WO2025110774A1 - Antiviral composition comprising nanoparticle carrier based on gold nanoparticle-nucleic acid conjugate - Google Patents

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 nanoparticle carrier based on gold nanoparticle-nucleic acid conjugate

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 of the gold nanoparticle; and ribonuclease H (RNase H) that induces cleavage of mRNA, and an antiviral composition comprising the same.

The coronavirus disease (COVID-19), which has spread worldwide, is caused by the highly contagious SARS-CoV-2 virus, causing millions of deaths and causing a global medical crisis and economic loss. SARS-CoV-2 is mainly transmitted through the respiratory route, and enters cells by binding to the ACE2 receptor in the human body through the viral spike protein (S protein). In the process, various variants appear, changing the transmissibility and mortality rate, so the effectiveness of the currently used vaccines and antiviral treatments is continuously being evaluated.

Existing antiviral agents mainly attempt to suppress viruses through protein-based approaches or chemical drugs, but these approaches have limitations such as reduced efficacy against mutant viruses, increased administration frequency, and high production costs. In contrast, technologies using the genetic material of viruses offer new possibilities by effectively delivering genes (DNA, RNA, etc.) that express specific viral antigens in the human body, inducing an immune response, and inhibiting viral replication. For these technologies, various genetic material delivery vehicles are being developed, and these have the characteristics of rapid adjustment to genetic material variants and various antiviral effects, enabling a rapid and extensive response, especially against mutant viruses such as SARS-CoV-2.

Accordingly, the inventors of the present invention, while conducting research to manufacture a genetic material delivery vehicle having antiviral activity, discovered that when an antisense oligonucleotide that complementarily binds to a nucleotide sequence derived from viral RNA, particularly included in a conserved 5'untranslated region, and ribonuclease H (RNase H), which binds to the antisense oligonucleotide and induces cleavage of mRNA, are delivered together with gold nanoparticles, an excellent antiviral effect against the virus can be achieved, thereby completing the present invention.

Accordingly, the purpose of the present invention is to provide a nanoparticle delivery vehicle 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.

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

In order to achieve the above-described purpose of the present invention, the present invention provides a nanoparticle delivery vehicle comprising a gold nanoparticle; 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.

In order to achieve another object of the present invention, the present invention provides an antiviral composition comprising the nano particle carrier.

Hereinafter, the present invention will be described in detail.

The present invention relates to a nanoparticle delivery vehicle 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.

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.

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). That is, RNaseH can recognize RNA/DNA hybrid duplex structures and induce mRNA cleavage and genome degradation (Bennett, Baker, Pham, Swayze, & Geary, 2017).

That is, the term "antisense oligonucleotide (ASO)" in the present invention means 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 by RNA-RNA interaction and modifies the activity of the target RNA. Antisense oligonucleotides can up-regulate or down-regulate the expression and/or function of a specific polynucleotide. It is intended that any foreign RNA or DNA molecule useful from a therapeutic, diagnostic, or other perspective be included in this definition. By way of example, antisense RNA or DNA molecules, interfering RNA (RNAi), micro RNA, bait 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.

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, which may target 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 region of SARS-CoV-2.

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

The above 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 the template DNA to form an RNA/DNA duplex, and the non-template strand is replaced by single-stranded DNA (ssDNA) to form a structure called R-loop (Thomas, White, & Davis, 1976). R-loops can help regulate transcription, but when they are not needed, 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 various organisms.

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, Thermos 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.

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

The gold nanoparticles of the present invention are gold particles having a diameter in the nanometer range, preferably 5-500 nm, more preferably 10-200 nm, which are easy to manufacture in the form of stable particles, are easy to control in 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 also the bonding between the gold surface, which does not have the characteristics of a nanomaterial, and functional groups such as thiol groups becomes weak, making it difficult to manufacture a carrier using gold nanoparticles.

The gold nanoparticles used in the present invention are not limited to the method, but can be manufactured as follows: HAuCl ₄ is used as a gold source, and HAuCl ₄ is reduced using sodium citrate as a reducing agent 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.

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.

The above 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 comprises one or more thiolated residues, through which it can directly bind to the surface of the gold nanoparticles.

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.

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 by 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 kind of aptamer of various tags or protein-specific aptamers, particularly for binding RNase H to the surface of the gold nanoparticles, and is not particularly limited.

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.

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.

The above gold nanoparticle carrier has an effect of treating viral infection by binding to a leader sequence present in the 5'UTR portion of the mRNA of the target virus through an antisense oligonucleotide sequence bound to the surface, thereby inducing mRNA degradation due to the action of RNaseH.

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

In the present invention, the virus is not limited to a type, but may be selected from the group including coronaviruses including SARS-CoV-2 (COVID-19), SARS-CoV (SARS), and MERS-CoV (MERS); influenza virus; Ebola virus; herpesviruses including HSV-1, HSV-2, VZV, and CMV; retroviruses including HIV, hepatitis viruses including HBV, HCV, and HDV; flaviviruses including dengue and Zika viruses; rotavirus; papillomavirus; measles virus; poliovirus; coxsackievirus; and adenovirus.

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

The pharmaceutical composition of the present invention can be used by being formulated in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injection solutions according to conventional methods, and may further include carriers or excipients necessary for the formulation. Pharmaceutically acceptable carriers, excipients, and diluents that may be additionally included in the effective 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, mineral oil, and the like. When formulating, it is usually prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants.

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, at least 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.

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

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, and can be selected in an appropriate form by a person skilled in the art.

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 matters. The pharmaceutical composition of the present invention may be administered individually or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. The dosage may be determined at a level that minimizes side effects by considering all of the above factors, and this may 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 may generally be administered in an amount of 0.001 to 150 mg per 1 kg of body weight, more preferably 0.01 to 100 mg, once to three times a day, every day or every other day. However, this is an example and the dosage may be set differently as needed.

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.

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

Figure 2 shows the results of simply mixing RNase H at the indicated concentrations (0, 1, 2, 4, and 8 μM) with AuNP- ^AptHis (5 nM) and reacting for 10 min, and the AuNP- ^AptHis -RNH (bound RNase H) was analyzed by SDS-polyacrylamide gel electrophoresis. The amount of RNase H bound to AuNP- ^AptHis was quantified as band intensity and compared to the known amount of RNase H (total RNase H). The results were quantified and displayed in a graph, and the data are expressed as the mean ± standard error of the mean (SEM) of at least three independent experiments.

Figure 3 shows the results of evaluating the cytotoxicity of AuNP materials by cell viability analysis, and the colors represent the AuNP untreated group (black), PBS treated group (red), AuNP-DNA oligo (yellow), and AuNP-DNA oligo-RNH (green). The relative cell viability is expressed as the percentage of viable cells compared to the viable cells of cells alone. The data are expressed as the mean ± SEM of three independent experiments. NS, unclear.

Figure 4 is a confocal fluorescence microscopy image of VERO-E6 cells treated with PBS, AuNP ^RNAI/His , ASO, RNase H, ASO loaded on AuNP ^RNAI/His , and/or RNase H, respectively, wherein each image shows Cy3-labeled ASO (red), Alexa 488-labeled RNase H (green), and nuclei stained with DAPI (blue). The graphs at the bottom are quantified fluorescence intensities of the images, and the fluorescence intensity quantification was expressed as corrected total cell fluorescence (CTCF) using lmage J software (NIH). [CTCF = integrated density - (area of selected cells × mean fluorescence of background readings)]. Data are presented as mean ± SEM of three independent experiments, and symbols (*,#) indicate statistical significance. (***, ###p < 0.001).

Figure 5 is a schematic diagram of the RNase H-dependent ASO mechanism.

Figure 6 is a real-time qRT-PCR analysis of positive-sense strand SARS-CoV-2 5'UTR mRNA in VERO-E6 cells 24 h after treatment with scrambled DNA, ASO, and/or RNase H loaded onto AuNP ^RNAI/His (1 nM). The untreated group (black), the group not treated with RNase H (red), and the group treated with RNase H (green) are shown, respectively. Data are presented as the mean ± SEM of three independent experiments. Asterisks indicate statistically significant values (***p < 0.001). NS, unclear.

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

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.

Figure 8 is a real-time qRT-PCR analysis of 5'UTR mRNA of negative-sense strand SARS-CoV-2 in VERO-E6 cells 24 h after treatment with Scrambled DNA, SO, and/or RNase H loaded on AuNP ^RNAI/His (1 nM) for 24 h. The untreated group (black), the group not treated with RNase H (red), and the group treated with RNase H (yellow) are shown, respectively. Data are presented as the mean ± SEM of three independent experiments. Asterisks indicate statistically significant values. NS, unclear.

Figure 9 is the real-time qRT-PCR analysis of 5'UTR mRNA of negative-sense strand SARS-CoV-2 in VERO-E6 cells 24 h after treatment with Scrambled DNA, SON, and/or RNase H loaded on AuNP ^RNAI/His (1 nM) for 24 h. The untreated group (black), the group not treated with RNase H (red), and the group treated with RNase H (purple) are shown, respectively. Data are presented as the mean ± SEM of three independent experiments. Asterisks indicate statistically significant values. NS, unclear.

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

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

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

Figure 13 shows the results of measuring the relative GFP fluorescence intensity after treating SARS-CoV-2 infected VERO-E6 cells with PBS (control), AuNP ^RNAI/His , scrambled DNA, ASO, and AuNP ^RNAI/His loaded with RNase H.

Figure 14 shows the results of confirming the expression of RdRP mRNA in the nasal conchae and lungs after treatment of SARS-CoV-2-infected mice by intranasal ^injection (A) and retroorbital injection (B) with PBS (control), AuNP RNAI ^{/His (2 nM), or AuNP RNAI /His} -ASO-RNH (ASO 0.2 μM and RNH-loaded AuNP RNAI/His 2 nM), respectively.

Hereinafter, examples will be described in detail to specifically explain the present specification. However, the embodiments according to the present specification may be modified in various different forms, and the scope of the present specification is not construed as being limited to the embodiments described below. The embodiments of the present specification are provided to more completely explain the present specification to a person having average knowledge in the art.

<Experimental methods and materials>

1. Plasmids and oligonucleotides

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

Plasmid explanation pMQ131-5´UTR-EGFP pBBR1 ori , Km ^r , CEN/ARS, URA3, (+) SARS-CoV-2-5´ UTR, EGFP pcDNA3.1-N gene SV40 ori , f1 ori, Amp ^r , Hyg ^r , T7, CMV, (+) SARS-CoV-2-N gene pMQ131-5´UTR-reversed-EGFP pBBR1 ori , Km ^r , CEN/ARS, URA3, (-) SARS-CoV-2-5´ UTR, EGFP pcDNA3.1-N gene-reversed SV40 ori , f1 ori, Amp ^r , Hyg ^r , T7, CMV, (-) SARS-CoV-2-N gene

* ori : origin of replication, Km ^r : kanamycin resistance, Amp ^r : ampicillin resistance, Hyg ^r : hygromycin resistance, (+): positive-sense strand, (-): negative-sense strand.

type Oligonucleotides Sequence (5’ to 3’) Sequence number For cloning pMQ131-5´ UTR-reversed 5´UTR-P1 (F) ACGCGTCGACCGGTCCGGGCCATT 1 5´UTR-P1 (R) GTAGGCGCCGGTCACAGCTT 2 5´UTR-P2 (F) AGTCGTCTCTCGCGAATTAAAGGTTTAATACCTTCC 3 5´UTR-Rev-P2-Sma I-F2 (R) TCCCCCGGGCGATCCAAGCTGTGACCGGCGCCTACACCTGTAAAACAGGCAAACT 4 For cloning pcDNA3.1-N gene-reversed N-BamH-Rev (F) CGGATCCTGCAGGGGCCTGAGTTGAGTCAGCAC 5 N-ApaI-Rev (R) TCGAGGGGCCCTTATCTGATAATGGACCCCCAAAA 6 For qRT-PCR 5´UTR-qR-F (F) TTTATACCTTCCCAGGTAAC 7 5´UTR-qR-R (R) TAATTATACTGCGTGAGTGC 8 N-qR-F (F) GGTTTACCCAATAATACTG 9 N-qR-R (R) CTTCGGTAGTAGCCAATTTG 10 hGAPDH-F (F) AGGGGCCATCCACAGTCTT 11 hGAPDH-R (R) AGCCAAAAGGGTCATCTCT 12 SARS-CoV-2 RdRP gene-F (F) GTGAAATGGTCATGTGTGGCGG 13 SARS-CoV-2 RdRP gene-R (R) CAAATGTTAAAAACACTATTAGCATA 14 For Antisense oligonucleotides ASO-RNA I-leader CAGAGCCGAGATACAAGAGATCGAAAGTTGGTT 15 SO-RNA I-leader CAGAGCCGAGATAACCAACTTTCGATCTCTTGT 16 SON-RNA I-leader CAGAGCCGAGATACCGCTCTCACTCAACATGG 17 For Scrambled DNA N-BamH-Rev (F) CGGATCCTGCAGGGGCCTGAGTTGAGTCAGCAC 18 N-ApaI-Rev (R) TCGAGGGGCCCTTATCTGATAATGGACCCCCAAAA 19 Rnh-Nde I (F) GGAATTCCATATGCTTAAACAGGTAGAAATT 20 For AuNP conjugates oligonucleotides RNA I oligos TCTCGGCTCTGCTAGCG-A10-Thiol 21 His-tagged aptamer GCTATGGGTGGTCTGGTTGGGATTGGCCCCGGGAGCTGGC-A10-Thiol 22 ASO-leader ACAAGAGATCGAAAGTTGGTT-A10-Thiol 23 SO-leader AACCAACTTTCGATCTCTTGT-A10-Thiol 24 SON leader ACCGCTCTCACTCAACATGG-A10-Thiol 25

2. Cell culture

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 (CO ₂ ).

3. Plasmid transfection

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 h 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 added sequentially to Opti-MEM medium, reacted for 10 minutes, and then added to cells and cultured for 24 h.

4. Preparation of AuNP-DNA oligo conjugates and AuNP ^RNAI/His - ASO-RNH complexes

AuNP-DNA oligo conjugates for intracellular delivery of ASO and RNase H were fabricated by the following method. AuNPs are 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 that specifically binds to ASO and the hexahistidine (His) tag of RNase H, respectively. (Fig. 1) The fabrication process is as follows.

First, citrate-capped AuNPs (diameter 15 nm) 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 of DTT in 0.01 M sodium acetate, pH 5.2) for 1 h at room temperature to cleave the disulfide bond. 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) and reacted at room temperature for 1 h, 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 until the final NaCl concentration became 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 ×g 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 ×g for 20 min, and washed twice with triple-distilled water (TDW). The mixture was redispersed in PB buffer (50 mM phosphate buffer based on 500 mM NaCl) until the final concentration of the fabricated AuNP ^RNAI/His became 50 nM.

AuNP ^RNAI/His was pre-incubated at 80°C for 5 min to prevent secondary structure formation 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 AuNP ^RNAI/His was added to the mixture. MgCl ₂ (0.05 mM) was added and the mixture was incubated at room temperature for 10 min.

(RNaseH sequence: MGSSHHHHHHSSGLVPRGSHMGSMLKQVEIFTDGSCLGNP GPGGYGAILRYRGREKTFSAGYTRTTNNRMELMAAIVALEALKEHCEVILSTDSQYVRQGITQWIHNWKKRGWKTADKKPVKNVDLWQRLDAALGQHQIKWEWVKGHAGHPENERCDELARAAAMNPTLEDTGYQVEV: SEQ ID NO: 26)

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

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.

5. Cytotoxicity test

VERO-E6 cells (5 × 10 ³ /well) were seeded in 96-well plates and cultured for 24 h. The cells were then incubated again for an additional 24 h in the medium with 2 nM of ASO, SO, SON, and/or RNase H loaded onto AuNP ^RNAI/His . Cell viability was measured using the CytoTox 96 ^® Non-Radioactive Cytotoxicity Assay (Promega, WI, USA). Colors are represented as cells only (black), PBS (red), AuNP-DNA oligos (yellow), and AuNP-DNA oligo-RNH (green), and relative cell viability is expressed as the percentage of viable cells compared to viable cells in cells only. Data are presented as the mean ± SEM from three independent experiments. (NS: unclear)

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

6. AuNP ^RNAI/His Intracellular delivery of ASO and RNase H by

To investigate the delivery efficiency of ASO and RNase H by AuNP ^RNAI/His in animal cells, confocal microscopy 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 shown in Fig. 4, high fluorescence signals were detected in VERO-E6 cells treated with AuNP ^RNAI/His -ASO, AuNP ^RNAI/His -RNH, and AuNP ^RNAI/His -ASO-RNH, respectively. In contrast, no significant fluorescence was detected in VERO-E6 cells treated with PBS, AuNP RNAI/ ^His , ASO, or RNH alone. These results demonstrate that ASO and RNase H were loaded onto AuNP ^RNAI/His and efficiently delivered into cells.

7. Inhibition of viral mRNA expression by AuNP ^RNAI/His -ASO-RNH

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

As a result, as confirmed in Fig. 6, when 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 -ASO-RNH complex (lane 8), it was confirmed that the mRNA expression level was reduced by about 30-40% compared to the control cells treated with buffer or other AuNP ^RNAI/His complexes (AuNP ^{RNAI /His} , AuNP ^{RNAI/His -ASO, AuNP RNAI/His} -RNH, and AuNP ^RNAI/His -scrambled DNA-RNH). In addition, in the case of AuNP ^His -ASO, in which ASO was directly bound to AuNP without hybridization with RNAI, RNase H did not affect the expression level of 5'UTR mRNA (compare lane 4 or 6), and it was confirmed that the expression inhibition efficiency was lower compared to AuNP ^RNAI/His -ASO.

In addition, as shown in Fig. 7, after transfection of the AuNP ^RNAI/His -ASO-RNH complex, the expression inhibition efficiency according to 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 incubation time elapsed, and this was also confirmed by separating the qRT-PCR product on a 1.5% agarose gel and visualizing it using ethidium bromide.

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 AuNP ^RNAI/His -SO-RNH or AuNP ^RNAI/His -SON-RNH complexes were not effective in knockdown of the target RNA (Fig. 8, Fig. 9).

Taken together, these results demonstrate that the AuNP ^RNAI/His -ASO-RNH complex can efficiently reduce the target mRNA levels of SARS-CoV-2 in VERO-E6 cells.

8. Target protein expression inhibition effect of AuNP ^RNAI/His -ASO-RNH complex

To determine whether the AuNP ^RNAI/His -ASO-RNH complex of the present invention 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 about 35% was observed. In addition, consistent with the mRNA expression level, no significant effect was observed when cells were treated with AuNP ^RNAI/His , AuNP ^RNAI/His -RNH, or AuNP RNAI/ ^His -scrambledDNA-RNH. In addition, similar results were confirmed from FACS analysis of GFP-expressing cells (Fig. 11). These results demonstrate that AuNP ^RNAI/His -ASO-RNH can knockdown the expression of the GFP reporter gene fused to the positive sense strand of SARS-CoV-2.

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

To further test the effect of AuNP ^RNAI/His -ASO-RNH in mammalian cells infected with SARS-CoV-2, VERO-E6 cells infected with SARS-CoV-2 were transfected with AuNP ^RNAI/His or AuNP ^RNAI/His -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.

As a result, as shown in Fig. 12, the nucleocapsid protein level was reduced by 30-40% in VERO-E6 (Fig. 17) treated with AuNP ^{RNAI/His (} 0.1 and 1 nM) and a lower concentration of AuNP ^RNAI/His -ASO-RNH (0.1 nM), no significant effect on the N protein level was observed. These results demonstrate that 1 nM of AuNP ^RNAI/His -ASO-RNH can effectively knockdown the expression of SARS-CoV-2 N protein in mammalian cells.

Furthermore, the ability of AuNP ^RNAI/His -ASO-RNH to inhibit viral replication was confirmed using VERO-E6 cells infected with recombinant SARS-CoV-2 capable of expressing GFP (SARS-CoV-2-mNeon). As a result, as shown in Fig. 13, a 30-40% decrease in GFP intensity was observed in cells treated with AuNP ^RNAI / ^His -ASO-RNH compared to cells treated with virus alone, AuNP RNAI ^/His , AuNP ^RNAI/His -RNH, or AuNP RNAI/His -Scrambled DNA-RNH. These results demonstrate that AuNP ^RNAI/His -ASO-RNH inhibits GFP translation through the cleavage of viral mRNA.

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

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

As a result, as shown in Fig. 14A, the expression level of RdRP mRNA was reduced by ~30% in the lung when AuNP ^RNAI/His -ASO-RNH was delivered intranasally compared to treatment with PBS or only AuNP-DNA oligos. Similarly, as shown in Fig. 14B, when AuNP ^RNAI/His -ASO-RNH was delivered retroorbitally, viral mRNA was reduced by ~20% in the nasal conchae and lung compared to treatment with PBS or only AuNP-DNA oligos. These data demonstrate that AuNP ^RNAI/His -ASO-RNH can be effectively delivered to the airways of infected animals and reduce the viral titer when administered to treat the SARS-CoV-2 virus.

The present invention has been described with reference to preferred embodiments thereof. Those skilled in the art will appreciate that the present invention may be implemented in modified forms without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is set forth in the claims, not in the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

In one aspect, the present invention relates to a nanoparticle delivery vehicle comprising a gold nanoparticle; and 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.

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

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

As an example, 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.

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

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

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, 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 (10)

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. In the first paragraph, A nanoparticle carrier wherein the above viral-derived sequence is a nucleotide sequence contained in the 5’untranslated region (UTR). In the first paragraph, A nanoparticle carrier wherein the above virus-derived sequence comprises a leader sequence. In the first paragraph, 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. In the first paragraph, A nanoparticle carrier wherein the antisense oligonucleotide (ASO) is bound to the gold nano surface via a moiety containing a functional group. In paragraph 5, A nanoparticle carrier wherein the residue containing the above functional group contains a thiol group or an amine group. In the first paragraph, A gold nanoparticle carrier wherein the above RNaseH is bound to the surface of the gold nano via an aptamer. In Article 7, The above aptamer is a gold nanoparticle carrier containing a histidine tag. An antiviral composition comprising the nano particle carrier of claim 1. 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) bound to the surface that induces cleavage of mRNA of the virus, 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 degrade the mRNA through RNase H.

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