Antiviral composition comprising nanoparticle carrier based on gold nanoparticle-nucleic acid conjugate

Abstract

The present invention relates to a nanoparticle carrier and an antiviral composition comprising same, the nanoparticle carrier comprising: gold nanoparticles; antisense oligonucleotides (ASOs) that complementarily bind to conserved RNA sequences derived from a viral genome; and ribonuclease H (RNase H) that binds to the ASOs to induce the cleavage of mRNA. The nanoparticle carrier and the antiviral composition comprising same can reduce mRNA expression of a virus by RNase H, and in particular, can have the effect of treating infectious diseases caused by a virus.

Description

Antiviral composition comprising a nanoparticle carrier based on a gold nanoparticle-nucleic acid conjugate

[0001] The present invention relates to a nanoparticle delivery vehicle comprising a gold nanoparticle and an antisense oligonucleotide complementarily binding to a viral RNA-derived sequence bound to the surface thereof; and ribonuclease H (RNase H) that induces cleavage of mRNA, and an antiviral composition comprising the same.

[0002] The coronavirus disease (COVID-19), which has become a global pandemic, is caused by the highly contagious SARS-CoV-2 virus, resulting in millions of deaths and triggering a global health crisis and economic losses. SARS-CoV-2 primarily spreads through the respiratory route, entering cells by binding to the ACE2 receptor on the human body via the viral spike protein (S protein). As various variants emerge, transmissibility and mortality rates change, and the effectiveness of currently available vaccines and antiviral treatments is continuously being evaluated.

[0003] Existing antiviral drugs primarily attempt to suppress viruses through protein-based approaches or chemical drugs. However, these approaches have limitations, such as reduced efficacy against mutant viruses, increased administration frequency, and high production costs. In contrast, technologies utilizing viral genetic material offer new possibilities, as they can effectively deliver genes (DNA, RNA, etc.) that express specific viral antigens within the body, inducing an immune response and inhibiting viral replication. To achieve this, various genetic material delivery systems are being developed. These vehicles enable rapid adaptation to genetic material variants and possess diverse antiviral properties, enabling a rapid and broad response, particularly against mutant viruses such as SARS-CoV-2.

[0004] Accordingly, the present inventors, while conducting research to manufacture a genetic material delivery vehicle having antiviral activity, discovered that an excellent antiviral effect against the virus can be achieved by combining an antisense oligonucleotide that complementarily binds to a nucleotide sequence derived from viral RNA, particularly included in a conserved 5'untranslated region, with a gold nanoparticle-conjugated delivery vehicle technology, and ribonuclease H (RNase H), which binds to the gold nanoparticle and causes cleavage of mRNA, and delivering the same, thereby completing the present invention.

[0005] Accordingly, the object of the present invention is to provide a nanoparticle delivery system comprising a gold nanoparticle and an antisense oligonucleotide (ASO) that complementarily binds to a viral RNA-derived sequence bound to the surface of the gold nanoparticle; and ribonuclease H (RNase H) that induces cleavage of mRNA.

[0006] Another object of the present invention is to provide an antiviral composition comprising the above nanoparticle carrier.

[0007] In order to achieve the above-described object of the present invention, the present invention provides a nanoparticle delivery system comprising a gold nanoparticle and an antisense oligonucleotide (ASO) that complementarily binds to a viral RNA-derived sequence bound to the surface of the gold nanoparticle; and ribonuclease H (RNase H) that induces cleavage of mRNA.

[0008] In order to achieve another object of the present invention, the present invention provides an antiviral composition comprising the above nanoparticle carrier.

[0009] Hereinafter, the present invention will be described in detail.

[0010] The present invention relates to a nanoparticle delivery system comprising a gold nanoparticle and an antisense oligonucleotide (ASO) that complementarily binds to a nucleotide sequence included in a viral RNA-derived sequence bound to the surface of the gold nanoparticle; and ribonuclease H (RNase H) that induces cleavage of mRNA.

[0011] The nanoparticle delivery vehicle of the present invention comprises an antisense oligonucleotide (ASO) that complementarily binds to a nucleotide sequence contained in a genomic RNA sequence located in the 5'untranslated region (UTR) derived from the viral genome.

[0012] Antisense oligonucleotides (ASOs) are single-stranded deoxyribonucleotides complementary to specific mRNAs, as well as noncoding RNAs such as microRNAs or long noncoding RNAs (Beermann, Piccoli, Viereck, & Thum, 2016; Bennett, 2019). Antisense oligonucleotides specifically bind to target RNA sequences through Watson–Crick base pairing and promote the degradation of bound RNA by endogenous nucleases such as RNaseH (Watts & Corey, 2012). Specifically, RNaseH can recognize RNA / DNA hybrid duplex structures and induce mRNA cleavage and genome degradation (Bennett, Baker, Pham, Swayze, & Geary, 2017).

[0013] That is, the term "antisense oligonucleotide (ASO)" in the present invention refers to an RNA or DNA molecule that binds to another RNA or DNA (target RNA, DNA). For example, if it is an RNA oligonucleotide, it binds to another RNA target through RNA-RNA interaction and modulates the activity of the target RNA. Antisense oligonucleotides can upregulate or downregulate the expression and / or function of a specific polynucleotide. This definition is intended to include any foreign RNA or DNA molecule that is useful from a therapeutic, diagnostic, or other perspective. As examples, antisense RNA or DNA molecules, interfering RNA (RNAi), microRNA, decoy RNA molecules, siRNA, enzymatic RNA, therapeutic editing RNA, and antisense oligomeric compounds, antisense oligonucleotides, external guide sequence (EGS) oligonucleotides, primers, probes, and other oligomeric compounds that hybridize to at least a portion of a target nucleic acid may be included. Therefore, these compounds can be introduced in the form of single-stranded, double-stranded, partially single-stranded, or circular oligomeric compounds.

[0014] As an example, the RNA-derived sequence of the virus of the present invention may be an antisense oligonucleotide (ASO) that complementarily binds to the 5' untranslated region (UTR) of the viral RNA and targets a nucleotide sequence included in the 5'UTR of the SARS-CoV-2 genomic RNA. The antisense oligonucleotide includes a sequence complementary to a nucleotide sequence included in the 5'UTR of the SARS-CoV-2 genomic RNA, and the nucleotide sequence may include a leader sequence, which is an RNaseH binding site, among the 5'UTR regions of SARS-CoV-2.

[0015] Additionally, the gold nanoparticle carrier of the present invention comprises ribonuclease RNase H, which induces cleavage of mRNA.

[0016] RNase H is an essential endonuclease involved in various genetic processes such as transcription and replication (Aguilera & Garcia-Muse, 2012; Crossley, Bocek, & Cimprich, 2019). During transcription, the RNA strand can combine with template DNA to form an RNA / DNA duplex, and the non-template strand is replaced by single-stranded DNA (ssDNA), forming a structure called an R-loop (Thomas, White, & Davis, 1976). R-loops can help regulate transcription, but when unnecessary, they can cause double-stranded DNA breaks (DSBs), chromosomal rearrangements, and hypermutations, all causes of genome instability (Richard & Manley, 2017). RNase H increases genome stability by degrading RNA in the RNA / DNA duplex and suppressing unnecessary R-loops (Nguyen et al., 2017). That is, "RNase H enzyme" is an enzyme that hydrolyzes RNA among RNA-DNA hybrids, and RNase H, which was first discovered in calf thymus, was subsequently discovered in many organisms.

[0017] The RNaseH bound to the gold nanoparticles of the present invention may be derived from various organisms, and is not limited to the derived organisms, but may be isolated from Pyrococcus furiosus, Pyrococcus horikoshi, Thermococcus litoralis, Thermus thermophilus, or E. coli. As an example, the RNaseH of the present invention may be RNaseH1, and may be an RNaseH having 50%, 60%, 70%, 80%, 90%, 95%, or 99% homology to the amino acid sequence of RNaseH1.

[0018] The antisense oligonucleotide (ASO) and RNaseH of the present invention are bound to the surface of gold nanoparticles.

[0019] The gold nanoparticles of the present invention are gold particles having a diameter in the nanometer unit, preferably 5-500 nm, more preferably 10-200 nm, which are easy to manufacture in the form of stable particles, easy to control the size, and are harmless to the human body unlike heavy metals such as manganese, aluminum, cadmium, lead, mercury, cobalt, nickel, and beryllium, and thus have high biocompatibility. When the diameter of gold nanoparticles increases to more than 500 nm, not only do their characteristics as nanoparticles disappear, but the bonding between the gold surface, which does not have the characteristics of a nanomaterial, and functional groups such as thiol groups becomes weak, so it is difficult to manufacture a carrier using gold nanoparticles.

[0020] The gold nanoparticles used in the present invention are not limited to the method, but can be manufactured as follows, for example: HAuCl4 is used as a gold source, and sodium citrate is used as a reducing agent to reduce HAuCl4 to manufacture gold nanoparticles. In this case, the size of the gold nanoparticles can be controlled by varying the amount of citrate added. That is, as the amount of citrate added increases, nucleation increases, and thus the size of the gold nanoparticles decreases.

[0021] The gold nanoparticles of the present invention include surface modification for binding antisense oligonucleotides and RNaseH to the surface. In addition, the antisense oligonucleotides and RNaseH may include one or more functionalities for binding to the surface of the gold nanoparticles.

[0022] The functional group may be a thiol group or an amine group, and may be included in one or more residues of the antisense oligonucleotide or the RNAI that binds to the antisense oligonucleotide. Although not limited thereto, the antisense oligonucleotide or the RNAI that binds to the antisense oligonucleotide may include one or more thiolated residues, thereby enabling direct binding to the surface of gold nanoparticles.

[0023] In one embodiment of the present invention, a thiolated RNAI oligo is bound to the surface of a gold nanoparticle, and the thiolated RNAI oligo binds the antisense oligonucleotide to the surface of the gold nanoparticle through specific binding to the antisense oligonucleotide sequence of the present invention.

[0024] In addition, the gold nanoparticles of the present invention may include an aptamer on the surface. In the present invention, the "aptamer" refers to a single-stranded nucleic acid (DNA, RNA, or modified nucleic acid) that has a stable tertiary structure in itself and has the characteristic of being able to bind to a target molecule with high affinity and specificity, and aptamers for various desired target substances (proteins, sugars, dyes, DNA, metal ions, cells, etc.) can be developed using a method called SELEX (Systematic Evolution of Ligands of Exponential Enrichment). In the present invention, the aptamer that binds to the gold nanoparticles may use any type of aptamer of various tags or protein-specific aptamer, particularly for binding RNase H to the surface of the gold nanoparticles, and is not particularly limited.

[0025] In one embodiment of the present invention, an aptamer including a histidine tag (His-tag) was bound to a gold nanoparticle, and RNaseH was bound to the surface of the gold nanoparticle through the histidine tag aptamer.

[0026] The gold nanoparticle carrier of the present invention comprises at least one antisense oligonucleotide bound to the surface of a gold nanoparticle and a histidine tag aptamer for RNaseH binding, wherein the antisense oligonucleotide can bind to the surface of the gold nanoparticle directly or through RNAI. That is, the total number of oligos including RNAI, antisense oligonucleotide, and histidine tag aptamer bound to the surface of the gold nanoparticle is not limited thereto, but may be 1 to 100, and preferably 40 to 50.

[0027] The above gold nanoparticle carrier has an antisense oligonucleotide sequence bound to its surface, which binds to a leader sequence present in the 5'UTR portion of the mRNA of the target virus, thereby inducing mRNA degradation due to the action of RNaseH, thereby having the effect of treating viral infection.

[0028] That is, the gold nanoparticle carrier of the present invention can be utilized as a therapeutic agent for infectious diseases caused by viruses.

[0029] In the present invention, the virus is not limited to a type thereof, but may be selected from the group including coronaviruses including SARS-CoV-2 (COVID-19), SARS-CoV (SARS), and MERS-CoV (MERS); influenza viruses; Ebola viruses; herpes viruses including HSV-1, HSV-2, VZV, and CMV; retroviruses including HIV, hepatitis viruses including HBV, HCV, and HDV; flaviviruses including dengue and Zika viruses; rotaviruses; papillomaviruses; measles viruses; polioviruses; coxsackieviruses; and adenoviruses.

[0030] The term “treatment” as used herein refers to any action that improves or benefits symptoms by administering the pharmaceutical composition according to the present invention.

[0031] The pharmaceutical composition of the present invention can be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injection solutions according to conventional methods, and may additionally include carriers or excipients necessary for the formulation. Pharmaceutically acceptable carriers, excipients, and diluents that may be additionally included in the active ingredient include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, and mineral oil. When formulating, it is usually prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants.

[0032] For example, solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations are prepared by mixing the extract or compound with at least one excipient, such as cotton, starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, syrups, etc., and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included.

[0033] Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solutions and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include witepsol, macrogol, Tween 61, cacao butter, laurin, and glycerogelatin.

[0034] The pharmaceutical composition of the present invention can be administered orally or parenterally (intravenously, subcutaneously, intraperitoneally, or topically) depending on the intended method, and the dosage varies depending on the patient's condition and weight, the degree of the disease, the drug form, the route of administration, and the time of administration, and can be selected in an appropriate form by a person skilled in the art.

[0035] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, the term "pharmaceutically effective amount" means a reasonable amount applicable to medical treatment and an amount sufficient to treat a disease, and the standard thereof may be determined according to the patient's disease, severity, drug activity, drug sensitivity, administration time, administration route and excretion rate, treatment period, concomitantly used ingredients, and other factors. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with another therapeutic agent, and may be administered sequentially or simultaneously with conventional therapeutic agents. The dosage may be determined at a level that can minimize side effects by taking all of the above factors into consideration, and this can be easily determined by a person skilled in the art. Specifically, the dosage of the pharmaceutical composition may vary depending on the patient's age, weight, severity, sex, etc., and generally, 0.001 to 150 mg per 1 kg of body weight, more preferably 0.01 to 100 mg, may be administered once to three times a day, daily or every other day. However, this is an example and the dosage may be set differently as needed.

[0036] The present invention relates to an antiviral gene carrier comprising a gold nanoparticle and an antisense oligonucleotide (ASO) that complementarily binds to a nucleotide sequence included in a viral RNA-derived sequence bound to the surface of the gold nanoparticle; and ribonuclease H (RNase H) that induces cleavage of mRNA, and an antiviral composition comprising the same, which can have a therapeutic effect on diseases caused by infection with various viruses, including SARS-CoV-2, by inhibiting viral mRNA synthesis.

[0037] Figure 1 is a schematic diagram showing the process of binding antisense oligonucleotides (ASOs) and RNaseH to the surface of gold nanoparticles through functionalized DNA oligos.

[0038] Figure 2 shows the AuNP-RNase H at the indicated concentrations (0, 1, 2, 4, and 8 μM). AptHis As a result of simple mixing with (5 nM) and reaction for 10 minutes, AuNP- AptHis -RNH (bound RNase H) was analyzed by SDS-polyacrylamide gel electrophoresis. AuNP- AptHis The amount of RNase H bound to was quantified by band intensity and compared to a known amount of RNase H (total RNase H). Results were quantified and graphed, and data are expressed as the mean ± standard error of the mean (SEM) of at least three independent experiments.

[0039] Figure 3 shows the results of a cell viability assay evaluating the cytotoxicity of AuNP materials. Colors represent the AuNP-untreated group (black), PBS-treated group (red), AuNP-DNA oligo (yellow), and AuNP-DNA oligo-RNH (green). Relative cell viability is expressed as the percentage of viable cells compared to viable cells alone. Data are presented as the mean ± SEM from three independent experiments. NS, unclear.

[0040] Figure 4: PBS, AuNP RNAI / His , ASO, RNase H, AuNP RNAI / HisConfocal fluorescence microscopy images of VERO-E6 cells treated with ASO and / or RNase H loaded in the well, respectively, showing Cy3-labeled ASO (red), Alexa 488-labeled RNase H (green), and nuclei stained with DAPI (blue). The graphs at the bottom quantify the fluorescence intensity of the images, and the quantification of fluorescence intensity was expressed as corrected total cell fluorescence (CTCF) using Image J software (NIH). [CTCF = Integrated density - (Area of ​​selected cells × Mean fluorescence of background readings)]. Data are presented as mean ± SEM from three independent experiments, and symbols (*,#) indicate statistically significant differences (***, ###p < 0.001).

[0041] Figure 5 schematically illustrates the RNase H-dependent ASO mechanism.

[0042] Figure 6 is AuNP RNAI / His Real-time qRT-PCR analysis of positive-sense strand SARS-CoV-2 5'UTR mRNA in VERO-E6 cells 24 hours after treatment with scrambled DNA, ASO, and / or RNase H loaded at 1 nM. The groups represent the untreated group (black), the group without RNase H treatment (red), and the group treated with RNase H (green), respectively. Data are presented as the mean ± SEM of three independent experiments. Asterisks indicate statistically significant values ​​(***p < 0.001). NS, unclear.

[0043] Figure 7A shows AuNP-mediated inhibition of 5'UTR mRNA expression of positive-sense strand SARS-CoV-2 in VERO-E6 cells. RNAI / His -Scrambled DNA-RNH (black) and AuNP RNAI / His-These results were confirmed by real-time qRT-PCR analysis at 4-hour intervals after injection of ASO-RNH (green). Data are presented as the mean ± SEM of three independent experiments. Asterisks indicate statistically significant values ​​(**p < 0.01; ***p < 0.001). NS, unclear.

[0044] Figure 7B shows the qRT-PCR product of Figure 7A separated on a 1.5% agarose gel and visualized using ethidium bromide. GAPDH was used as a loading control.

[0045] Figure 8: AuNP for 24 hours RNAI / His Real-time qRT-PCR analysis of negative-sense strand SARS-CoV-2 5'UTR mRNA in VERO-E6 cells 24 hours after treatment with scrambled DNA, SO, and / or RNase H loaded at 1 nM. The untreated group (black), the group without RNase H treatment (red), and the group treated with RNase H (yellow) are shown, respectively. Data are expressed as the mean ± SEM of three independent experiments. Asterisks indicate statistically significant values. NS, unclear.

[0046] Figure 9: AuNP for 24 hours RNAI / His Real-time qRT-PCR analysis of negative-sense strand SARS-CoV-2 5'UTR mRNA in VERO-E6 cells 24 hours after treatment with scrambled DNA, SON, and / or RNase H loaded at 1 nM. The groups represent the untreated group (black), the group without RNase H treatment (red), and the group treated with RNase H (purple), respectively. Data are presented as the mean ± SEM of three independent experiments. Asterisks indicate statistically significant values. NS, unclear.

[0047] Figure 10 shows the expression of a GFP reporter gene fused to the (+) 5'UTR of SARS-CoV-2 by AuNP loaded with PBS (control), scrambled DNA, ASO, and RNase H. RNAI / His (A) The results were confirmed by incubating VERO-E6 cells with 1 nM for 24 hours, and the graph below shows its quantification (B). The graph shows the results when the amount of PBS was set to 1, and the data are expressed as the mean ± SEM from three independent experiments. Asterisks indicate statistically significant values ​​(**p < 0.01). NS, unclear.

[0048] Figure 11 shows the expression of a GFP reporter gene fused to the (+) 5'UTR of SARS-CoV-2 by AuNP loaded with PBS (control), scrambled DNA, ASO, and RNase H. RNAI / His (1 nM) was cultured with VERO-E6 cells for 24 h and confirmed by FACS analysis. Cells were observed using a 40 X water-immersion objective (Scale bar = 20 μm). The number of GFP-expressing cells was measured using a flow cytometry plot.

[0049] Figure 12: PBS (control), AuNP RNAI / His , AuNP loaded with ASO and RNase H RNAI / HisRepresentative confocal fluorescence microscopy images (A) of SARS-CoV-2-infected VERO-E6 cells treated with , with proteins stained in green and nuclei in blue. Cells were observed using a 40 X water-immersion objective (Scale bar = 20 μm). Quantification of fluorescence intensity was expressed as corrected total cell fluorescence (CTCF) using Image J software (NIH) (B). [CTCF = Integrated density - (Area of ​​selected cells × Mean fluorescence of background readings)]. Data are presented as mean ± SEM from three independent experiments, and symbols (*) indicate statistical significance (*p < 0.05, ***p < 0.001).

[0050] Figure 13 shows SARS-CoV-2 infected VERO-E6 cells treated with PBS (control), AuNP RNAI / His , AuNP loaded with scrambled DNA, ASO, and RNase H RNAI / His After treatment, the relative GFP fluorescence intensity was measured.

[0051] Figure 14 shows the treatment of SARS-CoV-2 infected mice with PBS (control), AuNP RNAI / His (2nM) or AuNP RNAI / His -ASO-RNH (ASO 0.2 μM and RNH loaded AuNPs RNAI / His The results are as follows: After treatment by injecting 2 nM (200 μg / ml) into the nasal cavity (A) and the back of the orbit (B), the expression of RdRPmRNA was confirmed in the nasal conchae and lungs.

[0052] Hereinafter, examples will be described in detail to specifically explain this specification. However, the embodiments according to this specification may be modified in various different forms, and the scope of this specification is not limited to the embodiments described below. The embodiments of this specification are provided to more fully explain this specification to those of average skill in the art.

[0053]

[0054] Experimental Methods and Materials

[0055] 1. Plasmids and oligonucleotides

[0056] The plasmids and oligonucleotides used in the present invention are shown in Tables 1 and 2 below.

[0057] Plasmid description pMQ131-5´ UTR-EGFPpBBR1ori, Km r , CEN / ARS, URA3, (+) SARS-CoV-2-5´ UTR, EGFPpcDNA3.1-N geneSV40ori, f1ori,Amp r , Hyg r , T7, CMV, (+) SARS-CoV-2-N genepMQ131-5´ UTR-reversed-EGFPpBBR1ori, Km r , CEN / ARS, URA3, (-) SARS-CoV-2-5´ UTR, EGFPpcDNA3.1-N gene-reversedSV40ori, f1ori,Amp r , Hyg r , T7, CMV, (-) SARS-CoV-2-N gene

[0058] *ori: origin of replication, Km r : Kanamycin resistance, Amp r : Ampicillin resistance, Hyg r : Hygromycin resistance, (+): positive-sense strand, (-): negative-sense strand.

[0059] 종류올리고 뉴클레오티드서열 (5' to 3')서열번호For cloning pMQ131-5´ UTR-reversed5´UTR-P1 (F) (R)GTAGGCGCCGGTCACAGCTT25´UTR-P2 (F)AGTCGTCTCTCGCGAATTAAAGGTTTATACCTTCC35´UTR-Rev-P2-Sma I-F2 (R)TCCCCCGGGCGATCCAAGCTGTGACCGGCCTACCTGTAAAACAGGCAAACT4For cloning of pcNA gene-reversedN-BamH-Rev (F)CGGATCCTGCAGGGGCCTGAGTTGAGTCAGCAC5N-ApaI-Rev (R)TCGAGGGGCCCTTATCTGATAATGGACCCCAAAA6For qRT-PCR5´UTR-qR-F (F)TTTATACCTTCCCAGGTAAC75R-RUTR (R)TAATTATACTGCGTGAGTGC8N-qR-F (F)GGTTTACCCAATACTG9N-qR-R (R)CTTCGGTAGTAGCCAATTTG10hGAPDH-F (F)AGGGGCCATCCACAGTCTT11hGAPDH-R (R)AGCCAAAAGGGTCATCTCo-CRT1-2RFRSARPV-2 (F)GTGAAATGGTCATGTGTGGCGG13SARS-CoV-2 RdRP gene-R (R)CAAATGTTAAAAACACTATTAGCATA14For Antisense oligonucleotidesASO-RNA I-leaderCAGAGCCGAGATACAAGAGATCGAAAGTTGGTT15SO-RNA I-leaderCAGAGCCGAGATAACCAACTTTCGATCTCTTGT16SON-RNA I-leaderCAGAGCCGAGATACCGCTCTCACTCAACATGG17For Scrambled DNAN-BamH-Rev (F)CGGATCCTGCAGGGGCCTGAGTTGAGTCAGCAC18N-ApaI-Rev(R)TCGAGGGGCCCTTATCTGATAATGGACCCCAAAA19Rnh-Nde I (F)GGAATTCCATATGCTTAAACAGGTAGAAATT20For AuNP conjugates oligonucleotidesRNA I oligosTCTCGGCTCTGCTAGCG-A10-Thiol21His-tagged aptamerGCTATGGGGTGGTCTGGTTGGGATTGGCCCCGGGAGCTGGC-A10-Thiol22ASO-leaderACAAGAGATCGAAAGTTGGTT-A10-Thiol23SO-leaderAACCAACTTTCGATCTCTTGT-A10-Thiol24SON-leaderACCGCTCTCACTCAACATGG-A10-Thiol25

[0060]

[0061] 2. Cell culture

[0062] VERO-E6 (African green monkey kidney) and Calu-3 (Human Lung Adenocarcinoma) cells were cultured in Dulbecco's modified Eagle's medium (Welgene, Korea) containing 10% (v / v) FBS (Welgene) and 1% (v / v) penicillin-streptomycin (Welgene) at 37°C in a humidified atmosphere containing 5% (v / v) carbon dioxide (CO2).

[0063] 3. Plasmid transfection

[0064] Plasmids were extracted from E. coli strain DH5α using the NucleoBond Xtra Midi Preparation Kit (Macherey-Nagel, DR, Germany). VERO-E6 cells were seeded on plates 24 hours before transfection and transfected with the plasmids using Lipofectamine 3000 (Invitrogen, CA, USA). Plasmid: Lipofectamine 3000 (1 μg / μl): p3000 (1 μg / μl) were mixed in a ratio of 1:2:2.5 and sequentially added to Opti-MEM medium. After incubation for 10 minutes, the cells were treated and cultured for 24 hours.

[0065]

[0066] 4. AuNP-DNA oligo conjugates and AuNPs RNAI / His - Fabrication of ASO-RNH complex

[0067] AuNP-DNA oligo conjugates for intracellular delivery of ASO and RNase H were fabricated using the following method. AuNPs were linked to two types of DNA oligos, RNA I and aptamer (AptHis), which target the 5' UTR of the positive-sense strand of SARS-CoV-2, which specifically binds to ASO, and the hexahistidine (His) tag of RNase H, respectively. (Figure 1) The fabrication process is as follows.

[0068] First, citrate-capped AuNPs (15 nm in diameter) were purchased from BBI Life Science (Crumlin, UK). Thiolated RNA I, 6X His-tagged aptamer, and ASO were treated with 1 N dithiothreitol (1.545 g DTT in 0.01 M sodium acetate, pH 5.2) for 1 h at room temperature to cleave disulfide bonds. Excess DTT and unwanted thiol fragments were removed from the free thiol-modified oligonucleotide mixture by extraction with ethyl acetate. The cleaved oligonucleotides were purified using ethanol precipitation. AuNPs, thiolated DNA oligos (RNAI or ASO), and 6X His-tagged aptamer were mixed at a ratio of 1:150:150 in phosphate buffer (100 mM, pH 7.4), reacted for 1 h at room temperature, and then the mixture was sonicated for 5 min. Next, NaCl (2 M, based on 10 mM pH 7.4 phosphate buffer) was added dropwise while shaking simultaneously to make the final NaCl concentration 300 mM, three times at 4-h intervals, and incubated at room temperature for 12 h. Next, the AuNP-DNA oligo mixture was transferred to a 15 ml tube and centrifuged at 12,000 xg for 20 min. The supernatant was removed, and the AuNP-DNA oligo mixture was transferred to a 1.5 ml tube, centrifuged at 20,000 xg for 20 min, and washed twice with triple-distilled water (TDW). The mixture was redispersed in PB buffer (500 mM NaCl based 50 mM phosphate buffer), and the fabricated AuNP RNAI / His The final concentration was set to 50 nM.

[0069] AuNP RNAI / HisTo prevent secondary structure formation, the AuNPs were pre-incubated at 80°C for 5 min and cooled to room temperature. 1X PBS, 6X His-tagged RNase H (0.02 μM) (Prospec, NZ, Israel), ASO (0.1 μM), and TDW were mixed, and then AuNPs RNAI / His was added to the mixture. MgCl2 (0.05 mM) was added and the mixture was incubated at room temperature for 10 minutes.

[0070] (RNaseH sequence: MGSSHHHHHHSSGLVPRGSHMGSMLKQVEIFTDGSCLGNP GPGGYGAILRYRGREKTFSAGYTRTTNNRMELMAAIVALEALKEHCEVILSTDSQYVRQGITQWIHNWKKRGWKTADKKPVKNVDLWQRLDAALGQHQIKWEWVKGHAGHPENERCDELARAAAMNPTLEDTGYQVEV: SEQ ID NO: 26)

[0071] The above AuNP-Apt His The binding affinity of AuNP-Apt and RNase H (RNH) was measured as follows. His The amount of RNase H in the -RNH(5 nM) complex was analyzed on a 12% SDS-polyacrylamide gel, and the AuNP-Apt for RNase H His The dissociation constant (KD) of AuNP-Apt was 1.1 μM, which means that RNase H His It shows that it has a high binding affinity for (Fig. 2).

[0072] In addition, a sense oligonucleotide (SO) targeting the 5' UTR of the negative sense strand SARS-CoV-2 and a sense oligonucleotide (SON) targeting the N gene of the negative sense strand SARS-CoV-2 were synthesized and loaded onto AuNPs, and the results such as cytotoxicity and binding efficiency were compared.

[0073] 5. Cytotoxicity test

[0074] VERO-E6 cells (5×10 3 / well) were seeded in a 96-well plate and cultured for 24 h. The cells were further incubated in the medium with AuNP for 24 h. RNAI / His ASO, SO, SON and / or RNase H were added at 2 nM each and incubated again. Cell viability was measured using CytoTox 96 ® Cell viability was measured using the Non-Radioactive Cytotoxicity Assay (Promega, WI, USA). Colors represent cells alone (black), PBS (red), AuNP-DNA oligos (yellow), and AuNP-DNA oligo-RNH (green). Relative cell viability was expressed as the percentage of viable cells compared to cells alone. Data are presented as the mean ± SEM from three independent experiments. (NS: unclear)

[0075] As a result, as shown in Fig. 3, all substances having the indicated concentrations did not exhibit cytotoxicity against VERO-E6 cells.

[0076]

[0077] 6. AuNP RNAI / His Intracellular delivery of ASO and RNase H by

[0078] In animal cells, AuNPs RNAI / His To investigate the delivery efficiency of ASO and RNase H by AuNP, confocal microscopy analysis and fluorescence signal intensity analysis (Corrected Total Cell Fluorescence, CTCF) were performed. For visualization, the 3' end of ASO was labeled with cyanine 3 (Cy3) and the primary amine of RNase H was labeled with Alexa-488. As a result of observation, as shown in Figure 4, AuNP RNAI / His -ASO, AuNP RNAI / His -RNH and AuNP RNAI / His -High fluorescence signals were detected in VERO-E6 cells treated with ASO-RNH, respectively, and in contrast, PBS, AuNPRNAI / His , no significant fluorescence was detected in VERO-E6 cells treated with ASO or RNH alone. These results suggest that AuNP RNAI / His This shows that ASO and RNase H were loaded and efficiently delivered into cells.

[0079]

[0080] 7. AuNP RNAI / His - Inhibition of viral mRNA expression by ASO-RNH

[0081] AuNP RNAI / His To investigate whether the -ASO-RNH complex could affect the level of viral mRNA expression, VERO-E6 cells were treated with AuNP RNAI / His -ASO-RNH complex was transfected for 24 hours, and the 5' UTR mRNA expression level of SARS-CoV-2 was measured by qRT-PCR (Fig. 5).

[0082] As a result, as confirmed in Fig. 6, VERO-E6 cells expressing 5'UTR mRNA of positive sense strand SARS-CoV-2 (VERO-E6 cells pre-transfected with plasmid pMQ131-5' UTR-EGFP) were treated with AuNP RNAI / His -Results of treatment with ASO-RNH complex (lane 8) Buffer or other AuNP RNAI / His Complex (AuNP) RNAI / His , AuNP RNAI / His -ASO, AuNP RNAI / His -RNH and AuNP RNAI / His It was confirmed that the mRNA expression level was reduced by approximately 30-40% compared to the control cells treated with -scrambled DNA-RNH. In addition, AuNPs in which ASO was directly conjugated to AuNPs without hybridization with RNAI His -In the case of ASO, RNase H did not affect the expression level of 5'UTR mRNA (compare lane 4 or 6), and AuNP RNAI / His -It was confirmed that the expression inhibition efficiency was lower compared to ASO.

[0083] Also, as shown in Fig. 7, AuNP RNAI / His After transfection of the -ASO-RNH complex, the expression suppression efficiency over time was observed every 4 hours and analyzed by qRT-PCR. As a result, the expression level of 5' UTR mRNA gradually decreased as the culture time elapsed, and this was confirmed by separation of qRT-PCR products on a 1.5% agarose gel and visualization using ethidium bromide.

[0084] In addition, negative sense strand SARS-CoV-2 (plasmid pMQ131-5'UTR-reversed-EGFP or pcDNA3.1-N gene reversed) was tested in the same manner as above, but with AuNP RNAI / His -SO-RNH or AuNP RNAI / His -SON-RNH complex was not effective in knockdown of target RNA (Fig. 8, Fig. 9).

[0085] In summary, these results indicate that AuNP RNAI / His - Demonstrates that the ASO-RNH complex can efficiently reduce the target mRNA level of SARS-CoV-2 in VERO-E6 cells.

[0086]

[0087] 8. AuNP RNAI / His - Inhibitory effect of target protein expression of ASO-RNH complex

[0088] AuNP of the present invention RNAI / His To confirm whether the -ASO-RNH complex actually inhibits target protein expression, we further investigated whether treatment of VERO-E6 cells expressing GFP mRNA fused to the (+) 5'UTR of SARS-CoV-2 reduces the expression level of GFP. As a result, as shown in Fig. 10, a decrease in GFP expression of approximately 35% was observed. In addition, consistent with the mRNA expression level, AuNP RNAI / His , AuNP RNAI / His-RNH or AuNP RNAI / His -No significant effect was observed when cells were treated with scrambled DNA-RNH. Similar results were also confirmed from FACS analysis of GFP-expressing cells (Fig. 11). These results are consistent with AuNP RNAI / His - Showing that ASO-RNH can knockdown the expression of a GFP reporter gene fused to the positive sense strand of SARS-CoV-2.

[0089]

[0090] 9. AuNP RNAI / His -Effect of ASO-RNH complex on SARS-CoV-2 infected animal cells

[0091] AuNPs in SARS-CoV-2-infected mammalian cells RNAI / His -To further test the effect of ASO-RNH, AuNPs were added to VERO-E6 cells infected with SARS-CoV-2. RNAI / His or AuNP RNAI / His - Cells were transfected by injecting ASO-RNH at doses of 0.1 and 1 nM, respectively, and the nucleocapsid protein of SARS-CoV-2 was detected using immunocytochemistry (ICC) analysis.

[0092] As a result, as shown in Fig. 12, the nucleocapsid protein level was AuNP RNAI / His -ASO-RNH (1 nM) treated VERO-E6 (Fig. 17) showed a 30-40% decrease. The cells were treated with AuNP RNAI / His (0.1 and 1 nM) and lower concentrations of AuNPs RNAI / His - Treatment with ASO-RNH (0.1 nM) did not significantly affect the N protein level. These results were obtained by 1 nM AuNP RNAI / His -This demonstrates that ASO-RNH can effectively knockdown the expression of SARS-CoV-2 N protein in mammalian cells.

[0093] AuNPs that inhibit viral replication using VERO-E6 cells infected with recombinant SARS-CoV-2 capable of expressing GFP (SARS-CoV-2-mNeon) RNAI / His -The ability of ASO-RNH was confirmed. As a result, as shown in Figure 13, the virus alone, AuNP RNAI / His , AuNP RNAI / His -RNH, or AuNP RNAI / His -AuNP compared to cells treated with scrambled DNA-RNH RNAI / His A 30-40% decrease in GFP intensity was observed in cells treated with -ASO-RNH. These results are consistent with AuNP RNAI / His - Showing that ASO-RNH inhibits GFP translation through cleavage of viral mRNA.

[0094] 10. AuNP RNAI / His -Effect of ASO-RNH complex on SARS-CoV-2 infected animal cells

[0095] AuNP RNAI / His - To evaluate whether ASO-RNH can be used as a therapeutic agent for viral-induced respiratory diseases, AuNPs were tested in an established animal model of SARS-CoV-2 infection. RNAI / His -The in vivo efficacy of ASO-RNH was tested. K18-hACE2 mice were injected with 1 x 10 5 Infection with PFU SARS-CoV-2 Omicron variant, PBS, and AuNP RNAI / His or AuNP RNAI / His -ASO-RNH was injected intranasally (IN) or retroorbitally (RO) three times at 24-h intervals. Mice infected with the virus were euthanized on day 5, and lung and nasal turbinate tissues were collected. Viral RNA-dependent RNA polymerase (RdRP) mRNA was measured from these organs using qRT-PCR.

[0096] As a result, as shown in Figure 14A, the expression level of RdRP mRNA was higher in AuNP compared to that treated with PBS or AuNP-DNA oligo only. RNAI / His -ASO-RNH was delivered intranasally, and the lungs showed a ~30% reduction. Similarly, as shown in Figure 14B, AuNP RNAI / His -When ASO-RNH was delivered retro-orbitally, viral mRNA was reduced by ~20% in the nasal conchae and lungs compared to treatment with PBS or AuNP-DNA oligos alone. These data indicate that AuNP RNAI / His -These results show that when ASO-RNH is administered to treat the SARS-CoV-2 virus, it can be effectively delivered to the airways of infected animals and reduce the virus titer.

[0097]

[0098] The present invention has been described above, focusing on preferred embodiments thereof. Those skilled in the art will appreciate that the present invention can be implemented in modified forms without departing from its essential characteristics. Therefore, the disclosed embodiments should be considered illustrative rather than restrictive. The scope of the present invention is set forth in the claims, not the foregoing description, and all differences within the scope equivalent thereto should be construed as being encompassed by the present invention.

[0099]

[0100] In one aspect, the present invention relates to a nanoparticle delivery system comprising a gold nanoparticle; an antisense oligonucleotide (ASO) complementary to a virus-derived sequence bound to the surface of the gold nanoparticle; and ribonuclease H (RNase H) that induces cleavage of mRNA of the virus.

[0101] As an example, the viral sequence comprises a nucleotide sequence included in a 5'untranslated region (UTR), and the viral sequence comprises a leader sequence.

[0102] As an example, the virus may be selected from the group consisting of coronavirus, influenza virus, ebola virus, herpes virus, retrovirus, hepatitis virus, rotavirus, flavivirus, papillomavirus, measles virus, poliovirus, coxsackievirus, and adenovirus.

[0103] In one embodiment, the antisense oligonucleotide (ASO) is bound to the gold nano surface via a moiety comprising a functional group, wherein the moiety comprising a functional group comprises a thiol group or an amine group.

[0104] As an example, the RNaseH may be bound to the gold nano surface via an aptamer, and further, the aptamer may include a histidine tag.

[0105] In another aspect, the present invention relates to an antiviral composition comprising the gold nanoparticle carrier.

[0106] In another aspect, the present invention relates to a gold nanoparticle carrier for delivering into cells gold nanoparticles having an antisense oligonucleotide (ASO) complementary to a virus-derived sequence and ribonuclease H (RNase H) bound to the surface thereof, which induces cleavage of mRNA of the virus, and wherein the antisense oligonucleotide (ASO) complementary to the virus-derived sequence binds to the virus-derived mRNA sequence and degrades the mRNA through RNase H, thereby inhibiting the activity of the virus.

Claims

1. Gold nanoparticles; and A nanoparticle delivery vehicle comprising an antisense oligonucleotide (ASO) complementary to a virus-derived sequence bound to the surface of the gold nanoparticle and ribonuclease H (RNase H) that induces cleavage of mRNA of the virus.

2. In paragraph 1, A nanoparticle carrier, wherein the above viral-derived sequence is a nucleotide sequence contained in the 5'untranslated region (UTR).

3. In paragraph 1, A nanoparticle carrier wherein the sequence derived from the virus comprises a leader sequence.

4. In paragraph 1, A nanoparticle carrier, wherein the virus is selected from the group consisting of coronavirus, influenza virus, ebola virus, herpes virus, retrovirus, hepatitis virus, rotavirus, flavivirus, papillomavirus, measles virus, poliovirus, coxsackievirus and adenovirus.

5. In paragraph 1, A nanoparticle carrier wherein the antisense oligonucleotide (ASO) is bound to the gold nano surface via a moiety containing a functional group.

6. In paragraph 5, A nanoparticle carrier wherein the residue containing the above functional group contains a thiol group or an amine group.

7. In paragraph 1, A gold nanoparticle carrier wherein the above RNaseH is bound to the surface of the gold nano via an aptamer.

8. In paragraph 7, The above aptamer is a gold nanoparticle carrier containing a histidine tag.

9. An antiviral composition comprising the nano particle carrier of clause 1.

10. Use of a gold nanoparticle carrier to deliver into cells gold nanoparticles having an antisense oligonucleotide (ASO) complementary to a virus-derived sequence and ribonuclease H (RNase H) that induces cleavage of mRNA of the virus bound to the surface, and to inhibit the activity of the virus by causing the antisense oligonucleotide (ASO) complementary to the virus-derived sequence to bind to the virus-derived mRNA sequence and decompose the mRNA through RNase H.

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