DOUBLE-STRANDED OLIGONUCLEOTIDE AND COMPOSITION FOR TREATING COVID-19 CONTAINING SAME

Abstract

The present invention relates to: a double-stranded oligonucleotide which can highly specifically and efficiently inhibit the proliferation of Severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2), preferably a double-stranded oligonucleotide comprising a sequence in the form of RNA/RNA, DNA/DNA or a DNA/RNA hybrid; a double-stranded oligonucleotide structure and nanoparticles comprising the double-stranded oligonucleotide; and a use thereof for treating COVID-19.

Claims

1. A double-stranded oligonucleotide specific to SARS-Cov-2, comprising a sense strand comprising any one sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 and an antisense strand comprising a sequence complementary thereto.

2. The double-stranded oligonucleotide according to claim 1, wherein the sense strand or the antisense strand comprises 19 to 31 nucleotides.

3. The double-stranded oligonucleotide according to claim 1, wherein the oligonucleotide is siRNA, shRNA, or miRNA.

4. The double-stranded oligonucleotide according to claim 1, wherein each of the sense strand and the antisense strand is independently DNA or RNA.

5. The double-stranded oligonucleotide according to claim 1, wherein the sense strand or the antisense strand of the double-stranded oligonucleotide comprises a chemical modification.

6. The double-stranded oligonucleotide according to claim 5, wherein the chemical modification is at least one selected from the group consisting of: a modification in which a hydroxyl group (—OH) at a 2′ carbon position of a sugar structure in a nucleotide is substituted with any one selected from the group consisting of methyl (—CH.sub.3), methoxy (—OCH.sub.3), amine (—NH.sub.2), fluorine (—F), O-2-methoxyethyl, O-propyl, O-2-methylthioethyl, O-3-aminopropyl, O-3-dimethylaminopropyl, O—N-methylacetamido, and O-dimethylamidooxyethyl; a modification in which oxygen of the sugar structure in the nucleotide is substituted with sulfur; a modification in which a nucleotide bond is any one bond selected from the group consisting of a phosphorothioate bond, a boranophosphate bond, and a methyl phosphonate bond; and a modification into PNA (peptide nucleic acid), LNA (locked nucleic acid), or UNA (unlocked nucleic acid).

7. The double-stranded oligonucleotide according to claim 1, wherein at least one phosphate group is bound to a 5′ end of the antisense strand of the double-stranded oligonucleotide.

8. A double-stranded oligonucleotide construct specific to SARS-Cov-2, having a structure of Structural Formula (1) below:
A-X—R—Y—B  Structural Formula (1) wherein in Structural Formula (1), A is a hydrophilic material, B is a hydrophobic material, X and Y are each independently a simple covalent bond or a linker-mediated covalent bond, and R is the double-stranded oligonucleotide according to claim 1.

9. The double-stranded oligonucleotide construct according to claim 8, having a structure of Structural Formula (2) below:
A-X—S—Y—B AS  Structural Formula (2) in Structural Formula (2), S and AS respectively represent a sense strand and an antisense strand of the double-stranded oligonucleotide according to claim 8, and A, B, X, and Y are as defined in claim 8.

10. The double-stranded oligonucleotide construct according to claim 9, having a structure of Structural Formula (3) or Structural Formula (4) below:
A-X-5′ S 3′-Y—B AS  Structural Formula (3)
A-X-3′ S 5′-Y—B AS  Structural Formula (4) in Structural Formulas (3) and (4), A, B, X, Y, S, and AS are as defined in claim 9, and 5′ and 3′ respectively represent a 5′ end and a 3′ end of the sense strand of the double-stranded oligonucleotide.

11. The double-stranded oligonucleotide construct according to claim 8, wherein the hydrophilic material is selected from the group consisting of polyethylene glycol (PEG), polyvinylpyrrolidone, and polyoxazoline.

12. The double-stranded oligonucleotide construct according to claim 8, wherein the hydrophilic material has a structure of Structural Formula (5) or Structural Formula (6) below:
(A′.sub.m-J).sub.n  Structural Formula (5)
(J-A′.sub.m).sub.n  Structural Formula (6) in Structural Formula (5) or Structural Formula (6), A′ is a hydrophilic material monomer, J is a linker connecting m hydrophilic material monomers to each other or connecting m hydrophilic material monomers and a double-stranded oligonucleotide to each other, m is an integer of 1 to 15, n is an integer of 1 to 10, the hydrophilic material monomer A′ is any one compound selected from among compounds (1) to (3) below, and the linker (J) is selected from the group consisting of —PO.sub.3.sup.−—, —SO.sub.3—, and —CO.sub.2—: ##STR00005## in compound (1), G is selected from the group consisting of O, S, and NH; ##STR00006##

13. The double-stranded oligonucleotide construct according to claim 12, having a structure of Structural Formula (7) or Structural Formula (8) below:
(A′.sub.m-J).sub.n-X—R—Y—B  Structural Formula (7)
(J-A′.sub.m).sub.n-X—R—Y—B.  Structural Formula (8)

14. The double-stranded oligonucleotide construct according to claim 8, wherein the hydrophilic material has a molecular weight of 200 to 10,000, or wherein the hydrophobic material has a molecular weight of 250 to 1,000.

15. (canceled)

16. The double-stranded oligonucleotide construct according to claim 14, wherein the hydrophobic material is any one selected from the group consisting of a steroid derivative, glyceride derivative, glycerol ether, polypropylene glycol, C.sub.12 to C.sub.50 unsaturated or saturated hydrocarbon, diacyl phosphatidylcholine, fatty acid, phospholipid, lipopolyamine, lipid, tocopherol, and tocotrienol.

17. The double-stranded oligonucleotide construct according to claim 16, wherein the steroid derivative is any one selected from the group consisting of cholesterol, cholestanol, cholic acid, cholesteryl formate, cholestanyl formate, and cholesteryl amine, or wherein the glyceride derivative is any one selected from the group consisting of monoglycerides, diglycerides, and triglycerides.

18. (canceled)

19. The double-stranded oligonucleotide construct according to claim 8, wherein the covalent bond represented by X and Y is a non-degradable bond or a degradable bond.

20. The double-stranded oligonucleotide construct according to claim 19, wherein the non-degradable bond is an amide bond or a phosphate bond, or the degradable bond is any one selected from the group consisting of a disulfide bond, an acid-degradable bond, an ester bond, an anhydride bond, a biodegradable bond, and an enzyme-degradable bond.

21. (canceled)

22. Nanoparticles comprising the double-stranded oligonucleotide construct according to claim 8.

23. (canceled)

24. A method for treating COVID-19 comprising administering a pharmaceutical composition comprising the double-stranded oligonucleotide according to claim 1 or a double-stranded oligonucleotide construct having a structure of Structural Formula (1) below:
A-X—R—Y—B  Structural Formula (1) wherein in Structural Formula (1), A is a hydrophilic material, B is a hydrophobic material, X and Y are each independently a simple covalent bond or a linker-mediated covalent bond, and R is the double-stranded oligonucleotide according to claim 1, as an active ingredient.

25. A method for treating COVID-19 comprising administering a pharmaceutical composition comprising the nanoparticles according to claim 22 as an active ingredient.

26.-27. (canceled)

Description

DESCRIPTION OF DRAWINGS

[0137] FIG. 1 shows a process of selecting candidate sequences each composed of 19 nucleotides by applying a 1-base sliding-window algorithm to a genome in order to design SARS-CoV-2-specific oligonucleotide candidate sequences;

[0138] FIGS. 2A and 2B show results of measurement of E and Rdrp gene Ct values after infecting a SAMiRNA-treated Huh7 cell line with COVID-19;

[0139] FIG. 3 shows a process for evaluating the in-vivo antiviral efficacy of SAMiRNA-COVID19;

[0140] FIG. 4 shows changes in body weight and body temperature of ferrets in the evaluation of the in-vivo antiviral efficacy of SAMiRNA-COVID19; and

[0141] FIG. 5 shows results of measurement of SARS-CoV-2 viral RNA copy number and viral titer in the nasal wash sample of ferrets in the evaluation of the in-vivo antiviral efficacy of SAMiRNA-COVID19.

MODE FOR INVENTION

[0142] A better understanding of the present invention may be obtained through the following examples. These examples are merely set forth to illustrate the present invention and are not construed as limiting the scope of the present invention, as will be apparent to those skilled in the art. Therefore, the substantial scope of the present invention will be defined by the appended claims and equivalents thereto.

Example 1. Algorithm for Screening of Oligonucleotide Targeting SARS-CoV-2 and Selection of Candidate Sequences

[0143] siRNA-based drug high-throughput screening is a method for generating all possible candidate sequences by applying a sliding-window algorithm to an entire RNA virus genome sequence, removing unnecessary candidate sequences through homology filtering, and determining the extent of inhibition of virus replication by all of the finally selected oligonucleotides.

[0144] A design process for oligonucleotide candidate sequences for SARS-CoV-2 was performed in a manner in which a 1-base sliding-window algorithm was applied to the SARS-CoV-2 reference genome (NC_045512.2, 29,903 bp) to thus generate 29,885 candidate sequences each composed of 19 nucleotides (FIG. 1).

[0145] The generated oligonucleotide candidate sequence list was performed with a BLAST e-value of 100 or less for human total reference seq RNA, and as such, 6,885 candidate sequences having identities of 15 nucleotides or less to human genes and RNA sequences were selected. Thereamong, 560 candidate sequences having all 19 nucleotides identical to SARS-CoV (NC_004718.3, 29,751 bp) were selected. In consideration of siRNA efficiency, 480 candidate sequences with a GC content of 30 to 60 were finally selected.

Example 2. Synthesis of Double-Stranded Oligo RNA Construct

[0146] The double-stranded oligo RNA construct (SAMiRNA) produced in the present invention has a structure according to the following structural formula.

C.sub.24-5′ S 3′-(hexaethylene glycol-PO.sub.3.sup.−).sub.3-hexaethylene glycol AS 5′-PO.sub.4

[0147] The sense strand of a monoSAMiRNA (n=4) double-stranded oligo construct was synthesized as follows. Specifically, three dimethoxytrityl (DMT) hexaethylene glycol phosphoramidates, which are hydrophilic material monomers, were successively bound through the above reaction using 3,4,6-triacetyl-1-hexa(ethylene glycol)-N-acetyl galactosamine-CPG as a support, RNA or DNA synthesis was performed, and then C.sub.24(C.sub.6—S—S—C.sub.18) containing a disulfide bond, which is a hydrophobic material, was additionally bound to the 5′ end, thereby synthesizing the sense strand of monoSAMiRNA (n=4) in which NAG-hexaethylene glycol-(—PO.sub.3.sup.− hexaethylene glycol).sub.3 was bound to the 3′ end and C.sub.24 (C.sub.6—S—S—C.sub.18) was bound to the 5′ end.

[0148] After completion of synthesis, the synthesized single-stranded RNA and oligo (DNA or RNA)/polymer construct were separated from CPG using 28% (v/v) ammonia in a water bath at 60° C., followed by a deprotection reaction to remove protective residues. After removal of the protective residues, the single-stranded RNA and the oligo (DNA or RNA)/polymer construct were treated with N-methylpyrrolidone, triethylamine, and triethylamine trihydrofluoride at a volume ratio of 10:3:4 in an oven at 70° C. to remove 2′TBDMS (tert-butyldimethylsilyl). The single-stranded RNA, the oligo (DNA or RNA)/polymer construct, and the ligand-bound oligo (DNA or RNA)/polymer construct were separated from the reaction products through high-performance liquid chromatography (HPLC), and the molecular weights thereof were measured using a MALDI-TOF mass spectrometer (MALDI TOF-MS, SHIMADZU, Japan) to determine whether they matched the nucleotide sequence and the oligo/polymer construct to be synthesized. Thereafter, in order to produce each double-stranded oligo construct, the sense strand and the antisense strand were mixed in equal amounts, added to a 1× annealing buffer (containing 30 mM HEPES, 100 mM potassium acetate, and 2 mM magnesium acetate, pH 7.0-7.5), allowed to react in a constant-temperature water bath at 90° C. for 3 minutes, and further allowed to react at 37° C., thereby producing desired SAMiRNA. Annealing of the double-stranded oligo RNA constructs thus produced was confirmed through electrophoresis.

Example 3. High-Throughput Screening of RNAi-Inducing SAMiRNA Nanoparticles Targeting SARS-CoV-2

[0149] 3.1 Preparation of SAMiRNA Nanoparticles

[0150] 960 SAMiRNAs targeting the SARS-CoV-2 sequence synthesized in Example 2 were dissolved in a 1× Dulbecco's phosphate-buffered saline (DPBS) (WELGENE, KR), filtered with a 96-well Filtration Plate MultiScreen.sub.HTS GV Filter Plate, 0.22 μm, clear, sterile (MERCK, DE), and then used in the experiment for the present invention.

[0151] 3.2 Intracellular Processing of SAMiRNA Nanoparticles

[0152] In order to discover SAMiRNA that inhibits the expression of SARS-CoV-2, Huh7, which is a human-derived liver cancer cell line, was used, and Huh-7 cells (JCRB, JP) were seeded in a 96-well plate 24 hours before treatment with SAMiRNA. RPMI 1640 medium (sh30027.01) (Hyclone, US) was used as a cell culture medium, and 100 μl thereof was added to each well, followed by cell culture at 37° C. and 5% CO.sub.2. The RPMI 1640 medium was supplemented with 2.05 mM L-glutamine, 1% penicillin-streptomycin (Hyclone, US), and 10% fetal bovine serum (Hyclone, US). The cells were treated once with SAMiRNA candidates at a concentration of 10 μM with two medium replacements, followed by culture at 37° C. and 5% CO.sub.2 for 24 hours.

[0153] 3.3 Intracellular SARS-CoV-2 Infection

[0154] After removing the medium from the 96-well cell culture plate cultured in Example 3.2, each well was treated with the prepared virus at an MOI of 0.01 and infected for 1 hour and 30 minutes. Thereafter, the medium was replaced with an RPMI 1640 medium (virus growth medium) containing 2% fetal bovine serum, followed by cell culture at 37° C. and 5% CO.sub.2 for 2 days. Finally, the cell culture fluid was harvested from the cell culture plate and then mixed with Cartridge No. 1 (Lysis buffer) and Proteinase K of the Exiprep™ 96 Viral DNA/RNA Kit (BIONEER, KR), followed by culture at 65° C. for 2 hours.

[0155] 3.4 SAMiRNA Screening Through Analysis of SARS-CoV-2 gRNA Expression Inhibitory Efficacy

[0156] In order to extract gRNA from the cell culture fluid obtained in Example 3.3, ExiPrep™ 96 Lite (BIONEER, KR) and ExiPrep™ 96 Viral DNA/RNA Kit (BIONEER, KR), which are automated nucleic acid extraction instruments, were used. The extracted nucleic acids were analyzed for E and Rdrp genes using the Exicycler™ 96 Real-Time Quantitative Thermal Block (BIONEER, KR) according to the manufacturer's protocol of AccuPower® SARS-CoV-2 Real-Time RT-PCR Kit (BIONEER, KR). Here, RT-qPCR was performed 45 cycles of 30 minutes at 50° C., 10 minutes at 94° C., 15 seconds at 95° C., and 1 minute at 60° C.

[0157] All Ct values derived from biological/technical repeats of the E and Rdrp genes obtained from the virus-infected cell culture samples treated with SAMiRNA were determined. Based on the above results, the optimal SAMiRNA candidates for each gene region of SARS-CoV-2 were selected. The selected SAMiRNA candidates were compared with 7259 sequences registered in the COVID-19 database and tested for gene sequence variation.

[0158] Consequently, as shown in Table 2 below, 10 types of SAMiRNA having SARS-CoV-2 inhibitory efficacy were finally selected. The selected SAMiRNA candidates are SAMiRNAs having the sequences of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, respectively, in the order described. The selected SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 correspond to #161, #214, #233, #246, #249, #544, #625, #707, #715, and #758 of FIG. 2, respectively.

TABLE-US-00002 TABLE 2 SARS-CoV-2-specific oligonucleotide candidates  selected through 1-base sliding-window screening SEQ Acces- Sense SEQ Antisense ID sion Posi- strand ID strand Iden- NO: No. tion sequence NO: sequence tity  1 NC_ 15094- AATAGAGCTC 11 CUACGGUGCG  99.99% 045512.2 15112 GCACCGTAG AGCUCUAUU  2 NC_ 17088- TGGTACTGGT 12 AUGACUCUUA  99.83% 045512.2 17106 AAGAGTCAT CCAGUACCA  3 NC_ 18255- GTTTATCACC 13 UUCUUCGCGG  99.92% 045512.2 18273 CGCGAAGAA GUGAUAAAC  4 NC_ 20457- CATAACAGAT 14 UGUUUGCGCA  99.89% 045512.2 20475 GCGCAAACA UCUGUUAUG  5 NC_ 20776- ATAATGATGA 15 UUGCGACAUU  99.96% 045512.2 20794 ATGTCGCAA CAUCAUUAU  6 NC_ 10488- TTAAAACCAA 16 GUGGUAGUGU 100.00% 045512.2 10506 CACTACCAC UGGUUUUAA  7 NC_ 15038- ATAGTAGGGA 17 GUAAUGUCAU 100.00% 045512.2 15056 TGACATTAC CCCUACUAU  8 NC_ 17957- TCTCTATCAG 18 GCAUAAUGUC 100.00% 045512.2 17975 ACATTATGC UGAUAGAGA  9 NC_ 18257- GCTTCTTCGC 19 UUAUCACCCG  99.94% 045512.2 18275 GGGTGATAA CGAAGAAGC 10 NC_ 25509- CCATCCGAAA 20 CUCACUCCCU  99.71% 045512.2 25527 GGGAGTGAG UUCGGAUGG

Example 4. Evaluation of Symptom Relief Efficacy of SAMiRNA™ in SARS-CoV-2-Infected Ferret Model

[0159] 4.1 Preparation of SAMiRNA™ Nanoparticles

[0160] A mixture of 10 types of SAMiRNA™ targeting the SARS-CoV-2 sequence shown in Table 2 was dissolved in a 1× Dulbecco's phosphate-buffered saline (DPBS) (WELGENE, KR), filtered with a 96-well Filtration Plate MultiScreen.sub.HTS GV Filter Plate, 0.22 μm, clear, sterile (MERCK, DE), and then used in the experiment for the present invention.

[0161] 4.2 SARS-CoV-2-Infected Ferret Animal Test Method

[0162] A total of 11 female ferrets with an average age of 15 months (IDBio, Korea) were divided into a control group untreated after infection including 3 ferrets, a subcutaneous injection (sc) group including 4 ferrets, and a subcutaneous injection+intratracheal administration group including 4 ferrets.

[0163] 0.5 mL of SARS-CoV-2 (CBNU-nCoV-01) was inoculated at 1×10.sup.5.5 TCID50/mL into each of the nasal cavity and bronchi of ferrets. 24 hours after virus infection, SAMiRNA™ nanoparticles were administered once to each experimental animal through subcutaneous injection (50 mpk) and through subcutaneous injection (50 mpk)+intratracheal administration (500 μl).

[0164] Changes in body weight and body temperature of individual subjects in the control and test groups were measured on day 0 before virus infection and on days 2, 4, 6, 8, and 10 after infection, and nasal wash samples were collected (FIG. 3). The collected samples were subjected to viral titer measurement using a TCID50 method, or RNA was extracted therefrom and then absolute quantitative analysis of viral RNA copy number was performed through qRT-PCR according to the manufacturer's protocol of AccuPower® SARS-CoV-2 Real-Time RT-PCR Kit (BIONEER, KR).

[0165] Consequently, the body weight of ferrets after virus infection was reduced by 7-8% compared to before infection, and body temperature was returned to normal after an average increase of 0.5° C. or higher at 4 dpi, and there was no significant difference by SAMiRNA treatment (FIG. 4).

[0166] Based on the results of measurement of the SARS-CoV-2 copy number through qRT-PCR in the nasal wash of ferrets, the number of SARS-CoV-2 viruses was significantly reduced in the subcutaneous injection+intratracheal administration group at 8 dpi compared to the infection control group (FIG. 5A).

[0167] Based on the results of measurement of the viral titer using TCID50, the viral titer was statistically significantly reduced in both the subcutaneous injection group and the subcutaneous injection+intratracheal administration group at 4 dpi compared to the infection control group. Statistical significance was confirmed in the subcutaneous injection+intratracheal administration group even at 6 dpi compared to the infection control group (FIG. 5B).

[0168] Although specific embodiments of the present invention have been disclosed in detail above, it will be obvious to those skilled in the art that the description is merely of preferable exemplary embodiments and is not to be construed as limiting the scope of the present invention. Therefore, the substantial scope of the present invention will be defined by the appended claims and equivalents thereto. Simple modifications or changes of the present invention can be easily used by those of ordinary skill in the art, and all such modifications or changes can be considered to be included in the scope of the present invention.