A pharmaceutical composition for the prevention or treatment of cancer containing siRNA as an active ingredient.

Abstract

This invention relates to a pharmaceutical composition for the prevention or treatment of cancer containing siRNA as an active ingredient, which has been shown to exhibit excellent anticancer effects by significantly suppressing the cell proliferation effect of colorectal cancer or pancreatic cancer, and by reducing the expression level of KRAS mRNA. Furthermore, it can be usefully utilized as an excellent anticancer agent because it reduces tumor size and weight while maintaining the individual's body weight.

Description

Technical Field

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

[0002] This application claims priority based on Korean Patent Application Nos. 10-2023-0083349, 10-2023-0083355, and 10-2023-0083360 filed on June 28, 2023, and all the contents disclosed in the specification and drawings of the application are incorporated herein by reference.

[0003] In addition, this application claims priority based on Korean Patent Application No. 10-2024-0084878 filed on June 27, 2024, and all the contents disclosed in the specification and drawings of the application are incorporated herein by reference.

Background Art

[0004] Cancer is a genetic disease that forms tumors through abnormal cell division. When it deteriorates, it becomes a malignant tumor and metastasizes to surrounding tissues, threatening life. The main cause of cancer is the abnormal overexpression of genes related to cell growth and division or mutations in genes that control them.

[0005] There are various types of cancer depending on the organ where it occurs. According to the statistics of the Ministry of Health and Welfare, pancreatic cancer ranks 8th among all cancers in 2017. It is difficult to diagnose at an early stage and is likely to metastasize to other organs, so it is known as a cancer with a poor prognosis. In addition, the 5-year survival rate is about 12%, which is significantly lower compared to gastric cancer, colorectal cancer, and breast cancer, which are 76.5%, 75%, and 93% respectively. Currently used anticancer drugs such as gemcitabine and radiation therapy may cause various side effects in the vascular system, nervous system, blood system, skin, etc. Therefore, there is a need for a new therapeutic agent that minimizes these side effects and has a better therapeutic effect.

[0006] Colorectal cancer is a malignant tumor (adenocarcinoma) that develops in the colon or rectum, and is mostly a cancer that develops in the mucous membrane. Depending on the location where the cancer develops, it is called colon cancer if it develops in the colon, and rectal cancer if it develops in the rectum, and these are collectively called colorectal cancer. According to data from the Central Cancer Registry published in 2017, there were 214,701 cases of cancer in South Korea in 2015, of which 26,790 cases (12.5%) were colorectal cancer, ranking second in the total, with a sex ratio of 1.5:1, indicating that it occurred more frequently in men. In particular, the number of cases was high, with 15,911 cases in men, ranking third among cancers in men, and 10,879 cases in women, ranking third among cancers in women.

[0007] On the other hand, gene therapy is a technology that uses genes to treat or prevent diseases, and is being actively developed to treat intractable or incurable diseases such as cancer. One gene therapy technology for cancer treatment, siRNA (small interfering RNA), suppresses gene expression by forming a RISC complex with 21-23 bp long RNA duplexes and cleaving mRNA. By using siRNA to suppress the expression of genes essential for cancer development, cancer can be treated by inhibiting the growth of cancer cells or by killing cancer cells.

[0008] Nevertheless, there are still very few reported cases of effective siRNA therapies for treating cancer. [Overview of the project] [Problems that the invention aims to solve]

[0009] The object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of cancer, comprising as an active ingredient one or more siRNAs selected from the group consisting of 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 the prevention or treatment of cancer, comprising a composition containing as an active ingredient one or more siRNAs selected from the group consisting of any one nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 29, and instructions for use.

[0011] (blank)

[0012] However, the technical problems that this invention aims to solve are not limited to those mentioned above, and other problems not mentioned should be clearly understood by a person with ordinary skill in the art to which this invention pertains from the description below. [Means for solving the problem]

[0013] The present invention provides a pharmaceutical composition for the prevention or treatment of cancer, comprising as an active ingredient one or more siRNAs selected from the group consisting of any one nucleotide 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 19-25 nt in length and an antisense strand of 21-27 nt in length, but is not limited thereto:

[0015] 1) siRNA containing any one nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-5;

[0016] 2) siRNA containing the nucleotide sequence shown in Sequence ID No. 7;

[0017] 3) siRNA containing any one nucleotide sequence selected from the group consisting of SEQ ID NOs: 14, 15, and 17; and

[0018] 4) An siRNA containing any one nucleotide sequence selected from the group consisting of SEQ ID NOs. 20, 23, and 26-28.

[0019] In one embodiment of the present invention, the group consisting of siRNAs containing the nucleotide sequences represented by SEQ ID NO: 12, siRNAs containing the nucleotide sequences represented by SEQ ID NO: 13, siRNAs containing the nucleotide sequences represented by SEQ ID NO: 18, siRNAs containing the nucleotide sequences represented by SEQ ID NO: 21, siRNAs containing the nucleotide sequences represented by SEQ ID NO: 24, and siRNAs containing the nucleotide sequences represented by SEQ ID NO: 29 can be characterized by, but is not limited to, the following group:

[0020] a) The sense strand has a length of 19 - 25 nt, and the antisense strand has a length of 21 - 27 nt;

[0021] b) The sense strand has no tt overhang;

[0022] c) The sense strand has a chemically modified nucleotide of 2'-OME at the 7th position from the 5' end, and the nucleotide of the antisense strand binding to said position has no chemical modification of 2'-OME;

[0023] d) The nucleotide at the 9th position from the 5' end of the sense strand has no chemical modification of 2'-OME; and

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

[0025] In one embodiment of the present invention, the group consisting of siRNAs containing the nucleotide sequences shown in SEQ ID NO: 6, siRNAs containing the nucleotide sequences shown in SEQ ID NO: 8, siRNAs containing the nucleotide sequences shown in SEQ ID NO: 9, siRNAs containing the nucleotide sequences shown in SEQ ID NO: 10, siRNAs containing the nucleotide sequences shown in SEQ ID NO: 11, siRNAs containing the nucleotide sequences shown in SEQ ID NO: 16, siRNAs containing the nucleotide sequences shown in SEQ ID NO: 19, siRNAs containing the nucleotide sequences shown in SEQ ID NO: 22, and siRNAs containing the nucleotide sequences shown in SEQ ID NO: 25 can be characterized by, but not limited to, the following group:

[0026] a) The sense strand has a length of 19 - 25 nt, and the antisense strand has a length of 21 - 27 nt;

[0027] b) It has a chemically modified nucleotide of 2'-OME at the 7th position from the 5' end of the sense strand, and the nucleotide of the antisense strand binding to said position has no chemical modification of 2'-OME;

[0028] c) The nucleotide at the 9th position from the 5' end of the sense strand has no chemical modification of 2'-OME;

[0029] d) It has chemically modified nucleotides of 2'-OME 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 any one or more selected from the group consisting of pancreatic cancer, colorectal cancer, squamous cell carcinoma, lung cancer, adenocarcinoma of the lung, peritoneal cancer, skin cancer, cutaneous or intraocular melanoma, rectal cancer, anal cancer, esophageal cancer, small intestine cancer, endocrine adenocarcinoma, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, blood cancer, liver cancer, gastrointestinal cancer, glioblastoma, cervical cancer, ovarian cancer, bladder cancer, liver tumor, 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 suppress the proliferation of cancer cells, but is not limited thereto.

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

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

[0034] The present invention provides a kit for the prevention or treatment of cancer, comprising a composition containing as an active ingredient one or more siRNAs selected from the group consisting of any one nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 29, and instructions for use.

[0035] (blank)

[0036] Furthermore, the present invention provides a method for treating cancer, or a method for treating pancreatic cancer or colorectal cancer, which includes the step of administering a pharmaceutically effective amount of a composition containing, as an active ingredient, one or more siRNAs selected from the group consisting of any one nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 29, to an individual in need of it.

[0037] Furthermore, the present invention provides a composition containing, as an active ingredient, one or more siRNAs selected from the group consisting of any one nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 29, for use in the prevention or treatment of cancer, or for use in the prevention or treatment of pancreatic cancer or colorectal cancer.

[0038] Furthermore, the present invention provides a use for manufacturing a composition containing, as an active ingredient, one or more siRNAs selected from the group consisting of any one nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 29, for the prevention or treatment of cancer, or for the prevention or treatment of pancreatic cancer or colorectal cancer.

[0039] Furthermore, the present invention is

[0040] i) siRNAs containing any one nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-5; siRNAs containing the nucleotide sequence shown in SEQ ID NO: 7; siRNAs containing any one nucleotide sequence selected from the group consisting of SEQ ID NOs: 14, 15, and 17; siRNAs containing any one nucleotide sequence selected from the group consisting of SEQ ID NOs: 20, 23, and 26-28;

[0041] ii) A group consisting of siRNA containing the nucleotide sequence shown in SEQ ID NO: 12; siRNA containing the nucleotide sequence shown in SEQ ID NO: 13; siRNA containing the nucleotide sequence shown in SEQ ID NO: 18; siRNA containing the nucleotide sequence shown in SEQ ID NO: 21; siRNA containing the nucleotide sequence shown in SEQ ID NO: 24; and siRNA containing the nucleotide sequence shown in SEQ ID NO: 29; or

[0042] iii) In a group consisting of siRNAs containing the nucleotide sequence shown in SEQ ID NO: 6; siRNAs containing the nucleotide sequence shown in SEQ ID NO: 8; siRNAs containing the nucleotide sequence shown in SEQ ID NO: 9; siRNAs containing the nucleotide sequence shown in SEQ ID NO: 10; siRNAs containing the nucleotide sequence shown in SEQ ID NO: 11; siRNAs containing the nucleotide sequence shown in SEQ ID NO: 16; siRNAs containing the nucleotide sequence shown in SEQ ID NO: 19; siRNAs containing the nucleotide sequence shown in SEQ ID NO: 22; and siRNAs containing the nucleotide sequence shown in SEQ ID NO: 25,

[0043] The present invention provides a method for preventing or treating cancer, or a method for preventing or treating pancreatic cancer or colorectal cancer, comprising the step of administering a pharmaceutically effective amount of a composition containing one or more siRNAs selected from each group as an active ingredient to an individual in need of it.

[0044] Furthermore, the present invention provides a use for manufacturing a composition containing one or more siRNAs selected from each of the aforementioned groups as an active ingredient, for the prevention or treatment of cancer, or for the prevention or treatment of pancreatic cancer or colorectal cancer.

[0045] Furthermore, the present invention provides a use for manufacturing a composition containing one or more siRNAs selected from each of the aforementioned groups as an active ingredient, for the prevention or treatment of cancer, or for the prevention or treatment of pancreatic cancer or colorectal cancer. [Effects of the Invention]

[0046] A pharmaceutical composition for the prevention or treatment of cancer containing siRNA as an active ingredient exhibits excellent anticancer effects by significantly suppressing the cell proliferation effect of colorectal cancer or pancreatic cancer, and can reduce the expression level of KRAS mRNA. Furthermore, it can be usefully utilized as an excellent anticancer agent because it reduces tumor size and weight while maintaining the individual's body weight. [Brief explanation of the drawing]

[0047] [Figure 1a] Figure 1(a) shows a graph that ranks the cell death rates of 105 candidate siRNA substances after analyzing the results of treating three pancreatic cancer cell lines and three colorectal cancer cell lines with these substances.

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

[0049] [Figure 1c] Figure 1(c) shows the cell death effect observed when 105 candidate siRNA substances were treated on three colorectal cancer cell lines.

[0050] [Figure 1d] Figure 1(d) shows the sequences and modifications of the 29 candidate siRNA substances of the present invention.

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

[0052] [Figure 2a] Figures 2(a) to 2(g) show the cell death effects observed when 29 types of siRNA lead substances were treated with three types of pancreatic cancer cell lines. Figures 2(a) to 2(c) show the experimental results for Group 1, Figure 2(d) shows the results for Group 2, and Figures 2(e) to 2(g) show the results for Group 3 lead substances. [Figure 2b] Same as above. [Figure 2c] Same as above. [Figure 2d] Same as above. [Figure 2e] Same as above. [Figure 2f] Same as above. [Figure 2g] Same as above.

[0053] [Figure 3a] Figures 3(a) to 3(g) show the cell death effects observed when 29 types of siRNA lead substances were treated on three types of colorectal cancer cell lines. Figures 3(a) to 3(c) show the experimental results for Group 1, Figure 3(d) shows the results for Group 2, and Figures 3(e) to 3(g) show the results for Group 3 lead substances. [Figure 3b] Same as above. [Figure 3c] Same as above. [Figure 3d] Same as above. [Figure 3e] Same as above. [Figure 3f] Same as above. [Figure 3g] Same as above.

[0054] [Figure 4a] Figure 4(a) shows the KRAS mRNA expression inhibitory activity when some of the 29 siRNA lead substances of the present invention were treated on pancreatic cancer cell lines, and Figure 4(b) shows the experimental results for the control group. [Figure 4b] Same as above.

[0055] [Figure 5a]Figure 5(a) shows the effect of administering some of the 29 siRNA lead substances of the present invention to an animal model of pancreatic cancer on reducing tumor size and tumor weight. (Tumor volume: The highest point on the graph is relative to siScramble, Ctl, Exollence 25nt #1, and Exollence 25nt #6 in order.)

[0056] [Figure 5b] Figure 5(b) shows experimental results regarding changes in animal weight when some of the 29 siRNA lead substances of the present invention were administered to an animal model of pancreatic cancer. (The highest point on the graph is used as the reference point, and the models are in order: Exollence 25nt #1, Exollence 25nt #6, siScramble, and Ctl.) [Best Mode for Carrying Out the Invention]

[0057] The inventors of the present invention created novel siRNAs by making various modifications to existing siRNAs and treated pancreatic cancer or colorectal cancer cell lines with these siRNAs. As a result, they discovered 29 siRNAs that exhibited excellent inhibitory activity against KRAS G12D and thus superior anticancer effects. In particular, the 29 siRNAs of the present invention consist of a sense strand and an antisense strand, and the characteristics such as length, chemical deformation, and sequence deformation observed in the sense strand and antisense strand of a single siRNA differ. Therefore, the inventors confirmed that each siRNA exhibits the therapeutic activity for colorectal cancer and pancreatic cancer confirmed in the present invention by possessing all the characteristics of the sense strand sequence and antisense strand sequence, thus completing the present invention.

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

[0059] Furthermore, the present invention provides a pharmaceutical composition for the prevention or treatment of cancer, comprising as an active ingredient one or more selected from the group consisting of siRNAs containing any one base sequence selected from the group consisting of SEQ ID NOs: 30 to 58.

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

[0061] 1) The unmodified siKRAS G12D nucleotide sequence shown in Sequence ID No. 1;

[0062] 2) The unmodified siKRAS G12D-2 nucleotide sequence shown in Sequence ID No. 2;

[0063] 3) The unmodified siKRAS G12D-PS nucleotide sequence shown in Sequence ID No. 3;

[0064] 4) The unmodified siKRAS G12D-PS-2 nucleotide sequence is shown in Sequence ID No. 4;

[0065] 5) The base sequence shown in sequence number 5 is Pattern_unmodified siKRAS G12D;

[0066] 6) The base sequence displayed in sequence number 6 is Pattern_OME modified siKRAS G12D;

[0067] 7) The base sequence shown in sequence number 7 is Exollence_25nt #3;

[0068] 8) The base sequence shown in sequence number 8 is Exollence_25nt #4;

[0069] 9) The base sequence displayed in sequence number 9 is Exollence_25nt #5;

[0070] 10) The base sequence shown in sequence number 10 is Exollence_25nt #6;

[0071] 11) The base sequence shown in sequence number 11 is Exollence_25nt #7;

[0072] 12) The base sequence shown in sequence number 12 is Exollence_25nt #8;

[0073] 13) The base sequence shown in sequence number 13 is Exollence_25nt #9;

[0074] 14) The base sequence shown in sequence number 14 is Exollence_25nt #12;

[0075] 15) The base sequence shown in sequence number 15 is Exollence_25nt #13;

[0076] 16) The base sequence shown in sequence number 16 is Exollence_25nt #16;

[0077] 17) The base sequence displayed in sequence number 17 is Exollence_25nt #28;

[0078] 18) The base sequence shown in sequence number 18 is Exollence_25nt #29;

[0079] 19) The base sequence shown in sequence number 19 is Exollence_25nt #30;

[0080] 20) The base sequence shown in sequence number 20 is Exollence_25nt #32;

[0081] 21) The base sequence shown in sequence number 21 is Exollence_25nt #33;

[0082] 22) The base sequence shown in sequence number 22 is Exollence_25nt #34;

[0083] 23) The base sequence shown in sequence number 23 is Exollence_25nt #36;

[0084] 24) The base sequence shown in sequence number 24 is Exollence_25nt #37;

[0085] 25) The base sequence displayed in sequence number 25 is Exollence_25nt #38;

[0086] 26) The base sequence shown in sequence number 26 is Exollence_25nt #40;

[0087] 27) The base sequence displayed in sequence number 27 is Exollence_25nt #41;

[0088] 28) The base sequence shown in sequence number 28 is Exollence_25nt #42; and

[0089] 29) Exollence_25nt #44 is the base sequence displayed in sequence number 29.

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

[0091] 1) The unmodified siKRAS G12D nucleotide sequence shown in Sequence ID No. 30;

[0092] 2) The unmodified siKRAS G12D-2 nucleotide sequence shown in Sequence ID No. 31;

[0093] 3) The unmodified siKRAS G12D-PS nucleotide sequence shown in Sequence ID No. 32;

[0094] 4) The unmodified siKRAS G12D-PS-2 nucleotide sequence shown in Sequence ID No. 33;

[0095] 5) The base sequence shown in sequence number 34 is Pattern_unmodified siKRAS G12D;

[0096] 6) The base sequence displayed in sequence number 35 is Pattern_OME modified siKRAS G12D;

[0097] 7) The base sequence shown in sequence number 36 is Exollence_25nt #3;

[0098] 8) The base sequence shown in sequence number 37 is Exollence_25nt #4;

[0099] 9) The base sequence shown in sequence number 38 is Exollence_25nt #5;

[0100] 10) The base sequence shown in sequence number 39 is Exollence_25nt #6;

[0101] 11) The base sequence shown in sequence number 40 is Exollence_25nt #7;

[0102] 12) The base sequence shown in sequence number 41 is Exollence_25nt #8;

[0103] 13) The base sequence shown in sequence number 42 is Exollence_25nt #9;

[0104] 14) The base sequence shown in sequence number 43 is Exollence_25nt #12;

[0105] 15) The base sequence shown in sequence number 44 is Exollence_25nt #13;

[0106] 16) The base sequence shown in sequence number 45 is Exollence_25nt #16;

[0107] 17) The base sequence shown in sequence number 46 is Exollence_25nt #28;

[0108] 18) The base sequence shown in sequence number 47 is Exollence_25nt #29;

[0109] 19) The base sequence shown in sequence number 48 is Exollence_25nt #30;

[0110] 20) The base sequence shown in sequence number 49 is Exollence_25nt #32;

[0111] 21) The base sequence shown in sequence number 50 is Exollence_25nt #33;

[0112] 22) The base sequence shown in sequence number 51 is Exollence_25nt #34;

[0113] 23) The base sequence shown in sequence number 52 is Exollence_25nt #36;

[0114] 24) The base sequence shown in sequence number 53 is Exollence_25nt #37;

[0115] 25) The base sequence shown in sequence number 54 is Exollence_25nt #38;

[0116] 26) The base sequence shown in sequence number 55 is Exollence_25nt #40;

[0117] 27) The base sequence shown in sequence number 56 is Exollence_25nt #41;

[0118] 28) The base sequence shown in sequence number 57 is Exollence_25nt #42; and

[0119] 29) Exollence_25nt #44 is the base sequence shown in sequence number 58.

[0120] In this invention, 29 types of siRNA were created by applying modifications such as chemical modification, sequence length adjustment, and sequence shift to KRAS G12D siRNA as a base.

[0121] In this case, Sequence IDs 1 to 29 of the present invention are the sense strand sequences of each siRNA. Sequence IDs 30 to 58 are the antisense strand sequences of the siRNA. Therefore, the present invention provides a pharmaceutical composition for the prevention or treatment of cancer, comprising, as an active ingredient, one or more siRNAs selected from the group consisting of a sense strand, which is a base sequence represented by any one base sequence selected from the group consisting of Sequence IDs 1 to 29, and an antisense strand, which is a base sequence selected from the group consisting of Sequence IDs 30 to 58, which includes a complementary sequence to the sense strand.

[0122] In this invention, "sense strand" refers to a polynucleotide having the same nucleic acid sequence as the target nucleic acid, and which is identical in whole or in part to mRNA, a non-mRNA RNA sequence, or a coding or non-coding DNA sequence. "Antisense strand" refers to a polynucleotide that is substantially or 100% complementary to the target nucleic acid of interest, and is a general term for RNA having 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 mRNA (messenger RNA), a non-mRNA RNA sequence (microRNA, piwiRNA, tRNA, rRNA, and hnRNA), or a coding or non-coding DNA sequence.

[0123] Therefore, the siRNA defined as a sense strand in this invention can be understood as forming a double helix with the corresponding siRNA defined as an antisense strand, and since the siRNA is a double helix, it can be understood as having both sense strands and antisense strands. For example, in this 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 in relation to its antisense strand being the base sequence represented by SEQ ID NO: 30.

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

[0125] 1) Unmodified siKRAS G12D including the sense strand sequence shown in SEQ ID NO: 1 and the antisense strand sequence shown in SEQ ID NO: 30;

[0126] 2) Unmodified siKRAS G12D-2 including the sense strand sequence shown in SEQ ID NO: 2 and the antisense strand sequence shown in SEQ ID NO: 31;

[0127] 3) Unmodified siKRAS G12D-PS including the sense strand nucleotide sequence shown in SEQ ID NO: 3 and the antisense strand nucleotide sequence shown in SEQ ID NO: 32;

[0128] 4) Unmodified siKRAS G12D-PS-2 including the sense strand sequence shown in SEQ ID NO: 4 and the antisense strand sequence shown in SEQ ID NO: 33;

[0129] 5) Pattern_unmodified siKRAS G12D, which includes the sense strand nucleotide sequence shown in SEQ ID NO: 5 and the antisense strand nucleotide sequence shown in SEQ ID NO: 34;

[0130] 6) Pattern_OME modified siKRAS G12D, including the sense strand nucleotide sequence shown in SEQ ID NO: 6 and the antisense strand nucleotide sequence shown in SEQ ID NO: 35;

[0131] 7) Exollence_25nt #3, which includes the sense strand nucleotide sequence shown in SEQ ID NO: 7 and the antisense strand nucleotide sequence shown in SEQ ID NO: 36;

[0132] 8) Exollence_25nt #4, which includes the sense strand nucleotide sequence shown in SEQ ID NO: 8 and the antisense strand nucleotide sequence shown in SEQ ID NO: 37;

[0133] 9) Exollence_25nt #5, which includes the sense strand nucleotide sequence shown in SEQ ID NO: 9 and the antisense strand nucleotide sequence shown in SEQ ID NO: 38;

[0134] 10) Exollence_25nt #6, which includes the sense strand nucleotide sequence shown in SEQ ID NO: 10 and the antisense strand nucleotide sequence shown in SEQ ID NO: 39;

[0135] 11) Exollence_25nt #7, which includes the sense strand nucleotide sequence shown in SEQ ID NO: 11 and the antisense strand nucleotide sequence shown in SEQ ID NO: 40;

[0136] 12) Exollence_25nt #8, which includes the sense strand nucleotide sequence shown in SEQ ID NO: 12 and the antisense strand nucleotide sequence shown in SEQ ID NO: 41;

[0137] 13) Exollence_25nt #9, which includes the sense strand nucleotide sequence shown in SEQ ID NO: 13 and the antisense strand nucleotide sequence shown in SEQ ID NO: 42;

[0138] 14) Exollence_25nt #12, which includes the sense strand nucleotide sequence shown in SEQ ID NO: 14 and the antisense strand nucleotide sequence shown in SEQ ID NO: 43;

[0139] 15) Exollence_25nt #13, which includes the sense strand nucleotide sequence shown in SEQ ID NO: 15 and the antisense strand nucleotide sequence shown in SEQ ID NO: 44;

[0140] 16) Exollence_25nt #16, which includes the sense strand nucleotide sequence shown in SEQ ID NO: 16 and the antisense strand nucleotide sequence shown in SEQ ID NO: 45;

[0141] 17) Exollence_25nt #28, which includes the sense strand nucleotide sequence shown in SEQ ID NO: 17 and the antisense strand nucleotide sequence shown in SEQ ID NO: 46;

[0142] 18) Exollence_25nt #29, which includes the sense strand nucleotide sequence shown in SEQ ID NO: 18 and the antisense strand nucleotide sequence shown in SEQ ID NO: 47;

[0143] 19) Exollence_25nt #30, which includes the sense strand nucleotide sequence shown in SEQ ID NO: 19 and the antisense strand nucleotide sequence shown in SEQ ID NO: 48;

[0144] 20) Exollence_25nt #32, which includes the sense strand nucleotide sequence shown in SEQ ID NO: 20 and the antisense strand nucleotide sequence shown in SEQ ID NO: 49;

[0145] 21) Exollence_25nt #33, which includes the sense strand nucleotide sequence shown in SEQ ID NO: 21 and the antisense strand nucleotide sequence shown in SEQ ID NO: 50;

[0146] 22) Exollence_25nt #34, which includes the sense strand nucleotide sequence shown in SEQ ID NO: 22 and the antisense strand nucleotide sequence shown in SEQ ID NO: 51;

[0147] 23) Exollence_25nt #36, which includes the sense strand nucleotide sequence shown in SEQ ID NO: 23 and the antisense strand nucleotide sequence shown in SEQ ID NO: 52;

[0148] 24) Exollence_25nt #37, which includes the sense strand nucleotide sequence shown in SEQ ID NO: 24 and the antisense strand nucleotide sequence shown in SEQ ID NO: 53;

[0149] 25) Exollence_25nt #38, which includes the sense strand nucleotide sequence shown in SEQ ID NO: 25 and the antisense strand nucleotide sequence shown in SEQ ID NO: 54;

[0150] 26) Exollence_25nt #40, which includes the sense strand nucleotide sequence shown in SEQ ID NO: 26 and the antisense strand nucleotide sequence shown in SEQ ID NO: 55;

[0151] 27) Exollence_25nt #41; containing the sense strand nucleotide sequence shown in SEQ ID NO: 27 and the antisense strand nucleotide sequence shown in SEQ ID NO: 56;

[0152] 28) Exollence_25nt #42; and the sense strand nucleotide sequence shown in SEQ ID NO: 28 and the antisense strand nucleotide sequence shown in SEQ ID NO: 57

[0153] 29) Exollence_25nt #44, which includes the sense strand nucleotide sequence shown in SEQ ID NO: 29 and the antisense strand nucleotide sequence shown in SEQ ID NO: 58.

[0154] Technologies that suppress gene expression are important tools in the development of therapeutic agents and target validation for the treatment of diseases. Among these technologies, RNA interference (RNAi) has been shown to act on sequence-specific mRNA in various types of mammalian cells since its role was discovered. When a long-chain RNA double helix is ​​transmitted to a cell, the transmitted RNA double helix is ​​converted into short interfering RNA (siRNA) consisting of 21-23 base pairs (bp) by an endonuclease called Dicer. siRNA then 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] In other words, in the present invention, "siRNA (small interfering RNA)" is a small RNA fragment of 18-23 nucleotides in size produced when double-stranded RNA is cleaved by the Dicer enzyme, and can be used to specifically bind to mRNA having a complementary sequence and suppress the expression of that protein or mRNA.

[0156] Furthermore, the 29 types of siRNAs of the present invention can be transmitted to an individual in a form that includes a nucleic acid carrier.

[0157] In this invention, the term "nucleic acid delivery system" refers to a system designed to enhance the efficiency of delivery into the body. It has the advantages of not only being highly stable in the body but also having a simple manufacturing process as a pharmaceutical product.

[0158] The nucleic acid carrier may include, but is not limited to, viral vectors, nonviral vectors, liposomes, cationic polymers, micelles, emulsions, and solid lipid nanoparticles.

[0159] In other words, the present invention can provide the siRNA nucleic acid molecule in a form contained in an expression vector. The expression vector preferably encodes the miRNA of the present invention in an expressible form and can be introduced into a host by methods commonly used in the industry, in which case the expression vector can be used without limitation as long as it is for transmitting siRNA. Furthermore, the expression vector of the present invention preferably additionally contains a sorting marker to facilitate the sorting of transformed cells, but is not limited thereto. The present invention can provide a transformant obtained by introducing the expression vector into a host cell, but is not limited thereto.

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

[0161] When siRNA is included in a composition in the form of a complex with a nucleic acid transduction medium as described above, it efficiently promotes the delivery of siRNA to target cells, and even at relatively low concentrations, it can be delivered to target cells and exhibit high target gene expression regulatory function. Furthermore, it has the advantage of preventing nonspecific delivery of siRNA to organs and cells other than the target.

[0162] In the present invention, the siRNA may be chemically modified. Such chemical modification of the siRNA may be for the purpose of improving in vivo stability, conferring resistance to nucleases, and reducing nonspecific immune responses, but is not limited to these purposes.

[0163] In this invention, all 29 types of siRNA were confirmed to exhibit anticancer activity against colorectal cancer or pancreatic cancer. However, analysis of the commonalities in the sequences of the 29 types of siRNA revealed that they could be classified into three groups.

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

[0165] 1) siRNA containing any one nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-5;

[0166] 2) siRNA containing the nucleotide sequence shown in Sequence ID No. 7;

[0167] 3) siRNA containing any one nucleotide sequence selected from the group consisting of SEQ ID NOs: 14, 15, and 17; and

[0168] 4) An siRNA containing any one nucleotide sequence selected from the group consisting of SEQ ID NOs. 20, 23, and 26-28.

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

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

[0171] b) The sense chain does not have a tt overhang;

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

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

[0174] e) Having a chemically deformed nucleotide (nucleotide) of 2'-OME at the 18th and 20th positions from the 5' end of the antisense strand.

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

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

[0177] b) The sense strand has a 2'-OME chemically deformed nucleotide at the 7th position from the 5' end, and the nucleotide of the antisense strand bound to that position does not have the 2'-OME chemical deformation;

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

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

[0180] In this invention, "overhang" may refer to an unpaired nucleotide at the end of a DNA or RNA molecule. Such unpaired nucleotides can be present on any strand and can create 3' or 5' overhangs. Longer overhangs may be named aggregated or sticky ends, but are not limited to these terms. The 29 siRNAs of this invention differ in the presence or absence of overhangs on part of the sense strand or antisense strand, and thus can be classified into three groups. Therefore, in this invention, it can be understood that the therapeutic effect of siRNA against colorectal cancer or pancreatic cancer is determined by the presence or absence of overhangs, the presence or absence of sense or antisense strands, and the type of overhang.

[0181] In this invention, "-OME" may mean a methoxy group, which can be linked to a nucleotide and exhibit the effect of chemical modification. Chemical modification can exhibit different chemical properties depending on the type and order of nucleotides, and furthermore, can exhibit different effects on the therapeutic activity of colorectal cancer or pancreatic cancer. Therefore, it is self-evident that the 29 types of siRNA in this invention exhibit specific therapeutic activity for colorectal cancer or pancreatic cancer by applying chemical modification to a specific position and a specific type of nucleotide in either the sense strand or the antisense strand. The specialness and specificity of such modification can be understood by applying the same principle to sequence modification applied not only to chemical modification but also to sequence modification applied to the nucleotide sequence of the siRNA. In conclusion, the 29 types of siRNA in this invention, by applying sequence length adjustment, chemical modification, and sequence modification to one or more of either the sense strand or the antisense strand, are among the siRNAs that exhibit therapeutic effects on colorectal cancer or pancreatic cancer among the remarkably large number of siRNAs.

[0182] In this invention, it was confirmed that KRAS mRNA expression is suppressed when treated with 29 types of siRNA, particularly each siRNA whose base sequence is represented by SEQ ID NOs: 1-3 or SEQ ID NO: 6. Therefore, the siRNA of this invention can be applied to cancers that can be treated by suppressing KRAS mRNA expression. In one embodiment of this invention, the cancer may be one or more selected from the group consisting of pancreatic cancer, colorectal cancer, squamous cell carcinoma, lung cancer, lung adenocarcinoma, peritoneal cancer, skin cancer, melanoma of the skin or eye, rectal cancer, anal cancer, esophageal cancer, small intestine cancer, endocrine adenocarcinoma, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, hematological cancer, liver cancer, gastrointestinal cancer, glioblastoma, cervical cancer, ovarian cancer, bladder cancer, liver tumor, 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.

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

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

[0185] KRAS is one of the oncogenes that frequently mutate in human tumors. While normal KRAS performs essential functions in signal transduction in normal tissues, mutations in the KRAS gene are involved in the development of various cancers, and therefore KRAS gene mutations are known as an important therapeutic target.

[0186] KRAS gene mutations usually occur at codons, with mutations most common at codons 12, 13, and 61. Codon 12 is the most frequent site of mutations, and it is known that G12C accounts for approximately 40% of all KRAS gene mutations.

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

[0188] In this invention, it was confirmed that the siRNA produced in this invention exhibits a significant inhibitory effect on KRAS G12D expression among KRAS enzymes. Therefore, it has been demonstrated that the siRNA of this invention exhibits excellent preventive or therapeutic effects against pancreatic cancer and colorectal cancer, which are known to be highly associated with KRAS G12D, by suppressing KRAS G12D.

[0189] The patients to whom the composition of the present invention is administered may be mammals, preferably humans, monkeys, or rodents (mice, rats), and in particular, all mammals having pancreatic cancer or colorectal cancer, such as humans.

[0190] To increase the therapeutic effect of pancreatic cancer or colorectal cancer, the concentration of siRNA in the composition, or the concentration used or processed, 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 compositions of the present invention, including siRNAs containing the nucleotide sequences shown in SEQ ID NOs: 1 to 29, can achieve synergistic effects through combination therapy.

[0192] The compositions of the present invention may be prepared by adding one or more pharmaceutically acceptable carriers in addition to the above-mentioned active ingredients. The pharmaceutically acceptable carriers must be compatible with the active ingredients of the present invention and can be saline solution, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components in combination. Other common additives such as antioxidants, buffers, and bacteriostatic agents may be added as needed. Furthermore, diluents, dispersants, surfactants, binders, and lubricants may be added to formulate the compositions into injectable dosage forms such as aqueous solutions, suspensions, and emulsions. In addition, the compositions may be preferably formulated according to each disease or component by appropriate methods in the art or by methods disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA).

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

[0194] The compositions of the present invention can be administered orally or parenterally. The administration routes of the compositions according to the present invention are not limited to these, but include, for example, oral, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intestinal, sublingual, or topical administration. The dosage of the compositions according to the present invention varies widely depending on the patient's weight, age, sex, health status, diet, administration time, method, excretion rate, or disease severity, and can be easily determined by a typical expert in the art. Furthermore, the compositions of the present invention can be formulated into appropriate dosage forms 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. By combining the siRNA of the present invention with chemotherapy in this way, the sensitivity to chemotherapeutic agents can be enhanced, thereby maximizing therapeutic efficacy and reducing side effects.

[0196] Furthermore, the terms "percent identity," "sequence identity," "percent similarity," "sequence similarity," and "percent sequence identity" used herein in relation to amino acid sequences and / or nucleic acid sequences may refer to, but are not limited to, a measure that maximizes the similarity between aligned amino acid residues or nucleotides and is determined by comparing the degree of similarity between two sequences based on sequence alignment, which 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 sequence alignment.

[0197] A portion 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 in which the same nucleic acid base or amino acid residue appears in both sequences, calculating the number of matching positions, dividing the number of matching positions by the total number of positions using a comparison window, and multiplying the result by 100 to calculate the sequence identity percentage.

[0198] In relation to two or more nucleic acid or polypeptide sequences, the terms “identical” or percentage “identity” refer to two or more sequences or subsequences that are identical or have an identical percentage of embodied amino acid residues or nucleotides (i.e., approximately 60% identity with respect to the embodied region, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity) when measured using the BLAST or BLAST 2.0 sequence comparison algorithm with the default parameters described below, or by manual alignment and visual inspection (see, for example, the NCBI website http: / / www.ncbi.nlm.nih.gov / BLAST / ). Subsequently, these sequences will be referred to as “substantially identical.”

[0199] Furthermore, this definition refers to, or may be applied to, the completion of a test sequence. The definition also includes sequences with substitutions, as well as sequences with deletions and / or additions. Preferred algorithms, as described below, can account for gaps, etc. Preferably, identity exists over a region of at least about 25 amino acids or nucleotides in length, or more preferably over a region of 50 to 100 amino acids or nucleotides in length.

[0200] The "position" of an amino acid or nucleotide base is represented by a number that sequentially identifies each amino acid (or nucleotide base) in the reference sequence based on its position relative to the N-terminus (or 5'-terminus). Due to deletions, insertions, cleavages, and fusions that must be considered when determining optimal alignment, the number of amino acid residues in a test sequence 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 mutant has a deletion in an aligned reference sequence, the mutant will not have an amino acid at the location in the reference sequence corresponding to the deletion site. If there is an insertion in an aligned reference sequence, the insertion will not correspond to an amino acid position numbered in the reference sequence. In the case of cleavage or fusion, there may be an amino acid elongation in one of the reference or aligned sequences that does not correspond to any particular amino acid in the corresponding sequence.

[0201] When used in connection with the numbering of a given amino acid or polynucleotide sequence, the terms “numbered with respect to” or “corresponding to” refer to the numbering of residues in a reference sequence that is embodied when the given amino acid or polynucleotide sequence is compared to a reference sequence.

[0202] In this specification, "active ingredient" means a substance or group of substances whose pharmacological action, directly or indirectly, is expected to contribute to the efficacy and effects of the formulation, and which exhibits the desired activity on its own or can exhibit the desired activity together with a carrier or the like that which is inactive on its own. The component may be present in an amount of 1% by weight or more relative to the dry weight of the entire composition, for example, 10% to 100% by weight, 20% to 100% by weight, 30% to 100% by weight, 40% to 100% by weight, 50% to 100% by weight, 60% to 100% by weight, 70% to 100% by weight, 80% to 100% by weight, 90% to 100% by weight, 10% to 80% by weight, 20% to 80% by weight, 30% to 80% by weight, 40% to 80% by weight, 50% to 80% by weight, 60% to 80% by weight, or 70% to 80% by weight, but is not limited thereto.

[0203] As described above, in the present invention, when treated with the 29 types of siRNAs of the present invention, it was confirmed that they exhibited excellent therapeutic activity for colorectal cancer or pancreatic cancer. Therefore, the composition of the present invention can contain one or more of the 29 types of siRNAs and nucleic acid transduction mediators 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 excipient may be one or more selected from the group consisting of, for example, diluents, binders, disintegrants, lubricants, adsorbents, humectants, film-coating substances, and controlled-release additives.

[0205] The pharmaceutical compositions according to the present invention can be used in the form of external preparations such as powders, granules, sustained-release granules, enteric-coated granules, liquids, eye drops, elsilics, emulsions, suspensions, alcoholic preparations, lozenges, aromatic preparations, limonades, tablets, sustained-release tablets, enteric-coated tablets, sublingual tablets, hard capsules, soft capsules, sustained-release capsules, enteric-coated capsules, pills, tinctures, softened extracts, dried extracts, liquid extracts, injections, capsules, perfusion solutions, ointments, lotions, pastes, sprays, inhalants, patches, sterile injection solutions, or aerosols, respectively, by conventional methods. The external preparations may have dosage forms such as creams, gels, patches, sprays, ointments, lotions, liniments, pastes, or poultices.

[0206] Examples of carriers, excipients, and diluents that may be included in the pharmaceutical composition according to the present invention include lactose, dextrose, straw, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0207] When compounding, the drug is typically prepared using fillers, bulking agents, binders, wetting agents, disintegrants, surfactants, and other diluents or excipients.

[0208] Excipients for tablets, powders, granules, capsules, pills, and lozenges according to the present invention include: corn starch, potato starch, wheat starch, lactose, sucrose, glucose, fructose, D-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, monocalcium 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, Primozel, etc.; gelatin, gum arabic, ethanol, agar powder, cellulose phthalate acetate, carboxymethylcellulose, calcium carboxymethylcellulose, glucose, purified water, sodium casein Binders such as ammonium bicarbonate, glycerin, stearic acid, sodium carboxymethylcellulose, sodium methylcellulose, methylcellulose, microcrystalline cellulose, dextrin, hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, refined shellac, starch paste, hydroxypropylcellulose, hydroxypropyl methylcellulose, polyvinyl alcohol, and polyvinylpyrrolidone may be used; disintegrants such as hydroxypropyl methylcellulose, corn starch, agar powder, methylcellulose, bentonite, hydroxypropyl starch, sodium carboxymethylcellulose, sodium alginate, calcium carboxymethylcellulose, calcium citrate, sodium lauryl sulfate, anhydrous silicic acid, 1-hydroxypropylcellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde-treated casein and gelatin, alginic acid, amylose, guar gum, sodium bicarbonate, polyvinylpyrrolidone, calcium phosphate, gelled starch, acacia gum, amylopectin, pectin, sodium polyphosphate, ethylcellulose, sucrose, magnesium aluminum silicate, D-sorbitol solution, and hard anhydrous silicic acid;Lubricants such as calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopodium, 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 ethers, starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine, and hard anhydrous silicic acid may be used.

[0209] Possible additives for the liquid formulation according to the present invention include water, dilute hydrochloric acid, dilute sulfuric acid, sodium citrate, sucrose monostearate, polyoxyethylene sorbitol fatty acid esters (twin esters), polyoxyethylene monoalkyl ethers, lanolin ethers, lanolin esters, acetic acid, hydrochloric acid, aqueous ammonia, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethylcellulose, sodium carboxymethylcellulose, and the like.

[0210] The syrup according to the present invention may contain a solution of sucrose, other sugars, or sweeteners, and may, if necessary, contain fragrances, colorants, preservatives, stabilizers, suspending agents, emulsifiers, viscosity modifiers, etc.

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

[0212] The suspending agent according to the present invention may use suspending agents such as acacia, tragacantha, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropyl methylcellulose, HPMC1828, HPMC2906, HPMC2910, and, if necessary, surfactants, preservatives, stabilizers, colorants, and fragrances may be used.

[0213] The injectable preparations according to the present invention include solvents such as distilled water for injection, 0.9% sodium chloride injection, Ringer's injection, dextrose injection, dextrose + sodium chloride injection, PEG (PEG), Ringer's lactate injection, ethanol, propylene glycol, non-volatile oils - sesame oil, cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate, and benzene benzoate; solubilizers such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidinedione, propylene glycol, twins, nijonthinamide, hexamine, and dimethylacetamide; weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), and organic compounds. It may contain buffering agents such as compounds, proteins, albumin, peptones, and gums; isotonic agents such as sodium chloride; stabilizers such as sodium sulfite (NaHSO3), carbon dioxide gas, sodium metabisulfite (Na2S2O5), sodium sulfite (Na2SO3), nitrogen gas (N2), and ethylenediaminetetraacetic acid; sulfurizing agents such as 0.1% sodium bisulfide, sodium formaldehyde sulfoxylate, thiourea, disodium ethylenediaminetetraacetate, and sodium acetone bisulfite; analgesics such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; and suspending agents such as sodium CMC, sodium alginate, Twin 80, and aluminum monostearate.

[0214] The suppositories according to the present invention contain cocoa butter, lanolin, vitepsol, polyethylene glycol, glycerol gelatin, methylcellulose, carboxymethylcellulose, a mixture of stearic acid and oleic acid, Subanal, cottonseed oil, peanut oil, coconut oil, cocoa butter + cholesterol, lecithin, lanette wax, glycerol monostearate, twin or span, Imhausen, monolen (propylene glycol monostearate), glycerin, Adeps solidus, Buytyrum Tego-G, and Cebes Pharma 16. 16) Hexalide Base 95, Cotomar, Hydrocoat SP, S-70-XXA, S-70-XX75 (S-70-XX95), Hydrocoat 25, Hydrocoat 711, Idropostal, Massa Estralium Bases such as estrarium (A, AS, B, C, D, E, I, T), Masa-MF, Maspol, Maspol-15, Neospostal-en, Paramount-B, Sposyl (OSI, OSIX, A, B, C, D, H, L), Suppository base type IV (AB, B, A, BC, BBG, E, BGF, C, D, 299), Spostal (N, Es), Wecobi (W, R, S, M, Fs), and Tezestr triglyceride base (TG-95, MA, 57) may be used.

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

[0216] Liquid formulations for oral administration include suspensions, liquid preparations, emulsions, and syrups. Besides commonly used simple diluents such as water and liquid paraffin, various excipients may be included, such as humectants, sweeteners, fragrances, and preservatives. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspension solvents may 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, and the effective dose level may be determined by factors including the type and severity of the patient’s disease, the activity of the drug, the patient’s sensitivity to the drug, the time of administration, the route of administration and elimination ratio, the duration of treatment, drugs used concurrently, and other factors well known in the medical field.

[0218] The pharmaceutical compositions according to the present invention may be administered as individual therapeutic agents, in combination with other therapeutic agents, sequentially or simultaneously with conventional therapeutic agents, or administered as a single or multiple agent. Considering all of the above factors, it is important to administer an amount that provides the greatest effect with the minimum amount without side effects, which can be easily determined by a person of the skill in the art to which the present invention belongs.

[0219] The pharmaceutical compositions of the present invention can be administered to individuals by various routes. All possible methods of administration are predictable, but for example, they may be administered orally, by subcutaneous injection, intraperitoneal injection, intravenous injection, intramuscular injection, intradural injection, sublingual injection, buccal injection, rectal insertion, vaginal insertion, ocular injection, ear injection, nasal injection, inhalation, spraying through the mouth or nose, skin administration, transdermal administration, etc.

[0220] The pharmaceutical composition of the present invention is determined by the type of drug that is the active ingredient, along with various related factors such as the disease being treated, the route of administration, the patient's age, sex, weight, and the severity of the disease. Specifically, the effective amount of the composition according to the present invention can vary depending on the patient's age, sex, and weight, and is generally 0.001 to 150 mg per kg of body weight, preferably 0.01 to 100 mg, administered daily or every other day, or divided into 1 to 3 doses per day. However, the dosage can be increased or decreased depending on the route of administration, the severity of the disease, sex, weight, age, etc., so the aforementioned dosage does not limit the scope of the present invention in any way.

[0221] In this invention, "individual" means a subject requiring treatment for a disease, and more specifically, it 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 an individual by any appropriate method. In the present invention, “prevention” means all actions that suppress or delay the onset of the disease of interest; “treatment” means all actions by which the disease of interest and the metabolic abnormalities caused thereby are improved or favorably altered by the administration of a pharmaceutical composition according to the present invention; and “improvement” means all actions by which the parameters associated with the disease of interest, such as the severity of symptoms, are reduced by the administration of a composition according to the present invention.

[0223] (blank)

[0224] The present invention provides a kit for the prevention or treatment of cancer, comprising a composition containing as an active ingredient one or more siRNAs selected from the group consisting of any one nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 29, and instructions for use.

[0225] The "kit" of the present invention may include other components, devices, and substances that are typically necessary for treating cancer, in addition to the aforementioned substances. Furthermore, all components included in the kit can be used one or more times without limitation, there is no restriction on the order in which each substance is applied, and the application of each substance may proceed simultaneously or last.

[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 components, or to store and secure them. The material of the container may take the form of, for example, a bottle, tub, sachet, envelope, tube, ampoule, etc., and these may be formed in part or all from plastic, glass, paper, foil, wax, etc. The container may be fitted with a stopper that is initially part of the container or can be attached to the container by mechanical, adhesive or other means, and may be fitted with a stopper that allows access to the contents with an injection needle. The kit may include an outer package, which may include instructions on how to use the components, but is not limited thereto. <Modes for carrying out the invention>

[0227] The following are preferred embodiments to aid in understanding the present invention. However, the following embodiments are provided only to facilitate understanding of the present invention and do not limit the scope of the present invention.

[0228] (blank)

[0229] <Examples>

[0230] (blank) [Examples]

[0231] Example 1. Selection of candidate siRNA substances for cancer treatment and confirmation of lead substances.

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

[0233] To find RNA agonists with enhanced gene expression suppression effects, we secured siRNAs containing RNA sequences based on KRAS G12D siRNA, with chemical modifications introduced into the aforementioned sequence. The unmodified reference RNA sequence is as follows:

[0234] (blank)

[0235] [Table 1]

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

[0237] (blank)

[0238] [Table 2]

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

[0240] b. The sense chain has a form without a tt overhang.

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

[0242] d. PS deformation occurs at the first t from the 5' end in the tt overhang present in the antisense chain.

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

[0244] f-1. A nucleotide chemically deformed at the 7th position from the 5' end of the sense strand (the nucleotide of the antisense strand bound at this position remains deformed).

[0245] f-2. The nucleotide at the 9th position from the 5' end of the sense strand remains unchanged.

[0246] f-3. The 16th, 18th, and 20th nucleotides from the 5' end of the antisense chain are capable of 2'-OMe chemical deformation.

[0247] f-3-1. Does it deform?

[0248] f-3-2. Numbers 18 and 20 are all deformed.

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

[0250] *f-1 and f-2 are essential conditions, and f-3 results in three possible cases depending on the number of variations.

[0251] g-1. Replace the 20th nucleotide with A at the 5' end of the sense strand, and replace the 6th nucleotide with U at the 5' end of the antisense strand.

[0252] g-2. Replace the 20th nucleotide with U at the 5' end of the sense strand, and replace the 6th nucleotide with A at the 5' end of the antisense strand.

[0253] *This refers to cases g-1 or g-2, and does not necessarily include both conditions.

[0254] (blank)

[0255] As a result, 105 candidate siRNAs were ultimately selected.

[0256] (blank)

[0257] Examples 1-2. Confirmation of siRNA lead substances for cancer treatment.

[0258] To identify the leading substance among the 105 siRNAs selected in Example 1-1, the survival rate of cancer cells treated with each siRNA was examined, and the cell death effect was analyzed. Specifically, the cell survival rates obtained after treating three pancreatic cancer cell lines and three colorectal cancer cell lines with the 105 siRNAs were compiled and shown in a single graph (a graph analyzing a total of 18 values) in order of ranking. In this case, n=3 was obtained for each cell line.

[0259] (blank)

[0260] As a result, the findings were confirmed as shown in Figures 1(a) to 1(e), and the 29 types of siRNA listed in Tables 3 and 4 below were identified (Figures 1(d) and 1(e)). (In this case, PS modification means that G / C was changed to A / U, and is shown in italics (A: blue / U: green). Also, 2'OME modification is shown underlined (red).)

[0261] (blank)

[0262] [Table 3-1] [Table 3-2]

[0263] [Table 4-1] [Table 4-2]

[0264] Examples 1-3. Confirmation of three groups of lead materials having a common pattern.

[0265] It was analyzed whether the lead substances confirmed in Examples 1-2 have a common pattern. As a result, it was confirmed that they are classified into three groups (hereinafter, Groups 1 to 3) (Tables 5 to 7).

[0266] (Blank)

[0267]

Table 5

[0268] (Blank)

[0269]

Table 6

[0270] (Blank)

[0271]

Table 7

[0272] (Blank)

[0273] According to this, it was confirmed that in Group 1, the sense strand has a length of 19 to 25 nt, and the antisense strand has a length of 21 to 27 nt.

[0274] (Blank)

[0275] In group 2, the sense strand was 19–25 nt long, and the antisense strand was 21–27 nt long. The sense strand had no tt overhang, and chemical deformation was performed using the 2'-OMe method. A nucleotide that was chemically deformed by 2'-OMe was identified at the 7th position from the 5' end of the sense strand (the nucleotide of the antisense strand bound at this position was not deformed), and it was shown that the nucleotide at the 9th position from the 5' end of the sense strand was not deformed. Furthermore, it was confirmed that the nucleotides at the 18th and 20th positions from the 5' end of the antisense strand were chemically deformed by 2'-OMe.

[0276] (blank)

[0277] Finally, in group 3, the sense strand had a length of 19–25 nt, and the antisense strand had a length of 21–27 nt. Chemical deformation was performed using the 2'-OMe method. A nucleotide that was 2'-OME chemically deformed was observed at the 7th position from the 5' end of the sense strand (the antisense strand nucleotide bound at this position was not deformed), and it was shown that the nucleotide at the 9th position from the 5' end of the sense strand was not deformed. In addition, nucleotides at the 16th, 18th, and 20th positions from the 5' end of the antisense strand were confirmed to be 2'-OMe chemically deformed.

[0278] (blank)

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

[0280] The anticancer effects of the 29 siRNAs finally identified in Example 1 were confirmed when pancreatic cancer cell lines were treated with them. Specifically, pancreatic cancer cell lines were treated with 100 cm³ of siRNA. 2Cells were cultured in a dish using DMEM culture medium (10% FBS, 1% penicillin-streptomycin). Colorectal cancer cell lines were cultured using RPMI culture medium (10% FBS, 1% penicillin-streptomycin). Both pancreatic and colorectal cancer cell lines were cultured at 37°C and 5% CO2. Once the cells reached 70-80% density (confluency), they were washed with PBS, suspended using trypsin-EDTA, and then desuspended using culture medium. The cell suspension was then centrifuged (1,000 rpm, 5 minutes) to collect the cells. After centrifugation, only the collected cells were resuspended in fresh culture medium. Cell counts were measured to confirm cell viability, and then the cells were seeded into 96-well plates at a concentration of 10,000 cells / well. The cells were cultured for 24 hours at 37°C and 5% CO2 to allow adhesion.

[0281] The experimental siRNA and the control siRNA were mixed in 75 μL of Opti-MEM medium with the siRNA (prepared to a final concentration of 10 nM). In another new tube, 75 μL of Opti-MEM medium was mixed with 2.5 μL of Lipofectamine RNAiMAX. The two mixtures were combined and reacted 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 and 5% CO2 for 1 hour. The absorbance of each well was measured at 450 nm using a microplate reader, and the background absorbance was measured at 650 nm and corrected. The relative cell proliferation rate was calculated by comparing the absorbance values ​​of the treated samples with those of the control group using the following formula.

[0283] (blank)

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

[0285] (Blank)

[0286] As a result, the cell-killing effects on pancreatic cancer cell lines AsPC-1, HPAFII, and PANC-1 in Group 1 were confirmed as shown in (a) to (c) of FIG. 2. The results of the same experiment in Group 2 are the same as those in (d) of FIG. 2. Finally, the experimental results of Group 3 are the same as those in (e) to (g) of FIG. 2. It was confirmed that in each of Groups 1 to 3, pancreatic cancer cell growth decreased in the group treated with each lead substance compared to the control group treated with Scrambled siRNA (Bioneer).

[0287] (Blank)

[0288]

Table 8

[0289] Example 3. Confirmation of the anti-cancer effects of 29 siRNAs on colorectal cancer cell lines

[0290] The anti-cancer effects of 29 siRNAs on colorectal cancer were analyzed by applying the same experimental method as in Example 2.

[0291] (Blank)

[0292] As a result, the cell-killing effects on colorectal cancer cell lines SNU-C2B, LS174T, and LS513 in Group 1 were confirmed as shown in (a) to (c) of FIG. 3. The results of the same experiment in Group 2 are the same as those in (d) of FIG. 3. Finally, the experimental results of Group 3 are the same as those in (e) to (g) of FIG. 3. It was confirmed that in each of Groups 1 to 3, colorectal cancer cell growth decreased in the group treated with each lead substance compared to the control group treated with Scrambled siRNA.

[0293] (Blank)

[0294] Examples 2 and 3 confirmed that the 29 leading substances of the present invention exhibit anticancer effects against colorectal cancer and pancreatic cancer by inhibiting the growth rate of cancer cells.

[0295] (blank)

[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 when pancreatic cancer cell lines are treated with it was investigated. Specifically, to confirm the changes in intracellular KRAS mRNA expression due to siRNA treatment, the number of cells was measured, and then the cells were seeded in a 12-well plate at a concentration of 100,000 cells / well. The cells were cultured for 24 hours at 37°C and 5% CO2 to allow adhesion.

[0298] The siRNAs used in the experiment were unmodified siKRAS G12D, unmodified siKRAS G12D-2, unmodified siKRAS G12D-PS (Table 3, items 1-3), and Pattern_OME modified siKRAS G12D (Table 3, item 6). Alnylam_unmodified siKRAS G12 was used as the control group. Alnylam_unmodified siKRAS G12D was identified among the 105 siRNAs of the present invention, but was not selected by the final 29 lead substances. Alnylam_unmodified siKRAS G12D has the following characteristics: 21nt, no 2'OME modification, no overhang (SS), no overhang (AS), and no overhang PS modification.

[0299] (blank)

[0300] [Table 9]

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

[0302] After 24 hours of incubation, the existing culture medium was removed, and 1000 μL of QIAzol Lysis Reagent was added to each 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 and mixed vigorously for 15 seconds. The mixture was then left at room temperature for 2-3 minutes, and the mixture was centrifuged at 12,000 × g for 15 minutes at 4°C. The supernatant (water-soluble layer) was carefully transferred to a new tube. 2-propanol was added to the supernatant and mixed to precipitate the RNA, then centrifuged at 12,000 × g for 15 minutes at 4°C. The supernatant was removed, and the RNA was washed with 70% ethanol. The mixture was then centrifuged at 12,000 × g for 10 minutes at 4°C to completely remove the supernatant. The RNA pellet was dissolved in nuclease-free water, and the concentration and purity of the extracted RNA were measured using nanodrops.

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

[0304] (blank)

[0305] The results showed a significant decrease in KRAS mRNA expression in unmodified siKRAS G12D, unmodified siKRAS G12D-2, unmodified siKRAS G12D-PS (Table 3, items 1-3), and Pattern_OME modified siKRAS G12D (Table 3, item 6) (Figure 4(a)). On the other hand, the control group, Alnylam_unmodified siKRAS G12D, showed no significant effect on KRAS mRNA expression (Figure 4(b)). (In this case, siRNA 0nM refers to the control group and means siScramble siRNA 10nM.)

[0306] (blank)

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

[0308] We confirmed the tumor size suppression effect of the siRNA of the present invention when applied to an animal model of pancreatic cancer. Specifically, 1 × 10 KPC-luc cells 7 Prepare 1 x 10¹⁶ cells in 100 μl PBS, anesthetize 6-8 week old female nude mice, and insert 1 x 10¹⁶ cells subcutaneously into the thigh of the anesthetized mice. 7 100 μL of cells were injected subcutaneously between the skin and muscle layers using a 31-gauge syringe. After injection, mice were recovered in a recovery room. Following cell injection, the length (L) and width (W) of the tumor were measured using a digital caliper. The tumor volume was calculated using the following formula: tumor volume (mm²). 3 ) = L × W 2 ×0.5.

[0309] Tumor size: 100 mm 3 When it reaches a certain size, the treatment agent is injected, and the tumor size reaches 100 mm. 3Mice that reached a certain stage were randomly divided into two groups: a treatment group and a control group. The treatment group received a therapeutic agent via tail vein injection three times a week (Monday, Wednesday, and Friday). In this experiment, the treatment group used Exollence_25nt #6 (Table 3, 10) as the experimental group, and Exollence_25nt #1 (Table 10), siScramble (negative control group), and Ctl (control group) as the comparison groups. Exollence_25nt #1 was used as a negative control in this experiment because it did not show the cell-killing effect of KRAS G12D in experiments where it was treated with cells and cell viability was measured. Exollence_25nt #1 has the characteristics of 25nt, no 2'OME modification, overhang (SS)tt, overhang (AS)tt, and overhang PS modification (-).

[0310] (blank)

[0311] [Table 10]

[0312] The mice received an injection dose of siRNA 20 μg / 100 μL, while the control group received the same amount of sterile PBS at the same intervals. Before injecting the therapeutic agent, the mice were placed on a warming pad, their tails were warmed to dilate the veins, and the therapeutic agent was injected into the tail vein using a 29-gauge needle.

[0313] During the injection period, the length and width of the tumor were measured twice a week, and the tumor volume was calculated. The mice's body weight and general health were regularly monitored to check for side effects. After the final injection of the therapeutic agent, the tumor was removed and weighed.

[0314] (blank)

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

[0316] (blank)

[0317] The above description of the present invention is illustrative, and a person with ordinary skill in the art to which the present invention pertains will understand that it can be easily modified into other specific forms without altering the technical idea or essential features of the present invention. Therefore, the embodiments described above should be understood in all respects as illustrative and not limiting. [Industrial applicability]

[0318] This invention relates to a pharmaceutical composition for the prevention or treatment of cancer containing siRNA as an active ingredient, which has been shown to exhibit excellent anticancer effects by significantly suppressing the cell proliferation effect of colorectal cancer or pancreatic cancer, and by reducing the expression level of KRAS mRNA. Furthermore, it can be useful as an excellent anticancer agent because it reduces tumor size and weight while maintaining the individual's body weight, thus demonstrating industrial applicability.

Claims

1. A pharmaceutical composition for the prevention or treatment of cancer, characterized by containing as an active ingredient one or more siRNAs selected from the group consisting of siRNAs containing any one base sequence selected from the group consisting of SEQ ID NOs: 1 to 29.

2. The group consisting of the following siRNAs is 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, as described in claim 1: 1) siRNA containing any one base sequence selected from the group consisting of SEQ ID NOs: 1 to 5; 2) siRNA containing the base sequence shown in Sequence ID No. 7; 3) siRNA containing any one nucleotide sequence selected from the group consisting of SEQ ID NOs: 14, 15, and 17; and 4) siRNA containing any one base sequence selected from the group consisting of SEQ ID NOs: 20, 23, and 26-28.

3. The group consisting of siRNA containing the nucleotide sequence shown in SEQ ID NO: 12, siRNA containing the nucleotide sequence shown in SEQ ID NO: 13, siRNA containing the nucleotide sequence shown in SEQ ID NO: 18, siRNA containing the nucleotide sequence shown in SEQ ID NO: 21, siRNA containing the nucleotide sequence shown in SEQ ID NO: 24, and siRNA containing the nucleotide sequence shown in SEQ ID NO: 29 is characterized by the following group, the pharmaceutical composition according to claim 1: a) The sense strand is 19–25 nt in length, and the antisense strand is 21–27 nt in length; b) The sense chain does not have a tt overhang; c) The sense strand has a chemically deformed nucleotide (nucleotide) of 2'-OME at the 7th position from the 5' end, and the nucleotide of the antisense strand bound to that position does not have the chemical deformation of 2'-OME; d) The nucleotide at the 9th position from the 5' end of the sense strand does not have a chemical modification of 2'-OME; and e) Having chemically deformed nucleotides (nucleotides) of 2'-OME at the 18th and 20th positions from the 5' end of the antisense strand.

4. The group consisting of siRNA containing the nucleotide sequence shown in SEQ ID NO: 6, siRNA containing the nucleotide sequence shown in SEQ ID NO: 8, siRNA containing the nucleotide sequence shown in SEQ ID NO: 9, siRNA containing the nucleotide sequence shown in SEQ ID NO: 10, siRNA containing the nucleotide sequence shown in SEQ ID NO: 11, siRNA containing the nucleotide sequence shown in SEQ ID NO: 16, siRNA containing the nucleotide sequence shown in SEQ ID NO: 19, siRNA containing the nucleotide sequence shown in SEQ ID NO: 22, and siRNA containing the nucleotide sequence shown in SEQ ID NO: 25 is characterized by the following group, the pharmaceutical composition according to claim 1: a) The sense strand is 19–25 nt in length, and the antisense strand is 21–27 nt in length; b) Having a chemically modified nucleotide (nucleotide) of 2'-OME at the 7th position from the 5' end of the sense strand, the nucleotide of the antisense strand bound to the said position does not have the chemical modification of 2'-OME; c) The nucleotide at the 9th position from the 5' end of the sense strand does not have the chemical deformation of 2'-OME; d) Having chemically modified nucleotides (nucleotides) of 2'-OME at the 16th, 18th, and 20th positions from the 5' end of the antisense strand.

5. The pharmaceutical composition according to claim 1, characterized in that the cancer is one or more selected from the group consisting of pancreatic cancer, colorectal cancer, squamous cell carcinoma, lung cancer, adenocarcinoma of the lung, peritoneal cancer, skin cancer, melanoma of the skin or eye, rectal cancer, anal cancer, esophageal cancer, small intestine cancer, endocrine adenocarcinoma, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, hematological cancer, liver cancer, gastrointestinal cancer, glioblastoma, cervical cancer, ovarian cancer, bladder cancer, liver tumor, 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. The pharmaceutical composition according to claim 1, characterized in that the composition inhibits the proliferation of cancer cells.

7. The pharmaceutical composition according to claim 1, characterized in that the composition reduces the size and weight of a tumor.

8. The pharmaceutical composition according to claim 1, characterized in that the composition suppresses KRAS mRNA expression.

9. A kit for the prevention or treatment of cancer, comprising a composition containing as an active ingredient one or more siRNAs selected from the group consisting of one nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 29, and instructions.

10. A method for treating cancer, characterized by comprising the step of administering a pharmaceutically effective amount of a composition containing, as an active ingredient, one or more siRNAs selected from the group consisting of one nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 29, to an individual in need of it.

11. A composition containing one or more siRNAs selected from the group consisting of any single nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 29, as an active ingredient, for the purpose of preventing or treating cancer.

12. For use in manufacturing cancer prevention or therapeutic formulations of a composition containing, as an active ingredient, one or more siRNAs selected from the group consisting of any single nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 29.

Images