WO2021234647A1 - 이중가닥 올리고뉴클레오티드 및 이를 포함하는 코로나바이러스감염증-19(covid-19) 치료용 조성물 - Google Patents
이중가닥 올리고뉴클레오티드 및 이를 포함하는 코로나바이러스감염증-19(covid-19) 치료용 조성물 Download PDFInfo
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- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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Definitions
- Double-stranded oligonucleotide and composition for treating coronavirus infection-19 comprising the same
- the present invention is a double-stranded oligonucleotide capable of inhibiting the proliferation of SARS-Cov-2 (Severe acute respiratory syndrome coronavirus 2) with very specific and high efficiency, preferably RNA/RNA, DNA/DNA, or It relates to a double-stranded oligonucleotide comprising a sequence in the form of a DNA/RNA hybrid, a double-stranded oligonucleotide structure and nanoparticles comprising the double-stranded oligonucleotide, and a use thereof for the treatment of coronavirus infection-19.
- SARS-Cov-2 severe acute respiratory syndrome coronavirus 2
- RNAi RNA interference
- target genes such as cancer and genetic diseases that are the cause of overexpression of specific genes can be removed from the mRNA level, thereby developing therapeutic agents for disease treatment And it can be utilized as an important tool for target verification.
- a technique for introducing a transgene for a target gene is disclosed.
- RNA therapy targeting RNA is a method that removes the function of the gene by using an oligonucleotide for the target RNA.
- ASOs antisense oligonucleotides
- Antisense oligonucleotide (ASO, hereinafter referred to as 'ASO') is a short-length synthetic DNA designed to bind to a target gene according to the Watson-Crick base reaction, and can specifically inhibit the expression of a specific nucleotide sequence of a gene.
- Genetic 2021/234647 ?01/162021/054404 It has been used to study function and develop therapeutics that can treat diseases such as cancer at the molecular level.
- Such ASO has the advantage that it can be easily manufactured by setting various targets for suppressing gene expression, and there have been studies to use it to suppress the expression of oncogenic genes and the growth of cancer cells.
- ASO suppresses the expression of a specific gene is achieved by binding to a complementary mRNA sequence and inducing RNase H activity to remove mRNA or to interfere with the formation and progression of a ribosome complex for protein translation. It has also been reported that ASO binds to genomic DNA to form a triple-helix structure, thereby inhibiting gene transcription. ASO has the above potential, but in order to be used in clinical practice, it must be efficiently delivered into target tissues or cells to improve stability to nucleases and specifically bind to the nucleotide sequence of the target gene. .
- the secondary and tertiary structures of gene mRNA are important factors for specific binding of ASO, and since the portion with less secondary structure of mRNA is very advantageous for ASO to access, less secondary structure of mRNA is generated before synthesizing ASO. Efforts have been made to effectively achieve gene-specific inhibition not only in vitro but also in vivo by systematically analyzing the target region. These ASOs are very stable than siRNA, which is a kind of RNA, and have the advantage of being well soluble in water and physiological saline. Currently, three ASOs have been approved by the Federal Drug Administration (FDA) (Jessica, C, J Postdoc).
- FDA Federal Drug Administration
- siRNA small interfering RNA
- RISC RNA-induced silencing complex
- guide (antisense) strand recognizes and degrades the target mRNA.
- RISC RNA-induced silencing complex
- RISC RNA-induced silencing complex
- antisense RNA-induced silencing complex
- Sequence-specific inhibition of expression The gene expression suppression technology using siRNA suppresses the expression of a target gene in a target cell and observes the resulting change, which is useful for research to elucidate the function of a target gene in a target cell. In particular, inhibiting the function of a target gene in an infectious virus or cancer cell will be useful for developing a treatment method for the disease As a result of in vivo studies, it has been reported that siRNA can suppress the expression of a target gene.
- siRNA is complementary to [ ⁇ 1/ ⁇ and sequence-specifically inhibits the expression of the target gene.
- 2021/234647 ?01/162021/054404 It has the advantage of being able to dramatically expand the applicable target compared to the existing antibody-based drugs or small molecule drugs (MA Behlke, MOLECULAR THERAPY) 2006 13(4):664-670).
- siRNA Despite siRNA's excellent effect and wide range of uses, in order for siRNA to be developed as a therapeutic agent, siRNA must be effectively delivered to target cells by improving the stability of siRNA in the body and improving cell delivery efficiency (FY Xie, Drug) Discov. Today. 2006 Jan; 11(1-2):67-73).
- some nucleotides or backbone of siRNA are modified to have nuclease resistance or a viral vector, Research on this is being actively attempted, such as using a carrier such as liposomes or nanoparticles (nanopartide).
- a delivery system using a viral vector such as an adenovirus or a retrovirus has high transfection efficacy, but high immunogenicity and oncogenicity.
- a non-viral delivery system containing nanoparticles has lower cell delivery efficiency than a viral delivery system, but has poor stability in vivo ( ⁇ 1//1/0). It has the advantages of high, target-specific delivery, uptake and internalization of embedded RNAi oligonucleotides into cells or tissues, and little cytotoxicity and immune stimulation.
- viral transmission 2021/234647 -01/162021/054404 It is evaluated as a powerful delivery method compared to the system (Akhtar S, J Clin Invest. 2007 December 3; 117(12): 3623-3632).
- the method using a nanocarrier forms nanoparticles by using various polymers such as liposomes and cationic polymer complexes, and siRNA is loaded on these nanoparticles, that is, a nanocarrier.
- method of delivery to cells include polymer nanoparticles, pdymer micelles, and lipoplexes.
- the method using lipoplex is composed of cationic lipids It interacts with the anionic lipid of the endosome of the cell to induce a destabilizing effect of the endosome and deliver it into the cell.
- siRNA table H passenger (sense) strand high efficiency in vivo ( ⁇ 1//1/1/ ⁇ ) by linking chemicals to the terminal site of the siRNA table H passenger (sense) strand to have enhanced pharmacokinetics characteristics (J Soutschek, Nature 11; 432(7014):173-8, 2004), where siRNA sense (sense; Table H passenger) or antisense (guide) )
- siRNA sense (sense; Table H passenger) or antisense (guide) The stability of siRNA varies depending on the nature of the chemical compound attached to the end of the strand, for example, siRNA conjugated with a high molecular compound such as polyethylene glycol (PEG) can be used in the presence of a cationic material.
- PEG polyethylene glycol
- 2021/234647 -01/162021/054404 SH Kim, J Control Release 129(2):107-16, 200.
- micelles composed of polymer complexes are another system used as drug delivery vehicles, microspheres ( Microsphere) or nanoparticles have the advantage of being very small in size and uniform in distribution and spontaneously formed, so it is easy to control the quality of the preparation and secure the reproducibility.
- siRNA conjugate in which a hydrophilic material (eg, polyethylene glycol (PEG)), which is a biocompatible polymer, is conjugated to siRNA by a simple covalent bond or a linker-mediated covalent bond,
- a hydrophilic material eg, polyethylene glycol (PEG)
- PEG polyethylene glycol
- oligo RNA structure in which hydrophilic and hydrophobic substances are combined with oligonucleotides, particularly double-stranded oligo RNA such as siRNA, has been developed.
- the structure forms self-assembled nanoparticles called SAMiRNAä (self assembled micelle inhibitory RNA) by the hydrophobic interaction of hydrophobic substances (Korea Registered Patent Nos. 1,224, 828) It has the advantage of being able to obtain uniform (homogenous) nanoparticles with a very small size compared to that of the present invention.
- SAMiRNAä self assembled micelle inhibitory RNA
- PEG polyethylene 2021/234647 -01/162021/054404 glycol
- HEG Hexaethylene glycol
- PEG is a synthetic polymer and is often used in pharmaceuticals, especially proteins, to increase solubility and control pharmacokinetics.
- used for PEG is a polydisperse material, and the I polymer in one batch consists of the sum of different numbers of monomers, so the molecular weight shows a Gaussian curve shape, and the polydisperse value (Mw/Mn) represents the degree of homogeneity of a substance.
- PEG has a low molecular weight (3 ⁇ 5 kDa), it shows a polydispersity index of about 1.()1, and when it has a high molecular weight (20 kDa), it shows a high polydispersity index of about 1.2. relatively low characteristics. Therefore, when PEG is combined with pharmaceuticals, the polydispersity of PEG is reflected in the conjugate, which makes it difficult to verify a single substance.
- the size is smaller than the existing £ ! ⁇ /11[ ⁇ 1/ ⁇ ä 2021/234647 - 01/162021/054404
- a new type of carrier technology with dramatically improved polydispersity has been developed. Also, as it is already known that when siRNA is injected into the body, siRNA is rapidly degraded by various enzymes present in the blood, resulting in poor delivery efficiency to target cells or tissues. There was a variation in expression inhibition rate.
- the present inventors used an ASO DNA sequence as a guide and an RNA sequence as an antisense strand as a passenger. It has been confirmed that the expression inhibitory effect and stability of the target gene can be remarkably increased by applying the double-stranded oligonucleotide of the -RNA hybrid type.
- Coronavirus Infectious Disease-19 COVID-19 is a respiratory syndrome caused by SARS-Cov-2 (Severe Acute Respiratory Syndrome Coronavirus 2) infection.
- Respiratory symptoms such as coughing or shortness of breath, and pneumonia are the main symptoms, and it is spread by touching droplets (saliva) generated during coughing or sneezing or objects contaminated with SARS-Cov-2 and then touching your eyes, nose, and mouth. is known
- pandemic the highest level of infectious disease risk, means 'the global spread of a virus for which the majority of people do not have immunity', and the number of confirmed cases, deaths, and affected countries continues to increase.
- Coronavirus is a positive single-stranded (+ssRNA) virus characterized by the appearance of a crown-shaped Spike(S) protein in the envelope under an electron microscope, and is a circular or oval virus. Coronaviruses are classified into four genera: alphaCoV, betaCoV, deltaCoV, and gammaCoV. alphaCoV, which causes common respiratory infections, is HCoV-229E, HCoV-NL63, and betaCoV is HCoV-OC43 and HCoV-HKU1. It can also lead to infections and digestive problems. SARS-CoV (B lineage) and MERS-CoV (C lineage) belonging to betaCoV cause serious respiratory infectious diseases characterized by acute respiratory symptoms.
- the genomic sequence isolated from pneumonia patients in the early stages of COVID-19 was analyzed to have 89% nucleotide homology with bat SARS-like-CoVZXC21 and 82% homology with SARS-CoV, and was found to belong to the same betaCoV, SARS-CoV It was named -2.
- RT-PCR real time reverse
- 2021/234647 ?01/162021/054404 Transcription polymerase chain reaction is performing a diagnostic test for coronavirus infection-19.
- RNAi therapeutic agent and its delivery technology for coronavirus infection-19 is still insignificant, and a double-stranded oligonucleotide therapeutic agent capable of inhibiting the proliferation of SARS-Cov-2 with specific and high efficiency and The market demand for its delivery technology is very high.
- the present inventors selected a double-stranded oligonucleotide that targets SARS-Cov-2, and in particular, by inhibiting the proliferation of SARS-Cov-2, an RNAi therapeutic agent capable of effectively treating coronavirus infection-19 and its delivery system
- SARS-Cov-2 a double-stranded oligonucleotide that targets SARS-Cov-2
- an RNAi therapeutic agent capable of effectively treating coronavirus infection-19 and its delivery system
- Patent Literature U.S. Patent Nos. 5,034,323; U.S. Patent Nos. 5, 231,020; US Patent No. 5,283,184 Korean Registered Patent No. 8834 Korean Registered Patent No. 1224828 2021/234647 ?01/162021/054404
- An object of the present invention is a double-stranded oligonucleotide capable of inhibiting the proliferation of SARS-Cov-2 (Severe acute respiratory syndrome coronavirus 2) with very specific and high efficiency, preferably RNA/RNA, DNA
- SARS-Cov-2 severe acute respiratory syndrome coronavirus 2
- RNA/RNA DNA
- Another object of the present invention is the double-stranded oligonucleotide, comprising the 2021/234647 - 01/162021/054404
- a pharmaceutical composition for treating coronavirus infection-19 containing a double-stranded oligonucleotide structure and/or nanoparticles as an active ingredient.
- Another object of the present invention is to provide a coronavirus infection-19 treatment method comprising administering a pharmaceutical composition for the treatment of coronavirus infection-19 to an individual in need of treatment for coronavirus infection-19.
- Another object of the present invention is to provide the double-stranded oligonucleotide, the double-stranded oligonucleotide structure and/or nanoparticles comprising the same for use in the treatment of coronavirus infection-19.
- Another object of the present invention is to provide the pharmaceutical composition for use in the treatment of coronavirus infection-19.
- Another object of the present invention is to provide the use of the double-stranded oligonucleotide, the double-stranded oligonucleotide structure comprising the same, and nanoparticles for the manufacture of a drug for the treatment of coronavirus infection-19.
- the present invention provides a sense comprising any one sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10
- a sense comprising any one sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10
- a double-stranded oligonucleotide comprising a sense strand and an anti-sense strand comprising a sequence complementary thereto.
- the present invention also provides a double-stranded oligonucleotide comprising the above-mentioned double-stranded oligonucleotide.
- 2021/234647 -01/162021/054404 Oligonucleotide constructs and/or nanoparticles are provided.
- the present invention also provides 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 a sequence complementary thereto It provides a pharmaceutical composition for treating coronavirus infection-19 comprising a double-stranded oligonucleotide comprising an anti-sense strand, a double-stranded oligonucleotide structure comprising the double-stranded oligonucleotide, or nanoparticles .
- the present invention also provides a coronavirus infection-19 treatment method comprising administering the pharmaceutical composition for the treatment of coronavirus infection-19 to an individual in need of treatment for coronavirus infection-19.
- the double-stranded oligonucleotide according to the present invention is a concept including all substances having a general RNAKRNA interference) action, and the SARS-Cov-2 specific double-stranded oligonucleotide includes SARS-Cov-2 specific shRNA, etc. It will be apparent to those of ordinary skill in the art to which the present invention pertains. That is, the oligonucleotide may be characterized in that it is siRNA, shRNA or miRNA.
- the sense or antisense strand may independently be DNA or RNA, for example, the sense strand is DNA, the antisense strand is RNA, or the sense strand is RNA, and the antisense strand is DNA. hybrid) can be used.
- the sequences of SEQ ID NOs: 1 to 10 are described in the form of DNA, but when an RNA form is used, the sequences of SEQ ID NOs: 1 to 10 are the corresponding RNA sequences, That is, a sequence in which T is changed to Ni may be used.
- the double-stranded oligonucleotide according to the present invention is a nucleotide sequence 100% complementary to the binding site of the SARS-Cov-2 gene, i.e., complete, as long as the specificity for SARS-Cov-2 is maintained.
- the double-stranded oligonucleotide according to the present invention may include an overhang, which is a structure comprising one or more unpaired nucleotides at the 3' end of one or both strands.
- the sense strand or the antisense strand may preferably consist of 19 to 31 nucleotides, but is not limited thereto.
- the sense strand or antisense strand of the double-stranded oligonucleotide includes various chemical modifications to improve in vivo stability, or to confer nuclease resistance and reduce non-specific immune response.
- the chemical modification is a hydroxyl group (-OH) at the 2' carbon position of the sugar structure in the nucleotide is a methyl group (-on), a methoxy group (-OCH 3 ), 2021/234647 -01/162021/054404 amine group (bur), fluorine (-F), 0-2 -methoxyethyl group, 0-propyl group, 0-2 -methylthioethyl group, 0-3-aminopropyl group, a modification substituted with any one selected from the group consisting of 0-3 -dimethylaminopropyl group, 0-N-methylacetamido group and 0-dimethylamidooxyethyl; a modification in which the oxygen of the sugar structure in the nucleotide is replaced with sulfur; a modification in which the nucleotide bond is any one bond selected from the group consisting of a phosphorothioate bond, a bora
- phosphate groups are bonded to the 5' end of the antisense strand of the double-stranded oligonucleotide, preferably 1 to 3 phosphate groups are bonded. can do.
- the present invention provides a double-stranded structure comprising the following structural formula (1): 2021/234647 ?01/162021/054404 relates to an oligonucleotide structure, wherein / ⁇ is a hydrophilic material, 8 is a hydrophobic material, and X and V are each independently a simple covalent bond or a linker-mediated covalent bond in the following structural formula (1) means, and ⁇ means a double-stranded oligonucleotide.
- the double-stranded oligonucleotide construct including the specific sequence preferably has a structure as shown in Structural Formula (1).
- AXRYB Structural Formula (1) 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 SARS- Cov-2 specific It means a red double-stranded oligonucleotide.
- the double-stranded oligonucleotide according to the present invention is preferably a DNA-RNA hybrid, siRNA (short interfering RNA), shRNA (short hairpin RNA), and miRNA (microRNA), but is not limited thereto, and an antagonist for miRNA Single-stranded miRNA inhibitors that can act as antagonists are also included.
- the double-stranded oligonucleotide according to the present invention will be described with a focus on RNA, but other double-stranded oligonucleotides having the same properties as the double-stranded oligonucleotide of the present invention are described below. It is apparent to those skilled in the art that it can also be applied to 2021/234647 -01/162021/054404 double-stranded oligonucleotides. More preferably, the double-stranded oligonucleotide structure comprising £[1 ⁇ 2 ⁇ 0 ⁇ /-2 specific double-stranded oligonucleotide according to the present invention has the structure of the following structural formula (2).
- the double-stranded oligonucleotide structure comprising £ [1 ⁇ 2-0) ⁇ /-2 specific double-stranded oligonucleotide has the structure of the following structural formula (3) or (4). ⁇ - .X - 5 ' % 3 1 - V ⁇ ' : ⁇ Structural Formula (3) 2021/234647 ?01/162021/054404
- ⁇ ⁇ X and are the same as defined in Structural Formula (2) 5' and 3' are $ [1 ⁇ 2-(; 0 ⁇ /-2 refers to the 5' end and 3' end of the specific double-stranded oligonucleotide sense strand, wherein the hydrophilic material is selected from the group consisting of polyethylene glycol, polyvinylpyrrolidone and polyoxazoline.
- the double-stranded oligonucleotide construct comprising the $ [1 ⁇ 2-[0 ⁇ /-2 specific double-stranded oligonucleotide in Structural Formulas (1) to (4) is antisense One to three phosphate groups ⁇ 05 6 a01 ) may be bonded to the end of the strand, and it is apparent to those of ordinary skill in the art that ⁇ [ 1/ ⁇ may be used instead of []. did it
- the hydrophilic material in Structural Formulas (1) to (4) is preferably a polymer material having a molecular weight of 200 to 10,000, and more preferably a polymer material having a molecular weight of 1,000 to 2,000.
- the hydrophilic material (/ ⁇ ) in Structural Formulas (1) to (4) may be used in the form of a hydrophilic material block ⁇ 10 ⁇ ) in the form of the following Structural Formula (5) or Structural Formula (6).
- the double-stranded oligonucleotide construct comprising the specific double-stranded oligonucleotide may have a structure as shown in the following Structural Formula (7) or Structural Formula (8).
- Structural formula (7) Structural formula (8)
- X, ⁇ and 8 are in the structural formula (1) 2021/234647 - 01/162021/054404 It is the same as the definition, /V,", this and n are the same as the definition in Structural Formula (5) and Structural Formula (6).
- the hydrophilic material monomer (/ V) can be used without limitation as long as it meets the purpose of the present invention among the monomers of the nonionic hydrophilic polymer, preferably the compounds described in Table 1 ( A monomer selected from 1) to compound (3), more preferably a monomer of compound (1) may be used, and 6 in compound (1) may be preferably selected from 0, £ and .
- the monomer represented by compound (1) can introduce various functional groups, has good in vivo compatibility and has excellent biocompatibility (ratio 0-[absorption ratio %), such as inducing less immune response.
- the hydrophilic material in the structural formulas (5) to (8) has a total molecular weight in the range of 1,000 to 2,000.
- hexaethylene glycol ( ⁇ 16X36 ⁇ ⁇ 6116 is) according to compound (1) that is, when a substance in which (3 is 0 and 171 is 6) is used, hexa Since the molecular weight of the ethylene glycol spacer a) is 344, it is preferable that the number of repeating characters is 3 to 5.
- the present invention provides the above structural formulas (5) to structural formulas as needed.
- the repeating element I of the hydrophilic group represented by 0- ⁇ that is, the hydrophilic material block ⁇ 10 ⁇
- n. / ⁇ which is a hydrophilic material monomer and a linker, included in each of the hydrophilic material blocks may be independently the same or different between each hydrophilic material block.
- the first block contains the hydrophilic material monomer according to compound (1)
- the second block contains the hydrophilic material monomer according to compound (2)
- the third block contains the compound
- a different hydrophilic material monomer may be used for every hydrophilic material block, such as using the hydrophilic material monomer according to 3), and any one selected from the hydrophilic material monomers according to compounds (1) to (3) for all hydrophilic material blocks
- a hydrophilic substance monomer may be used equally.
- the same linker may be used for each block of the hydrophilic material, or a different linker may be used for each block of the hydrophilic material as a linker for mediating the binding of the hydrophilic material monomer.
- the linker (J) is preferably selected from the group consisting of -P0 3- -, -S0 3 - and -C0 2 -, but is not limited thereto, depending on the monomer of the hydrophilic material used, etc. It will be apparent to those skilled in the art that any linker may be used as long as it is suitable for the purpose of the present invention.
- the hydrophobic material (B) in Structural Formulas (1) to (4), Structural Formula (7) and Structural Formula (8) is formed through hydrophobic interaction between Structural Formulas (1) to Structural Formulas (4), Structural Formulas (J) and Structural Formulas ( 8) serves to form nanoparticles composed of the double-stranded oligonucleotide structure according to the method.
- the hydrophobic material preferably has a molecular weight of 250 to 1,000, a steroid derivative, a glyceride derivative, glycerol ether, polypropylene glycol, C 12 to Cso unsaturated or saturated Hydrocarbons, diacyl phosphatidylcholine, fatty acids, phospholipids, lipopolyamines, etc. may be used, but are not limited thereto, 2021/234647 -01/162021/054404 It is obvious to those skilled in the art that any hydrophobic material can be used as long as it meets the object of the present invention.
- the steroid derivative may be selected from the group consisting of cholesterol, cholestanol, cholic acid, cholesteryl formate, cotestanyl formate and colistanylamine, and the glyceride derivative is mono-, di- and tri-glyceride.
- the fatty acid of the glyceride is preferably a C 12 to C 50 unsaturated or saturated fatty acid.
- the hydrophobic material is bound to the distal end of the hydrophilic material, and may be bound to any position of the sense strand or the antisense strand of the siRNA.
- the hydrophilic or hydrophobic material in Structural Formulas (1) to (4), Structural Formulas (J) and (8) according to the present invention and the SARS-Cov-2 specific double-stranded oligonucleotide are mediated by a simple covalent bond or a linker. It is bound by a covalent bond (X or ).
- the linker mediating the covalent bond is a hydrophilic substance or a hydrophobic substance and $ [1 ⁇ 2-(; 0 ⁇ /-2) at the end of the specific double-stranded oligonucleotide.
- the linker binds to activate £ [1 ⁇ 2-[0 ⁇ /-2 specific double-stranded oligonucleotide and/or a hydrophilic substance (or hydrophobic substance)) in the manufacturing process of the double-stranded oligonucleotide construct according to the present invention. Any compound may be used.
- the covalent bond may be either a non-degradable bond or a degradable bond.
- the non-degradable bond includes an amide bond or a phosphorylation bond
- the degradable bond includes a disulfide bond, an acid-decomposable bond, an ester bond, an anhydride bond, a biodegradable bond, or an enzyme-degradable bond, but is not limited thereto.
- Structural Formulas (1) to (4) Structural Formulas (7) and Structural Formulas (8) (or £ and expressed £ [1 ⁇ 2-(; 0 ⁇ /-2) specific double-stranded oligonucleotides are $ [1 ⁇ 2 -All double-stranded oligonucleotides having a characteristic capable of specifically binding to [0 ⁇ /-2] can be used without limitation, and preferably, in the present invention, SEQ ID NOs: 1, 2, It consists of a sense strand comprising any one sequence selected from the group consisting of 3, 4, 5, 6, 7, 8, 9 and 10 and an antisense strand comprising a sequence complementary thereto.
- the double-stranded oligonucleotide structure comprising £ [1 ⁇ 2-0) ⁇ /-2 specific double-stranded oligonucleotide according to 2021/234647 -01/162021/054404
- An amine group or a polyhistidine group may be additionally introduced. This is for facilitating the intracellular introduction and endosomal escape of the delivery system of the double-stranded oligonucleotide construct containing the SARS-Cov-2 specific double-stranded oligonucleotide according to the present invention, and has already been used in Quantum dot, Dendrimer, liposome, etc.
- the introduction of an amine group and a polyhistidine group can be used and its effect to facilitate the intracellular introduction of the transporter and the escape of the endosome.
- the primary amine group modified at the end or outside of the carrier forms a conjugate by electrostatic interaction with a negatively charged gene while being protonated at pH in vivo.
- the transporter can be protected from degradation of lysosomes as the escape of endosomes is facilitated due to the internal tertiary amine having (gene transfer and expression inhibition using a polymer-based hybrid material. Polymer Sci. TechnoL, Vol.
- histidine one of the non-essential amino acids, has an imidazoling (pKa3 6.04) at the residue (-R), so it has the effect of increasing the buffering capacity in endosomes and lysosomes. It is known that histidine modification can be used to increase the endosomal escape efficiency in non-viral gene carriers, including liposomes (Novel histidine-conjugated galactosylated cationic liposomes for efficient hepatocyte selective gene transfer) in human hepatoma 2021/234647 ?01/162021/054404
- the amine group or polyhistidine group may be connected to the hydrophilic material or the hydrophilic material block through one or more linkers. If oligonucleotide duplexes according to the structural formula (1) of the present invention in which the amine group or a polyhistidine ⁇ the hydrophilic material of the nucleotide structure, four) groups introduced may have a structure such as the formula (9).
- _) 2 is a simple covalent bond or 0 3 - , Preferably, but not limited to this.
- a polyhistidine group is introduced, in Structural Formula (9) 2021/234647 1 ⁇ (:1 ⁇ 2021/054404
- the hydrophilic material of the double-stranded oligonucleotide structure according to Structural Formula (9) is a hydrophilic material block according to Structural Formula (5) or Structural Formula (6), an amine group or a polyhistidine group is introduced. It may have a structure such as Structural Formula (10) or Structural Formula (11). and n is the same as defined in Structural Formula (7) or (8), and ⁇ is the same as defined in Structural Formula (9).
- the hydrophilic material is 0) ⁇ /-2
- the specific double-stranded oligonucleotide is preferably bound to the 3' end of the sense strand.
- X, [IXmin ⁇ / ⁇ ', , and ⁇ are the structural formulas Same as the definitions in (9) to (11), 5' and 3' mean $ [1 ⁇ 2-(; 0 ⁇ /- 2) the 5' end and 3' end of the double-stranded oligonucleotide sense strand
- amine group that can be introduced in the present invention primary to tertiary amine groups can be used, and it is particularly preferable that primary amine groups are used.
- a salt of a primary amine group may exist in the form of 1 ⁇ 3 + .
- the polyhistidine group that can be introduced in the present invention preferably includes 3 to 10 histidines, particularly preferably 5 to 8, and most preferably 6 histidines. Additionally, one or more cysteines may be included in addition to histidine.
- a targeting moiety is provided in a double-stranded oligonucleotide construct comprising a specific double-stranded oligonucleotide and nanoparticles formed therefrom, it can efficiently promote delivery to a target cell, and can be delivered to a target cell even at a relatively low dose. It can exhibit high target gene expression control function by being delivered, and can prevent the non-specific delivery of $ [1 ⁇ 2-0) ⁇ /-2 specific double-stranded oligonucleotides to other organs and cells.
- the present invention according to the above structural formula (I) to Formula (4), structural formula ligand structure according to (7) and Formula (8) (1_), especially receptor-mediated nested action (Hey-to 171601 ⁇ 601 0
- a structure for example, a double-stranded oligo according to Structural Formula (1) [The form in which the ligand is bound to the structure is
- A, B, ) ( and are the same as defined in structural formula (1) above, and L is receptor-mediated endocytosis, RME) refers to a ligand having a property of specifically binding to a receptor that promotes internalization of a target cell, and i is an integer from 1 to 5, preferably an integer from 1 to 3.
- the ligand is preferably a target receptor-specific antibody, aptamer, or peptide having RME properties to promote target cell-specific cell internalization; or folate (generally folate and folic acid cross each other)
- folate generally folate and folic acid cross each other
- folic acid refers to folate in a natural state or an activated state in the human body
- hexoamine such as N-acetyl galactosamine (NAG), and glucose (glucose) , mannose, but may be selected from chemical substances such as sugars or carbohydrates, etc., but is not limited thereto.
- the hydrophilic substance A in Structural Formula (15) is Structural Formula (5) and Structural Formula (6) ) according to the hydrophilic material block.
- the present invention provides a method for preparing a double-stranded oligonucleotide construct comprising a SARS-Cov-2 specific double-stranded oligonucleotide.
- the process of preparing a double-stranded oligonucleotide construct comprising a SARS-Cov-2 specific double-stranded oligonucleotide according to the present invention is, for example,
- the solid support in the present invention is preferably Controlled Pore Glass (CPG), but is not limited thereto, and polystyrene (PS), 2021/234647 ?01/162021/054404 of polymethyl methacrylate
- CPG Controlled Pore Glass
- PS polystyrene
- Silica gel cellulose paper, etc.
- the diameter is preferably 40 ni 80 !, and it is preferable to have a pore size of 500 to 3000.
- the purified [-polymer structure and oligonucleo For single-stranded nucleotides the molecular weight is measured with a mass spectrometer to confirm whether the desired oligonucleotide-polymer structure and oligonucleotide single-stranded are prepared.
- the step (4) of synthesizing the oligonucleotide single-stranded sequence complementary to the oligonucleotide single-stranded sequence synthesized in (4) may be performed before step (1) or during any one of steps (1) to (5).
- a production method characterized in that the oligonucleotide single strand synthesized in step (2) and the oligonucleotide single strand comprising a complementary sequence are used in a form in which a phosphate group is attached to the end may also be used.
- a method for preparing a double-stranded oligonucleotide construct comprising a specific double-stranded oligonucleotide comprises, for example,
- preparing a ligand-double-stranded oligonucleotide structure through annealing of the prepared ligand-oligonucleotide-polymer structure and an oligonucleotide single-stranded complementary sequence may be included.
- the ligand-oligonucleotide-polymer structure and the oligonucleotide single-strand of the complementary sequence are separated and purified, and the molecular weight is determined by mass spectrometry 1/1/ ⁇ 1_i-1 By measuring, it can be confirmed whether the desired ligand-oligonucleotide-polymer structure and complementary oligonucleotide have been prepared.
- step (4) of synthesizing an oligonucleotide single-stranded sequence complementary to the sequence of the oligonucleotide single-strand synthesized in step (3) is an independent synthesis process before step (1) or step (1) to (6) may be performed during any one of the steps.
- the present invention relates to nanoparticles comprising the double-stranded oligonucleotide construct according to the present invention.
- self-assembled nanoparticles are formed by the hydrophobic interaction of hydrophobic substances (Korean Patent Publication No. 1224828). as well as the stability is extremely excellent, because it is excellent in uniformity of the particle size ( ⁇ ⁇ times i a) is easy and the manufacturing process is simple as a drug.
- the nanoparticles may be characterized in that one or more, preferably two or more kinds of double-stranded oligonucleotide structures comprising different double-stranded oligonucleotides are mixed, for example, the nanoparticles is SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 comprising a sense strand comprising any one sequence selected from the group consisting of and an antisense strand comprising a complementary sequence thereto
- the present invention relates to a pharmaceutical composition for treating coronavirus infection-19 containing the double-stranded oligonucleotide, the double-stranded oligonucleotide structure or the nanoparticles according to the present invention as an active ingredient.
- composition of the present invention may be prepared by including one or more pharmaceutically acceptable carriers in addition to the active ingredients described above for administration.
- a pharmaceutically acceptable carrier must be compatible with the active ingredient of the present invention, and contains saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or two or more of these components. They can be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostats can be added as needed.
- diluents, dispersants, surfactants, binders and lubricants may be additionally added to form an injectable formulation such as an aqueous solution, suspension, emulsion, and the like.
- an injectable formulation such as an aqueous solution, suspension, emulsion, and the like.
- a method commonly known in the art to which the present invention pertains may be used, and a stabilizer for freeze-drying may be used. 2021/234647 ?01/162021/054404 may be added.
- composition of the present invention can be determined by one of ordinary skill in the art based on the symptoms of the ordinary patient and the severity of the disease. In addition, it can be formulated in various forms such as powders, tablets, capsules, liquids, injections, ointments, syrups, etc. .
- the composition of the present invention can be administered orally or parenterally.
- the route of administration of the composition according to the present invention is not limited thereto, but for example, oral, inhalational, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, Enteral, sublingual or topical administration is possible.
- Coronavirus Infectious Disease-19 is a respiratory disease
- intranasal administration preferably intranasal administration using a spray method, or administration to the lungs through intrabronchial instillation is possible, but is not limited thereto.
- the dosage of the composition according to the present invention varies depending on the patient's weight, age, sex, health condition, diet, administration time, method, excretion rate or severity of disease, etc.
- composition of the present invention may be formulated into a suitable dosage form for clinical administration using known techniques.
- the SAMiRNAä targeting the SARS-CoV-2 sequence according to the present invention is administered to the ferret infected with the SARS-CoV-2 virus, and the ferret
- the virus titer was statistically significantly decreased in both the subcutaneous injection group and the subcutaneous injection and bronchial administration group compared to the infection control group (Fig.
- the present invention relates to a formulation in a lyophilized form comprising the pharmaceutical composition according to the present invention.
- the present invention relates to a method for treating coronavirus infection-19, comprising administering a pharmaceutical composition for treatment of coronavirus infection-19 according to the present invention to an individual in need of treatment for coronavirus infection-19.
- the present invention relates to the double-stranded oligonucleotide, the double-stranded oligonucleotide structure and nanoparticles comprising the same for use in the treatment of coronavirus infection-19.
- Another aspect of the present invention relates to said pharmaceutical composition for use in the treatment of Coronavirus Infectious Disease-19.
- the present invention is for the treatment of coronavirus infection-19 2021/234647 01/162021/054404 to the use of the double-stranded oligonucleotide, the double-stranded oligonucleotide construct comprising the same and nanoparticles for the manufacture of a drug.
- the double-stranded oligonucleotide construct comprising the SARS-Cov-2 specific double-stranded oligonucleotide according to the present invention and a pharmaceutical composition comprising the same as an active ingredient can express SARS-Cov-2 with high efficiency without side effects. can be suppressed, so it can have an excellent effect in the treatment of COVID-19.
- Figure 1 shows a candidate sequence consisting of 19 bases by applying a 1-base sliding window algorithm in the genome for the design of a SARS-CoV-2 specific oligonucleotide candidate sequence. represents the selection process.
- 2A and 2B show the measurement results of the E and Rdrp gene Ct values after COVID-19 infection in the SAMiRNA-treated Huh7 cell line.
- 3 shows a procedure for evaluating the in-vivo antiviral efficacy of SAMiRNA-COVID19.
- FIG. 4 shows changes in body weight and body temperature of weasels in the evaluation of in-vivo antiviral efficacy of SAMiRNA-COVID19.
- Figure 5 shows the specific results of SARS-CoV-2 virus RNA copy number and virus titer in the nasal lavage sample of weasel in the evaluation of the in-vivo antiviral efficacy of SAMiRNA-COVID19.
- Algorithm and candidate sequence selection for oligonucleotide screening targeting SARS-CoV-2 siRNA-based drug high-throughput screening is a sliding window algorithm ( sliding window algorithm) to generate all possible candidate sequences and 2021/234647 ?01/162021/054404 This is a method to remove unnecessary candidate sequences through homology filtering, and to check the degree of inhibition of replication of the virus for all finally selected oligonucleotides.
- the design process for the oligonucleotide candidate sequence for SARS-CoV-2 was carried out by applying the 1-base sliding window algorithm to the SARS-CoV-2 reference genome (NC_045512.2, 29,903 bp) 19 29, 885 candidate sequences consisting of nucleotides were generated (FIG. 1).
- the generated oligonucleotide candidate sequence list was performed with a BLAST e-value of 100 or less for human total reference seq RNA, and 6,885 identities of 15 bases or less with respect to human genes and RNA sequences were performed. Candidate sequences were selected.
- C 24 (C 6 -SSC 18 ) containing a disulfide bond which is a hydrophobic material, was additionally bonded to the 5' end, and NAG-hexaethylene glycol- (- P0 3 hexaethylene) was bonded to the 3' end.
- RNA single-stranded and oligo (DNA or RNA)-polymer constructs synthesized by treatment with 28% (v/v) ammonia in ba ⁇ i) were separated from CPG I, through deprotection reaction Protecting residues removed
- the RNA single-stranded and oligo (DNA or RNA)-polymer structures from which the protective residues have been removed were prepared with N-methylpyrrolidone and triethylamine in an oven at 70°C. and triethylamine trihydrofluoride
- RNA single-stranded, oligo (DNA or RNA)-polymer structures and ligand-bound oligo (DNA or RNA)-polymer structures are separated by chromatography (High Performance Liquid Chromatography, HPLC), , and the molecular weight was measured with a MALDI-T0F mass spectrometer (MALDI T0F-MS, SHIMADZU, Japan), and it was confirmed whether the nucleotide sequence and the oligo-polymer structure to be synthesized matched.
- MALDI-T0F mass spectrometer MALDI T0F-MS, SHIMADZU, Japan
- each double-stranded oligo structure the sense strand and the antisense strand were mixed in equal amounts, followed by 1X annealing buffer (30 mM HEPES, 100 mM potassium acetate), 2 mM magnesium acetate (Magnesium acetate), pH. 7.0 ⁇ 7.5), reacted at 90°C for 3 minutes in a water bath, and then reacted again at 37°C to prepare the desired SAMiRNA. Annealing of the prepared double-stranded oligo RNA constructs was confirmed through electrophoresis.
- Example 3 High-Speed Bulk Screening of SAMiRNA Nanoparticles Inducing RNAi by Targeting SARS-CoV-2
- Human-derived liver cancer for the discovery of SAMiRNA that suppresses the expression of SARS-CoV-2
- Huh-7 cells Huh-7 cells (JCRB, JP) were inoculated (seeding) in a 96-well plate 24 hours before SAMiRNA treatment.
- RPMI 1640 (sh30027.01) (Hyclone, US) medium as a cell culture medium, put 100ul per well, and incubate the cells at 37°C, 5% C0 2 conditions.
- composition of the medium 2.05 mM L-Glutamine, 1% Penicillin-Streptomycin (Hyclone, US), and 10% Fetal Bovine Serum (Hyclone, US) were added to RPMI 1640. SAMiRNA candidates were treated once at a concentration of 10 uM with medium replacement in two repetitions, and then cells were incubated at 37 °C and 5% CO 2 for 24 hours.
- the E and Rdrp genes were transfected with an Exicyclerä 96 Real-Time Quantitative Thermal Block (BIONEER, KR) at 50°C for 30 min, 94°C for 10 min, and then at 95°C. It was analyzed by RT-qPCR with 45 repetitions of 15 s and 1 min at 60 °C.
- 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.
- Selected SEQ ID NOs 1 to 10 correspond to #161, #214, #233, #246, #249, #544, #625, #707, #715, and #758 of FIG. 2, respectively.
- SARS-CoV-2 specific oligonucleotide candidates selected by 1-base sliding window screening 2021/234647 1 ⁇ (:1 ⁇ 2021/054404 Example 4. 5 ⁇ [ «- ⁇ : 0 ⁇ /-2 Infected ferret sub model Efficacy evaluation of symptom relief 2021/234647 ?01/162021/054404
- each experimental animal was administered a single dose of SAMiRNAä nanoparticles by subcutaneous (50mpk), subcutaneous (50mpk), and bronchial (500ul) routes.
- Changes in body weight and body temperature of individual subjects in the control and test groups were measured on day 0 before viral infection and on days 2, 4, 6, 8, and 10 post-infection, and nasal wash samples were retaken ( FIG. 3 ).
- the recollected sample was analyzed for viral titer using the TCID50 method, or RNA was extracted and then viral RNA was performed by qRT-PCR according to the manufacturer's manual of Accu Power ® SARS-CoV-2 Real-Time RT-PCR Kit (BIONEER, KR). Absolute quantitative analysis of copy number.
- the virus titer was statistically significantly decreased compared to the infection control group in both the subcutaneous injection group and the subcutaneous injection group and the bronchial injection group at 4 dpi.
- the subcutaneous injection and the bronchial administration group confirmed the statistical significance compared to the infection control group even at 6 dpi (Fig. 5B).
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Abstract
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MX2022014496A MX2022014496A (es) | 2020-05-22 | 2021-05-21 | Oligonucleótido de doble hebra y composición para el tratamiento de la covid-19 que contiene el mismo. |
BR112022023408A BR112022023408A2 (pt) | 2020-05-22 | 2021-05-21 | Oligonucleotídeo de fita dupla e composição para tratar covid-19 contendo o mesmo |
EP21809288.0A EP4183879A1 (en) | 2020-05-22 | 2021-05-21 | Double-stranded oligonucleotide and composition for treating covid-19 containing same |
JP2022570238A JP2023533124A (ja) | 2020-05-22 | 2021-05-21 | 二本鎖オリゴヌクレオチド及びこれを含むコロナウイルス感染症-19(covid-19)治療用組成物 |
US17/999,399 US20230203491A1 (en) | 2020-05-22 | 2021-05-21 | Double-stranded oligonucleotide and composition for treating covid-19 containing same |
CA3179016A CA3179016A1 (en) | 2020-05-22 | 2021-05-21 | Double-stranded oligonucleotide and composition for treating covid-19 containing same |
CN202180047294.1A CN116157521A (zh) | 2020-05-22 | 2021-05-21 | 双链寡核苷酸和用于治疗covid-19的含有其的组合物 |
AU2021275643A AU2021275643A1 (en) | 2020-05-22 | 2021-05-21 | Double-stranded oligonucleotide and composition for treating covid-19 containing same |
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BR112022023408A2 (pt) | 2023-01-31 |
AU2021275643A1 (en) | 2022-12-15 |
CN116157521A (zh) | 2023-05-23 |
US20230203491A1 (en) | 2023-06-29 |
MX2022014496A (es) | 2023-03-10 |
CA3179016A1 (en) | 2021-11-25 |
KR20210144601A (ko) | 2021-11-30 |
JP2023533124A (ja) | 2023-08-02 |
EP4183879A1 (en) | 2023-05-24 |
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