Pharmaceutical composition for preventing or treating cancer comprising sirna as active ingredient

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating cancer, the composition comprising siRNA as an active ingredient. The pharmaceutical composition was found to significantly inhibit the cell proliferation effect of colorectal cancer or pancreatic cancer, thus having an excellent anticancer effect, and reduce the expression level of KRAS mRNA. In addition, the composition maintains the body weight of an individual even while reducing the size and weight of a tumor, and thus can be effectively used as an excellent anticancer therapeutic agent.

Description

Pharmaceutical composition for preventing or treating cancer containing SIRNA as an active ingredient

[0001] The present invention relates to a pharmaceutical composition for preventing or treating cancer, comprising siRNA as an active ingredient.

[0002] This application claims priority to Korean Patent Application Nos. 10-2023-0083349, 10-2023-0083355, and 10-2023-0083360, filed on June 28, 2023, the entire contents of which are incorporated herein by reference.

[0003] Additionally, this application claims priority to Korean Patent Application No. 10-2024-0084878, filed June 27, 2024, the entire disclosure of which is incorporated herein by reference.

[0004] Cancer is a genetic disease that forms tumors through abnormal division. If left untreated, it can become malignant, metastasize to surrounding tissues, and pose a life-threatening threat. The primary cause of cancer is the abnormal overexpression of genes involved in cell growth and division, or mutations in the genes that control these processes.

[0005] Cancer varies depending on the organ where it occurs. According to statistics from the Ministry of Health and Welfare, pancreatic cancer ranked 8th among all cancers in 2017. It is known to be difficult to diagnose early and has a poor prognosis due to its tendency to metastasize to other organs. Furthermore, the five-year survival rate is approximately 12%, which is significantly lower than the 76.5%, 75%, and 93% rates for stomach cancer, colon cancer, and breast cancer, respectively. Currently used anticancer drugs such as gemcitabine and radiation therapy can cause various side effects in the vascular, nervous, hematological, and skin systems. Therefore, new treatments that minimize these side effects and provide better therapeutic effects are needed.

[0006] Colorectal cancer is a malignant tumor (adenocarcinoma) that develops in the colon or rectum, most often in the mucosa. Depending on the location of the cancer, it is called colon cancer if it develops in the colon, and rectal cancer if it develops in the rectum. These are collectively referred to as colorectal cancer. According to data from the Central Cancer Registry published in 2017, there were 214,701 new cancer cases in Korea in 2015. Of these, colorectal cancer accounted for 12.5% ​​of the total, with 26,790 cases in both men and women, ranking second. The sex ratio is 1.5:1, indicating a higher incidence in men. The number of new cases is particularly high, with 15,911 cases in men, ranking third among male cancers, and 10,879 cases in women, ranking third among female cancers.

[0007] Meanwhile, gene therapy, a technology that uses genes to treat or prevent disease, is actively being developed to treat intractable or terminal diseases such as cancer. One such gene therapy technology for cancer treatment is small interfering RNA (siRNA), a 21-23 bp RNA duplex that forms a RISC complex to cleave mRNA, thereby suppressing gene expression. Using siRNA to suppress the expression of genes essential for cancer development can lead to cancer treatment through inhibition of cancer cell growth or cell death.

[0008] Despite this, few effective siRNA therapeutics for treating cancer have been reported to date.

[0009] The purpose of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, which comprises as an active ingredient at least one selected from the group consisting of siRNAs comprising any one base sequence selected from the group consisting of SEQ ID NOs: 1 to 29.

[0010] Another object of the present invention is to provide a kit for preventing or treating cancer, comprising a composition comprising at least one selected from the group consisting of siRNAs comprising any one base sequence selected from the group consisting of SEQ ID NOs: 1 to 29 as an active ingredient, and an instruction manual.

[0011]

[0012] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by a person having ordinary skill in the technical field to which the present invention belongs from the description below.

[0013] The present invention provides a pharmaceutical composition for preventing or treating cancer, comprising as an active ingredient at least one selected from the group consisting of siRNAs comprising any one base sequence selected from the group consisting of SEQ ID NOs: 1 to 29.

[0014] In one embodiment of the present invention, the group consisting of the following siRNAs may have a sense strand of 19 to 25 nt in length and an antisense strand of 21 to 27 nt in length, but is not limited thereto:

[0015] 1) siRNA comprising any one base sequence selected from the group consisting of sequence numbers 1 to 5;

[0016] 2) siRNA containing the base sequence represented by sequence number 7;

[0017] 3) siRNA comprising any one base sequence selected from the group consisting of sequence numbers 14, 15, and 17; and

[0018] 4) siRNA comprising any one base sequence selected from the group consisting of sequence numbers 20, 23, and 26 to 28.

[0019] In one embodiment of the present invention, the group consisting of siRNA including the base sequence represented by SEQ ID NO: 12, siRNA including the base sequence represented by SEQ ID NO: 13, siRNA including the base sequence represented by SEQ ID NO: 18, siRNA including the base sequence represented by SEQ ID NO: 21, siRNA including the base sequence represented by SEQ ID NO: 24, and siRNA including the base sequence represented by SEQ ID NO: 29 may be characterized by the group consisting of, but is not limited to:

[0020] a) the sense strand is 19 to 25 nt in length and the antisense strand is 21 to 27 nt in length;

[0021] b) There is no tt overhang in the sense strand;

[0022] c) a 2'-OME chemically modified nucleotide at the 7th position from the 5' end of the sense strand, and the nucleotide of the antisense strand binding to that position does not have a 2'-OME chemical modification;

[0023] d) the nucleotide at the 9th position from the 5' end of the sense strand does not have a 2'-OME chemical modification; and

[0024] e) having 2'-OME chemically modified nucleotides at the 18th and 20th positions from the 5' end of the antisense strand.

[0025] In one embodiment of the present invention, a group consisting of siRNA including a base sequence represented by SEQ ID NO: 6, siRNA including a base sequence represented by SEQ ID NO: 8, siRNA including a base sequence represented by SEQ ID NO: 9, siRNA including a base sequence represented by SEQ ID NO: 10, siRNA including a base sequence represented by SEQ ID NO: 11, siRNA including a base sequence represented by SEQ ID NO: 16, siRNA including a base sequence represented by SEQ ID NO: 19, siRNA including a base sequence represented by SEQ ID NO: 22, and siRNA including a base sequence represented by SEQ ID NO: 25 may be characterized by a group consisting of, but is not limited to:

[0026] a) the sense strand is 19 to 25 nt in length and the antisense strand is 21 to 27 nt in length;

[0027] b) having a 2'-OME chemically modified nucleotide at the 7th position from the 5' end of the sense strand, and the nucleotide of the antisense strand binding to that position does not have a 2'-OME chemical modification;

[0028] c) the nucleotide at the 9th position from the 5' end of the sense strand does not have a 2'-OME chemical modification; and

[0029] d) having 2'-OME chemically modified nucleotides at the 16th, 18th, and 20th positions from the 5' end of the antisense strand.

[0030] In one embodiment of the present invention, the cancer may be at least one selected from the group consisting of pancreatic cancer, colon cancer, squamous cell carcinoma, lung cancer, lung adenocarcinoma, peritoneal cancer, skin cancer, cutaneous or intraocular melanoma, rectal cancer, anal cancer, esophageal cancer, small intestine cancer, endocrine cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, blood cancer, liver cancer, gastrointestinal cancer, glioblastoma, cervical cancer, ovarian cancer, bladder cancer, hepatoma, breast cancer, colon cancer, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, head and neck cancer, and brain cancer, but is not limited thereto.

[0031] In one embodiment of the present invention, the composition can inhibit the proliferation of cancer cells, but is not limited thereto.

[0032] In one embodiment of the present invention, the composition can reduce the size and weight of a tumor, but is not limited thereto.

[0033] In one embodiment of the present invention, the composition can inhibit KRAS mRNA expression, but is not limited thereto.

[0034] The present invention provides a kit for preventing or treating cancer, comprising a composition comprising at least one selected from the group consisting of siRNAs comprising any one base sequence selected from the group consisting of SEQ ID NOs: 1 to 29 as an active ingredient, and an instruction manual.

[0035]

[0036] In addition, the present invention provides a method for treating cancer, or a method for treating pancreatic cancer or colon cancer, comprising a step of administering to a subject in need thereof a pharmaceutically effective amount of a composition comprising, as an active ingredient, at least one selected from the group consisting of siRNAs comprising any one base sequence selected from the group consisting of SEQ ID NOs: 1 to 29.

[0037] In addition, the present invention provides a composition comprising at least one selected from the group consisting of siRNAs comprising any one base sequence selected from the group consisting of SEQ ID NOs: 1 to 29 as an active ingredient for use in preventing or treating cancer, or for use in preventing or treating pancreatic cancer or colon cancer.

[0038] In addition, the present invention provides a use for manufacturing a composition for preventing or treating cancer, or a preparation for preventing or treating pancreatic cancer or colon cancer, comprising as an active ingredient at least one selected from the group consisting of siRNAs comprising any one base sequence selected from the group consisting of SEQ ID NOs: 1 to 29.

[0039] In addition, the present invention,

[0040] i) a group consisting of an siRNA comprising any one base sequence selected from the group consisting of SEQ ID NOs: 1 to 5; an siRNA comprising a base sequence represented by SEQ ID NO: 7; an siRNA comprising any one base sequence selected from the group consisting of SEQ ID NOs: 14, 15, and 17; and an siRNA comprising any one base sequence selected from the group consisting of SEQ ID NOs: 20, 23, and 26 to 28;

[0041] ii) a group consisting of siRNA comprising a base sequence represented by SEQ ID NO: 12; siRNA comprising a base sequence represented by SEQ ID NO: 13; siRNA comprising a base sequence represented by SEQ ID NO: 18; siRNA comprising a base sequence represented by SEQ ID NO: 21; siRNA comprising a base sequence represented by SEQ ID NO: 24; and siRNA comprising a base sequence represented by SEQ ID NO: 29; or

[0042] iii) in the group consisting of siRNA comprising a base sequence represented by SEQ ID NO: 6; siRNA comprising a base sequence represented by SEQ ID NO: 8; siRNA comprising a base sequence represented by SEQ ID NO: 9; siRNA comprising a base sequence represented by SEQ ID NO: 10; siRNA comprising a base sequence represented by SEQ ID NO: 11; siRNA comprising a base sequence represented by SEQ ID NO: 16; siRNA comprising a base sequence represented by SEQ ID NO: 19; siRNA comprising a base sequence represented by SEQ ID NO: 22; and siRNA comprising a base sequence represented by SEQ ID NO: 25;

[0043] A method for preventing or treating cancer, or a method for preventing or treating pancreatic cancer or colon cancer, is provided, comprising a step of administering a pharmaceutically effective amount of a composition comprising at least one siRNA selected from each group as an active ingredient to a subject in need thereof.

[0044] In addition, the present invention provides a use for producing a composition comprising at least one siRNA selected from each of the above groups as an active ingredient for preventing or treating cancer, or for preventing or treating pancreatic cancer or colon cancer.

[0045] In addition, the present invention provides a use for preparing a composition for preventing or treating cancer, or a preparation for preventing or treating pancreatic cancer or colon cancer, comprising at least one siRNA selected from each of the above groups as an active ingredient.

[0046] A pharmaceutical composition for cancer prevention or treatment containing siRNA as an active ingredient has been shown to significantly inhibit cell proliferation in colon or pancreatic cancer, exhibiting excellent anticancer effects and reducing KRAS mRNA expression levels. Furthermore, it can be useful as an excellent anticancer treatment because it reduces tumor size and weight while maintaining body weight.

[0047] Figure 1a is a graph that summarizes the results of analyzing the cell death rate when 105 siRNA candidate substances were treated on three pancreatic cancer cell lines and three colon cancer cell lines, and ranks them.

[0048] Figure 1b shows the cell death effect when 105 types of siRNA candidate substances were treated on three types of pancreatic cancer cell lines.

[0049] Figure 1c shows the cell death effect when 105 types of siRNA candidate substances were treated on three types of colon cancer cell lines.

[0050] Figure 1d shows the sequences and modification details of 29 siRNA candidate substances of the present invention.

[0051] Figure 1e shows the sequences (ss strand and as strand) of 29 siRNAs identified as leading substances among 105 siRNA candidate substances.

[0052] Figures 2a to 2g show the cell death effects of 29 types of siRNA lead materials when treated with three types of pancreatic cancer cell lines. Figures 2a to 2c show the experimental results for lead materials of Group 1, Figure 2d shows the experimental results for lead materials of Group 2, and Figures 2e to 2g show the experimental results for lead materials of Group 3, respectively.

[0053] Figures 3a to 3g show the cell death effects observed when 29 types of siRNA lead materials were treated on three types of colon cancer cell lines. Figures 3a to 3c show the experimental results for the lead materials of Group 1, Figure 3d shows the experimental results for Group 2, and Figures 3e to 3g show the experimental results for the lead materials of Group 3, respectively.

[0054] Figure 4a shows the KRAS mRNA expression inhibitory activity when some of the 29 siRNA leading substances of the present invention were treated to a pancreatic cancer cell line, and Figure 4b shows the experimental results of the control group.

[0055] Figure 5a shows the effect of reducing tumor size and tumor weight when some of the 29 siRNA leading substances of the present invention were administered to a pancreatic cancer animal model (tumor volume: siScramble, Ctl, Exollence 25nt #1, Exollence 25nt #6 in that order based on the highest point of the graph).

[0056] Figure 5b shows the experimental results of changes in animal weight when some of the 29 siRNA leading substances of the present invention were administered to a pancreatic cancer animal model (in order from the highest point of the graph, they are Exollence 25nt #1, Exollence 25nt #6, siScramble, and Ctl).

[0057] The inventors of the present invention made various modifications to siRNA to produce novel siRNAs, and when the siRNAs were treated with pancreatic cancer or colon cancer cell lines, they discovered 29 types of siRNAs with excellent inhibitory activity against KRAS G12D and thus anticancer effects. In particular, the 29 types of siRNAs of the present invention are composed of a sense strand and an antisense strand, and the sense strand and antisense strand of each siRNA have different characteristics such as length, chemical modification, and sequence modification. Therefore, the inventors of the present invention completed the present invention by confirming that each siRNA exhibits the colon cancer and pancreatic cancer therapeutic activity confirmed in the present invention by having the characteristics of both the sense strand sequence and the antisense strand sequence.

[0058] Accordingly, the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising as an active ingredient at least one selected from the group consisting of siRNAs comprising any one base sequence selected from the group consisting of SEQ ID NOs: 1 to 29.

[0059] In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising as an active ingredient at least one selected from the group consisting of siRNAs comprising any one base sequence selected from the group consisting of SEQ ID NOs: 30 to 58.

[0060] In the present invention, siRNA (ss strand) comprising any one base sequence selected from the group consisting of SEQ ID NOs: 1 to 29 is as follows (Table 3):

[0061] 1) Unmodified siKRAS G12D, which is the base sequence represented by sequence number 1;

[0062] 2) Unmodified siKRAS G12D-2, which is the base sequence represented by sequence number 2;

[0063] 3) Unmodified siKRAS G12D-PS, which is the base sequence represented by sequence number 3;

[0064] 4) Unmodified siKRAS G12D-PS-2, the base sequence represented by sequence number 4;

[0065] 5) Pattern_unmodified siKRAS G12D, a base sequence represented by sequence number 5;

[0066] 6) Pattern_OME modified siKRAS G12D, a base sequence represented by sequence number 6;

[0067] 7) Exollence_25nt #3, a base sequence represented by sequence number 7;

[0068] 8) Exollence_25nt #4, a base sequence represented by sequence number 8;

[0069] 9) Exollence_25nt #5, a base sequence represented by sequence number 9;

[0070] 10) Exollence_25nt #6, a base sequence represented by sequence number 10;

[0071] 11) Exollence_25nt #7, a base sequence represented by sequence number 11;

[0072] 12) Exollence_25nt #8, a base sequence represented by sequence number 12;

[0073] 13) Exollence_25nt #9, a base sequence represented by sequence number 13;

[0074] 14) Exollence_25nt-#12, a base sequence represented by sequence number 14;

[0075] 15) Exollence_25nt #13, a base sequence represented by sequence number 15;

[0076] 16) Exollence_25nt #16, a base sequence represented by sequence number 16;

[0077] 17) Exollence_25nt #28, a base sequence represented by sequence number 17;

[0078] 18) Exollence_25nt #29, a base sequence represented by sequence number 18;

[0079] 19) Exollence_25nt #30, a base sequence represented by sequence number 19;

[0080] 20) Exollence_25nt #32, a base sequence represented by sequence number 20;

[0081] 21) Exollence_25nt #33, a base sequence represented by sequence number 21;

[0082] 22) Exollence_25nt #34, a base sequence represented by sequence number 22;

[0083] 23) Exollence_25nt #36, a base sequence represented by sequence number 23;

[0084] 24) Exollence_25nt #37, a base sequence represented by sequence number 24;

[0085] 25) Exollence_25nt #38, a base sequence represented by sequence number 25;

[0086] 26) Exollence_25nt #40, a base sequence represented by sequence number 26;

[0087] 27) Exollence_25nt #41, a base sequence represented by sequence number 27;

[0088] 28) Exollence_25nt #42, which is the base sequence represented by sequence number 28; and

[0089] 29) Exollence_25nt #44, a base sequence represented by sequence number 29.

[0090] In addition, in the present invention, siRNA (as strand) comprising any one base sequence selected from the group consisting of SEQ ID NOs: 30 to 58 is as follows (Table 4):

[0091] 1) Unmodified siKRAS G12D, the base sequence represented by sequence number 30;

[0092] 2) Unmodified siKRAS G12D-2, the base sequence represented by sequence number 31;

[0093] 3) Unmodified siKRAS G12D-PS, which is the base sequence represented by sequence number 32;

[0094] 4) Unmodified siKRAS G12D-PS-2, the base sequence represented by sequence number 33;

[0095] 5) Pattern_unmodified siKRAS G12D, a base sequence represented by sequence number 34;

[0096] 6) Pattern_OME modified siKRAS G12D, a base sequence represented by sequence number 35;

[0097] 7) Exollence_25nt #3, a base sequence represented by sequence number 36;

[0098] 8) Exollence_25nt #4, a base sequence represented by sequence number 37;

[0099] 9) Exollence_25nt #5, a base sequence represented by sequence number 38;

[0100] 10) Exollence_25nt #6, a base sequence represented by sequence number 39;

[0101] 11) Exollence_25nt #7, a base sequence represented by sequence number 40;

[0102] 12) Exollence_25nt #8, a base sequence represented by sequence number 41;

[0103] 13) Exollence_25nt #9, a base sequence represented by sequence number 42;

[0104] 14) Exollence_25nt-#12, a base sequence represented by sequence number 43;

[0105] 15) Exollence_25nt #13, a base sequence represented by sequence number 44;

[0106] 16) Exollence_25nt #16, a base sequence represented by sequence number 45;

[0107] 17) Exollence_25nt #28, a base sequence represented by sequence number 46;

[0108] 18) Exollence_25nt #29, a base sequence represented by sequence number 47;

[0109] 19) Exollence_25nt #30, a base sequence represented by sequence number 48;

[0110] 20) Exollence_25nt #32, a base sequence represented by sequence number 49;

[0111] 21) Exollence_25nt #33, a base sequence represented by sequence number 50;

[0112] 22) Exollence_25nt #34, a base sequence represented by sequence number 51;

[0113] 23) Exollence_25nt #36, a base sequence represented by sequence number 52;

[0114] 24) Exollence_25nt #37, a base sequence represented by sequence number 53;

[0115] 25) Exollence_25nt #38, a base sequence represented by sequence number 54;

[0116] 26) Exollence_25nt #40, a base sequence represented by sequence number 55;

[0117] 27) Exollence_25nt #41, a base sequence represented by sequence number 56;

[0118] 28) Exollence_25nt #42, which is the base sequence represented by sequence number 57; and

[0119] 29) Exollence_25nt #44, a base sequence represented by sequence number 58.

[0120] In the present invention, 29 types of siRNAs were produced by applying modifications such as chemical modification, sequence length adjustment, and sequence movement based on KRAS G12D siRNA.

[0121] At this time, SEQ ID NOs. 1 to 29 of the present invention are the sense strand sequences of each siRNA. In addition, SEQ ID NOs. 30 to 58 are the antisense strand sequences of the siRNA. Accordingly, the present invention provides a pharmaceutical composition for preventing or treating cancer, which comprises at least one selected from the group consisting of siRNAs, which are composed of a sense strand, which is a base sequence represented by any one base sequence selected from the group consisting of SEQ ID NOs. 1 to 29, and an antisense strand, which is any one base sequence selected from the group consisting of SEQ ID NOs. 30 to 58, which includes a complementary sequence thereto, as an active ingredient.

[0122] In the present invention, the “sense strand” refers to a polynucleotide having the same nucleic acid sequence as the target nucleic acid, and is a polynucleotide that is identical in whole or in part to an mRNA, non-mRNA RNA sequence, or a coding or non-coding DNA sequence. In addition, the “antisense strand” is a general term for an RNA that is substantially or 100% complementary to the target nucleic acid of interest, and has a base sequence complementary to the nucleic acid sequence of the sense strand. For example, it may be complementary in whole or in part to messenger RNA (mRNA), non-mRNA RNA sequences (microRNA, piwiRNA, tRNA, rRNA, and hnRNA), or a coding or non-coding DNA sequence.

[0123] Therefore, in the present invention, the siRNA defined as the sense strand can be understood to form a double strand with the siRNA defined as the corresponding antisense strand, and since the siRNA is a double strand, it can be understood to have both a sense strand and an antisense strand. For example, in the present invention, when referring to any one siRNA selected from the group consisting of SEQ ID NOs: 1 to 29 based on the sense strand, the siRNA represented by SEQ ID NO: 1 can be understood to be associated with the base sequence represented by SEQ ID NO: 30 as its antisense strand.

[0124] Accordingly, the present invention provides an siRNA comprising any one sense strand base sequence selected from the group consisting of SEQ ID NOs: 1 to 29, and any one antisense strand base sequence selected from the group consisting of SEQ ID NOs: 30 to 58:

[0125] 1) unmodified siKRAS G12D comprising a sense strand sequence represented by SEQ ID NO: 1 and an antisense strand sequence represented by SEQ ID NO: 30;

[0126] 2) unmodified siKRAS G12D-2 comprising a sense strand sequence represented by SEQ ID NO: 2 and an antisense strand sequence represented by SEQ ID NO: 31;

[0127] 3) unmodified siKRAS G12D-PS comprising a sense strand sequence represented by SEQ ID NO: 3 and an antisense strand sequence represented by SEQ ID NO: 32;

[0128] 4) Unmodified siKRAS G12D-PS-2 comprising a sense strand sequence represented by SEQ ID NO: 4 and an antisense strand sequence represented by SEQ ID NO: 33;

[0129] 5) Pattern_unmodified siKRAS G12D comprising a sense strand base sequence represented by SEQ ID NO: 5 and an antisense strand base sequence represented by SEQ ID NO: 34;

[0130] 6) Pattern_OME modified siKRAS G12D comprising a sense strand base sequence represented by SEQ ID NO: 6 and an antisense strand base sequence represented by SEQ ID NO: 35;

[0131] 7) Exollence_25nt #3 comprising a sense strand sequence represented by SEQ ID NO: 7 and an antisense strand sequence represented by SEQ ID NO: 36;

[0132] 8) Exollence_25nt #4 comprising a sense strand sequence represented by SEQ ID NO: 8 and an antisense strand sequence represented by SEQ ID NO: 37;

[0133] 9) Exollence_25nt #5 comprising a sense strand sequence represented by SEQ ID NO: 9 and an antisense strand sequence represented by SEQ ID NO: 38;

[0134] 10) Exollence_25nt #6 comprising a sense strand sequence represented by SEQ ID NO: 10 and an antisense strand sequence represented by SEQ ID NO: 39;

[0135] 11) Exollence_25nt #7 comprising a sense strand base sequence represented by SEQ ID NO: 11 and an antisense strand base sequence represented by SEQ ID NO: 40;

[0136] 12) Exollence_25nt #8 comprising a sense strand sequence represented by SEQ ID NO: 12 and an antisense strand sequence represented by SEQ ID NO: 41;

[0137] 13) Exollence_25nt #9 comprising a sense strand sequence represented by SEQ ID NO: 13 and an antisense strand sequence represented by SEQ ID NO: 42;

[0138] 14) Exollence_25nt-#12 comprising a sense strand base sequence represented by SEQ ID NO: 14 and an antisense strand base sequence represented by SEQ ID NO: 43;

[0139] 15) Exollence_25nt #13 comprising a sense strand sequence represented by SEQ ID NO: 15 and an antisense strand sequence represented by SEQ ID NO: 44;

[0140] 16) Exollence_25nt #16 comprising a sense strand sequence represented by SEQ ID NO: 16 and an antisense strand sequence represented by SEQ ID NO: 45;

[0141] 17) Exollence_25nt #28 comprising a sense strand sequence represented by SEQ ID NO: 17 and an antisense strand sequence represented by SEQ ID NO: 46;

[0142] 18) Exollence_25nt #29 comprising a sense strand sequence represented by SEQ ID NO: 18 and an antisense strand sequence represented by SEQ ID NO: 47;

[0143] 19) Exollence_25nt #30 comprising a sense strand base sequence represented by SEQ ID NO: 19 and an antisense strand base sequence represented by SEQ ID NO: 48;

[0144] 20) Exollence_25nt #32 comprising a sense strand sequence represented by SEQ ID NO: 20 and an antisense strand sequence represented by SEQ ID NO: 49;

[0145] 21) Exollence_25nt #33 comprising a sense strand sequence represented by SEQ ID NO: 21 and an antisense strand sequence represented by SEQ ID NO: 50;

[0146] 22) Exollence_25nt #34 comprising a sense strand sequence represented by SEQ ID NO: 22 and an antisense strand sequence represented by SEQ ID NO: 51;

[0147] 23) Exollence_25nt #36 comprising a sense strand sequence represented by SEQ ID NO: 23 and an antisense strand sequence represented by SEQ ID NO: 52;

[0148] 24) Exollence_25nt #37 comprising a sense strand sequence represented by SEQ ID NO: 24 and an antisense strand sequence represented by SEQ ID NO: 53;

[0149] 25) Exollence_25nt #38 comprising a sense strand sequence represented by SEQ ID NO: 25 and an antisense strand sequence represented by SEQ ID NO: 54;

[0150] 26) Exollence_25nt #40 comprising a sense strand sequence represented by SEQ ID NO: 26 and an antisense strand sequence represented by SEQ ID NO: 55;

[0151] 27) Exollence_25nt #41 comprising a sense strand sequence represented by SEQ ID NO: 27 and an antisense strand sequence represented by SEQ ID NO: 56;

[0152] 28) Exollence_25nt #42 comprising a sense strand sequence represented by SEQ ID NO: 28 and an antisense strand sequence represented by SEQ ID NO: 57; and

[0153] 29) Exollence_25nt #44 comprising a sense strand sequence represented by SEQ ID NO: 29 and an antisense strand sequence represented by SEQ ID NO: 58.

[0154] Technology that suppresses gene expression is an important tool in the development of therapeutics and target verification for disease treatment. Among these technologies, RNA interference (hereinafter referred to as 'RNAi') has been discovered to act on sequence-specific mRNAs in various types of mammalian cells since its role was discovered. When a long RNA duplex is delivered to a cell, the delivered RNA duplex is processed into 21 to 23 base pairs (bp) duplexes by the endonuclease Dicer, resulting in short interfering RNA (hereinafter referred to as 'siRNA'). The siRNA binds to the RNA-induced silencing complex (RISC), and the guide (antisense) strand recognizes and degrades the target mRNA, thereby sequence-specifically inhibiting the expression of the target gene.

[0155] That is, in the present invention, “siRNA (small interfering RNA)” is a small RNA fragment of 18 to 23 nucleotides in size generated by cleavage of double-stranded RNA by the Dicer enzyme, and can be used to specifically bind to mRNA having a complementary sequence and suppress the expression of the protein or mRNA.

[0156] Additionally, the 29 types of siRNA of the present invention can be delivered to an individual in a form including a nucleic acid delivery vehicle.

[0157] In the present invention, the term “nucleic acid delivery system” is intended to increase the delivery efficiency into the body, and has the advantage of not only excellent stability in the body but also a simple manufacturing process as a drug.

[0158] The nucleic acid delivery vehicle may include, but is not limited to, viral vectors, non-viral vectors, liposomes, cationic polymers, micelles, emulsions, solid lipid nanoparticles, and the like.

[0159] That is, in the present invention, the siRNA nucleic acid molecule can be provided in a form included in an expression vector. The expression vector, preferably encoding the miRNA of the present invention in an expressible form, can be introduced into a host according to a method generally used in the art. At this time, the expression vector in the present invention can be used without limitation as long as it is for delivering siRNA. In addition, in the present invention, it may be preferable to additionally include a selection marker to facilitate selection of transformed cells, but the present invention is not limited thereto. In the present invention, the expression vector can be introduced into a host cell and provided as a transformed transformant, but the present invention is not limited thereto.

[0160] Viral vectors have the advantages of high delivery efficiency and long duration, and include retroviral vectors, adenoviral vectors, vaccinia virus vectors, adeno-associated viral vectors, and oncolytic viral vectors. Nonviral vectors may include plasmids. In addition, various formulations such as liposomes, cationic polymers, micelles, emulsions, and solid lipid nanoparticles can be used. Cationic polymers for nucleic acid delivery may include natural polymers such as chitosan, atelocollagen, and cationic polypeptides, as well as synthetic polymers such as poly(L-lysin), linear or branched polyethylene imine (PEI), cyclodextrin-based polycations, and dendrimers.

[0161] When siRNA is included in a composition in the form of a complex with a nucleic acid delivery vehicle as described above, the siRNA can be efficiently delivered to target cells, resulting in high target gene expression regulation even at relatively low doses. Furthermore, it has the advantage of preventing non-specific siRNA delivery to organs and cells other than the target.

[0162] In the present invention, siRNA may be chemically modified. Such chemical modification of siRNA may be intended to enhance in vivo stability, impart resistance to nucleases, and reduce nonspecific immune responses, but is not limited thereto.

[0163] In the present invention, all 29 types of siRNA were confirmed to exhibit anticancer activity against colon cancer or pancreatic cancer, and as a result of analyzing the commonalities in the sequences of the 29 types of siRNA, it was confirmed that they were classified into three groups.

[0164] First, the first group and commonalities are as follows. In one embodiment of the present invention, the group consisting of the following siRNAs may have a sense strand of 19 to 25 nt in length and an antisense strand of 21 to 27 nt in length, but is not limited thereto:

[0165] 1) siRNA comprising any one base sequence selected from the group consisting of sequence numbers 1 to 5;

[0166] 2) siRNA containing the base sequence represented by sequence number 7;

[0167] 3) siRNA comprising any one base sequence selected from the group consisting of sequence numbers 14, 15, and 17; and

[0168] 4) siRNA comprising any one base sequence selected from the group consisting of sequence numbers 20, 23, and 26 to 28.

[0169] The second group and commonalities are as follows. In one embodiment of the present invention, the group consisting of siRNA comprising the base sequence represented by SEQ ID NO: 12, siRNA comprising the base sequence represented by SEQ ID NO: 13, siRNA comprising the base sequence represented by SEQ ID NO: 18, siRNA comprising the base sequence represented by SEQ ID NO: 21, siRNA comprising the base sequence represented by SEQ ID NO: 24, and siRNA comprising the base sequence represented by SEQ ID NO: 29 may be characterized by the group consisting of, but is not limited to:

[0170] a) the sense strand is 19 to 25 nt in length and the antisense strand is 21 to 27 nt in length;

[0171] b) There is no tt overhang in the sense strand;

[0172] c) a 2'-OME chemically modified nucleotide at the 7th position from the 5' end of the sense strand, and the nucleotide of the antisense strand binding to that position does not have a 2'-OME chemical modification;

[0173] d) the nucleotide at the 9th position from the 5' end of the sense strand does not have a 2'-OME chemical modification; and

[0174] e) having 2'-OME chemically modified nucleotides at the 18th and 20th positions from the 5' end of the antisense strand.

[0175] Finally, the third group and commonalities are as follows. In one embodiment of the present invention, the group consisting of siRNA including the base sequence represented by SEQ ID NO: 6, siRNA including the base sequence represented by SEQ ID NO: 8, siRNA including the base sequence represented by SEQ ID NO: 9, siRNA including the base sequence represented by SEQ ID NO: 10, siRNA including the base sequence represented by SEQ ID NO: 11, siRNA including the base sequence represented by SEQ ID NO: 16, siRNA including the base sequence represented by SEQ ID NO: 19, siRNA including the base sequence represented by SEQ ID NO: 22, and siRNA including the base sequence represented by SEQ ID NO: 25 may be characterized by the group consisting of, but is not limited to:

[0176] a) the sense strand is 19 to 25 nt in length and the antisense strand is 21 to 27 nt in length;

[0177] b) having a 2'-OME chemically modified nucleotide at the 7th position from the 5' end of the sense strand, and the nucleotide of the antisense strand binding to that position does not have a 2'-OME chemical modification;

[0178] c) the nucleotide at the 9th position from the 5' end of the sense strand does not have a 2'-OME chemical modification; and

[0179] d) having 2'-OME chemically modified nucleotides at the 16th, 18th, and 20th positions from the 5' end of the antisense strand.

[0180] In the present invention, "overhang" may refer to an unpaired nucleotide at the end of a DNA or RNA molecule. Such unpaired nucleotides may be present on either strand and may form a 3' or 5' overhang. Longer overhangs may be referred to as cohesive ends or sticky ends, but are not limited thereto. The 29 siRNAs of the present invention differ in the presence or absence of overhangs in portions of the sense strand or antisense strand, and are thus classified into three groups. Therefore, in the present invention, it can be understood that the therapeutic effect of siRNAs against colon or pancreatic cancer is determined by the presence or absence of overhangs, whether they are present in the sense strand or antisense strand, and the type of overhang.

[0181] In the present invention, “-OME” may refer to a methoxy group and may exhibit the effect of being chemically modified by being linked to a nucleotide. Chemical modifications may exhibit different chemical characteristics depending on the type and sequence of nucleotides, and further, may exhibit different effects on the therapeutic activity for colon cancer or pancreatic cancer. Therefore, it is self-evident that the 29 types of siRNAs of the present invention exhibit unique therapeutic activity for colon cancer or pancreatic cancer by adding chemical modifications to specific types of nucleotides at specific positions in either the sense strand or the antisense strand. The specificity and specificity of such modifications can be understood by equally applying not only chemical modifications but also sequence modifications applied to the nucleotide sequence of the siRNA. In conclusion, the 29 types of siRNAs of the present invention are siRNAs that exhibit therapeutic activity for colon cancer or pancreatic cancer among a remarkably large number of siRNAs by applying length adjustments, chemical modifications, and sequence modifications to either the sense strand or the antisense strand, respectively.

[0182] In the present invention, it was confirmed that when 29 types of siRNA, particularly each siRNA having a base sequence represented by SEQ ID NO: 1 to 3 or SEQ ID NO: 6, was treated, the expression of KRAS mRNA was suppressed. Therefore, the siRNA of the present invention can be applied to cancers that can be treated by suppressing KRAS mRNA expression. In one embodiment of the present invention, the cancer may be at least one selected from the group consisting of pancreatic cancer, colon cancer, squamous cell carcinoma, lung cancer, lung adenocarcinoma, peritoneal cancer, skin cancer, cutaneous or intraocular melanoma, rectal cancer, anal cancer, esophageal cancer, small intestine cancer, endocrine cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, blood cancer, liver cancer, gastrointestinal cancer, glioblastoma, cervical cancer, ovarian cancer, bladder cancer, hepatoma, breast cancer, colon cancer, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, head and neck cancer, and brain cancer, but is not limited thereto.

[0183] In one embodiment of the present invention, the composition can inhibit the proliferation of cancer cells, but is not limited thereto.

[0184] In one embodiment of the present invention, the composition can reduce the size and weight of a tumor, but is not limited thereto.

[0185] KRAS is one of the most frequently mutated oncogenes in human tumors. While normal KRAS performs essential functions in signaling in normal tissues, mutations in the KRAS gene are implicated in the development of various cancers, making it an important therapeutic target.

[0186] KRAS gene mutations typically occur in codons, with mutations most commonly occurring at positions 12, 13, and 61. Mutations are particularly prevalent in codon 12, with G12C accounting for approximately 40% of all KRAS mutations.

[0187] In one embodiment of the present invention, the composition can inhibit KRAS mRNA expression, but is not limited thereto.

[0188] In the present invention, it was confirmed that the siRNA produced in the present invention has a remarkable expression suppression effect against KRAS G12D, among KRAS. Therefore, it was proven that the siRNA of the present invention exhibits excellent preventive or therapeutic effects against pancreatic cancer and colon cancer, which are known to be highly related to KRAS G12D, by suppressing KRAS G12D.

[0189] The target patient for administration of the composition of the present invention may be a mammal, preferably a human, a monkey, or a rodent (mouse, rat), and in particular, any mammal having pancreatic cancer or colon cancer, such as a human.

[0190] In order to increase the therapeutic effect of pancreatic cancer or colon cancer, the concentration of siRNA in the composition, or the concentration used or treated, may be 0.001 to 1000 nM, preferably 0.01 to 100 nM, and more preferably 0.1 to 10 nM, but is not limited thereto.

[0191] The composition of the present invention comprises siRNA comprising the base sequence represented by SEQ ID NO: 1 to 29, and can achieve a synergistic effect through combination therapy.

[0192] The composition of the present invention can be manufactured by including, in addition to the above-mentioned effective ingredient, one or more pharmaceutically acceptable carriers. The pharmaceutically acceptable carrier must be compatible with the effective ingredient of the present invention, and may be used as a mixture of saline solution, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components. If necessary, other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate the composition into an injectable formulation such as an aqueous solution, suspension, or emulsion. Furthermore, the composition can be preferably formulated according to each disease or component using an appropriate method in the art or a method disclosed in Remington's Pharmaceutical Science (Mack Publishing company, Easton PA).

[0193] The content and administration method of the active ingredients, etc. included in the composition of the present invention can be determined by a person skilled in the art based on the symptoms and severity of the disease of a typical patient. Furthermore, the composition can be formulated in various forms, such as powders, tablets, capsules, liquids, injections, ointments, syrups, etc., and can also be provided in unit-dose or multi-dose containers, such as sealed ampoules and bottles.

[0194] 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 to these, but for example, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, enteral, sublingual, or topical administration is possible. 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 disease severity, and can be easily determined by a person skilled in the art. In addition, the composition of the present invention can be formulated into a suitable dosage form using known techniques for clinical administration.

[0195] The siRNA of the present invention can be introduced into cells in vivo or ex vivo for the treatment of cancer. Thus, by combining the siRNA of the present invention with chemotherapy, sensitivity to chemotherapeutic agents can be increased, maximizing therapeutic efficacy and reducing side effects.

[0196] Additionally, the terms “percent identity,” “sequence identity,” “percent similarity,” “sequence similarity,” “percent sequence identity,” and the like, as used herein with respect to amino acid sequences and / or nucleic acid sequences in the present invention, may refer to, but are not limited to, a measure determined by comparing the degree of similarity of two sequences based on alignment of the sequences that maximizes the similarity between aligned amino acid residues or nucleotides and is a function of the number of identical or similar residues or nucleotides, the total number of residues or nucleotides, and the presence and length of gaps in the sequence alignment.

[0197] Portions of a polynucleotide or polypeptide sequence may contain additions or deletions (i.e., gaps) compared to a reference sequence (which does not contain additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the same nucleic acid base or amino acid residue appears in both sequences to yield the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to yield the percent sequence identity.

[0198] The term “identical” or percent “identity” in relation to two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or represented region) as measured using the BLAST or BLAST 2.0 sequence comparison algorithms using the default parameters described below or by manual alignment and visual inspection (see, e.g., the NCBI website at http: / / www.ncbi.nlm.nih.gov / BLAST / ). These sequences are then referred to as “substantially identical.”

[0199] This definition also refers to, or can be applied to, complementation of a test sequence. This definition also encompasses sequences with deletions and / or additions, as well as substitutions. As described below, preferred algorithms can account for gaps, etc. Preferably, identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably, over a region that is 50 to 100 amino acids or nucleotides in length.

[0200] Amino acid or nucleotide base “positions” are represented by numbers that sequentially identify each amino acid (or nucleotide base) in the reference sequence based on its position relative to the N-terminus (or 5'-end). Due to deletions, insertions, truncations, fusions, etc., which must be considered when determining optimal alignment, the number of amino acid residues in a test sequence, generally determined simply by counting from the N-terminus, will not necessarily be the same as the number of corresponding positions in the reference sequence. For example, if a variant has a deletion relative to the aligned reference sequence, the amino acid corresponding to the position in the reference sequence at the deletion site will be absent in the variant. If there is an insertion in the aligned reference sequence, the insertion will not correspond to an amino acid position numbered in the reference sequence. In the case of truncations or fusions, there may be a stretch of amino acids in either the reference or aligned sequence that does not correspond to any amino acid in the corresponding sequence.

[0201] The terms “numbered with respect to” or “corresponding to” when used in reference to the numbering of a given amino acid or polynucleotide sequence refers to the numbering of residues in the reference sequence specified when the given amino acid or polynucleotide sequence is compared to the reference sequence.

[0202] In this specification, “active ingredient” means a substance or group of substances whose pharmacological action, directly or indirectly, is expected to be the efficacy and effect of a preparation, and which exhibits the intended activity alone or can exhibit the intended activity together with a carrier or the like that is inactive in itself. The component may be included in an amount of 1 wt% or more based on the dry weight of the entire composition, for example, 10 wt% to 100 wt%, 20 wt% to 100 wt%, 30 wt% to 100 wt%, 40 wt% to 100 wt%, 50 wt% to 100 wt%, 60 wt% to 100 wt%, 70 wt% to 100 wt%, 80 wt% to 100 wt%, 90 wt% to 100 wt%, 10 wt% to 80 wt%, 20 wt% to 80 wt%, 30 wt% to 80 wt%, 40 wt% to 80 wt%, 50 wt% to 80 wt%, 60 wt% to 80 wt%, or 70 wt% to 80 wt%, but is not limited thereto.

[0203] In the present invention, as described above, it was confirmed that the therapeutic activity of colon cancer or pancreatic cancer was excellent when treated with 29 types of siRNA of the present invention, and thus the composition of the present invention can contain any one or more of the 29 types of siRNA and a nucleic acid delivery vehicle as active ingredients.

[0204] The "pharmaceutical composition" according to the present invention may further include suitable carriers, excipients, and diluents commonly used in the manufacture of pharmaceutical compositions. The excipients may be, for example, one or more selected from the group consisting of diluents, binders, disintegrants, lubricants, adsorbents, moisturizers, film-coating materials, and controlled-release additives.

[0205] The pharmaceutical composition according to the present invention may be formulated and used in the form of external preparations such as powders, granules, sustained-release granules, enteric-coated granules, liquids, eye drops, ellipsoids, emulsions, suspensions, alcohols, troches, aromatic waters, limonades, tablets, sustained-release tablets, enteric-coated tablets, sublingual tablets, hard capsules, soft capsules, sustained-release capsules, enteric capsules, pills, tinctures, soft extracts, dry extracts, fluid extracts, injections, capsules, irrigation solutions, ointments, lotions, pastes, sprays, inhalants, patches, sterile injection solutions, or aerosols, according to a conventional method, and the external preparations may have formulations such as creams, gels, patches, sprays, ointments, ointments, lotions, liniments, pastes, or cataplasmas.

[0206] Carriers, excipients and diluents that may be included in the pharmaceutical composition according to the present invention include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

[0207] When formulating, it is usually prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants.

[0208] The additives of the tablets, powders, granules, capsules, pills, and troches according to the present invention include excipients such as corn starch, potato starch, wheat starch, lactose, sucrose, glucose, fructose, D-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, calcium hydrogen phosphate, calcium sulfate, sodium chloride, sodium bicarbonate, purified lanolin, microcrystalline cellulose, dextrin, sodium alginate, methylcellulose, sodium carboxymethylcellulose, kaolin, urea, colloidal silica gel, hydroxypropyl starch, hydroxypropyl methylcellulose (HPMC) 1928, HPMC 2208, HPMC 2906, HPMC 2910, propylene glycol, casein, calcium lactate, and Primogel; Gelatin, gum arabic, ethanol, agar powder, cellulose acetate phthalate, carboxymethylcellulose, calcium carboxymethylcellulose, glucose, purified water, sodium caseinate, glycerin, stearic acid, sodium carboxymethylcellulose, sodium methylcellulose, methylcellulose, microcrystalline cellulose, dextrin, hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, refined shellac, starch starch, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl alcohol, polyvinyl pyrrolidone, and binders such as hydroxypropyl methylcellulose, corn starch, agar powder, methylcellulose, bentonite, hydroxypropyl starch, sodium carboxymethylcellulose, sodium alginate, Disintegrants such as carboxymethylcellulose calcium, calcium citrate, sodium lauryl sulfate, anhydrous silicic acid, 1-hydroxypropyl cellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde-treated casein and gelatin, alginic acid, amylose, guar gum, baking soda, polyvinylpyrrolidone, calcium phosphate, gelled starch, gum arabic, amylopectin, pectin, sodium polyphosphate, ethylcellulose, sucrose, magnesium aluminum silicate, di-sorbitol solution, and light anhydrous silicic acid;Lubricants such as calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopodium dentata, kaolin, petrolatum, sodium stearate, cocoa butter, sodium salicylate, magnesium salicylate, polyethylene glycol 4000, PEG 6000, liquid paraffin, hydrogenated soybean oil (Lubri wax), aluminum stearate, zinc stearate, sodium lauryl sulfate, magnesium oxide, macrogol, synthetic aluminum silicate, anhydrous silicic acid, higher fatty acids, higher alcohols, silicone oil, paraffin oil, polyethylene glycol fatty acid ether, starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine, and light anhydrous silicic acid can be used.;

[0209] As additives of the liquid formulation according to the present invention, water, dilute hydrochloric acid, dilute sulfuric acid, sodium citrate, monostearate sucrose, polyoxyethylene sorbitol fatty acid esters (twin esters), polyoxyethylene monoalkyl ethers, lanolin ethers, lanolin esters, acetic acid, hydrochloric acid, ammonia water, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethylcellulose, sodium carboxymethylcellulose, etc. can be used.

[0210] The syrup according to the present invention may include a solution of white sugar, other sugars, or sweeteners, and may also include a fragrance, a coloring agent, a preservative, a stabilizer, a suspending agent, an emulsifier, a viscosity modifier, and the like, as needed.

[0211] Purified water may be used in the emulsion according to the present invention, and emulsifiers, preservatives, stabilizers, fragrances, etc. may be used as needed.

[0212] The suspension according to the present invention may include suspending agents such as acacia, tragacanth, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropylmethylcellulose, HPMC 1828, HPMC 2906, and HPMC 2910, and, if necessary, surfactants, preservatives, stabilizers, colorants, and fragrances may be used.

[0213] The injection according to the present invention includes a solvent such as distilled water for injection, 0.9% sodium chloride injection, Ringer's injection, dextrose injection, dextrose + sodium chloride injection, PEG, lactated Ringer's injection, ethanol, propylene glycol, non-volatile oils - sesame oil, cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate, benzene benzoate; a solubilizing agent such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidine, propylene glycol, tween, nitrile acid amide, hexamine, dimethylacetamide; a buffer such as weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, albumins, peptones, gums; It may include isotonic agents such as sodium chloride; stabilizers such as sodium bisulfite (NaHSO3), carbon dioxide gas, sodium metabisulfite (Na2S2O5), sodium sulfite (Na2SO3), nitrogen gas (N2), and ethylenediaminetetraacetic acid; oxidizing agents such as sodium bisulfite 0.1%, sodium formaldehyde sulfoxylate, thiourea, disodium ethylenediaminetetraacetic acid disodium, and acetone sodium bisulfite; analgesics such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; and suspending agents such as sodium cis-methylenediamine, sodium alginate, Tween 80, and aluminum monostearate.

[0214] The suppository according to the present invention comprises cocoa butter, lanolin, withepsol, polyethylene glycol, glycerogelatin, methylcellulose, carboxymethylcellulose, a mixture of stearic acid and oleic acid, Subanal, cottonseed oil, peanut oil, palm oil, cocoa butter + cholesterol, lecithin, ranet wax, glycerol monostearate, Tween or Span, Imhausen, monolene (propylene glycol monostearate), glycerin, Adeps solidus, Buytyrum Tego-G, Cebes Pharma 16, hexalide base 95, Cotomar, Hydroxycote SP, S-70-XXA, S-70-XX75 (S-70-XX95), Hydroxycote Mechanisms such as (Hydrokote) 25, Hydrokote 711, Idropostal, Massa estrarium (A, AS, B, C, D, E, I, T), Massa-MF, Masupol, Masupol-15, Neosupostal-N, Paramound-B, Suposiro (OSI, OSIX, A, B, C, D, H, L), Suppository base type IV (AB, B, A, BC, BBG, E, BGF, C, D, 299), Supostal (N, Es), Wecovi (W, R, S, M, Fs), Tezester triglyceride base (TG-95, MA, 57) can be used.

[0215] Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid dosage forms are prepared by mixing the extract with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.

[0216] Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

[0217] The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment. The effective dosage level can be determined based on factors including the type and severity of the patient's disease, drug activity, drug sensitivity, administration time, administration route and excretion rate, treatment period, concurrently used drugs, and other factors well known in the medical field.

[0218] The pharmaceutical composition according to the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents. It can be administered sequentially or simultaneously with conventional therapeutic agents, or in single or multiple doses. Taking all of the above factors into account, it is important to administer an amount that achieves maximum efficacy with minimal side effects. This amount can be readily determined by those skilled in the art to which the present invention pertains.

[0219] The pharmaceutical composition of the present invention can be administered to a subject via various routes. All modes of administration are conceivable, including oral ingestion, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, intrathecal injection, sublingual administration, buccal mucosa administration, rectal insertion, vaginal insertion, ocular administration, otic administration, nasal administration, inhalation, oral or nasal spraying, dermal administration, and transdermal administration.

[0220] The pharmaceutical composition of the present invention is determined according to the type of drug as an active ingredient, along with various related factors such as the disease to be treated, the route of administration, the patient's age, sex, weight, and the severity of the disease. Specifically, the effective dose of the composition according to the present invention may vary depending on the patient's age, sex, and weight, and is generally 0.001 to 150 mg per 1 kg of body weight, preferably 0.01 to 100 mg, administered daily or every other day, or divided into 1 to 3 times a day. However, since the dosage may increase or decrease depending on the route of administration, the severity of the disease, sex, weight, age, etc., the above dosage does not limit the scope of the present invention in any way.

[0221] In the present invention, “subject” means a subject requiring treatment for a disease, and more specifically, means a mammal such as a human or non-human primate, mouse, rat, dog, cat, horse, and cow.

[0222] In the present invention, “administration” means providing a predetermined composition of the present invention to a subject by any suitable method. In the present invention, “prevention” means any action that inhibits or delays the onset of a target disease, “treatment” means any action that improves or beneficially changes a target disease and its resulting metabolic abnormality symptoms by administering a pharmaceutical composition according to the present invention, and “improvement” means any action that reduces a parameter related to a target disease, for example, the severity of a symptom, by administering a composition according to the present invention.

[0223]

[0224] The present invention provides a kit for preventing or treating cancer, comprising a composition comprising at least one selected from the group consisting of siRNAs comprising any one base sequence selected from the group consisting of SEQ ID NOs: 1 to 29 as an active ingredient, and an instruction manual.

[0225] In addition to the above components, the "kit" of the present invention may include other components, devices, materials, etc. typically required for treating cancer. Furthermore, all components included in the kit may be used more than once without limitation on the number of times, there are no restrictions on the order in which each material is used, and the application of each material may be performed simultaneously or sequentially.

[0226] The kit of the present invention may include a container in addition to the formulation and instructions. The container may serve to package the composition, and may also serve to store and secure the composition. The container may be formed of a material such as a bottle, a tub, a sachet, an envelope, a tube, an ampoule, or the like, and may be formed partially or wholly from plastic, glass, paper, foil, wax, or the like. The container may be equipped with a completely or partially detachable stopper, which may initially be part of the container or may be attached to the container by mechanical, adhesive, or other means, and may also be equipped with a stopper that allows access to the contents by means of a syringe needle. The kit may include an outer package, which may include, but is not limited to, instructions for the use of the components.

[0227] Hereinafter, preferred examples are presented to aid in understanding the present invention. However, the following examples are provided solely to facilitate a better understanding of the present invention, and the scope of the present invention is not limited by the following examples.

[0228]

[0229] [Example]

[0230]

[0231] Example 1. Selection of siRNA candidates for cancer treatment and identification of lead compounds.

[0232] Example 1-1. Selection of siRNA candidates for cancer treatment

[0233] To identify RNA agents with enhanced gene expression suppression effects, we developed siRNAs with RNA sequences based on KRAS G12D siRNA, with chemical modifications introduced to the sequence. The unmodified reference RNA sequence is as follows.

[0234]

[0235] No.NAMESEQUENCE59base RNA_SSGUU GGA GCU GAU GGC GUA G=tt60base RNA_ASCUA CGC CAU CAG CUC CAA C=tt

[0236] Specifically, the siRNA modification condition test items for securing effective candidate substances are as follows.

[0237]

[0238] no.modificationcontentasiRNA length (nt)19 ~ 25 ntbPresence of tt overhang in SS(-)cPresence of tt overhang in AS(+)dPresence Phosphorothioate (PS) modification of tt overhand(+)eType of nucleotides chemical modification*2'-OMEfNumber of modified nucleotides (Pattern)0, 3, 4gSequence of the 20 th If nucleotide is G / C, change to A / U

[0239] (a. The sense strand is 19–25 nt long and the antisense strand is 21–27 nt long.

[0240] b. The sense strand has no tt overhang

[0241] c. The antisense strand has a tt overhang

[0242] d. PS modification at the first t at the 5' end of the tt overhang present in the antisense strand

[0243] e. Chemical modification using the 2'-OMe method

[0244] f-1. 2'-OME chemically modified nucleotide at position 7 from the 5' end of the sense strand (the nucleotide of the antisense strand that binds at this position is not modified)

[0245] f-2. The 9th nucleotide from the 5' end of the sense strand is not modified.

[0246] f-3. The 16th, 18th, and 20th nucleotides from the 5' end of the antisense strand can be chemically modified with 2'-OMe.

[0247] f-3-1. Do not transform or

[0248] f-3-2. Both numbers 18 and 20 are transformed

[0249] f-3-3. Numbers 16, 18, and 20 are all transformed.

[0250] *f-1 and f-2 are mandatory conditions, and f-3 has three cases depending on the number of variations.

[0251] g-1. Substitution of the 20th nucleotide from the 5' end of the sense strand with A and the 6th nucleotide from the 5' end of the antisense strand with U.

[0252] g-2. Substitute the 20th nucleotide from the 5' end of the sense strand with U and the 6th nucleotide from the 5' end of the antisense strand with A.

[0253] *This refers to cases where g-1 or g-2 is true, but does not include both conditions.)

[0254]

[0255] As a result, 105 siRNA candidates were finally selected.

[0256]

[0257] Example 1-2. Identification of a Leading siRNA for Cancer Treatment

[0258] To identify the leading substance among the 105 siRNAs selected in Example 1-1, the survival rate of cancer cells was determined when treated with them, and the cell killing effect was analyzed for this purpose. Specifically, the cell survival rates obtained after treating three pancreatic cancer cell lines and three colon cancer cell lines with 105 siRNAs were summarized and presented in a single graph (a graph analyzing a total of 18 values) according to the ranking. At this time, the obtained values ​​are n=3 for each cell line.

[0259]

[0260] As a result, it was confirmed as shown in Fig. 1a to Fig. 1c, and accordingly, 29 types of siRNAs in Tables 3 and 4 below were confirmed (Fig. 1d and Fig. 1e). (At this time, PS modification means that G / C is changed to A / U, and is indicated in italics (A: blue / U: green). In addition, the 2' OME modification is indicated by an underline (red).)

[0261]

[0262] No. IDSS sequence1unmodified siKRAS G12DGUU GGA GCU GAU GGC GUA G=tt2unmodified siKRAS G12D-2AGU UGG AGC UGA UGG CGU A=tt3unmodified siKRAS G12D-PSGUU GGA GAU GGC sitRASKAn G=t G12D-PS-2AGU UGG AGC UGA UGG CGU A=t*t5Pattern_unmodified siKRAS G12DGUU GGA GCU GAU GGC GUA GGC AAG A6Pattern_OME modified siKRAS G12DGUU Ance33 GAU GAU GGC A2E GAGGC # GGC GUA GGC AAG A8Exollence_25nt #4GUU GGAGCU GAU GGC GUA GGC AAG A=tt9Exollence_25nt #5GUU GGAGCU GAU GGC GUA GGC AAG A=t*t10Exollence_25nce #6GUU AAG2A_15GAU GAUGC GGAGCU GAU GGC GUA GAC AAG A12Exollence_25nt #8GUU GGAGCU GAU GGC GUA GGC AAG A13Exollence_25nt #9GUU GGAGCU GAU GGC GUA GGC AAG A14Exollence_25nt-#12GUECO GAUGC GGA GCUll22 #13GUU GGA GCU GAU GGC GUA GAC AAG A16Exollence_25nt #16GUU GGAGCU GAU GGC GUA GAC AAG A17Exollence_25nt #28GUU GGA GCU GAU GGC GUA GGC AAG A18Exollence_25nt GAU GAU #GUGGUAA19Exollence_25nt #30EXOLLENCE_25nt #32Exollence_25nt #32Exollence_25nt #32Exollence_25nt #32Exollence_25nt #32Exollence_25nt GGC GUA GUC AAG A23Exollence_25nt #36GUA GGA GCU GAU GGC GUA GUC AAG A24Exollence_25nt #37GUA GUC AAG A25Exollence_25nt #38GUA GCU AAG A26Exollence_25nt #40AGU UGG AGC UGA UGG CGU A27Exollence_25nt #41AGU UGG AGC UGA UGG CGU A28Exollence_25nt #42AGU UGG AGC UGA UGG CGU AGG CAA G29Exollence_25nt #44AGU UGG AGC UGA UGG CGU AGG CAA G

[0263] No. IDAS sequence30unmodified siKRAS G12DCUA CGC CAU CAG CUC CAA C=tt31unmodified siKRAS G12D-2UAC GCC AUC AGC UCC AAC U=tt32unmodified siKRAS G12D-PSCUA CGC CAU CAG CUC CAA C=t*t31unmodified siKRAS-2 GCC AUC AGC UCC AAC U=t*t34Pattern_unmodified siKRAS G12DUCU UGC CUA CGC CAU CAG CUC CAA C=tt35Pattern_OME modified siKRAS G12DUCU UGC CUA CGC CAUCAGCCUC CAA C=tt36Exollence_25nt #3 C=t*t37Exollence_25nt #4UCU UGC CUA CGC CAUCAGCUC CAA C=tt38Exollence_25nt #5UCU UGC CUA CGC CAUCAGCUC CAA C=t*t39Exollence_25nt #6UCU UGC CUA CGC CAUCAGCUC CAA C=t*xollence CUA #25nt CGC CAUCAGCCUC CAA C=tt41Exollence_25nt #8UCU UGC CUA CGC CAU CAGCUC CAA C=tt42Exollence_25nt #9UCU UGC CUA CGC CAU CAGCUC CAA C=t*t43Exollence_25nt-#12UCU UGUA CAGCA CUCCA CUCCA Cllence_25nt Cllence_25nt #13UCU UGUCUA CGC CAU CAG CUC CAA C=t*t45Exollence_25nt #16UCU UGUCUA CGC CAUCAGCUC CAA C=tt46Exollence_25nt #28UCU UGC CUA CGC CAU CUC CAA C47Exollence_25nt #29U CUC UGCUACGC CAU CAGCUC CAA C48Exollence_25nt #30UCU UGC CUA CGC CAUCAGCUC CAA C49Exollence_25nt #32UCU UGUCUA CGC CAU CAG CUC CAA C50Exollence_25nt #33UCU UGUCUA CGC CAU CAGCUC CAA C51Exollence_25nt #34UCU UGUCUA CGC CAUCAGCUC CAA C52Exollence_25nt #36UCU UGACUA CGC CAU CAG CUC CAA C53Exollence_25nt #37UCU UGACUA CGC CAU CAGCUC CAA C54Exollence_25nt #38UCU UGACUA CGC CAUCAGCUC CAA C55Exollence_25nt #40UAC GCC AUC AGC UCC AAC U=tt56Exollence_25nt #41UAC GCC AUC AGC UCC AAC U=t*t57Exollence_25nt #42CUU GCC UAC GCC AUC AGC UCC AAC U=tt58Exollence_25nt #44CUU GCC UAC GCC AUC AGCUCC AAC U=tt

[0264] Example 1-3. Identification of three groups of leading substances with common patterns.

[0265] The leading materials identified in Example 1-2 were analyzed to determine whether they had a common pattern. As a result, they were confirmed to be classified into three groups (hereinafter referred to as Groups 1 to 3) (Tables 5 to 7).

[0266]

[0267]

[0268]

[0269]

[0270]

[0271]

[0272]

[0273] According to this, group 1 was confirmed to have a sense strand of 19 to 25 nt in length and an antisense strand of 21 to 27 nt in length.

[0274]

[0275] In addition, group 2 has a sense strand of 19-25 nt in length and an antisense strand of 21-27 nt in length. The sense strand has no tt overhang, and the chemical modification was performed using the 2'-OMe method. A 2'-OME chemically modified nucleotide was confirmed at the 7th position from the 5' end of the sense strand (the nucleotide of the antisense strand binding to this position was not modified), and the nucleotide at the 9th position from the 5' end of the sense strand was found to be unmodified. In addition, the 18th and 20th nucleotides from the 5' end of the antisense strand were confirmed to be chemically modified with 2'-OMe.

[0276]

[0277] Finally, group 3 has a sense strand of 19–25 nt in length and an antisense strand of 21–27 nt in length. The chemical modification was performed using the 2'-OMe method. A 2'-OME chemically modified nucleotide was identified at the 7th position from the 5' end of the sense strand (the antisense strand nucleotide binding at this position was not modified), and the 9th nucleotide from the 5' end of the sense strand was found to be unmodified. In addition, the 16th, 18th, and 20th nucleotides from the 5' end of the antisense strand were confirmed to be chemically modified with 2'-OMe.

[0278]

[0279] Example 2. Confirmation of the anticancer effect of 29 types of siRNA on pancreatic cancer cell lines.

[0280] The anticancer effects of the 29 siRNAs finally confirmed in Example 1 were confirmed when treated to pancreatic cancer cell lines. Specifically, pancreatic cancer cell lines were cultured in a 100 cm² dish using DMEM medium (10% FBS, 1% penicillin-streptomycin). In addition, colon cancer cell lines were cultured using RPMI medium (10% FBS, 1% penicillin-streptomycin). Both pancreatic cancer and colon cancer cell lines were cultured under conditions of 37°C and 5% CO₂. When the cells reached 70-80% confluency, the cells were washed with PBS, suspending them using trypsin-EDTA, and stopping the cell suspension using the medium. The cell suspension was then centrifuged (1,000 rpm, 5 minutes) to collect the cells. After centrifugation, the collected cells were resuspended in fresh culture medium, counted to determine cell viability, and seeded into 96-well plates at a concentration of 10,000 cells / well. Cells were cultured for 24 hours at 37°C and 5% CO₂ to allow attachment.

[0281] The siRNA to be tested and the control siRNA were each mixed with 75 μL of Opti-MEM medium and siRNA (prepared to a final concentration of 10 nM). In a separate new tube, 75 μL of Opti-MEM medium and 2.5 μL of Lipofectamine RNAiMAX were mixed. The two mixtures were combined and incubated at room temperature for 20 minutes to form siRNA-Lipofectamine complexes, which were then added to each well. Cells were cultured at 37°C and 5% CO₂.

[0282] Every 48 hours, 10 μL of WST-8 reagent was added to each well, and the plates were incubated at 37°C, 5% CO₂ for 1 hour. The absorbance of each well was measured at 450 nm using a microplate reader and corrected for background absorbance at 650 nm. The absorbance values ​​of the treated samples were compared to the control group, and the relative cell proliferation rate was calculated using the following formula.

[0283]

[0284] Calculation formula: (absorbance of treated sample / absorbance of control) × 100

[0285]

[0286] As a result, the cell death effect on the pancreatic cancer cell lines AsPC-1, HPAFⅡ, and PANC-1 of Group 1 was confirmed as shown in Figures 2a to 2c. The results for the same experiment in Group 2 are as shown in Figure 2d. Finally, the results of the experiment in Group 3 are as shown in Figures 2e to 2g. In all Groups 1 to 3, it was confirmed that the proliferation of pancreatic cancer cells was reduced in the groups treated with each lead substance compared to the control group treated with Scrambled siRNA (Bionier).

[0287]

[0288] No.NAMESEQUENCE61siScramble siRNA_SSGAG GUG UAG GGC UGU ACG U=tt62siScramble siRNA_ASACG UAG AGC CCU AGA CCU C=tt

[0289] Example 3. Confirmation of the anticancer effect of 29 types of siRNA on colon cancer cell lines.

[0290] The same experimental method as in Example 2 was applied to analyze the anticancer effects of 29 types of siRNA on colon cancer.

[0291]

[0292] As a result, the cell death effect on the colon cancer cell lines SNU-C2B, LS174T, and LS513 of Group 1 was confirmed as shown in Figures 3a to 3c. The results for the same experiment in Group 2 are as shown in Figure 3d. Finally, the results of the experiment in Group 3 are as shown in Figures 3e to 3g. In all Groups 1 to 3, it was confirmed that colon cancer cell proliferation was reduced in the groups treated with each lead substance compared to the control group treated with Scrambled siRNA.

[0293]

[0294] According to Examples 2 and 3, the 29 leading substances of the present invention were confirmed to exhibit anticancer effects against colon cancer and pancreatic cancer by inhibiting the growth rate of cancer cells.

[0295]

[0296] Example 4. Confirmation of KRAS mRNA expression levels in pancreatic cancer cell lines.

[0297] The effect of the siRNA of the present invention on KRAS mRNA expression was confirmed when it was treated in pancreatic cancer cell lines. Specifically, to determine changes in KRAS mRNA expression within cells due to siRNA treatment, cell number was measured, and cells were seeded into 12-well plates at a concentration of 100,000 cells / well. Cells were cultured for 24 hours at 37°C and 5% CO₂ to allow attachment.

[0298] The siRNAs to be tested were unmodified siKRAS G12D, unmodified siKRAS G12D-2, unmodified siKRAS G12D-PS (Table 3, Nos. 1 to 3), and Pattern_OME modified siKRAS G12D (Table 3, No. 6). In addition, Alnylam_unmodified siKRAS G12 was used as a control. Alnylam_unmodified siKRAS G12D is an siRNA that was identified in 105 species of the present invention, but was not selected as a final 29 lead substance. Alnylam_unmodified siKRAS G12D has the following characteristics: 21nt, no 2'OME modification, no overhang (SS), overhang (AS) tt, and overhang PS modification (-).

[0299]

[0300] No.NAMESEQUENCE63Alnylam_unmodified siKRAS G12D_SSGUU GGA GCU GAU GGC GUA GGC64Alnylam_unmodified siKRAS G12D_ASGCC UAC GCC AUC AGC UCC AAC=tt

[0301] The experimental siRNA and control siRNA were each mixed with 75 μL of Opti-MEM medium and siRNA (prepared to a final concentration of 10 nM). In a separate new tube, 75 μL of Opti-MEM medium and 2.5 μL of Lipofectamine RNAiMAX were mixed. The two mixtures were combined and incubated at room temperature for 20 minutes to form siRNA-Lipofectamine complexes. The siRNA-Lipofectamine complexes were added to each well, and the cells were incubated at 37°C in 5% CO₂.

[0302] After 24 hours of incubation, the existing medium was removed, and 1000 μL of QIAzol Lysis Reagent was added to the well to lyse the cells. The cells were completely homogenized by pipetting, and the resulting solution was transferred to a 1.5 mL tube and left at room temperature for 5 minutes to induce complete lysis. 200 μL of chloroform was added, mixed vigorously for 15 seconds, and the mixture was left at room temperature for 2–3 minutes. The mixture was centrifuged at 12,000 x g for 15 minutes at 4°C, and the supernatant (aqueous layer) was carefully transferred to a new tube. 2-Propanol was added to the supernatant, mixed, and RNA was precipitated, followed by centrifugation at 12,000 x g for 15 minutes at 4°C. The supernatant was removed, and the RNA was washed with 70% ethanol, centrifuged at 12,000 x g for 10 minutes at 4°C, and the supernatant was completely removed. The RNA pellet was dissolved in nuclease-free water, and the concentration and purity of the extracted RNA were measured using a nanodrop.

[0303] cDNA was synthesized using a cDNA Synthesis Kit using extracted RNA (1 μg). Real-time PCR was performed using designed target gene and housekeeping gene primers (GAPDH was used as the housekeeping gene). PCR was performed using a real-time PCR machine under the following conditions: Initial denaturation: 95°C for 2 min, 40 cycles: 95°C for 15 s, 60°C for 1 min. The relative expression level of the target gene was calculated using the ΔΔCt value for analysis of the results.

[0304]

[0305] As a result, the KRAS mRNA expression levels of unmodified siKRAS G12D, unmodified siKRAS G12D-2, unmodified siKRAS G12D-PS (Table 3, Nos. 1 to 3), and Pattern_OME modified siKRAS G12D (Table 3, No. 6) were found to be significantly reduced (Fig. 4a). In contrast, the control group, Alnylam_unmodified siKRAS G12D, was found to have no significant effect on KRAS mRNA expression (Fig. 4b). (Here, 0 nM siRNA refers to 10 nM siScramble siRNA as a control group.)

[0306]

[0307] Example 5. Confirmation of tumor size suppression effect in pancreatic cancer animal model

[0308] When the siRNA of the present invention was administered to an animal model of pancreatic cancer, the tumor size suppression effect was confirmed. Specifically, 1 × 10 KPC-luc cells 7 Prepare 100 μl of PBS, anesthetize 6-8 week old female nude mice, and inject 1×10 subcutaneously into the thigh of the anesthetized mouse. 7 / 100μL of cells were injected subcutaneously between the skin and muscle layers using a 31-gauge syringe. After injection, the mice were allowed to recover in a recovery room. After cell injection, the length (L) and width (W) of the tumors were measured using a digital caliper. The tumor volume was calculated using the following formula: Tumor volume (mm³) = L × W 2 ×0.5.

[0309] When the tumor size reached 100 mm³, the treatment agent was injected, and the mice whose tumor size reached 100 mm³ were randomly divided into two groups, the treatment group and the control group, and the treatment group was injected with the treatment agent through the tail vein three times a week (Monday, Wednesday, Friday). At this time, the treatment group used Exollence_25nt #6 (10 in Table 3) as the experimental group, and Exollence_25nt #1 (Table 10), siScramble (negative control group), and Ctl (control group) were used as the comparative group. At this time, Exollence_25nt #1 was used as a negative control in this experiment because it was a substance that did not show the cell death effect of KRAS G12D in an experiment measuring cell viability by treating cells. Exollence_25nt #1 has the characteristics of 25nt, no 2'OME modification, overhang (SS) tt, overhang (AS) tt, and overhang PS modification (-).

[0310]

[0311] No.NAMESEQUENCE65Exollence_25nt #1_SSGUU GGA GCU GAU GGC GUA GGC AAG A=tt66Exollence_25nt #1_ASUCU UGC CUA CGC CAU CAG CUC CAA C=tt

[0312] The injection dose was 20 μg / 100 μL of siRNA, and the control group was injected with the same volume of sterile PBS at the same interval. When injecting the treatment, the mice were placed on a warming pad, the tail was warmed to dilate the vein, and the treatment was injected into the tail vein using a 29-gauge needle.

[0313] During the injection period, tumor length and width were measured twice weekly, and tumor volume was calculated. The body weight and general health of the mice were regularly monitored to monitor for adverse effects. After the final treatment injection, the tumors were removed and weighed.

[0314]

[0315] As a result, in the case of Exollence_25nt #1, siScramble, and the control group, the tumor size in the pancreatic cancer cell line (KPC-luc) xenograft mouse model was not reduced at all. On the other hand, when treated with Exollence_25nt #6 (10 in Table 3), the tumor size and weight were reduced by approximately 60% (Fig. 5a). At the same time, when treated with Exollence_25nt #6 (10 in Table 3), no change in body weight was observed, similar to other comparative groups including the control group (Fig. 5b), demonstrating the anticancer effect of the lead substance of the present invention in animal experiments.

[0316]

[0317] The foregoing description of the present invention is provided for illustrative purposes only. Those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

[0318] The present invention relates to a pharmaceutical composition for cancer prevention or treatment, comprising siRNA as an active ingredient. It has been shown to exhibit excellent anticancer effects by significantly inhibiting cell proliferation in colon or pancreatic cancer and to reduce the expression level of KRAS mRNA. Furthermore, it can be usefully utilized as an excellent anticancer treatment because it reduces tumor size and weight while maintaining body weight, and thus, its industrial applicability is recognized.

Claims

1. A pharmaceutical composition for preventing or treating cancer, comprising as an active ingredient at least one selected from the group consisting of siRNAs comprising any one base sequence selected from the group consisting of sequence numbers 1 to 29.

2. In paragraph 1, A pharmaceutical composition comprising the following siRNAs, characterized in that the sense strand has a length of 19 to 25 nt and the antisense strand has a length of 21 to 27 nt: 1) siRNA comprising any one base sequence selected from the group consisting of sequence numbers 1 to 5; 2) siRNA containing the base sequence represented by sequence number 7; 3) siRNA comprising any one base sequence selected from the group consisting of sequence numbers 14, 15, and 17; and 4) siRNA comprising any one base sequence selected from the group consisting of sequence numbers 20, 23, and 26 to 28.

3. In paragraph 1, A pharmaceutical composition characterized by a group consisting of siRNA comprising a base sequence represented by SEQ ID NO: 12, siRNA comprising a base sequence represented by SEQ ID NO: 13, siRNA comprising a base sequence represented by SEQ ID NO: 18, siRNA comprising a base sequence represented by SEQ ID NO: 21, siRNA comprising a base sequence represented by SEQ ID NO: 24, and siRNA comprising a base sequence represented by SEQ ID NO: 29, a) the sense strand is 19 to 25 nt in length and the antisense strand is 21 to 27 nt in length; b) There is no tt overhang in the sense strand; c) a 2'-OME chemically modified nucleotide at the 7th position from the 5' end of the sense strand, and the nucleotide of the antisense strand binding to that position does not have a 2'-OME chemical modification; d) the nucleotide at the 9th position from the 5' end of the sense strand does not have a 2'-OME chemical modification; and e) having 2'-OME chemically modified nucleotides at the 18th and 20th positions from the 5' end of the antisense strand.

4. In paragraph 1, A pharmaceutical composition characterized by a group consisting of siRNA comprising a base sequence represented by SEQ ID NO: 6, siRNA comprising a base sequence represented by SEQ ID NO: 8, siRNA comprising a base sequence represented by SEQ ID NO: 9, siRNA comprising a base sequence represented by SEQ ID NO: 10, siRNA comprising a base sequence represented by SEQ ID NO: 11, siRNA comprising a base sequence represented by SEQ ID NO: 16, siRNA comprising a base sequence represented by SEQ ID NO: 19, siRNA comprising a base sequence represented by SEQ ID NO: 22, and siRNA comprising a base sequence represented by SEQ ID NO: 25, a) the sense strand is 19 to 25 nt in length and the antisense strand is 21 to 27 nt in length; b) having a 2'-OME chemically modified nucleotide at the 7th position from the 5' end of the sense strand, and the nucleotide of the antisense strand binding to that position does not have a 2'-OME chemical modification; c) the nucleotide at the 9th position from the 5' end of the sense strand does not have a 2'-OME chemical modification; and d) having 2'-OME chemically modified nucleotides at the 16th, 18th, and 20th positions from the 5' end of the antisense strand.

5. In paragraph 1, A pharmaceutical composition, wherein the cancer is at least one selected from the group consisting of pancreatic cancer, colon cancer, squamous cell carcinoma, lung cancer, lung adenocarcinoma, peritoneal cancer, skin cancer, cutaneous or intraocular melanoma, rectal cancer, anal cancer, esophageal cancer, small intestine cancer, endocrine cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, blood cancer, liver cancer, gastrointestinal cancer, glioblastoma, cervical cancer, ovarian cancer, bladder cancer, hepatoma, breast cancer, colon cancer, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, head and neck cancer, and brain cancer.

6. In paragraph 1, The above composition is a pharmaceutical composition that inhibits the proliferation of cancer cells.

7. In paragraph 1, A pharmaceutical composition, wherein the composition reduces the size and weight of a tumor.

8. In paragraph 1, A pharmaceutical composition wherein the composition inhibits KRAS mRNA expression.

9. A kit for preventing or treating cancer, comprising a composition comprising at least one selected from the group consisting of siRNAs comprising any one base sequence selected from the group consisting of sequence numbers 1 to 29 as an active ingredient, and an instruction manual.

10. A method for treating cancer, comprising administering to a subject in need thereof a pharmaceutically effective amount of a composition comprising at least one siRNA comprising any one base sequence selected from the group consisting of sequence numbers 1 to 29 as an active ingredient.

11. A composition for the prevention or treatment of cancer comprising, as an active ingredient, at least one selected from the group consisting of siRNAs comprising any one base sequence selected from the group consisting of sequence numbers 1 to 29.

12. Use for producing a cancer prevention or treatment agent comprising a composition comprising at least one selected from the group consisting of siRNAs comprising any one base sequence selected from the group consisting of sequence numbers 1 to 29 as an active ingredient.

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