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WO2020122534A1 - Arnsi asymétrique pour inhiber l'expression de snai1 - Google Patents

Arnsi asymétrique pour inhiber l'expression de snai1 Download PDF

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WO2020122534A1
WO2020122534A1 PCT/KR2019/017332 KR2019017332W WO2020122534A1 WO 2020122534 A1 WO2020122534 A1 WO 2020122534A1 KR 2019017332 W KR2019017332 W KR 2019017332W WO 2020122534 A1 WO2020122534 A1 WO 2020122534A1
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sirna
antisense strand
group
fibrosis
snai1
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이동기
박준현
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Olix Pharmaceuticals Inc
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Olix Pharmaceuticals Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • 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

Definitions

  • the present invention relates to an asymmetric siRNA that inhibits the expression of SNAI1 (Snail1) and uses thereof, and more specifically, an antisense strand comprising a sequence complementary to an mRNA encoding SNAI1, and a complementary bond with the antisense strand It relates to an asymmetric siRNA comprising the sense strand and a pharmaceutical composition for preventing or treating fibrosis or cancer comprising the asymmetric siRNA.
  • Fibrosis refers to a phenomenon that a normal tissue is wounded and hardened as a function decreases, and examples include lung fibrosis, subretinal fibrosis, and liver fibrosis.
  • PF pulmonary fibrosis
  • Idiopathic pulmonary fibrosis IPF is a very fatal disease with a life expectancy of 2-6 years after diagnosis due to the progression of PF as a cause of unknownness.
  • IPF Idiopathic pulmonary fibrosis
  • EMT epithelial-mesenchymal transition
  • E-cadherin E-cadherin
  • N-cadherin N-cadherin.
  • E-cadherin is a membranous glycoprotein, and the extracellular region binds with the E-cadherin molecule of adjacent cells to maintain adhesion between cells, and the intracellular region binds ⁇ -, ⁇ -, and p120 catenin to polarize epithelial cells and cells. It forms a skeleton.
  • E-cadherin When E-cadherin is lost by EMT, the tight junction between epithelial cells is loosened, and the cytoskeleton is restructured, resulting in changes in actin and actin stress fibers, resulting in loss of cellular polarity, and decomposition of extracellular matrix by matrix metalloprotease (MMP).
  • MMP matrix metalloprotease
  • Epithelial cells migrate to the epilepsy.
  • the transcription factor Snail1 (SNAI1) is recognized as a key factor of EMT by functioning to suppress the expression of E-cadherin.
  • the present inventors selected SNAI1 target asymmetric siRNA capable of inhibiting the expression of SNAI1, delivered to the cell without the help of a transporter, and as a result of earnest research efforts to develop a highly resistant to nucleic acid hydrolase, SNAI1 as a target SiRNAs were designed, siRNAs that most effectively inhibit SNAI1 were selected through screening, and it was confirmed that the intracellular delivery problem could be overcome through modification, and the present invention was completed.
  • An object of the present invention is to provide an asymmetric shorter duplex siRNA (asiRNA) that specifically inhibits the expression of SNAI1 (Snail1).
  • siRNA asymmetric shorter duplex siRNA
  • Another object of the present invention is to provide a pharmaceutical composition for preventing or treating fibrosis containing the asymmetric siRNA, or a method for preventing or treating fibrosis.
  • Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, or a method for preventing or treating cancer, including the asymmetric siRNA.
  • Another object of the present invention is to provide the use of the asymmetric siRNA for the prevention or treatment of fibrosis or cancer.
  • Another object of the present invention is to provide the use of the asymmetric siRNA for the manufacture of a medicament for the prevention or treatment of fibrosis or cancer.
  • the present invention comprises an antisense strand comprising a sequence complementary to an mRNA encoding SNAI1 (Snail1), a sense strand forming a complementary bond with the antisense strand, and 5 of the antisense strand.
  • The'terminal and 3'end of the sense strand provides an siRNA characterized by forming a blunt end.
  • the present invention also provides a pharmaceutical composition for preventing or treating fibrosis comprising the siRNA.
  • the present invention also provides a pharmaceutical composition for the prevention or treatment of cancer comprising the siRNA.
  • the present invention also provides a method of preventing or treating fibrosis comprising administering the siRNA to an individual.
  • the present invention also provides a method of preventing or treating cancer comprising administering the siRNA to an individual.
  • the present invention also provides the use of the asymmetric siRNA for the prevention or treatment of fibrosis or cancer.
  • the present invention also provides the use of the asymmetric siRNA for the manufacture of a medicament for the prevention or treatment of fibrosis or cancer.
  • Figure 1 shows the gene inhibition efficiency of asiRNA for 43 sequences targeting SNAI1, SNAI1 using qRT-PCR after 24 hours after transfection of 0.3nM asiRNA targeting each nucleotide sequence to A549 cells The expression level of mRNA was measured.
  • 1A and 1B are independent experimental data, respectively.
  • Figure 2 shows the gene suppression efficiency of asiRNA for four sequences targeting SNAI1, #41 was used as a negative control.
  • the expression level of SNAI1 protein was measured by western blot 48 hours after 10nM transfection of asiRNA targeting each nucleotide sequence in Panc-1 cells and A549 cells.
  • 2A is western blot data
  • FIG. 2B is a graph showing the amount of expression of SNAI1 protein relative to a normal sample after normalizing the expression amount of SNAI1 protein to vinculin, a housekeeping gene, using ImageJ for western blot data of FIG. 2A. .
  • Figure 3 shows the efficiency of inhibiting protein expression of the SNAI1 target asiRNA length variant, measuring the expression level of SNAI1 protein using western blot 48 hours after 5nM transfection of asiRNA targeting each nucleotide sequence to Panc-1 cells.
  • Did. 3A and 3B are western blot data
  • FIG. 3C is a graph showing the relative amount of SNAI1 expression for a non-treated sample after normalizing the western blot data of FIGS. 3A and 3B to housekeeping gene vinculin using ImageJ. .
  • Figure 4 shows the gene inhibition efficiency of 32 cp-asiRNAs with various chemical modifications targeting SNAI1, qRT after 24 hours after 1 ⁇ M incubation of cp-asiRNAs targeting each base sequence in A549 cells. -PCR was used to measure the expression level of SNAI1 mRNA. The graph shows the mean and SD of 2 replicates.
  • Figure 5 shows the gene inhibition efficiency of cp-asiRNA targeting SNAI1, after 48 hours of incubation of 1 ⁇ M-3 ⁇ M cp-asiRNA targeting each nucleotide sequence in A549 cells, Western blot of SNAI1 protein was performed after 48 hours. The expression level was measured.
  • Figure 6 shows a schematic diagram and conditions of animal experiments for confirming the SNAI1 protein inhibitory effect of cp-asiRNA targeting SNAI1.
  • RNAi RNA interference
  • dsRNA double-stranded RNA
  • RNA small interfering RNA
  • dsRNA short double-stranded RNA
  • a “antisense strand” is a polynucleotide that is substantially or 100% complementary to a target nucleic acid of interest, eg, mRNA (messenger RNA), RNA sequence other than mRNA (eg, microRNA, piwiRNA) , tRNA, rRNA and hnRNA) or coding or non-coding DNA sequences, in whole or in part.
  • mRNA messenger RNA
  • RNA sequence other than mRNA eg, microRNA, piwiRNA
  • tRNA, rRNA and hnRNA coding or non-coding DNA sequences, in whole or in part.
  • a “sense strand” is a polynucleotide having a nucleic acid sequence identical to a target nucleic acid, and is an mRNA (messenger RNA), an RNA sequence other than mRNA (eg, microRNA, piwiRNA, tRNA, rRNA and hnRNA) or coding or noncoding. Refers to a polynucleotide as a whole or part of a DNA sequence.
  • mRNA messenger RNA
  • RNA sequence other than mRNA eg, microRNA, piwiRNA, tRNA, rRNA and hnRNA
  • Gene should be considered in the broadest sense and can encode a structural or regulatory protein.
  • the regulatory protein includes a transcription factor, a heat shock protein or a protein involved in DNA/RNA replication, transcription and/or translation.
  • the target gene targeted for suppression of expression is inherent in the viral genome, and may be integrated into an animal gene or exist as an extrachromosomal component.
  • the target gene can be a gene on the HIV genome.
  • siRNA molecules are useful for inactivating the translation of HIV genes in mammalian cells.
  • epithelial-mesenchymal transition of the present invention means that epithelial cells lose cell polarity and cell-cell adhesion and acquire metastatic and invasive properties. It refers to the process of mesenchymal cellization, a multipotent stromal cell that can differentiate into various types of cells.
  • the EMT is essential for many developmental processes, including mesoderm formation and neural tube formation. EMT is also known to be involved in the initiation of metastasis in wound healing, tissue fibrosis and cancer progression.
  • EMT E-cadherin
  • Many transcription factors TFs
  • TFs transcription factors
  • SNAI1/Snail 1, SNAI2/Snail 2, ZEB1, ZEB2, E47 and KLF8 bind to the E-cadherin promoter to inhibit its transcription, while Twist, Goosecoid, E2.2 (aka TCF4), homeobox protein SIX1 And FOXC2 indirectly inhibits E-cadherin.
  • TCF4 homeobox protein SIX1 And FOXC2 indirectly inhibits E-cadherin.
  • other signaling pathways inducing EMT include those involved in TGF- ⁇ , FGF, EGF, HGF, Wnt/beta-catenin and notch.
  • an asiRNA targeting the transcription factor SNAI1 capable of suppressing the expression of E-cadherin was designed and asiRNA having the best knockdown efficiency was selected.
  • sequence length of the selected asiRNA was optimized, and by modifying, asiRNA was modified to have cell penetrating ability and resistance to nucleic acid hydrolase, cp-asiRNA capable of efficiently suppressing SNAI1 expression was selected. .
  • the present invention includes an antisense strand comprising a sequence complementary to an mRNA encoding SNAI1 (Snail1), a sense strand forming a complementary bond with the antisense strand, and 5'of the antisense strand.
  • the 3'end of the terminal and sense strands relates to siRNA, characterized in that it forms a blunt end.
  • RNAi is an intracellular gene regulation method first discovered in Caenorhabditis elegans by the Fire Research Group in 1998.
  • the mechanism of action is that the antisense strand of RNA double strands injected into the cell complementarily binds to the mRNA of the target gene to induce target gene degradation. It is said that.
  • synthetic RNA interference is one of the methods to suppress gene expression in " in vitro ".
  • the 19-21bp siRNA is a technology that can be developed as a therapeutic agent for various gene-related diseases, such as cancer, rare diseases, fibrosis, and viral infection, in theory because it can selectively inhibit almost all genes.
  • the first attempt to treat in vivo with siRNA in mammals was in mid 2002. Since then, more than 90 papers have been reported on in vivo treatment with many attempts at applied research.
  • siRNA is an effective method to directly regulate the expression of a target gene, these problems have made it difficult to develop a therapeutic agent.
  • the applicant of the present invention has developed an asymmetric shorter duplex siRNA (asiRNA) structure-related technology (WO 2009/078685).
  • asiRNA is an asymmetric RNAi-inducing structure with a short double helix length compared to the 19+2 structure.
  • the present invention presents an asymmetric siRNA comprising a sense strand and an antisense strand complementary to the sense strand, and the siRNA according to the present invention has an off-target effect, saturation of RNAi mechanism, immune response by TLR3, etc. It is possible to effectively suppress the expression of the SNAI1 target gene to a desired degree while stably maintaining high delivery efficiency without causing a problem.
  • the siRNA may be characterized in that the sense strand has a length of 15-17nt, and the antisense strand has a length of 16nt or more.
  • the antisense strand may be characterized as having a length of 16-31nt, and preferably may have a length of 19-26nt. More preferably, the length of the sense strand is 16nt, and the length of the complementary antisense strand is 19nt, 24nt or 26nt, but is not limited thereto.
  • the 3'end of the sense strand and the 5'end of the antisense strand form a blunt end.
  • the 3'end of the antisense strand may include, for example, an overhang of 1-15nt.
  • 43 asiRNAs were designed to suppress the expression of SNAI1, and inhibition of mRNA levels and protein levels was confirmed.
  • the sense strand is SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39 , 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83 and 85 It can be characterized by being selected.
  • siRNA comprising a sense strand selected from the group consisting of SEQ ID NOs: 35, 47, 49, and 51, and an antisense strand complementary to the sense strand, has the best SNAI1 expression inhibitory effect.
  • the antisense strand is SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40 , 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 91, 92 , 93, 94, 95, 96, 97, 98, 99, 100, 101, and 102.
  • the antisense strand is selected from the group consisting of SEQ ID NO: 36, 48, 50, 52, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 and 102 can do.
  • the sense strand is SEQ ID NO: 35 or 51
  • the antisense strand may be characterized by being SEQ ID NO: 92, 100 or 101, but is not limited thereto. More preferably, the sense strand is SEQ ID NO: 51, the antisense strand may be characterized in that SEQ ID NO: 100 or 101.
  • the sense strand or the antisense strand of the siRNA may be characterized by including one or more chemical modifications (chemical modification).
  • siRNA cannot pass through the cell membrane due to high negative charge and high molecular weight due to the phosphate backbone structure, and it is difficult to deliver sufficient amounts for RNAi induction to actual target sites due to rapid decomposition and removal in blood.
  • many high-efficiency delivery methods using cationic lipids and cationic polymers have been developed, but in vivo , it is difficult to deliver siRNA with inefficiencies as high as in vitro , and various proteins present in vivo. There is a problem in that siRNA delivery efficiency is reduced by interaction.
  • the present inventors have developed an asiRNA construct (cp-asiRNA) having an autotransfer capability capable of effectively and intracellular delivery without a separate carrier by introducing a chemical modification into an asymmetric siRNA structure.
  • the chemical modification in the sense strand or the antisense strand may include one or more selected from the group consisting of: -OH group at the 2'carbon position of the sugar structure in the nucleotide -CH3 (methyl), -OCH3 (methoxy), -NH2, -F(fluorine), -O-2-methoxyethyl -O-propyl, -O-2-methylthioethyl, -O-3-aminopropyl,- Substituted with O-3-dimethylaminopropyl; Oxygen in the sugar structure in the nucleotide is substituted with sulfur; Nucleotide bonds are modified with phosphorothioate, boranophosphate, or methyl phosphonate; Modification to peptide nucleic acid (PNA), locked nucleic acid (LNA) or unlocked nucleic acid (UNA) form; And phosphate groups, lipophilic compounds, or cell-penetrating peptide nucleic
  • the lipophilic compound may be selected from the group consisting of cholesterol, tocopherol, and long-chain fatty acids having 10 or more carbon atoms. Preferably it can be characterized as being cholesterol, but is not limited thereto.
  • the chemical modification is a modification in which the -OH group is substituted with -OCH3 (methoxy) or -F (fluorine) at the 2'carbon position of the sugar structure in two or more nucleotides of the sense strand or the antisense strand; 10% or more nucleotide bonds in the sense or antisense strand are modified with phosphorothioate; Cholesterol binding to the 3'end of the sense strand; Alternatively, a phosphate group may be attached to the 5'end of the antisense strand. Through this, it is possible to improve the stability of siRNA in vivo.
  • the -OH group is -OCH3 at the 2'carbon position of the sugar structure in the nucleotide located at the 5'end of the sense strand (methoxy).
  • a 2'-O-methyl nucleoside in which the -OH group is substituted with -OCH3 (methoxy) at the 2'carbon position of the sugar structure from the 5'end to the 3'end of the sense strand. May be included continuously or discontinuously.
  • a 2'-O-methylated nucleoside may be alternately included with an unmodified nucleoside.
  • 2, 3, 4, 5, 6, 7, 8 consecutive 2′-O-methyl nucleosides may be alternately included with the unmodified nucleoside in the sense strand.
  • 2′-O-methyl nucleosides in the sense strand for example 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 2-8 or Eight can exist.
  • 2'-O-methylated nucleosides may be continuously or discontinuously included in the 5'end to 3'end direction of the antisense strand.
  • a 2'-O-methylated nucleoside may be alternately included with an unmodified nucleoside.
  • 2, 3, 4, 5, 6, 7 and 8 consecutive 2′-O-methyl nucleosides may be alternately included with the unmodified nucleoside.
  • 2′-O-methyl nucleosides in the antisense strand for example, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 2-7 Can be.
  • the sense strand is any one selected from the group consisting of (a) to (i) in the following table
  • the antisense strand is any one selected from the group consisting of (j) to (s) in the following table.
  • * is a phosphorothioate bond
  • m is 2'-O-methyl (Methyl)
  • 2'-F- is 2'-Fluoro
  • /chol/ is cholesterol
  • P- is 5'-Phosphate group.
  • the sense strand is (e), (g) or (i) of the table
  • the antisense strand may be characterized in that (o), (p), (r) or (s) of the table. have.
  • the siRNA of the present invention may be characterized by being selected from the group consisting of the following sense strands and antisense strands, but is not limited to: sense strands (e) and antisense strands (p) ); (G) of the sense strand above and (o) of the antisense strand above; (E) of the sense strand above and (r) of the antisense strand above; And sense strand (i) in the above table and antisense strand (s) in the above table.
  • one to three phosphate groups may be attached to the 5'end of the antisense strand, but is not limited thereto.
  • the present invention relates to a pharmaceutical composition for preventing or treating fibrosis comprising the siRNA.
  • the fibrosis may be characterized by subretinal fibrosis, lung fibrosis, liver fibrosis, myocardial fibrosis or renal fibrosis, but is not limited thereto, and is a composition for preventing or treating fibrosis known to be involved in EMT Can be used without restrictions.
  • the present invention relates to a pharmaceutical composition for the prevention or treatment of cancer comprising the siRNA.
  • the cancer may be characterized as lung cancer, breast cancer, colon cancer or prostate cancer, but is not limited thereto, and can be used without limitation as a composition for preventing or treating cancer known to be involved in EMT.
  • the pharmaceutical composition may be prepared by including one or more pharmaceutically acceptable carriers in addition to siRNA as an active ingredient.
  • the pharmaceutically acceptable carrier should be compatible with the active ingredient of the present invention, and may include saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these ingredients. It can be used in combination, and if necessary, other conventional additives such as antioxidants, buffers, bacteriostatic agents can be added.
  • diluents, dispersants, surfactants, binders, and lubricants can be added in addition to formulated into injectable formulations such as aqueous solutions, suspensions, and emulsions.
  • a formulation in a lyophilized form it is preferable to provide a formulation in a lyophilized form.
  • a method commonly known in the art to which the present invention pertains may be used, and a stabilizer for lyophilization may be added.
  • the method of administration of the pharmaceutical composition can be determined by a person skilled in the art based on the symptoms and severity of the disease in a typical patient.
  • powders, tablets, capsules, liquids, injections, ointments, syrups, etc. can be formulated in various forms and can be provided in unit-dose or multi-dose containers, for example, sealed ampoules and bottles. .
  • the pharmaceutical 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 to these, for example, oral cavity, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intestinal, sublingual Or topical administration is possible.
  • the dosage amount of the composition according to the present invention varies in its range according to the patient's weight, age, sex, health condition, diet, administration time, method, excretion rate, or severity of disease, and is easy for a person skilled in the art. Can decide.
  • the compositions of the present invention can be formulated into suitable formulations using known techniques for clinical administration.
  • the present invention relates to a method for preventing or treating fibrosis, comprising administering a therapeutically effective amount of the siRNA to a patient in need of treatment.
  • the present invention relates to a method for preventing or treating cancer, comprising administering a therapeutically effective amount of the siRNA to a patient in need of treatment.
  • the present invention relates to the use of said siRNA for the prevention or treatment of fibrosis.
  • the invention relates to the use of said siRNA for the manufacture of a medicament for the prevention or treatment of fibrosis.
  • the present invention relates to the use of said siRNA for the prevention or treatment of cancer.
  • the present invention relates to the use of said siRNA for the manufacture of a medicament for the prevention or treatment of cancer.
  • Example 1 Screening of 43 RNAi-derived double-stranded nucleic acid molecules targeting SNAI1
  • RNAi interference targeting SNAI1 To secure double-stranded nucleic acid molecules that induce high-efficiency RNAi interference targeting SNAI1, asiRNA was designed after target sequencing for the SNAI1 gene.
  • the asiRNA structure has a different secondary structure, GC contents (%), 5'end stability difference compared to the commonly known siRNA, so it is optimized asiRNA design when designing the base sequence of asiRNA using a general siRNA design program. This can be rather difficult. Therefore, the asiRNA of this study was conducted as follows. SNAI1 gene information was obtained through NCBI database search (mRNA accession number: NM_005985).
  • Example 2 Screening of RNAi-induced double-stranded nucleic acid molecule targeting SNAI1
  • the expression level of SNAI1 mRNA was measured by performing qRT-PCR after transfection to the A549 cell line using 43 selected asiRNAs at a concentration of 0.3 nM.
  • the A549 cell line was cultured in F-12K Nutrient Mixture (Gibco), 10% fetal bovine serum (FBS, Gibco), 100 units/ml Penicillin 100 ⁇ g/ml Streptomycin.
  • A549 cells are seeded in a 24-well plate at 3x10 4 cells/well, and asviRNA (0.3nM, OliX Inc.) and RNAiMax (2 ⁇ l/ml, Invitrogen Inc.) are used to provide Invitrogen in a total volume of 500 ⁇ l Opti-MEM. According to the transfection was performed. After 24 hours, total RNA was extracted using Tri-RNA reagent (FAVORGEN), and cDNA was synthesized using a high-capacity cDNA reverse transcription kit (Applied Biosystems). (Applied Biosystems) and using the Primer of Table 2 was confirmed the degree of SNAI1 gene expression.
  • the Panc-1 cell line was cultured in Dulbecco's Modified Eagle Medium (DMEM, Gibco), 10% fetal bovine serum (FBS, Gibco), 100 units/ml Penicillin 100 ⁇ g/ml Streptomycin.
  • DMEM Dulbecco's Modified Eagle Medium
  • FBS 10% fetal bovine serum
  • Penicillin 100 ⁇ g/ml Streptomycin 100 units/ml Penicillin 100 ⁇ g/ml Streptomycin.
  • A549 and Panc-1 cells were seeded in a 6-well plate at 9x10 4 cells/well, using asiRNA (10 nM, OliX Inc.) and RNAiMax (2 ⁇ l/ml, Invitrogen Inc.) in a total volume of 2 ml Opti-MEM. Transfection was performed according to the protocol provided by Invitrogen. After 48 hours, cell lysis was performed using RIPA buffer (Sigma), and proteins were quantified using a BCA protein assay kit (Invitrogen). After 15% SDS-PAGE was used to separate 20 ⁇ g protein from each sample for 30 minutes at 60 V and 1 hour at 110 V, it was transferred to a PVDF membrane (Bio-rad) at 300 mA for 2 hours.
  • Example 3 Optimization of nucleotide sequence length through 16 design of RNAi-induced double-stranded nucleic acid molecule targeting SNAI1
  • the length of the antisense sequence was set to 24 mer, the length used for screening, 19 and 26 mer, the possible lengths, and the predicted 21 mer, which is expected to show the maximum efficiency. 16 base sequences were designed.
  • Example 5 Screening of 32 cp-asiRNAs with cell-penetrating ability targeting the SNAI1 gene
  • cp-asiRNA with 16 modification patterns is selected according to the position and number of 2'OMe, 2'F (Fluoro), and PS (phosphothioate bond). After design, it was synthesized by Dharmacon Inc. (USA). cp-asiRNA enhances endocytosis efficiency and stability, and can suppress the expression of target genes by penetrating the cell membrane with high efficiency without the aid of a delivery vehicle.
  • the synthesized cp-asiRNA was annealed after incubation at 95°C for 5 minutes and 37°C for 25 minutes, followed by QC through ChemiDoc UV transilluminator (BioRad) after 12% Polyacrylamide Gel Electrophoresis (PAGE).
  • Example 6 Screening of cp-asiRNA with cell penetrating ability targeting SNAI1 gene
  • the expression level of SNAI1 was measured by performing incubation (free uptake) at a concentration of 1 ⁇ M in the A549 cell line using the above 32 cp-asiRNAs and performing qRT-PCR.
  • A549 cells were seeded in a 24-well plate at 3x10 4 cells/well, and 32 cp-asiRNAs were incubated for 24 hours at 1 ⁇ M in Opti-MEM media, and total RNA was extracted using Tri-RNA reagent (FAVORGEN).
  • FAVORGEN Tri-RNA reagent
  • cDNA was synthesized using a high-capacity cDNA reverse transcription kit (Applied Biosystems), and the level of SNAI1 expression was confirmed using a power SYBR green PCR master Mix (Applied Biosystems) and primers in Table 2 with a StepOne real-time PCR system machine. ( Figure 4).
  • candidates of the top 12 (#1, 5, 8, 9, 13, 19, 21, 24, 25, 26, 27, 28) with high efficacy were selected.
  • Example 7 Confirmation of SNAI1 inhibitory effect of cp-asiRNA with cell penetrating ability targeting SNAI1 gene
  • Example 6 considering that the effect of inhibiting SNAI1 expression of cp-asiRNA by introducing chemical modification into asiSNAI1 26 (26 mer) is high, asiSNAI1 26 (19 mer) (sense sequence and sequence of SEQ ID NO: 51 of Table 4) It was also expected that the effect of cp-asiRNA, which introduced a chemical modification to the antisense sequence of No. 101), would be high. Accordingly, asiSNAI1 26 (19 mer) was introduced by chemical modification in the same manner as in Example 5 to further synthesize OLX201D-026-33 and OLX201D-026-34, cp-asiRNAs having cell penetrating ability (Table 7). .
  • OLX201D-026-33 and OLX201D-026-34 of Table 7 and OLX201D-026-26 selected in Example 6 were evaluated for target protein inhibitory effect in mouse skin tissue.
  • the target animals were purified for 1 week in C57BL/6 (6 week old, male, OrientBio) mice in the experimental animal room of the Orix Research Institute, and 7 week old animals were used for the test.
  • Each candidate substance was prepared as a 10 mg/ml stock solution using 0.5x PBS as a vehicle solvent, diluted to three concentrations of 0.3, 1.0 and 2.0 mg/100 ⁇ l and administered once in 100 ⁇ l to the mouse skin. Inter-site spacing was maintained at a minimum of 3 cm.
  • the mice were sacrificed on the third day of administration, and skin tissue at the administration site was collected using an 8-mm biopsy punch (BP-80F, Kai medical) (FIG. 6 and Table 8).
  • the membrane was blocked for 1 hour in 5% skim-milk, reacted with SNAI1 antibody (Cell Signaling) 1:1000 for 12 hours at 4°C, and reacted for 1 hour with anti-rabbit Goat antibody-HRP (Santacruz) 1:5000. Then, the level of SNAI1 protein expression was confirmed using ChemiDoc (BioRad).
  • an asymmetric siRNA that regulates the transcription factor SNAI1 (Snail1) which is a key factor of EMT, which is closely related to tissue regeneration and fibrosis, cancer development and metastasis by functioning to suppress the expression of E-cadherin

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Abstract

La présente invention concerne un ARNsi asymétrique permettant d'inhiber l'expression de SNAI1 (Snail1), et son utilisation, plus spécifiquement, un ARNsi asymétrique, qui comporte un brin antisens comprenant une séquence complémentaire de l'ARNm codant pour SNAI1, et un brin sens formant une liaison complémentaire avec le brin antisens; et une composition pharmaceutique pour prévenir ou traiter la fibrose ou le cancer, contenant l'ARNsi asymétrique. La présente invention permet la sélection d'ARNsi asymétrique modulant le facteur de transcription SNAI1 (Snail1), un facteur clé de l'EMT étroitement lié à la régénération tissulaire et la fibrose et à l'apparition du cancer et de la métastase, en fonctionnant pour inhiber l'expression de la E-cadhérine, et permet une modification chimique de telle sorte que la cytotoxicité provoquée par un porteur est éliminée et une inhibition plus efficace de l'expression génique in vivo est activée, ce qui permet d'être efficacement utilisable en tant qu'agent pour prévenir ou traiter la fibrose ou le cancer.
PCT/KR2019/017332 2018-12-10 2019-12-10 Arnsi asymétrique pour inhiber l'expression de snai1 Ceased WO2020122534A1 (fr)

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CN118638794A (zh) * 2024-08-12 2024-09-13 中国药科大学 一种用于调节蛋白酶激活受体4基因表达的双链siRNA、shRNA、药物组合物及应用

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US20060134787A1 (en) * 2004-12-22 2006-06-22 University Of Massachusetts Methods and compositions for enhancing the efficacy and specificity of single and double blunt-ended siRNA
KR20180128423A (ko) * 2016-04-11 2018-12-03 올릭스 주식회사 연결 조직 성장 인자를 표적화하는 rna 복합체를 사용한 특발성 폐 섬유증의 치료 방법

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KR20180128423A (ko) * 2016-04-11 2018-12-03 올릭스 주식회사 연결 조직 성장 인자를 표적화하는 rna 복합체를 사용한 특발성 폐 섬유증의 치료 방법

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118638794A (zh) * 2024-08-12 2024-09-13 中国药科大学 一种用于调节蛋白酶激活受体4基因表达的双链siRNA、shRNA、药物组合物及应用

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