WO2020122534A1 - Asymmetric sirna for inhibiting expression of snai1 - Google Patents

Abstract

The present invention relates to asymmetric siRNA for inhibiting the expression of SNAI1 (Snail1), and a use thereof and, more specifically, to: asymmetric siRNA, which comprises an antisense strand comprising a sequence complementary to mRNA encoding SNAI1, and a sense strand forming a complementary bond with the antisense strand; and a pharmaceutical composition for preventing or treating fibrosis or cancer, containing the asymmetric siRNA. The present invention allows the selection of asymmetric siRNA which modulates transcription factor SNAI1 (Snail1), a key factor of EMT closely related to tissue regeneration and fibrosis and cancer occurrence and metastasis, by functioning to inhibit the expression of E-cadherin, and allows chemical modification so that cytotoxicity caused by a carrier is removed and more efficient inhibition of gene expression in vivo is enabled, thereby being effectively usable as an agent for preventing or treating fibrosis or cancer.

Description

Asymmetric SIRNA that inhibits the expression of SNAI1

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 (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. Typically, pulmonary fibrosis (PF) is a respiratory disease in which lung tissue becomes inflamed, which causes the lung tissue to harden and cause respiratory failure. When the fibrosis becomes severe, the oxygen exchange function of the lungs decreases, and as the symptoms progress, the alveoli are destroyed and the lungs do not function normally. 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. Currently, the only practical treatment for this is unmet due to lung transplant surgery. Medical needs are very high.

The process of transforming epithelial cells into cells with metastatic and infiltrating capacity is called epithelial-mesenchymal transition (EMT). EMT is a form of cell plasticity in which epithelial cells are transformed into mesenchymal cells, and one cell differentiates into another cell (Zeisberg M, Neilson EG.Biomarkers for epithelial-mesenchymal transitions. J Clin Invest 2009; 119:1429-1437). This EMT is considered a basic phenomenon of morphological development, including the development of tissues and organs during embryogenesis in both vertebrates and invertebrates. Similar phenomena are found in the wound healing process in adults, and recently, it has been revealed that EMT plays a variety of roles in tissue regeneration and fibrosis, as well as cancer development and metastasis, in addition to the formation and differentiation of tissues in the early stages.

In the process of cancer, local invasion and metastasis of tumor cells occur, which is accompanied by EMT of epithelial tumor cells. It is known that in this process, the transcription program of a specific gene changes to exhibit the mesenchymal phenotype, the mobility of cells increases, and the interaction between cells-cells or cells-extracellular matrix changes.

The earliest and most important process in EMT is the cadherin switch that converts E-cadherin (E-cadherin) to 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 α-, β-, and p120 catenin to polarize epithelial cells and cells. It forms a skeleton. 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). 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.

Accordingly, 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.

The above information described in the background section is only for improving the understanding of the background of the present invention, and therefore does not include information forming prior art already known to those of ordinary skill in the art. It may not.

Summary of the invention

An object of the present invention is to provide an asymmetric shorter duplex siRNA (asiRNA) that specifically inhibits the expression of SNAI1 (Snail1).

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.

In order to achieve the above object, 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, and 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, and 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 μM 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 1μM-3μM 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.

7 shows the effect of inhibiting SNAI1 protein expression of cp-asiRNA targeting SNAI1.

Detailed description of the invention and preferred embodiments

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which the present invention pertains. In general, the nomenclature used herein is well known in the art and is commonly used.

Definitions of key terms used in the detailed description of the present invention are as follows.

“RNAi (RNA interference)” refers to a double-stranded RNA (dsRNA) composed of a strand having a sequence homologous to the mRNA of a target gene and a strand having a sequence complementary to that of a target gene, and is introduced into a cell or the like of the target gene mRNA. It means a mechanism that suppresses the expression of the target gene by inducing degradation.

“SmRNA (small interfering RNA)” refers to short double-stranded RNA (dsRNA) that mediates sequence-specific efficient gene silencing.

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.

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.

"Gene" should be considered in the broadest sense and can encode a structural or regulatory protein. At this time, the regulatory protein includes a transcription factor, a heat shock protein or a protein involved in DNA/RNA replication, transcription and/or translation. In the present invention, 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. For example, the target gene can be a gene on the HIV genome. In this case, siRNA molecules are useful for inactivating the translation of HIV genes in mammalian cells.

The term "epithelial-mesenchymal transition (EMT)" 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.

One of the important phenomena in EMT is the loss of E-cadherin. Many transcription factors (TFs) are known that can directly or indirectly inhibit the E-cadherin. 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. In addition, other signaling pathways inducing EMT include those involved in TGF-β, FGF, EGF, HGF, Wnt/beta-catenin and notch.

In one embodiment of the present invention, 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. In addition, the 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. .

Accordingly, in one aspect, 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.

The siRNA in the present invention is a concept including all substances having a general RNAi (RNA interference) action. 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. Among them, synthetic RNA interference (siRNA) 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.

Contrary to the possibility, however, the side effects and disadvantages of siRNA techniques have been reported continuously, and in order to develop a therapeutic agent based on RNAi, 1) absence of an effective delivery system 2) off-target effect 3) induction of immune response 4) intracellular RNAi There is a need to overcome barriers such as instrument saturation. Although 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. In this regard, 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. It is a technology that overcomes problems such as the off-target effect of siRNA, saturation of RNAi mechanism, and immune response by TLR3, and can be a promising platform for developing new RNAi drugs. In addition, chemical and structural modifications (chemical, structural modification) is added, so it is possible to develop as an effective next-generation treatment by overcoming the problems of the existing siRNA delivery system by enabling self-transmission into cells without a separate delivery system.

Based on this, 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.

In the present invention, 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. Without being limited thereto, 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.

In one embodiment of the present invention, 43 asiRNAs were designed to suppress the expression of SNAI1, and inhibition of mRNA levels and protein levels was confirmed.

In the present invention, 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.

Specifically, it was confirmed that a 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.

In the present invention, 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. Specifically, 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.

More specifically, the sense strand is SEQ ID NO: 35 or 51, and 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.

In the present invention, the sense strand or the antisense strand of the siRNA may be characterized by including one or more chemical modifications (chemical modification).

In general, 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. Currently, in the case of in vitro delivery, 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.

Accordingly, 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.

In the present invention, 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 peptides.

In the present invention, 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.

In an embodiment of the present invention, 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. When the -OH group is replaced with -OCH3 (methoxy) at the 2'carbon position of the sugar structure or the nucleotide bond is modified with phosphorothioate, resistance to the nuclease can be increased, and by binding to the cell membrane through cholesterol binding SiRNA delivery into cells can be facilitated.

Specifically, 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 antisense strand; 10% or more nucleotide bonds in the sense or antisense strand are modified with phosphorothioate; And a cholesterol bond at the 3'end of the sense strand; and one or more modifications selected from the group consisting of.

In relation to the modification in which the -OH group is replaced with -OCH3 (methoxy) at the 2'carbon position of the sugar structure in the nucleotide, 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). In addition, 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. In the sense strand, 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. In the antisense strand, a 2'-O-methylated nucleoside may be alternately included with an unmodified nucleoside. In the antisense strand, 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.

10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55 between the nucleotide strand and the ribonucleotide in relation to the modification of the nucleotide bond to phosphorothioate %, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or more can be modified with phosphorothioate. In some cases, all (100%) of the bonds between the ribonucleotides in the sense strand can be modified with phosphorothioates.

10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% between ribonucleotides in the antisense strand , 80%, 85%, 90% or 95% or more can be modified with phosphorothioates. In some cases, all of the binding (100%) between the antisense strand and the ribonucleotide can be modified with phosphorothioate.

Preferably, the sense strand is any one selected from the group consisting of (a) to (i) in the following table, and the antisense strand is any one selected from the group consisting of (j) to (s) in the following table. Can be.

In the above sequence, * is a phosphorothioate bond, m is 2'-O-methyl (Methyl), 2'-F- is 2'-Fluoro, /chol/ is cholesterol, and P- is 5'-Phosphate group.

More preferably, the sense strand is (e), (g) or (i) of the table, and the antisense strand may be characterized in that (o), (p), (r) or (s) of the table. have.

Most preferably, 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.

In the present invention, one to three phosphate groups may be attached to the 5'end of the antisense strand, but is not limited thereto.

In another aspect, the present invention relates to a pharmaceutical composition for preventing or treating fibrosis comprising the siRNA.

In the present invention, 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.

In another aspect, the present invention relates to a pharmaceutical composition for the prevention or treatment of cancer comprising the siRNA.

In the present invention, 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. In addition, diluents, dispersants, surfactants, binders, and lubricants can be added in addition to formulated into injectable formulations such as aqueous solutions, suspensions, and emulsions. In particular, it is preferable to provide a formulation in a lyophilized form. For the preparation of a lyophilized formulation, 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. In addition, 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. In addition, the compositions of the present invention can be formulated into suitable formulations using known techniques for clinical administration.

In another aspect, 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.

In another aspect, 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.

In another aspect, the present invention relates to the use of said siRNA for the prevention or treatment of fibrosis.

In another aspect, the invention relates to the use of said siRNA for the manufacture of a medicament for the prevention or treatment of fibrosis.

In another aspect, the present invention relates to the use of said siRNA for the prevention or treatment of cancer.

In another aspect, the present invention relates to the use of said siRNA for the manufacture of a medicament for the prevention or treatment of cancer.

Since the configuration included in the prevention or treatment method, use and use according to the present invention is the same as the configuration included in the above-described invention, the above description may be equally applied to the prevention or treatment method, use and use.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

Example 1: Screening of 43 RNAi-derived double-stranded nucleic acid molecules 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). Considering animal experiments, first select a nucleotide sequence showing 100% homology based on a nucleotide sequence having a common target with a mouse, and then remove the seed region from the antisense strand (bases 2-8 to 5') A total of 43 asiRNAs were selected and further synthesized on a 10 nmole scale by OliX Inc. (Korea). The synthesized asiRNA was annealing 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 2: Screening of RNAi-induced double-stranded nucleic acid molecule targeting SNAI1

In order to confirm the efficiency of gene inhibition at the mRNA level, 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 μg/ml Streptomycin. A549 cells are seeded in a 24-well plate at 3x10 ⁴ cells/well, and asviRNA (0.3nM, OliX Inc.) and RNAiMax (2μl/ml, Invitrogen Inc.) are used to provide Invitrogen in a total volume of 500μl 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 efficiency of gene inhibition of asiRNA for 43 sequences targeting SNAI1 is shown in FIG. 1. As a result, four candidates (#18, 24, 25, and 26) that showed mRNA knockdown efficacy of 50% or more at a minimum of n=2 were selected to proceed with the step of confirming the degree of gene suppression efficiency at the protein level.

After transfection at 10 nM concentration, 9 species (#15, 17, 18, 22, 24, 25, 26, 27, 28) that showed a knockdown effect of at least 50% at a minimum of n=1 through 43 asiRNA screening results were selected. Protein knockdown was confirmed by western blot. 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 μg/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 μl/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 μg 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. After transfer, the membrane was blocked for 1 hour in 5% skim milk, and then reacted with SNAI1 antibody (Cell Signaling) 1:1000 at 4°C for 12 hours. The next day, after reacting with anti-rabbit Goat antibody-HRP (Santa cruz) 1:5000 for 1 hour, ChemiDoc (BioRad) was used to compare SNAI1 protein expression.

In this experiment, it was confirmed that 4 types (#18, 24, 25, 26) asiRNA material knocked down SNAI1 protein expression by about 70% (FIG. 2), and it was confirmed that the tendency was maintained through repeated experiments. Proceeding to the length optimization step using the selected 4 kinds of asiRNA nucleotide sequences.

Example 3: Optimization of nucleotide sequence length through 16 design of RNAi-induced double-stranded nucleic acid molecule targeting SNAI1

Based on the four asiRNA base sequences derived in Example 2, 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 4: Length optimization through RNAi-induced double-stranded nucleic acid molecule screening targeting SNAI1

Seed Panc-1 cells in a 6-well plate at 9x10 ⁴ cells/well, and provide asviRNA (10 nM, OliX Inc.) and RNAiMax (2 μl/ml, Invitrogen Inc.) using total volume 2 ml Opti-MEM conditions Transfection was performed according to the protocol. After 48 hours, cell lysis was performed using RIPA buffer (Sigma), and proteins were quantified using a BCA protein assay kit (Invitrogen). 20 μg protein for each sample was separated for 30 minutes at 60 V and 1 hour at 110 V using 15% SDS-PAGE, and then transferred to PVDF membrane (Bio-rad) for 2 hours at 300 mA. After transfer, the membrane was blocked for 1 hour in 5% skim milk, and then reacted with SNAI1 antibody (Cell Signaling) 1:1000 at 4°C for 12 hours. The next day, after reacting with anti-rabbit Goat antibody-HRP (Santa cruz) 1:5000 for 1 hour, the amount of SNAI1 protein expression was confirmed using ChemiDoc UV transilluminator (BioRad) (FIG. 3 ). As a result, three candidates (asiSNAI1 18 (19 mer), asiSNAI1 26 (26 mer), and asiSNAI1 26 (19 mer)) were selected (Table 4).

Example 5: Screening of 32 cp-asiRNAs with cell-penetrating ability targeting the SNAI1 gene

The position of cholesterol is fixed in three asiRNAs targeting SNAI1, and SNAI1 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

In order to confirm the efficiency of gene inhibition at the mRNA level, the expression level of SNAI1 was measured by performing incubation (free uptake) at a concentration of 1 μM 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 μM in Opti-MEM media, and total RNA was extracted using Tri-RNA reagent (FAVORGEN). Next, 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). As a result, candidates of the top 12 (#1, 5, 8, 9, 13, 19, 21, 24, 25, 26, 27, 28) with high efficacy were selected.

After selecting the top 12 species (#1, 5, 8, 9, 13, 19, 21, 24, 25, 26, 27, 28) through 32 ap-asiRNA screening results, after processing 1, 1.5, 3 μM concentration Western blot was performed.

After seeding A549 cells in a 12-well plate at 4x10 ⁴ cells/well and incubating 12 cp-asiRNAs with 1, 1.5 and 3 μM in Opti-MEM media for 24 hours, F-12K Nutrient Mixture (Gibco), Media was replaced with 10% fetal bovine serum (FBS, Gibco), 100 units/ml Penicillin 100 μg/ml Streptomycin, and SNAI1 expression was confirmed at protein level 24 hours after media replacement (FIG. 5). As a result of the experiment, the top two criteria (#19, #26) were selected (Table 6).

Example 7: Confirmation of SNAI1 inhibitory effect of cp-asiRNA with cell penetrating ability targeting SNAI1 gene

In 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 µl and administered once in 100 µl 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).

After washing the collected tissue 3 times in 1x PBS, RIPA buffer (Sigma-Aldrich) was added, tissue lysis was performed using a homogenizer, and proteins were quantified using a BCA protein assay kit (Invitrogen). For protein analysis through Western blotting, 30 μg protein for each sample was separated for 30 minutes at 60 V and 1 hour at 110 V using 10% SDS-PAGE, and then transferred to PVDF membrane (Bio-Rad) for 2 hours at 250 mA. . After transfer, 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).

As a result, it was confirmed that all three cp-asiRNAs effectively inhibit the production of the target protein SNAI1 (FIG. 7 ).

According to the present invention, 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 By selecting and chemically modifying to remove cytotoxicity caused by the carrier and enabling more efficient suppression of gene expression in vivo , it can be usefully used as a preventive or therapeutic agent for fibrosis or cancer.

Since the specific parts of the present invention have been described in detail above, it will be apparent to those of ordinary skill in the art that these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereby. will be. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Electronic file attached.

Claims (19)

1. The antisense strand comprising a sequence complementary to an mRNA encoding SNAI1 (Snail1), the sense strand forming a complementary bond with the antisense strand, the 5'end of the antisense strand and the 3'end of the sense strand are SiRNA characterized in that it forms a blunt end.
2. The siRNA according to claim 1, wherein the sense strand has a length of 15-17nt, and the antisense strand has a length of 16nt or more.
3. The siRNA according to claim 2, wherein the antisense strand has a length of 16-31nt.
4. 3. The siRNA of claim 2, wherein the antisense strand has a length of 19-26nt.
5. The method of claim 1, wherein 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, Group consisting of 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 SiRNA, characterized in that selected from.
6. According to claim 5, The sense strand is siRNA, characterized in that selected from the group consisting of SEQ ID NO: 35, 47, 49 and 51.
7. According to claim 1, wherein the antisense strand 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, SiRNA, characterized in that selected from the group consisting of 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 and 102.
8. According to claim 7, 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 Characterized by siRNA.
9. The siRNA of claim 1, wherein the sense strand or the antisense strand of the siRNA comprises one or more chemical modifications.
10. The method of claim 9,

    The chemical modification is siRNA characterized in that it comprises 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 (propyl), -O-2-methylthioethyl, -O-3-aminopropyl, -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 group, lipophilic compound or cell-penetrating peptide binding.
11. The siRNA according to claim 10, wherein the lipophilic compound is selected from the group consisting of cholesterol, tocopherol, and long-chain fatty acids having 10 or more carbon atoms.
12. The method of claim 9,

    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; And

    SiRNA, characterized in that it comprises at least one modification selected from the group consisting of a phosphate group (phosphate group) bond to the 5'end of the antisense strand.
13. The method of claim 12,

    The sense strand is any one selected from the group consisting of (a) to (i) in the table below,

    The antisense strand is siRNA, characterized in that any one selected from the group consisting of (j) to (s) in the table below.

    

    

    (In the above sequence, * is a phosphorothioate bond, m is 2'-O-methyl (Methyl), 2'-F- is 2'-Fluoro, /chol/ is cholesterol, P- Means 5'-Phosphate group)
14. The method of claim 13,

    The sense strand is any one selected from the group consisting of (e), (g) and (i) in the table, and the antisense strand is a group consisting of (o), (p), (r) and (s) in the table. SiRNA, characterized in that any one selected from.
15. The siRNA according to claim 1, wherein the siRNA has a cell-penetrating ability.
16. A pharmaceutical composition for the prevention or treatment of fibrosis comprising siRNA according to any one of claims 1 to 15.
17. The pharmaceutical composition for preventing or treating fibrosis according to claim 16, wherein the fibrosis is subretinal fibrosis, lung fibrosis, liver fibrosis, myocardial fibrosis or renal fibrosis.
18. A pharmaceutical composition for the prophylaxis or treatment of cancer comprising the siRNA according to any one of claims 1 to 15.
19. The pharmaceutical composition for preventing or treating cancer according to claim 18, wherein the cancer is lung cancer, breast cancer, colon cancer or prostate cancer.

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