the surface of the gold nanoparticles for expression. Furthermore, the size of the bound double-stranded DNAs is not limited and can be greater than 10 bp, greater than 100 bp, or greater than 500 bp.

[0017] In one embodiment of the present invention, the double-stranded DNA is a 720 bp RBD DNA sequence derived from a SARS-CoV vaccine or a 1500 bp or longer DNA sequence derived from human papillomavirus (HPV), but is not limited to these types.

[0018] In the present invention, the term "vaccine" refers to an immunogenic or antigenic substance, which is a biological preparation containing an antigen that provides immunity to an individual, and is administered to humans or animals by injection or oral administration to produce immunity to prevent diseases.

[0019] The vaccines described herein may be DNA vaccines. "DNA vaccines" are vaccines that induce an immune response by artificially replicating partial genes of pathogens or viruses and administering them. Compared to existing protein vaccines, these DNA vaccines offer several advantages: i) Since synthetic production requires only the genetic information of the pure target pathogen antigen, there is no need to directly handle the dangerous pathogen; ii) Since only the partial genes required to induce toxicity are used, there is no concern about the expression of any other toxicity even when administered to subjects; and iii) Since they are simply composed of DNA, vaccines can be developed to rapidly respond to sudden outbreaks of various infectious diseases.

[0020] The vaccine composition can develop immunity against various infectious diseases or cancers. The vaccine composition can be injected into an individual in a variety of forms. The "injection" can be performed by any method selected from the group consisting of subcutaneous injection, intramuscular injection, intradermal injection, intraperitoneal injection, nasal administration, oral administration, transdermal administration, and oral administration. More preferably, the vaccine composition can be administered by any route suitable for administering a DNA vaccine, such as subcutaneous injection, intramuscular injection, intraperitoneal injection, or intravenous injection.

[0021] In addition, the vaccine composition may include one or more adjuvants to improve or enhance the immune response. Suitable adjuvants may include peptides, aluminum hydroxide, aluminum phosphate, aluminum oxide and mineral oil (e.g., Marcol 52) or vegetable oil and a composition consisting of one or more emulsifiers or a surfactant (e.g., lysophospholipids, polycations, polyanions) and the like.

[0022] In one embodiment of the present invention, the double-stranded DNA contained in the vaccine composition can be used to encode an antigen, which includes a protein, peptide, or fragment thereof of a coronavirus (SARS-CoV2). The coronavirus antigen can be, but is not limited to, a SARS-CoV2 spike protein, a SARS-CoV2 nucleocapsid protein, a SARS-CoV2 envelope protein, a SARS-CoV2 matrix protein, a fragment thereof, a variant thereof, or a combination thereof. The antigen can be used to treat, prevent, and / or prevent SARS-CoV-2-based pathologies by preventing and treating any number of SARS-CoV2 strains.

[0023] The vaccine composition of the present invention can significantly induce an immune response in the subject after administration, and can simultaneously induce humoral immune response and cellular immune response, thereby achieving the effect of preventing and treating SARS-CoV-2 infection.

[0024] The gold nanoparticles contained in the vaccine composition of the present invention are nanometer-sized gold particles, preferably 5 to 500 nm in diameter, more preferably 10 to 200 nm in diameter. They are easily formed into stable particles, their size is easily controlled, and unlike heavy metals such as manganese, aluminum, cadmium, lead, mercury, cobalt, nickel, and beryllium, they are harmless to the human body, thus exhibiting high biocompatibility. However, when the diameter of gold nanoparticles exceeds 500 nm, they not only lose their nanoparticle properties, but also weaken the binding force between the gold surface, which lacks the properties of a nanomaterial, and functional groups such as thiol groups, making it difficult to manufacture delivery materials using gold nanoparticles.

[0025] Effects of the Invention

[0026] The present invention relates to a vaccine composition comprising double-helix DNA delivered into cells via gold nanoparticles. More specifically, the double-helix DNA expresses antigens to induce immune responses, which can prevent and treat various viral and bacterial infectious diseases and cancers. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The figure shows the structure of a nucleic acid molecule comprising a target gene bound to gold nanoparticles according to one embodiment of the present invention.

[0028] Figure 2 Results showing the binding efficiency of nucleic acid molecules expressing SARS-CoV-2-RBD bound to the gold nanoparticles of the present invention via thiolated residues.

[0029] Figure 3 Shown are the results of delivering double-stranded DNA expressing SARS-CoV-2-RBD into cells for expression.

[0030] Figure 4 The results of in vivo injection of SARS-CoV-2-RBD DNA bound to gold nanoparticles and expression of antigens are shown.

[0031] FIG5 shows the results of whether SARS-CoV-2 antibodies are produced after in vivo injection of SARS-CoV-2-RBD DNA bound to gold nanoparticles.

[0032] Figure 6 Results are shown for the binding efficiency of nucleic acid molecules expressing HPV 16_L1 and HPV 18_L1 bound to the gold nanoparticles of the present invention via thiolated residues.

[0033] Figure 7 The results show whether HPV 16_L1 and HPV 18_L1 double-stranded DNAs are expressed after being delivered to cells.

[0034] Figure 8 The results show whether antibodies are produced after in vivo injection of HPV 16_L1 and HPV 18_L1 DNA bound to gold nanoparticles. DETAILED DESCRIPTION

[0035] In order to specifically illustrate this specification, the following will be described in detail by way of examples. However, the embodiments of this specification can be modified in various forms, and the scope of this specification should not be understood to be limited to the following examples. The embodiments of this specification are intended to more fully illustrate this specification to those skilled in the art.

[0036] Example 1. Preparation of gold nanoparticles bound to dsDNA (SARS-CoV2)

[0037] Double-helix DNA derived from coronavirus (SARS-CoV2) is combined with gold nanoparticles to create NESDNA that can be delivered and expressed inside cells. Figure 1 A schematic diagram thereof is shown.

[0038] 1-1. Production of dsDNA capable of expressing antigens in cells

[0039] The gene to be delivered was cloned between the CMV promoter and the bGH terminator of the plasmid pcDNA3.1. Thiolated residues of the gene were detected using thiolated primers to synthesize double-stranded DNA with thiolation at the 5' end, 3' end or internal residues by PCR.

[0040] The double-stranded DNA used in the synthesis is a sequence encoding the RBD domain of the spike protein of SARS-CoV2 (δ) (SEQ ID NO. 1). As the gene to be delivered, the SARS-CoV2 (δ) RBD gene of SEQ ID NO. 1 was cloned by the method described above, and the thiolated double-stranded DNA was synthesized by PCR using thiolated primers.

[0041] 1-2. Pretreatment of thiolated duplex DNA

[0042] The synthesized thiolated duplex DNA was dissolved in water to a final concentration of 1 μM. Then, 20 μL of 3M sodium acetate (pH 5.2) and 30 μL of 1N dithiothreitol (DTT) were added to 150 μL of the thiolated duplex DNA and allowed to react at room temperature for 60 minutes. To remove unwanted DTT containing thiol groups, 200 μL of ethyl acetate was added and mixed, followed by centrifugation and removal of the supernatant. This process was repeated three times. The thiolated duplex DNA was then precipitated using EtOH.

[0043] 1-3. Fabrication of Double-Helix dsDNA-Functionalized Gold Nanoparticles (NESDNA)

[0044] The thiolated duplex DNA precipitated in steps 1-2 above was dissolved in water and added to gold nanoparticles for binding via salt aging. Specifically, the thiolated duplex DNA was added to 7 nM gold nanoparticles (AuNP:thiolated duplex DNA = 1:40), mixed thoroughly, and then NaCl was added to a concentration of 0.1 M and mixed for 4 hours. After 4 hours, NaCl was added to a concentration of 0.2 M and mixed for 4 hours. After 4 hours, NaCl was added to a concentration of 0.3 M and mixed for 12 hours.

[0045] After 12 hours, the mixture of thiolated duplex DNA and gold was collected by centrifugation at approximately 10,000 × g for 20 minutes, and the unreacted duplex DNA in the supernatant was removed. This process was repeated three times.

[0046] The final AuNP-thiolated double-stranded DNA complex (AuNP-thiolated dsDNA) was dispersed in 10mM sodium phosphate buffer (pH 7.4) containing 0.1M NaCl. The prepared AuNP-thiolated double-stranded DNA complex was analyzed by electrophoresis on a 1% agarose gel. The results showed that each gold nanoparticle was bound to 10 to 15 thiolated double-stranded DNA ( Figure 2 ).

[0047] Example 2. In vitro identification of RBD gene protein expression

[0048] To verify the intracellular expression of the duplex DNA produced in Example 1, an experiment was conducted. First, plasmid DNA and the thiolated duplex DNA from Example 1 were transfected into HeLa cells using Lipofectamine 3000 (Invitrogen). Twenty-four hours later, the cells were washed with 1× PBS, harvested with a scraper, and lysed using NP-40 lysis buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 1% NP-40) containing a 1× protease inhibitor cocktail (GenDEPOT).

[0049] After centrifugation, the total protein concentration in the sample supernatant was measured using a BCA assay kit (Thermo Scientific). 30 μg of total protein in each sample was mixed with protein stain (6× standard, 0.3 M Tris-HCl (pH 6.8), 12% SDS, 60% glycerol, 0.6 M β-mercaptoethanol, 0.0025% bromophenol blue), subjected to electrophoresis on a 12% SDS-PAGE gel, and analyzed by Western blotting.

[0050] The results showed that thiolated double-stranded DNA normally expressed RBD protein in cells ( Figure 3).

[0051] Example 3. Characterization of gene delivery and expression of NESDNA (SARS-CoV2 RBDδ) in small animal models

[0052] To identify the antigen expression ability of double-helix DNA (SARS-CoV2 RBDδ: NESDNA) delivered into cells by binding to gold nanocarriers, the following experiments were performed.

[0053] NES DNA (SARS-CoV2 RBDδ) was injected into the gastrocnemius muscles of 6-week-old Balb / c mice (Central Lab Animal Inc, Korea). Thirty-six hours later, the gastrocnemius muscles were isolated and fixed with 4% PFA. The tissues were cryopreserved in 1× PBS containing 10%, 20%, and 30% sucrose, respectively. The cryopreserved tissues were rapidly frozen in FSC22 OCT compound (Leica Microsystems), and 20 μm-thick cryosections were prepared.

[0054] Using immunofluorescence staining, the protein expression in the slices was observed under a fluorescence microscope. Figure 4 As shown, the expression of RBDδ protein in the gastrocnemius muscle tissue injected with NESDNA (SARS-CoV2 RBDδ) was confirmed.

[0055] Example 4. In vivo identification of antibodies produced by NESDNA (SARS-CoV2 RBDδ)

[0056] In vivo experiments were performed to measure the concentration of binding antibody titers and competitive binding assays for detecting neutralizing antibody titer activity. First, Balb / c mice were vaccinated twice with the NESDNA (SARS-CoV2 RBDδ) vaccine, two weeks apart. Orbital blood was collected from the mice, and the blood was coagulated at room temperature and centrifuged to separate the serum.

[0057] To measure the binding antibodies against spike RBDδ produced in mouse blood, the separated serum was applied to a 96-well plate coated with RBD protein and identified. Competitive binding of the antibodies in the serum was performed by ELISA using a 96-well plate coated with human ACE2 protein and HRP-conjugated RBDδ protein.

[0058] The results are shown in FIG5 , which confirmed that binding antibodies and neutralizing antibodies against RBD were detected in the serum of mice vaccinated with the NESDNA of the present invention.

[0059] Example 5. Preparation of gold nanoparticles bound to double-stranded DNA (HPV)

[0060] To confirm whether intracellular delivery and expression can be achieved by conjugating double-helical DNA derived from human papillomavirus (HPV) to gold nanoparticles, the following experiments were performed.

[0061] First, HPV 16_L1 and HPV 18_L1 (Table 2) were synthesized, which were HPV virus-derived DNA sequences of SEQ ID NO. 2 and SEQ ID NO. 3, respectively. Then, the surface of gold nanoparticles was functionalized using the same method as in Example 1 to produce NESDNA-HPV 16_L1 and NESDNA-HPV 18_L1, respectively. Figure 1 A schematic diagram thereof is shown.

[0062] The binding efficiency of DNA bound to the gold nanoparticle surface was confirmed. The results showed that in the case of HPV-derived DNA, each gold nanoparticle was bound to 10 to 15 thiolated double-helical DNAs ( Figure 6 ).

[0063] Example 6. In vitro identification of protein expression levels of HPV16 L1 and HPV18 L1 genes

[0064] To confirm the intracellular expression of the double-stranded DNA HPV 16_L1 and HPV 18_L1 prepared in Example 5, the plasmid DNA and thiolated double-stranded DNA were transfected into HeLa cells using Lipofectamine 3000 (Invitrogen). The HPV 16 L1 and HPV 18 L1 proteins expressed in this manner were analyzed by Western blotting using 12% SDS PAGE gels.

[0065] The results are as follows Figure 7 As shown, the normal expression of HPV16 L1 and HPV18 L1 proteins was confirmed.

[0066] Example 7. In vivo identification of antibodies produced by NES DNA (HPV16 L1, HPV18 L1)

[0067] In order to analyze the concentration of the antibody titer binding to the gold nanoparticles-bound DNA (NESDNA-HPV 16_L1, NESDNA-HPV 18_L1) synthesized in Example 5, the following experiment was performed.

[0068] Balb / c mice were vaccinated with NESDNA (HPV16 L1, HPV18 L1) vaccine twice, two weeks apart. Orbital blood was collected from the mice, and the blood was coagulated at room temperature and then centrifuged to separate the serum.

[0069] The above serum was used as a test, and the binding antibodies against HPV L1 produced in the mouse blood were detected by ELISA using a 96-well plate coated with HPV16 L1 VLP and HPV18 L1 VLP proteins.

[0070] The results are as follows Figure 8 As shown, it was confirmed that mice administered with NESDNA (HPV16L1, HPV18L1) vaccine developed antibodies compared to the non-administered group (control).

[0071] Implementation Method

[0072] In one aspect, the present invention relates to a vaccine composition comprising: gold nanoparticles; and double-helix DNA, which is bound to the surface of the gold nanoparticles via residues comprising one or more functional groups and expresses antigens alone in cells.

[0073] As an example, the antigen includes a viral, bacterial or cancer antigen.

[0074] As an example, the double-stranded DNA alone expresses the antigen without integrating into the genome of the cell into which it is injected.

[0075] As an example, the double-helical DNA is derived from the SARS-CoV2 virus.

[0076] As an example, the double-helical DNA is derived from human papillomavirus (HPV).

[0077] As an example, the residue comprising a functional group includes a thiol group or an amine group.

[0078] As an example, the residue containing the functional group is one or more residues at the 3' end, the 5' end, or within the base sequence of the double-stranded DNA.

[0079] As an example, the size of the gold nanoparticles is 5 nm to 500 nm.

[0080] In another aspect, the present invention relates to a method for expressing viral, bacterial or cancer antigens by delivering double-helical DNA into cells, wherein the double-helical DNA is bound to the surface of gold nanoparticles via residues containing one or more functional groups.

Claims

1. A vaccine composition, characterized in that Include: Gold nanoparticles; as well as The double-helix DNA is bound to the surface of the gold nanoparticles via residues containing one or more functional groups and expresses the antigen alone in the cell.

2. The vaccine composition according to claim 1, characterized in that The antigens include viral, bacterial or cancer antigens.

3. The vaccine composition according to claim 1, characterized in that The double-stranded DNA alone expresses the antigen without integrating into the genome of the cell into which it is injected.

4. The vaccine composition according to claim 1, characterized in that The double-helix DNA is derived from the SARS-CoV2 virus.

5. The vaccine composition according to claim 1, characterized in that The double-helix DNA is derived from human papillomavirus.

6. The vaccine composition according to claim 1, characterized in that The residue comprising a functional group includes a thiol group or an amine group.

7. The vaccine composition according to claim 1, characterized in that The residues containing the functional group are one or more residues at the 3' end, the 5' end or within the base sequence of the double-stranded DNA.

8. The vaccine composition according to claim 1, characterized in that The size of the gold nanoparticles is 5 nm to 500 nm.

9. A method for expressing viral, bacterial or cancer antigens by delivering double-stranded DNA into cells, characterized in that The double-helical DNA is bound to the surface of the gold nanoparticles via residues containing one or more functional groups.

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