Sirna for inhibiting KRAS gene expression, and modified form thereof and use thereof

Abstract

The present application belongs to the field of biomedicine. Disclosed are an siRNA for inhibiting KRAS gene expression, and a modified form thereof and the use thereof. Provided in the present application is a double-stranded RNA molecule targeting KRAS, wherein the molecule can treat KRAS-associated diseases such as metastatic non-small cell lung cancer, metastatic pancreatic ductal adenocarcinoma, KRAS G12C+ non-small cell lung cancer, diabetic retinopathy, leukemia, cholangiocarcinoma, metastatic colorectal cancer, gastric cancer, advanced non-small cell lung cancer, stage-IV non-small cell lung cancer, gastric adenocarcinoma, sepsis, KRAS G12C-mutated advanced non-small cell lung cancer and gallbladder cancer.

Description

A siRNA for inhibiting KRAS gene expression and its modifications and applications Technical Field

[0001] This application belongs to the field of biomedicine and relates to an siRNA for inhibiting KRAS gene expression, its modifications, and its applications. Background Technology

[0002] The KRAS gene is a member of the rat sarcoma virus oncogene family (RAS), which also includes two other subtypes in humans: HRAS and NRAS. HRAS was discovered in 1982, isolated from a human bladder cancer cell line, and identified as a human homolog of the RAS gene, hence the name HRAS. In the same year, another homolog was discovered in human lung cancer cells, named KRAS. NRAS was discovered in human neuroblastoma. The RAS protein contains 6 β chains and 5 α helices, forming two main domains: the catalytic domain (G domain) and the hypervariable region (HVR).

[0003] There are two KRAS genes in the human genome. One is KRAS1, located on the short arm of chromosome 6; the other is KRAS2, located on the short arm of chromosome 12. KRAS1 is a "pseudogene," meaning it cannot be transcribed into RNA and therefore has no function. KRAS2, however, is the "true gene," capable of being transcribed and translated into protein, possessing biological activity. The KRAS genes encode two highly related protein isoforms, KRAS-4B and KRAS-4A, composed of 188 and 189 amino acids respectively, due to differences in the splicing of the fourth exon. KRAS usually refers to KRAS-4B because KRAS-4B mRNA levels are high in cells. The KRAS protein has a molecular weight of 21.6 kDa.

[0004] The switching between inactive and activated states of KRAS is regulated by two classes of factors. One class consists of guanine nucleotide exchange factors (GEFs), which catalyze the binding of KRAS to GTP, thereby promoting KRAS activation. This includes SOS proteins (belonging to GEFs / guanosine releasing factors / guanylate exchange factors). The other class consists of GTPase activators (GAPs), which promote the hydrolysis of GTP bound to KRAS into GDP, terminating the active state and thus inhibiting KRAS activity. Because KRAS occupies a central position on the axes of many important cellular signaling networks, they are associated with numerous cancer markers.

[0005] RAS is the most frequently mutated oncogene in human cancers, with constitutive activating mutations found in approximately one-fifth of human tumors. KRAS mutations account for 85% of all RAS gene mutations (NRAS (12%) is second, and HRAS (3%) is the least common). In human cancers, the mutation rate of the KRAS gene is close to 90% in pancreatic cancer, 30-40% in colon cancer, 17% in endometrial cancer, and 15-20% in lung cancer (mostly NSCLC). Furthermore, KRAS mutations can be found in other cancer types such as cholangiocarcinoma, cervical cancer, bladder cancer, liver cancer, and breast cancer. Therefore, a high proportion of KRAS gene mutations are present in various cancers.

[0006] Currently, there are two marketed drugs targeting KRAS G12C: SOTORASIB, used to treat metastatic and advanced non-small cell lung cancer (NSCLC); and adagrasib, used to treat advanced NSCLC, KRAS g12c-mutated metastatic NSCLC, colorectal cancer, metastatic colorectal cancer, and KRAS g12c-mutated locally advanced NSCLC. However, SOTORASIB failed to achieve median overall survival (OS) in the Phase III confirmatory CodeBreaK 200 study, and the approved indications for SOTORASIB are controversial. Another small molecule drug targeting KRAS G12C, Adagrasib, received accelerated approval from the FDA, also based on Phase II results (KRYSTAL-1 study Phase II cohort A (NSCLC cohort) results), and Phase III clinical trials are currently underway globally.

[0007] Although two small-molecule drugs targeting the KRAS G12C mutation currently exist, their Phase III clinical trial results are either unsatisfactory or remain to be determined. Both marketed drugs interfere with the pro-tumorigenic effects of KRAS G12C by binding to it. siRNA drugs can potently inhibit the pro-tumorigenic effects of mRNA-encoded proteins by degrading mRNA at the post-transcriptional regulatory level. Therefore, the search for KRAS siRNAs has become a current research direction. Summary of the Invention

[0008] The technical problem addressed by this application is to provide siRNA that efficiently inhibits KRAS expression.

[0009] To address the aforementioned technical problems, a first aspect of this application provides a double-stranded RNA molecule, which is any one of 150 siRNAs, wherein the siRNA comprises a sense strand and an antisense strand that at least partially form a double-stranded region, and the sense strand comprises any odd-numbered sequence from sequence 1 to sequence 300.

[0010] In the double-stranded RNA molecule described above, the siRNA includes a sense strand and an antisense strand that form the double-stranded region, and the sense strand includes any odd-numbered sequence from sequence 1 to sequence 300.

[0011] In the double-stranded RNA molecule described above, the length of the double-stranded region is 15-30bp, 23-27bp, 21-23bp, 19-21bp, 17-25bp, 17-23bp, or 17-19bp.

[0012] In the double-stranded RNA molecule described above, the length of the sense strand does not exceed 30 nucleotides, and / or the length of the antisense strand does not exceed 30 nucleotides.

[0013] Furthermore, each strand (sense strand or antisense strand) of the double-stranded RNA molecule has 15-30 nucleotides.

[0014] Furthermore, each strand (sense strand or antisense strand) of the double-stranded RNA molecule has 19-30 nucleotides.

[0015] In the double-stranded RNA molecule described above, the length of the sense strand does not exceed 19 nucleotides, and / or the length of the antisense strand does not exceed 21 nucleotides.

[0016] In the double-stranded RNA molecule described above, at least one of the sense strands and the antisense strand contains a 3' overhang with at least one nucleotide, or at least one of the strands contains a 3' overhang with at least two nucleotides.

[0017] In the double-stranded RNA molecules described above, the nucleotide sequences of the antisense strands of the 150 siRNAs each include any even-numbered sequence from sequence 1 to sequence 300.

[0018] In the above text, the nucleotide sequence of the sense strand of the 150 siRNAs is numbered n, and the nucleotide sequence of the antisense strand of the 150 siRNAs is numbered n+1, where n is any odd number from 1 to 300.

[0019] The nucleotide sequences of the sense strand of the 150 siRNAs mentioned above are as follows:

[0020] A1) A sequence numbered with any odd number from sequence 1 to sequence 300;

[0021] A2) The sequence that has more than 90% homology with any of the odd-numbered sequences from Sequence 1 to Sequence 300.

[0022] The nucleotide sequences of the antisense strands of the 150 siRNAs mentioned above are as follows:

[0023] A3) A sequence numbered with any even number from sequence 1 to sequence 300;

[0024] A4) The sequence that has more than 90% homology with any of the even-numbered sequences in Sequences 1 to 300.

[0025] Secondly, this application provides a double-stranded RNA molecule modifier, which is a compound containing a modified nucleotide obtained by modifying at least one nucleotide in the double-stranded RNA molecule described in the first aspect.

[0026] In the double-stranded RNA molecule modifications described above, at least one nucleotide in the sense strand or antisense strand of the double-stranded RNA molecule modification is a modified nucleotide.

[0027] In the double-stranded RNA molecule modifications described above, the modified nucleotide is a compound formed by replacing the 2' hydroxyl group of the ribosyl group of the nucleotide with other groups, or a compound formed by modifying the bases on the nucleotide.

[0028] The double-stranded RNA molecule modification described above further includes linking the modified nucleotides via phosphate thioesters.

[0029] In the double-stranded RNA molecule modifications described above, the modified nucleotide is selected from at least one of the following groups: 2'-methoxy-modified nucleotides, 2'-methyl-modified nucleotides, 2'-fluorine-modified nucleotides, deoxy-nucleotides, 2'-deoxy-nucleotides, 3'-terminal deoxy-thymidine (dT) nucleotides, 2'-deoxy-modified nucleotides, locked nucleotides, non-locked nucleotides, configuration-restricted nucleotides, restricted ethyl nucleotides, base-free nucleotides, 2'-amino-modified nucleotides, 2'-O-allyl-modified nucleotides, 2'-C-alkyl-modified nucleotides. 2'-hydroxy-modified nucleotides, 2'-methoxyethyl-modified nucleotides, 2'-O-alkyl-modified nucleotides, morpholinoyl nucleotides, nucleotides containing non-natural bases, tetrahydropyran-modified nucleotides, 1,5-dehydrohexyl-modified nucleotides, cyclohexenyl-modified nucleotides, glycerol nucleotides (GNA), open-ring nucleotides (UNA), nucleotides containing methyl phosphate, nucleotides containing 5'-(E)vinyl phosphate, nucleotides containing 5'-phosphate, and nucleotides containing 5'-phosphate mimics.

[0030] In the double-stranded RNA molecule modification described above, the double-stranded RNA molecule modification includes a 2'-methoxy-modified nucleotide, the 2'-methoxy-modified nucleotide being located in the antisense strand and the sense strand of the double-stranded RNA molecule modification, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 1-6 and 10-19 of the sense strand are 2'-methoxy-modified nucleotides; and at least the nucleotides at positions 1, 3-5, 7-13, 15, and 17-21 of the antisense strand are 2'-methoxy-modified nucleotides;

[0031] Furthermore, the double-stranded RNA molecule modifier also includes 2'-fluorinated nucleotides located in the antisense and sense strands of the double-stranded RNA molecule modifier, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 14 and 16 of the antisense strand are 2'-fluorinated nucleotides.

[0032] In the double-stranded RNA molecule modification described above, the double-stranded RNA molecule modification includes a 2'-methoxy-modified nucleotide, the 2'-methoxy-modified nucleotide being located in the antisense strand and the sense strand of the double-stranded RNA molecule modification, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 1-6 and 10-19 of the sense strand are 2'-methoxy-modified nucleotides; and at least the nucleotides at positions 1, 3-5, 7-13, 15, and 17-21 of the antisense strand are 2'-methoxy-modified nucleotides.

[0033] Furthermore, the double-stranded RNA molecule modifier also includes 2'-fluorinated nucleotides located in the antisense and sense strands of the double-stranded RNA molecule modifier, and, in the direction from the 5' end to the 3' end, at least the 7th, 8th, and 9th nucleotides of the sense strand are 2'-fluorinated nucleotides, and at least the 2nd, 6th, 14th, and 16th nucleotides of the antisense strand are 2'-fluorinated nucleotides.

[0034] The double-stranded RNA molecule modification further includes at least one phosphate-thioester modified backbone, wherein the phosphate-thioester modification is located in the antisense strand and the sense strand of the double-stranded RNA molecule modification, and, in the direction from the 5' end to the 3' end, the phosphate backbone between at least the 1st to 3rd nucleotides of the sense strand is phosphate-thioester modified (i.e., at least the 1st and 2nd nucleotides, and the 2nd and 3rd nucleotides are linked by phosphate-thioester), and the phosphate backbone between at least the 1st to 3rd nucleotides and the 19th to 21st nucleotides of the antisense strand is phosphate-thioester modified (i.e., at least the 1st and 2nd nucleotides, the 2nd and 3rd nucleotides, the 19th and 20th nucleotides, and the 20th and 21st nucleotides are linked by phosphate-thioester).

[0035] In the double-stranded RNA molecule modifications mentioned above, the double-stranded RNA molecule modifications are any one of the following 150 modifications: the nucleotide sequences of the positive strand of the 150 modifications are any odd-numbered sequences from sequence 301 to sequence 600;

[0036] And / or, the nucleotide sequences of the antisense strands of the 150 modifiers are sequences numbered with any even number from sequence 301 to sequence 600.

[0037] The nucleotide sequence of the sense strand of the 150 modifiers is numbered m, and the nucleotide sequence of the antisense strand is numbered m+1, where m is any odd number from 301 to 600.

[0038] The double-stranded RNA molecule modification is further specifically selected from any one of the following: Kras-1M1, Kras-4M1, Kras-5M1, Kras-8M1, Kras-9M1, Kras-10M1, Kras-11M1, Kras-12M1, Kras-13M1, Kras-14M1, Kras-15M1, Kras-17M1, Kras-21M1, Kras-23M1, Kras-2 5M1, Kras-27M1, Kras-29M1, Kras-30M1, Kras-31M1, Kras-33M1, Kras-34M1, Kras-35M1, Kras-36M1 , Kras-38M1, Kras-39M1, Kras-42M1, Kras-43M1, Kras-50M1, Kras-53M1, Kras-54M1, Kras-55M1, Kr as-57M1, Kras-59M1, Kras-62M1, Kras-63M1, Kras-72M1, Kras-74M1, Kras-76M1, Kras-81M1, Kras- 84M1, Kras-85M1, Kras-89M1, Kras-94M1, Kras-96M1, Kras-103M1, Kras-106M1, Kras-107M1, Kras- 109M1, Kras-113M1, Kras-114M1, Kras-118M1, Kras-123M1, Kras-124M1, Kras-126M1, Kras-128M1, Kras-129M1, Kras-134M1, Kras-141M1, Kras-32M1, Kras-49M1, Kras-86M1, Kras-95M1, Kras-130M1.

[0039] The double-stranded RNA molecule modification is further specifically selected from any one of the following: KRAS-76M1, KRAS-10M1, KRAS-85M1, KRAS-13M1, KRAS-113M1, KRAS-96M1, KRAS-12M1, KRAS-109M1, KRAS-81M1, KRAS-21M1, KRAS-25M1, KRAS-14M1, KRAS-27M1, KRAS-94M1, KRAS-39M1, KRAS-17M1, KRAS-74M1, KRAS-103M1, KRAS-42M1, KRAS-31M1, KRAS-5M1, KRAS-89M1, KRAS-32M1, KRAS-34M1, and KRAS-38M1.

[0040] The double-stranded RNA molecule modification is further specifically selected from any one of the following: KRAS-94M1, KRAS-113M1, KRAS-21M1, KRAS-39M1, KRAS-96M1, KRAS-10M1, and KRAS-13M1.

[0041] In the double-stranded RNA molecule modification described above, a ligand is attached to any nucleotide at the 3'-end or 5'-end of the positive strand or in the middle of the positive strand.

[0042] In the double-stranded RNA molecule modifications described above, the ligand is a small molecule, antibody, polypeptide, protein, or aptamer.

[0043] Among the double-stranded RNA molecule modifications described above, the antibody is, for example, HER2.

[0044] In the double-stranded RNA molecule modifications described above, the polypeptide is, for example, RGD.

[0045] In the double-stranded RNA molecule modification described above, the small molecule is GalNAc.

[0046] In the double-stranded RNA molecule modification described above, the protein is albumin, but it can also be other proteins.

[0047] In the double-stranded RNA molecule modification described above, the ligand is linked to the double-stranded RNA molecule modification via a linker.

[0048] Thirdly, this application provides the use of the double-stranded RNA molecule described in the first aspect or the double-stranded RNA molecule modification described in the second aspect in any of the following:

[0049] D1) Prepare a composition that inhibits KRAS gene expression;

[0050] D2) Inhibits KRAS gene expression;

[0051] D3) Treatment of KRAS-target-related diseases;

[0052] D4) Prepare compositions for treating KRAS target-related diseases;

[0053] D5) Preparation of compositions for treating tumors.

[0054] Fourthly, this application provides a composition for inhibiting KRAS expression, wherein the active ingredient of the composition is the double-stranded RNA molecule described in the first aspect or the double-stranded RNA molecule modification described in the second aspect.

[0055] The composition described above also includes a pharmaceutically acceptable carrier.

[0056] The compositions described above include lipid formulations, nanoformulations, and lipid-containing vesicles.

[0057] In the composition described above, the lipid formulation is an LNP formulation.

[0058] In the composition described above, the nano-formulation is polymer nanoparticles;

[0059] In the composition described above, the lipid-containing vesicles are exosomes;

[0060] Furthermore, the exosomes are artificially modified exosomes.

[0061] The composition is a pharmaceutical composition.

[0062] The pharmaceutical composition described above also comprises an unbuffered solution.

[0063] The unbuffered solution in the pharmaceutical composition described above is physiological saline or water.

[0064] The pharmaceutical composition described above also includes a buffer solution.

[0065] The buffer solution in the pharmaceutical composition described above comprises acetate, citrate, alcohol-soluble gluten, carbonate, or phosphate, or any combination thereof.

[0066] The buffer solution in the drug composition described above is phosphate-buffered saline (PBS).

[0067] The pharmaceutically acceptable carrier may be an excipient, stabilizer, suspending agent, or diluent, as is well known to those skilled in the art.

[0068] Fifthly, this application provides a cell comprising the double-stranded RNA molecule described in the first aspect, the double-stranded RNA molecule modification described in the second aspect, or the composition described in the fourth aspect.

[0069] In a sixth aspect, this application provides a method for treating KRAS target-related diseases, the method comprising the steps of administering to a subject suffering from a KRAS target-related disease the double-stranded RNA molecule described in the first aspect, the double-stranded RNA molecule modifier described in the second aspect, or the composition described in the fourth aspect.

[0070] In a seventh aspect, this application provides a method for treating tumors, the method comprising the steps of administering to a subject suffering from a tumor the double-stranded RNA molecule described in the first aspect, the double-stranded RNA molecule modifier of the second aspect, or the composition described in the fourth aspect.

[0071] Eighthly, this application provides a method for inhibiting KRAS expression in cells, the method comprising:

[0072] (a) Contacting the cells with the double-stranded RNA molecule described in the first aspect, the double-stranded RNA molecule modification described in the second aspect, or the composition described in the fourth aspect.

[0073] (b) Maintain the cells produced in step (a) for a period of time sufficient for the degradation of the mRNA transcript of the KRAS gene, thereby inhibiting KRAS expression in the cells.

[0074] In the method described above, the cells are located within the subject.

[0075] In the method described above, the subject suffers from a KRAS-related disease.

[0076] In the method described above, the KRAS gene expression is suppressed by at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, or about 100%.

[0077] The term "subject" as used in this article may refer to an animal, such as a mammal, including primates (e.g., humans or non-human primates, such as monkeys or chimpanzees), and non-primates (e.g., cattle, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, mice, horses, or whales). In some embodiments, the subject is a mouse.

[0078] The diseases caused by abnormal KRAS expression mentioned above can be diseases caused by upregulation of KRAS expression.

[0079] Furthermore, the diseases caused by the upregulation of KRAS expression are tumors, including but not limited to bile duct cancer, cervical cancer, bladder cancer, liver cancer, and breast cancer.

[0080] In this application, "modified nucleotide" refers to a nucleotide or nucleotide analog formed by replacing the 2'-hydroxyl group and / or the 5'-hydroxyl group of the ribosyl group with other groups, or a nucleotide whose bases are modified bases. "Methoxy-modified nucleotide" refers to a nucleotide formed by replacing the 2'-hydroxyl group of the ribosyl group with a methoxy group. "Fluoro-modified nucleotide" refers to a nucleotide formed by replacing the 2'-hydroxyl group of the ribosyl group with fluorine. "Nucleotide analog" refers to a group that can replace a nucleotide in nucleic acids but has a structure different from adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, or thymine deoxyribonucleotide. Examples include isonucleotides, bridged nucleic acids (BNAs), or acyclic nucleotides.

[0081] In one embodiment of this application, "fluorinated nucleotide" refers to a nucleotide formed by replacing the hydroxyl group at the 2' position of the ribosyl group with fluorine, having the structure shown in formula (1). The non-fluorinated nucleotide is independently selected from nucleotides or nucleotide analogs formed by replacing the hydroxyl group at the 2' position of the ribosyl group with a non-fluorinated group.

[0082] In one embodiment of this application, the nucleotide formed by replacing the hydroxyl group at the 2' position of the ribosome with a non-fluorinated group is well known to those skilled in the art. These nucleotides may be selected from one of the following: 2'-alkoxy modified nucleotides, 2'-substituted alkoxy modified nucleotides, 2'-alkyl modified nucleotides, 2'-substituted alkyl modified nucleotides, 2'-amino modified nucleotides, 2'-substituted amino modified nucleotides, and 2'-deoxynucleotides.

[0083] In one embodiment of this application, the 2'-alkoxy modified nucleotide is a 2'-methoxy (2'-OMe) modified nucleotide, as shown in formula (2), i.e., methoxy modified; the 2'-substituted alkoxy modified nucleotide, for example, can be a 2'-O-methoxyethyl (2'-MOE) modified nucleotide, as shown in formula (3), the 2'-amino (2'-NH2) modified nucleotide is shown in formula (4), and the 2'-deoxynucleotide (DNA) is shown in formula (5), where Base represents the modified or unmodified nucleotide base A, U, G, C, T or other nucleotide bases.

[0084] In one embodiment of this application, the nucleotide analogue refers to a group that can replace a nucleotide in nucleic acid, but whose structure is different from that of adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, or thymine deoxyribonucleotide.

[0085] In one embodiment of this application, the nucleotide analog may be an isonucleotide, a bridged nucleotide, or an acyclic nucleotide.

[0086] In one embodiment of this application, the bridged nucleic acid (BNA) refers to a restricted or inaccessible nucleotide. The BNA may contain a bridging structure with a "fixed" C3'-endoglucan condensation, such as a five-membered ring, a six-membered ring, or a seven-membered ring. Typically, the bridge is incorporated into the 2'- or 4'-position of the ribose to provide a 2',4'-BNA nucleotide.

[0087] In one embodiment of this application, the BNA may be LNA, ENA, cET BNA, etc., wherein LNA is as shown in formula (6), ENA is as shown in formula (7), and cET BNA is as shown in formula (8), where Base represents modified or unmodified nucleotide bases A, U, G, C, T or other nucleotide bases.

[0088] In one embodiment of this application, at least a portion of the phosphate ester in the phosphate-sugar backbone of at least one single strand of the sense and antisense strands of the siRNA is a phosphate ester with a modifying group.

[0089] In one embodiment of this application, the phosphate ester with the modified group is a thiophosphate ester formed by replacing at least one oxygen atom in the phosphate diester bond of the phosphate ester with a sulfur atom.

[0090] In one embodiment of this application, the phosphate ester having the modifying group is a thiophosphate ester having the structure shown in formula (9). In one embodiment of this application, the nucleotide linked to the thiophosphate ester is shown in formula (10), and the thiophosphate ester linkage is present at least at one of the following positions: between the first and second nucleotides at either end of the sense or antisense strand; between the second and third nucleotides at either end of the sense or antisense strand; or any combination thereof.

[0091] In one embodiment of this application, the modified nucleotide is a vinyl phosphate modified (VP-modified) nucleotide. In one embodiment of this application, the VP-modified and methoxy-modified nucleotide, namely a 5'-(E)-vinyl-2'-methoxy-modified phosphate (5'-(E)-VP-2'-OMe) modified nucleotide, is shown in formula (11); where Base represents the modified or unmodified nucleotide base A, U, G, C, T or other nucleotide bases.

[0092] In the siRNA preparation methods referred to in this application, unless otherwise specified, nucleoside monomer refers to the modified or unmodified RNAphosphoramidites (sometimes also called nucleoside phosphoramidites) used in phosphoramidite solid-phase synthesis, depending on the type and sequence of nucleotides in the siRNA to be prepared. Phosphoramidite solid-phase synthesis is a method known to those skilled in the art for the synthesis of siRNA. All nucleoside monomers used in this application are commercially available.

[0093] It is worth noting that the raw materials used in this application are all ordinary commercially available products, and their sources are not specifically limited.

[0094] The meanings of the abbreviations for nucleotide monomers used in this application are as follows: Error! No source found. It should be noted that when these monomers are present in oligonucleotides, they are interconnected by 5'-3'-phosphodiester bonds; and it should be understood that when a nucleotide contains a 2'-fluorine modification, the fluorine replaces the hydroxyl group at that position in the parent nucleotide (i.e., it is a 2'-deoxy-2'-fluoronucleotide).

[0095] Table 1. Abbreviations of nucleotide monomers used in this application.

[0096] This application also provides the following technical solutions:

[0097] 1. A double-stranded RNA molecule, wherein the double-stranded RNA molecule comprises a sense strand and an antisense strand that at least partially form a double-stranded region, the antisense strand comprising 17-21 consecutive nucleotides of any even-numbered nucleotide sequence in SEQ ID NO. 1-300, and the sense strand comprising 17-19 consecutive nucleotides of any odd-numbered nucleotide sequence in SEQ ID NO. 1-300.

[0098] 2. The double-stranded RNA molecule according to claim 1, wherein the length of the sense strand is no more than 30 nucleotides, and / or the length of the antisense strand is no more than 30 nucleotides, preferably, the length of the sense strand is no more than 21 nucleotides, and / or the length of the antisense strand is no more than 23 nucleotides, more preferably, the length of the sense strand is no more than 19 nucleotides, and / or the length of the antisense strand is no more than 21 nucleotides.

[0099] 3. The double-stranded RNA molecule according to claim 1 or 2, wherein the sense strand and antisense strand of said double-stranded RNA molecule comprise or are selected from any combination of the following:

[0100] The sense strand with nucleotide sequence number n and the antisense strand with nucleotide sequence number n+1, where n is an odd number between 1 and 300.

[0101] 4. The double-stranded RNA molecule according to claim 3, wherein the sense strand and antisense strand of said double-stranded RNA molecule comprise or are selected from any combination of the following:

[0102] The positive chain shown in SEQ ID NO.1 and the negative chain shown in SEQ ID NO.2;

[0103] The positive chain shown in SEQ ID NO.7 and the negative chain shown in SEQ ID NO.8;

[0104] The positive chain shown in SEQ ID NO.9 and the negative chain shown in SEQ ID NO.10;

[0105] The positive chain shown in SEQ ID NO.15 and the negative chain shown in SEQ ID NO.16;

[0106] The positive chain shown in SEQ ID NO.17 and the negative chain shown in SEQ ID NO.18;

[0107] The positive chain shown in SEQ ID NO.19 and the negative chain shown in SEQ ID NO.20;

[0108] The positive chain shown in SEQ ID NO.21 and the negative chain shown in SEQ ID NO.22;

[0109] The positive chain shown in SEQ ID NO.23 and the negative chain shown in SEQ ID NO.24;

[0110] The positive chain shown in SEQ ID NO.25 and the negative chain shown in SEQ ID NO.26;

[0111] The positive chain shown in SEQ ID NO.27 and the negative chain shown in SEQ ID NO.28;

[0112] The positive chain shown in SEQ ID NO.29 and the negative chain shown in SEQ ID NO.30;

[0113] The positive chain shown in SEQ ID NO.33 and the negative chain shown in SEQ ID NO.34;

[0114] The positive chain shown in SEQ ID NO.41 and the negative chain shown in SEQ ID NO.42;

[0115] The positive chain shown in SEQ ID NO.45 and the negative chain shown in SEQ ID NO.46;

[0116] The positive chain shown in SEQ ID NO.49 and the negative chain shown in SEQ ID NO.50;

[0117] The positive chain shown in SEQ ID NO. 53 and the negative chain shown in SEQ ID NO. 54;

[0118] The positive chain shown in SEQ ID NO. 57 and the negative chain shown in SEQ ID NO. 58;

[0119] The positive chain shown in SEQ ID NO. 59 and the negative chain shown in SEQ ID NO. 60;

[0120] The positive chain shown in SEQ ID NO. 61 and the negative chain shown in SEQ ID NO. 62;

[0121] The positive chain shown in SEQ ID NO. 65 and the negative chain shown in SEQ ID NO. 66;

[0122] The positive chain shown in SEQ ID NO. 67 and the negative chain shown in SEQ ID NO. 68;

[0123] The positive chain shown in SEQ ID NO. 69 and the negative chain shown in SEQ ID NO. 70;

[0124] The positive chain shown in SEQ ID NO. 71 and the negative chain shown in SEQ ID NO. 72;

[0125] The positive chain shown in SEQ ID NO.75 and the negative chain shown in SEQ ID NO.76;

[0126] The positive chain shown in SEQ ID NO.77 and the negative chain shown in SEQ ID NO.78;

[0127] The positive chain shown in SEQ ID NO. 83 and the negative chain shown in SEQ ID NO. 84;

[0128] The positive chain shown in SEQ ID NO. 85 and the negative chain shown in SEQ ID NO. 86;

[0129] The positive chain shown in SEQ ID NO. 99 and the negative chain shown in SEQ ID NO. 100;

[0130] The positive chain shown in SEQ ID NO. 105 and the negative chain shown in SEQ ID NO. 106;

[0131] The positive chain shown in SEQ ID NO. 107 and the negative chain shown in SEQ ID NO. 108;

[0132] The positive chain shown in SEQ ID NO. 109 and the negative chain shown in SEQ ID NO. 110;

[0133] The positive chain shown in SEQ ID NO. 113 and the negative chain shown in SEQ ID NO. 114;

[0134] The positive chain shown in SEQ ID NO. 117 and the negative chain shown in SEQ ID NO. 118;

[0135] The positive chain shown in SEQ ID NO. 123 and the negative chain shown in SEQ ID NO. 124;

[0136] The positive chain shown in SEQ ID NO. 125 and the negative chain shown in SEQ ID NO. 126;

[0137] The positive chain shown in SEQ ID NO. 143 and the negative chain shown in SEQ ID NO. 144;

[0138] The positive chain shown in SEQ ID NO. 147 and the negative chain shown in SEQ ID NO. 148;

[0139] The positive chain shown in SEQ ID NO. 151 and the negative chain shown in SEQ ID NO. 152;

[0140] The positive chain shown in SEQ ID NO.161 and the negative chain shown in SEQ ID NO.162;

[0141] The positive chain shown in SEQ ID NO. 167 and the negative chain shown in SEQ ID NO. 168;

[0142] The positive chain shown in SEQ ID NO. 169 and the negative chain shown in SEQ ID NO. 170;

[0143] The positive chain shown in SEQ ID NO. 177 and the negative chain shown in SEQ ID NO. 178;

[0144] The positive chain shown in SEQ ID NO. 187 and the negative chain shown in SEQ ID NO. 188;

[0145] The positive chain shown in SEQ ID NO.191 and the negative chain shown in SEQ ID NO.192;

[0146] The positive chain shown in SEQ ID NO. 205 and the negative chain shown in SEQ ID NO. 206;

[0147] The positive chain shown in SEQ ID NO. 211 and the negative chain shown in SEQ ID NO. 212;

[0148] The positive chain shown in SEQ ID NO. 213 and the negative chain shown in SEQ ID NO. 214;

[0149] The positive chain shown in SEQ ID NO. 217 and the negative chain shown in SEQ ID NO. 218;

[0150] The positive chain shown in SEQ ID NO. 225 and the negative chain shown in SEQ ID NO. 226;

[0151] The positive chain shown in SEQ ID NO. 227 and the negative chain shown in SEQ ID NO. 228;

[0152] The positive chain shown in SEQ ID NO. 235 and the negative chain shown in SEQ ID NO. 236;

[0153] The positive chain shown in SEQ ID NO. 245 and the negative chain shown in SEQ ID NO. 246;

[0154] The positive chain shown in SEQ ID NO. 247 and the negative chain shown in SEQ ID NO. 248;

[0155] The positive chain shown in SEQ ID NO. 251 and the negative chain shown in SEQ ID NO. 252;

[0156] The positive chain shown in SEQ ID NO. 255 and the negative chain shown in SEQ ID NO. 256;

[0157] The positive chain shown in SEQ ID NO. 257 and the negative chain shown in SEQ ID NO. 258;

[0158] The positive chain shown in SEQ ID NO.267 and the negative chain shown in SEQ ID NO.268;

[0159] The positive chain shown in SEQ ID NO. 281 and the negative chain shown in SEQ ID NO. 282;

[0160] The positive chain shown in SEQ ID NO. 63 and the negative chain shown in SEQ ID NO. 64;

[0161] The positive chain shown in SEQ ID NO.97 and the negative chain shown in SEQ ID NO.98;

[0162] The positive chain shown in SEQ ID NO. 171 and the negative chain shown in SEQ ID NO. 172;

[0163] The positive chain shown in SEQ ID NO. 189 and the negative chain shown in SEQ ID NO. 190;

[0164] The positive chain shown in SEQ ID NO.259 and the negative chain shown in SEQ ID NO.260.

[0165] 5. The double-stranded RNA molecule according to claim 3, wherein the sense strand and antisense strand of said double-stranded RNA molecule comprise or are selected from any combination of the following:

[0166] The positive chain shown in SEQ ID NO. 151 and the negative chain shown in SEQ ID NO. 152;

[0167] The positive chain shown in SEQ ID NO.19 and the negative chain shown in SEQ ID NO.20;

[0168] The positive chain shown in SEQ ID NO. 169 and the negative chain shown in SEQ ID NO. 170;

[0169] The positive chain shown in SEQ ID NO.25 and the negative chain shown in SEQ ID NO.26;

[0170] The positive chain shown in SEQ ID NO. 225 and the negative chain shown in SEQ ID NO. 226;

[0171] The positive chain shown in SEQ ID NO.191 and the negative chain shown in SEQ ID NO.192;

[0172] The positive chain shown in SEQ ID NO.23 and the negative chain shown in SEQ ID NO.24;

[0173] The positive chain shown in SEQ ID NO. 217 and the negative chain shown in SEQ ID NO. 218;

[0174] The positive chain shown in SEQ ID NO.161 and the negative chain shown in SEQ ID NO.162;

[0175] The positive chain shown in SEQ ID NO.41 and the negative chain shown in SEQ ID NO.42;

[0176] The positive chain shown in SEQ ID NO.49 and the negative chain shown in SEQ ID NO.50;

[0177] The positive chain shown in SEQ ID NO.27 and the negative chain shown in SEQ ID NO.28;

[0178] The positive chain shown in SEQ ID NO. 53 and the negative chain shown in SEQ ID NO. 54;

[0179] The positive chain shown in SEQ ID NO. 187 and the negative chain shown in SEQ ID NO. 188;

[0180] The positive chain shown in SEQ ID NO.77 and the negative chain shown in SEQ ID NO.78;

[0181] The positive chain shown in SEQ ID NO.33 and the negative chain shown in SEQ ID NO.34;

[0182] The positive chain shown in SEQ ID NO. 147 and the negative chain shown in SEQ ID NO. 148;

[0183] The positive chain shown in SEQ ID NO. 205 and the negative chain shown in SEQ ID NO. 206;

[0184] The positive chain shown in SEQ ID NO. 83 and the negative chain shown in SEQ ID NO. 84;

[0185] The positive chain shown in SEQ ID NO. 61 and the negative chain shown in SEQ ID NO. 62;

[0186] The positive chain shown in SEQ ID NO.9 and the negative chain shown in SEQ ID NO.10;

[0187] The positive chain shown in SEQ ID NO. 177 and the negative chain shown in SEQ ID NO. 178;

[0188] The positive chain shown in SEQ ID NO. 63 and the negative chain shown in SEQ ID NO. 64;

[0189] The positive chain shown in SEQ ID NO. 67 and the negative chain shown in SEQ ID NO. 68;

[0190] The positive chain shown in SEQ ID NO.75 and the negative chain shown in SEQ ID NO.76.

[0191] 6. The double-stranded RNA molecule according to claim 3, wherein the sense strand and antisense strand of said double-stranded RNA molecule comprise or are selected from any combination of the following:

[0192] The positive chain shown in SEQ ID NO. 187 and the negative chain shown in SEQ ID NO. 188;

[0193] The positive chain shown in SEQ ID NO. 225 and the negative chain shown in SEQ ID NO. 226;

[0194] The positive chain shown in SEQ ID NO.41 and the negative chain shown in SEQ ID NO.42;

[0195] The positive chain shown in SEQ ID NO.77 and the negative chain shown in SEQ ID NO.78;

[0196] The positive chain shown in SEQ ID NO.191 and the negative chain shown in SEQ ID NO.192;

[0197] The positive chain shown in SEQ ID NO.19 and the negative chain shown in SEQ ID NO.20;

[0198] The positive chain shown in SEQ ID NO.25 and the negative chain shown in SEQ ID NO.26.

[0199] 7. A modified double-stranded RNA molecule comprising any one of claims 1-6, wherein at least one nucleotide is chemically modified, preferably, the chemical modification comprising replacing the 2' hydroxyl group of the ribosyl group of the nucleotide with another group, and / or modifying a base on the nucleotide, and / or replacing the 5' hydroxyl group of the ribosyl group of the nucleotide with another group, more preferably, the chemically modified nucleotide is selected from at least one of the following: 2'-methoxy modified nucleotides, 2'-fluoro modified nucleotides, 5'-(E)-vinylphosphonate modified nucleotides, 2'-methyl modified nucleotides, deoxy-nucleotides, 2'-deoxy-nucleotides, 3'-terminal deoxy-thymidine (dT) nucleotides, 2'-deoxy-modified nucleotides, locked nucleotides, non-locked nucleotides, configuration-restricted nucleotides, restricted ethyl nucleotides, The chemically modified nucleotides include: baseless nucleotides, 2'-amino-modified nucleotides, 2'-O-allyl-modified nucleotides, 2'-C-alkyl-modified nucleotides, 2'-hydroxy-modified nucleotides, 2'-methoxyethyl-modified nucleotides, 2'-O-alkyl-modified nucleotides, morpholinyl nucleotides, nucleotides containing non-natural bases, tetrahydropyran-modified nucleotides, 1,5-dehydrohexyl alcohol-modified nucleotides, cyclohexenyl-modified nucleotides, glycerol nucleotides (GNA), open-ring nucleotides (UNA), nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphate esters, and nucleotides containing 5'-phosphate ester mimics. More preferably, the chemically modified nucleotides are selected from at least one of the following: 2'-methoxy-modified nucleotides, 2'-fluoro-modified nucleotides, and 5'-(E)-vinylphosphonate-modified nucleotides.

[0200] 8. The modified double-stranded RNA molecule according to claim 7, further comprising modification of the phosphodiester between nucleotides, preferably, the modification of the phosphodiester is a thiophosphate diester modification.

[0201] 9. A modified double-stranded RNA molecule according to claim 7 or 8, wherein the modified double-stranded RNA molecule has the following modification pattern:

[0202] Following the direction from the 5' end to the 3' end, the nucleotides at positions 1-6 and 10-19 of the sense strand are 2'-methoxy modified nucleotides, and the nucleotides at positions 7-9 are 2'-fluorinated modified nucleotides. The nucleotides at positions 1 and 2, and positions 2 and 3 are linked by thiophosphate. The nucleotides at positions 1, 3-5, 7-13, 15, and 17-21 of the antisense strand are 2'-methoxy modified nucleotides, and the nucleotides at positions 2, 6, 14, and 16 are 2'-fluorinated modified nucleotides. The nucleotides at positions 1 and 2, 2 and 3, 19 and 20, and 20 and 21 are linked by thiophosphate.

[0203] 10. The double-stranded RNA molecule described in any of items 1-6 or the modified double-stranded RNA molecule described in any of items 7-9, further coupled with a ligand, preferably, the ligand being selected from one or more small molecule compounds, polypeptides, short peptides, proteins, and antibodies.

[0204] 11. A pharmaceutical composition comprising a double-stranded RNA molecule as described in any one of items 1-6 or a modified double-stranded RNA molecule as described in any one of items 7-10, and a pharmaceutically acceptable carrier.

[0205] 12. The use of any of the double-stranded RNA molecules described in items 1-6, or any of the modified double-stranded RNA molecules described in items 7-10, or the pharmaceutical composition described in item 11, in any of the following:

[0206] D1) Application in the preparation of drugs that inhibit KRAS gene expression;

[0207] Application of D2 in inhibiting KRAS gene expression;

[0208] Application of D3 in the treatment of diseases related to KRAS gene targets;

[0209] Application of D4 in the preparation of drugs for treating diseases related to KRAS gene targets.

[0210] 13. A method for treating a disease related to a KRAS gene target, comprising administering to a subject a therapeutically effective amount of any of the double-stranded RNA molecules described in items 1-6, or any of the modified double-stranded RNA molecules described in items 7-10, or the pharmaceutical composition described in item 11.

[0211] 14. A method for inhibiting KRAS gene expression in cells, the method comprising:

[0212] The cells are brought into contact with any of the double-stranded RNA molecules described in items 1-6, or any of the modified double-stranded RNA molecules described in items 7-10, or the pharmaceutical composition described in item 11.

[0213] Maintaining the cell for a sufficient period of time to allow for the degradation of the KRAS gene mRNA transcript, thereby inhibiting KRAS gene expression in the cell;

[0214] Preferably, the cells are located inside or outside the subject's body;

[0215] More preferably, the subject suffers from a KRAS gene target-related disease.

[0216] 15. The application according to item 12, or the method according to item 13 or 14, wherein the KRAS gene target-related disease is selected from one or more of the following group:

[0217] Metastatic non-small cell lung cancer, metastatic pancreatic ductal adenocarcinoma, KRAS G12C+ non-small cell lung cancer, diabetic retinopathy, leukemia, cholangiocarcinoma, metastatic colorectal cancer, gastric cancer, advanced non-small cell lung cancer, stage IV non-small cell lung cancer, gastric adenocarcinoma, sepsis, KRAS G12C-mutated advanced non-small cell lung cancer, gallbladder cancer, age-related macular degeneration, malignant solid tumors, metastatic pancreatic cancer, non-squamous non-small cell lung cancer, pancreatic cancer, fibrosis, metastatic solid tumors, KRAS G12C-mutated metastatic non-small cell lung cancer, bone metastases, EGFR-mutated advanced non-small cell lung cancer, non-small cell lung cancer, brain metastases, advanced solid tumors, lung cancer, gastroesophageal junction adenocarcinoma, esophageal cancer, KRAS G12D-mutated advanced solid tumors, KRAS / G12C-mutated advanced malignant solid tumors, scleroderma, colorectal cancer, pancreatic ductal adenocarcinoma, colorectal cancer, ovarian cancer, esophageal adenocarcinoma, KRAS G12C mutation metastatic solid tumors.

[0218] This application provides a novel double-stranded RNA molecule targeting KRAS. In vitro and in vivo experiments demonstrate that this double-stranded RNA molecule can effectively inhibit KRAS expression, with an inhibition rate exceeding 85%. This indicates that the double-stranded RNA molecule provided has significant drug development potential and application value in diseases with abnormal KRAS expression, such as tumors. After entering the cell, the double-stranded RNA molecule mediates the formation of an siRNA silencing inducible complex (RISC). The activated RISC localizes to KRAS mRNA through base pairing, causing efficient degradation of KRAS mRNA and a significant reduction in KRAS protein levels.

[0219] The double-stranded RNA molecule of this application can be used for the prevention and treatment of diseases associated with KRAS overexpression or abnormal activation, including but not limited to metastatic non-small cell lung cancer, metastatic pancreatic ductal adenocarcinoma, KRAS G12C+ non-small cell lung cancer, diabetic retinopathy, leukemia, cholangiocarcinoma, metastatic colorectal cancer, gastric cancer, advanced non-small cell lung cancer, stage IV non-small cell lung cancer, gastric adenocarcinoma, sepsis, KRAS G12C-mutated advanced non-small cell lung cancer, gallbladder cancer, age-related macular degeneration, malignant solid tumors, metastatic pancreatic cancer, non-squamous non-small cell lung cancer, pancreatic cancer, fibrosis, metastatic solid tumors, KRAS G12C-mutated metastatic non-small cell lung cancer, bone metastases, EGFR-mutated advanced non-small cell lung cancer, non-small cell lung cancer, brain metastases, advanced solid tumors, lung cancer, gastroesophageal junction adenocarcinoma, esophageal cancer, and KRAS. Advanced solid tumors with G12D mutations, advanced malignant solid tumors with KRAS / G12C mutations, scleroderma, colorectal cancer, pancreatic ductal adenocarcinoma, colorectal cancer, ovarian cancer, esophageal adenocarcinoma, and metastatic solid tumors with KRAS G12C mutations. Detailed Implementation

[0220] The present application is further illustrated below with reference to embodiments. It should be understood that the embodiments are only used to further illustrate and explain the present application and are not intended to limit the present application.

[0221] Unless otherwise defined, technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art. While similar or identical methods and materials may be applied in experimental or practical applications, materials and methods are described herein. In case of conflict, the definitions included herein shall prevail. Furthermore, materials, methods, and examples are for illustrative purposes only and are not intended to be limiting. The present application is further described below with reference to specific embodiments, but is not intended to limit the scope of the application.

[0222] definition

[0223] In this application, "siRNA" is defined as a class of double-stranded RNA molecules that can mediate the silencing of a target RNA (e.g., mRNA, such as a transcript of a gene encoding a protein) that is complementary to it. siRNA comprises an antisense strand complementary to the target RNA and a sense strand complementary to that antisense strand. For convenience, such mRNA is also referred to herein as the mRNA to be silenced. Such genes are also referred to as target genes. Typically, the RNA to be silenced is an endogenous gene or a pathogen gene. Additionally, RNA other than mRNA (e.g., tRNA) and viral RNA can also be targeted.

[0224] In this application, "antisense strand" refers to a strand of siRNA that contains regions that are fully or substantially complementary to the target sequence. As used herein, the term "complementary region" refers to a region on the antisense strand that is substantially complementary to a sequence (e.g., the target sequence). When the complementary region is not fully complementary to the target sequence, the mismatch typically occurs in the internal or terminal regions of the molecule. In some embodiments, the double-stranded nucleic acid molecule includes nucleotide mismatches in the antisense strand.

[0225] In this application, as understood by those skilled in the art, the term “complementary” when used to describe the first nucleoside sequence relative to the second nucleoside sequence refers to the ability of an oligonucleotide containing the first nucleoside sequence to hybridize with an oligonucleotide containing the second nucleoside sequence under certain conditions and form a double-stranded structure.

[0226] In the application, the term "sense chain" refers to a chain of siRNA that includes regions substantially complementary to the region defined herein as the antisense chain.

[0227] In this application, the term "protrusion" refers to at least one unpaired nucleotide that protrudes from the double-stranded structure of the siRNA. A nucleotide protrusion exists, for example, when the 3' end of one strand of the siRNA extends beyond the 5' end of the other strand, or vice versa. The siRNA may contain a protrusion having at least one nucleotide; alternatively, the protrusion may contain at least two, three, four, five, or more nucleotides. The nucleotide protrusion may contain or consist of a nucleotide / nucleoside analogue (including deoxynucleotides / nucleosides). One or more protrusions may be located on the sense strand, the antisense strand, or any combination thereof. Additionally, one or more nucleotides of the protrusion may be present at the 5' end, 3' end, or both ends of the antisense strand or the sense strand of the siRNA.

[0228] In this application, complementary sequences within a double-stranded RNA molecule include base pairings along the entire length of the sense and antisense strands. Such sequences may be referred to as “perfectly complementary” to each other in this application. However, when the sense strand is referred to as “fundamentally complementary” or “partially complementary” relative to the antisense strand herein, the two sequences may be perfectly complementary, or they may form one or more mismatched base pairs, such as 2, 4, or 5 mismatched base pairs, but preferably no more than 5, while maintaining the ability to hybridize under the conditions most relevant to its final application. In determining complementarity, overhangs should not be considered mismatches. For example, a double-stranded RNA molecule containing a 19-nucleotide sense strand and a 21-nucleotide antisense strand, where the longer nucleotide contains a 19-nucleotide sequence perfectly complementary to the shorter nucleotide, can still be referred to as “perfectly complementary.”

[0229] In this application, "modified nucleotide" refers to a nucleotide or nucleotide analog formed by replacing the 2'-hydroxyl group of the ribosyl group with another group, or a nucleotide or nucleotide analog formed by replacing the 5'-hydroxyl group of the ribosyl group with another group, or a nucleotide in which the bases are modified bases. "Methoxy-modified nucleotide" refers to a nucleotide formed by replacing the 2'-hydroxyl group of the ribosyl group with a methoxy group. "Fluoro-modified nucleotide" refers to a nucleotide formed by replacing the 2'-hydroxyl group of the ribosyl group with fluorine. "Nucleotide analog" refers to a group that can replace a nucleotide in nucleic acids but has a structure different from adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, or thymine deoxyribonucleotide. Examples include isonucleotides, bridged nucleic acids (BNA), or acyclic nucleotides.

[0230] In this application, "ligand" refers to a chemical moiety conjugated to siRNA that can alter the distribution, targeting, or lifetime of the siRNA. In a preferred embodiment, such a ligand provides enhanced affinity for selected targets (e.g., molecules, cells or cell types), compartments (e.g., cell or organ compartments, tissues, organs, or regions of the body) compared to siRNA, for example, siRNA without such a ligand.

[0231] In this application, Base represents a modified or unmodified nucleotide base A, U, G, C, T or other nucleotide base.

[0232] In this application, the term “suppression” is used interchangeably with “reduction,” “silence,” “downregulation,” and other similar terms, and includes any level of suppression.

[0233] In this application, "inhibiting KRAS expression" means inhibiting the expression of any KRAS gene and its variants or mutants. Therefore, the KRAS gene can be a wild-type KRAS gene, a mutant KRAS gene, or a transgenic KRAS gene in the case of genetically manipulated cells, cell groups, or organisms.

[0234] In this application, "inhibition of KRAS gene expression" includes inhibition of any level of the KRAS gene, such as at least partial repression of KRAS gene expression. KRAS gene expression can be assessed based on the level or level change of any variable associated with KRAS gene expression, such as KRAS mRNA level, KRAS protein level, or lipid level. This level can be assessed in individual cells or in a group of cells (including, for example, samples derived from a subject).

[0235] Inhibition can be assessed by a decrease in the absolute or relative level of one or more variables associated with KRAS expression compared to a control level. A control level can be any type of control level utilized in the art, such as baseline levels before administration or levels determined from similar untreated or controlled (e.g., buffer-only control or inert agent control) subjects, cells, or samples.

[0236] In this application, "treatment" means a beneficial or desired outcome, including but not limited to the relief or improvement of one or more symptoms related to gene expression. "Treatment" can also mean extended survival compared to expected survival without treatment. Treatment may include prevention of the development of comorbidities, such as reducing liver injury in individuals with liver infections.

[0237] In this application, "therapeutic effective amount" is intended to include an amount of nucleic acid (e.g., siRNA) that, when administered to a patient to treat a subject with a disease, is sufficient to achieve treatment of the disease (e.g., by alleviating, improving, or maintaining an existing disease, or one or more symptoms of the disease or its associated comorbidities).

[0238] In this application, "pharmaceutically acceptable" means a compound, material, composition, or dosage form suitable for tissue contact with human and animal subjects without excessive toxicity, irritation, allergic reactions, or other problems or complications, and with a reasonable benefit / risk ratio.

[0239] In this application, "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or medium, such as a liquid or solid filler, diluent, excipient, manufacturing aid, or solvent encapsulating material, for carrying or transporting a target compound from one organ or body part to another. Each carrier must be "acceptable," meaning it is compatible with other components in the formulation and will not cause harm to the subject receiving treatment.

[0240] Double-stranded RNA molecules

[0241] On one hand, this application provides an unmodified double-stranded RNA molecule, specifically an siRNA, wherein the double-stranded RNA molecule comprises a sense strand and an antisense strand that at least partially form a double-stranded region, the antisense strand comprising 17-21 (e.g., 17, 18, 19, 20, or 21) consecutive nucleotides of any even-numbered nucleotide sequence in SEQ ID NO. 1-300, and the sense strand comprising 17-19 (e.g., 17, 18, or 19) consecutive nucleotides of any odd-numbered nucleotide sequence in SEQ ID NO. 1-300.

[0242] In some embodiments, the length of the double-stranded region is 15-30 bp, for example, it can be 15 bp, 16 bp, 17 bp, 18 bp, 19 bp, 20 bp, 21 bp, 22 bp, 23 bp, 24 bp, 25 bp, 26 bp, 27 bp, 28 bp, 29 bp, or 30 bp. In some embodiments, the length of the double-stranded region is 23-27 bp. In some embodiments, the length of the double-stranded region is 21-23 bp. In some embodiments, the length of the double-stranded region is 19-21 bp. In some embodiments, the length of the double-stranded region is 17-25 bp. In some embodiments, the length of the double-stranded region is 17-23 bp. In some embodiments, the length of the double-stranded region is 17-19 bp. In some embodiments, the length of the double-stranded region is 19 bp.

[0243] In some embodiments, the length of the positive strand does not exceed 30 nucleotides, and / or the length of the negative strand does not exceed 30 nucleotides. In some embodiments, the length of the positive strand does not exceed 21 nucleotides, and / or the length of the negative strand does not exceed 23 nucleotides. In some embodiments, the length of the positive strand does not exceed 19 nucleotides, and / or the length of the negative strand does not exceed 21 nucleotides.

[0244] In some embodiments, at least one of the sense and antisense strands includes a 3' overhang having at least one or at least two nucleotides.

[0245] In some embodiments, the sense strand of the double-stranded RNA molecule has a nucleotide sequence with any odd number in SEQ ID NO. 1-300, and the antisense strand has a nucleotide sequence with any even number in SEQ ID NO. 1-300.

[0246] In some embodiments, the sense and antisense strands of the double-stranded RNA molecule include or are any combination of those listed in Table 3 of this application.

[0247] In some embodiments, the sense and antisense strands of the double-stranded RNA molecule comprise or are any combination selected from the following:

[0248] The following chains of reference are listed: SEQ ID NO.1 (forward chain) and SEQ ID NO.2 (antisense chain); SEQ ID NO.7 (forward chain) and SEQ ID NO.8 (antisense chain); SEQ ID NO.9 (forward chain) and SEQ ID NO.10 (antisense chain); SEQ ID NO.15 (forward chain) and SEQ ID NO.16 (antisense chain); SEQ ID NO.17 (forward chain) and SEQ ID NO.18 (antisense chain); SEQ ID NO.19 (forward chain) and SEQ ID NO.20 (antisense chain); SEQ ID NO.21 (forward chain) and SEQ ID NO.22 (antisense chain); SEQ ID NO.23 (forward chain) and SEQ ID NO.24 (antisense chain); SEQ ID NO.25 (forward chain) and SEQ ID NO.26 (antisense chain); SEQ ID NO.27 (forward chain) and SEQ ID NO.28 (antisense chain); SEQ ID NO.29 (forward chain) and SEQ ID NO.30 (antisense chain); SEQ ID NO.33 (forward chain) and SEQ ID NO. The following are examples of SEQ ID NO. 34: the antisense chain; SEQ ID NO. 41: the right-hand chain and SEQ ID NO. 42: the antisense chain; SEQ ID NO. 45: the right-hand chain and SEQ ID NO. 46: the antisense chain; SEQ ID NO. 49: the right-hand chain and SEQ ID NO. 50: the antisense chain; SEQ ID NO. 53: the right-hand chain and SEQ ID NO. 54: the antisense chain; SEQ ID NO. 57: the right-hand chain and SEQ ID NO. 58: the antisense chain; SEQ ID NO. 59: the right-hand chain and SEQ ID NO. 60: the antisense chain; SEQ ID NO. 61: the right-hand chain and SEQ ID NO. 62: the antisense chain; SEQ ID NO. 65: the right-hand chain and SEQ ID NO. 66: the antisense chain; SEQ ID NO. 67: the right-hand chain and SEQ ID NO. 68: the antisense chain; SEQ ID NO. 69: the right-hand chain and SEQ ID NO. 70: the antisense chain; SEQ ID NO. 71: the right-hand chain and SEQ ID NO. 72: the antisense chain; SEQ ID The positive chain shown in SEQ ID NO. 75 and the negative chain shown in SEQ ID NO. 76; the positive chain shown in SEQ ID NO. 77 and the negative chain shown in SEQ ID NO. 78; the positive chain shown in SEQ ID NO. 83 and the negative chain shown in SEQ ID NO. 84; SEQ ID NO.The following are examples of SEQ ID NO. 85 and SEQ ID NO. 86: the positive chain and the negative chain; SEQ ID NO. 99 and SEQ ID NO. 100: the positive chain and the negative chain; SEQ ID NO. 105 and SEQ ID NO. 106: the positive chain and the negative chain; SEQ ID NO. 107 and SEQ ID NO. 108: the positive chain and the negative chain; SEQ ID NO. 109 and SEQ ID NO. 110: the positive chain and the negative chain; SEQ ID NO. 113 and SEQ ID NO. 114: the positive chain and the negative chain; SEQ ID NO. 117 and SEQ ID NO. 118: the positive chain; SEQ ID NO. 123 and SEQ ID NO. 124: the positive chain; SEQ ID NO. 125 and SEQ ID NO. 126: the positive chain; SEQ ID NO. 143 and SEQ ID NO. 144: the positive chain; SEQ ID NO. 147 and SEQ ID NO. 85: the positive chain and the negative chain; SEQ ID NO. 146: the positive chain and the negative chain; SEQ ID NO. 147: the positive chain and the negative chain; SEQ ID NO. 148: the positive chain and the negative chain; SEQ ID NO. 149: the positive chain and the negative chain; SEQ ID NO. 140 ... The following are examples of SEQ ID NO. 148: the antisense chain; SEQ ID NO. 151: the right-hand chain and SEQ ID NO. 152: the antisense chain; SEQ ID NO. 161: the right-hand chain and SEQ ID NO. 162: the antisense chain; SEQ ID NO. 167: the right-hand chain and SEQ ID NO. 168: the antisense chain; SEQ ID NO. 169: the right-hand chain and SEQ ID NO. 170: the antisense chain; SEQ ID NO. 177: the right-hand chain and SEQ ID NO. 178: the antisense chain; SEQ ID NO. 187: the right-hand chain and SEQ ID NO. 188: the antisense chain; SEQ ID NO. 191: the right-hand chain and SEQ ID NO. 192: the antisense chain; SEQ ID NO. 205: the right-hand chain and SEQ ID NO. 206: the antisense chain; SEQ ID NO. 211: the right-hand chain and SEQ ID NO. 212: the antisense chain; SEQ ID NO. 213: the right-hand chain and SEQ ID NO. 214: the antisense chain; SEQ ID NO. The positive chain shown in SEQ ID NO. 217 and the negative chain shown in SEQ ID NO. 218; the positive chain shown in SEQ ID NO. 225 and the negative chain shown in SEQ ID NO. 226; the positive chain shown in SEQ ID NO. 227 and the negative chain shown in SEQ ID NO. 228; the positive chain shown in SEQ ID NO. 235 and the negative chain shown in SEQ ID NO. 236; SEQ ID NO.The following are examples of SEQ ID NO. 245 and SEQ ID NO. 246: the positive chain and the negative chain; SEQ ID NO. 247 and SEQ ID NO. 248: the positive chain and the negative chain; SEQ ID NO. 251 and SEQ ID NO. 252: the positive chain and the negative chain; SEQ ID NO. 255 and SEQ ID NO. 256: the positive chain and the negative chain; SEQ ID NO. 257 and SEQ ID NO. 258: the positive chain and the negative chain; SEQ ID NO. 267 and SEQ ID NO. 268: the positive chain and the negative chain; SEQ ID NO. 281 and SEQ ID NO. 282: the positive chain; SEQ ID NO. 63 and SEQ ID NO. 64: the positive chain; SEQ ID NO. 97 and SEQ ID NO. 98: the positive chain; SEQ ID NO. 171 and SEQ ID NO. 172: the positive chain; SEQ ID NO. 189 and SEQ ID NO. 245: the positive chain and the negative chain; SEQ ID NO. 245: the positive chain and the negative chain; SEQ ID NO. 246: the positive chain and the negative chain; SEQ ID NO. 247: the positive chain and the negative chain; SEQ ID NO. 248: the positive chain and the negative chain; SEQ ID NO. 249 ... The antisense chain shown in NO. 190; the right chain shown in SEQ ID NO. 259; and the antisense chain shown in SEQ ID NO. 260.

[0249] In some embodiments, the sense and antisense strands of the double-stranded RNA molecule comprise or are any combination selected from the following:

[0250] The following are examples of the same sequence of numbers: SEQ ID NO. 151 (forward chain) and SEQ ID NO. 152 (antisense chain); SEQ ID NO. 19 (forward chain) and SEQ ID NO. 20 (antisense chain); SEQ ID NO. 169 (forward chain) and SEQ ID NO. 170 (antisense chain); SEQ ID NO. 25 (forward chain) and SEQ ID NO. 26 (antisense chain); SEQ ID NO. 225 (forward chain) and SEQ ID NO. 226 (antisense chain); SEQ ID NO. 191 (forward chain) and SEQ ID NO. 192 (antisense chain); SEQ ID NO. 23 (forward chain) and SEQ ID NO. 24 (antisense chain); SEQ ID NO. 217 (forward chain) and SEQ ID NO. 218 (antisense chain); SEQ ID NO. 161 (forward chain) and SEQ ID NO. 162 (antisense chain); SEQ ID NO. 41 (forward chain) and SEQ ID NO. 42 (antisense chain); SEQ ID NO. 49 (forward chain) and SEQ ID NO. 50 (antisense chain); SEQ ID NO. The following are examples of SEQ ID NO. 27 and SEQ ID NO. 28: the right chain and the wrong chain; SEQ ID NO. 53 and SEQ ID NO. 54: the right chain and the wrong chain; SEQ ID NO. 187 and SEQ ID NO. 188: the right chain and the wrong chain; SEQ ID NO. 77 and SEQ ID NO. 78: the right chain and the wrong chain; SEQ ID NO. 33 and SEQ ID NO. 34: the right chain and the wrong chain; SEQ ID NO. 147 and SEQ ID NO. 148: the right chain and the wrong chain; SEQ ID NO. 205 and SEQ ID NO. 206: the right chain and the wrong chain; SEQ ID NO. 83 and SEQ ID NO. 84: the right chain and the wrong chain; SEQ ID NO. 61 and SEQ ID NO. 62: the right chain and the wrong chain; SEQ ID NO. 9 and SEQ ID NO. 10: the right chain; SEQ ID NO. 177 and SEQ ID NO. 178: the right chain and the wrong chain; SEQ ID The positive chain shown in NO. 63 and the negative chain shown in SEQ ID NO. 64; the positive chain shown in SEQ ID NO. 67 and the negative chain shown in SEQ ID NO. 68; the positive chain shown in SEQ ID NO. 75 and the negative chain shown in SEQ ID NO. 76.

[0251] In some embodiments, the sense and antisense strands of the double-stranded RNA molecule comprise or are any combination selected from the following:

[0252] The positive chain shown in SEQ ID NO. 187 and the negative chain shown in SEQ ID NO. 188; the positive chain shown in SEQ ID NO. 225 and the negative chain shown in SEQ ID NO. 226; the positive chain shown in SEQ ID NO. 41 and the negative chain shown in SEQ ID NO. 42; the positive chain shown in SEQ ID NO. 77 and the negative chain shown in SEQ ID NO. 78; the positive chain shown in SEQ ID NO. 191 and the negative chain shown in SEQ ID NO. 192; the positive chain shown in SEQ ID NO. 19 and the negative chain shown in SEQ ID NO. 20; the positive chain shown in SEQ ID NO. 25 and the negative chain shown in SEQ ID NO. 26.

[0253] On the other hand, this application also provides modified double-stranded RNA molecules, comprising any of the aforementioned double-stranded RNA molecules, wherein at least one nucleotide is chemically modified. That is, it is obtained by modifying at least one nucleotide of any of the aforementioned unmodified double-stranded RNA molecules, resulting in a compound containing the modified nucleotide.

[0254] The chemical modification can be the substitution of the 2' hydroxyl group of the ribosyl group of the nucleotide by other groups, or the modification of the bases on the nucleotide, or the substitution of the 5' hydroxyl group of the ribosyl group of the nucleotide by other groups, or the modification of the phosphodiester between nucleotides, or any combination of these types of modifications.

[0255] In some embodiments, the chemical modification includes replacing the 2' hydroxyl group of the nucleotide with another group, and / or modifying the bases on the nucleotide, and / or replacing the 5' hydroxyl group of the nucleotide with another group.

[0256] In some embodiments, the modified nucleotide is selected from at least one of the following:

[0257] 2'-Methoxy-modified nucleotides, 2'-fluoro-modified nucleotides, 5'-(E)-vinylphosphonate-modified nucleotides, 2'-methyl-modified nucleotides, deoxy-nucleotides, 2'-deoxy-nucleotides, 3'-terminal deoxy-thymidine (dT) nucleotides, 2'-deoxy-modified nucleotides, locked nucleotides, non-locked nucleotides, configuration-restricted nucleotides, restricted ethyl nucleotides, base-free nucleotides, 2'-amino-modified nucleotides, 2'-O-allyl-modified nucleotides, 2'-C 2'-alkyl-modified nucleotides, 2'-hydroxy-modified nucleotides, 2'-methoxyethyl-modified nucleotides, 2'-O-alkyl-modified nucleotides, morpholinonucleotides, nucleotides containing non-natural bases, tetrahydropyran-modified nucleotides, 1,5-dehydrohexyl-modified nucleotides, cyclohexenyl-modified nucleotides, glycerol nucleotides (GNA), open-ring nucleotides (UNA), nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphate esters, and nucleotides containing 5'-phosphate ester mimics.

[0258] In some embodiments, the chemically modified nucleotide is selected from at least one of the following: 2'-methoxy modified nucleotides, 2'-fluoro modified nucleotides, and 5'-(E)-vinylphosphonate modified nucleotides.

[0259] In some embodiments, the chemical modification further includes modification of the phosphodiester between the nucleotides. In some embodiments, the modification of the phosphodiester is a thiophosphate diester modification.

[0260] In one embodiment of this application, "fluorinated nucleotide" refers to a nucleotide formed by replacing the hydroxyl group at the 2' position of the ribosome with fluorine. The non-fluorinated nucleotide is independently selected from nucleotides or nucleotide analogs formed by replacing the hydroxyl group at the 2' position of the ribosome with a non-fluorinated group.

[0261] In one embodiment of this application, the nucleotide formed by replacing the hydroxyl group at the 2' position of the ribosome with a non-fluorinated group is well known to those skilled in the art. These nucleotides may be selected from one of the following: 2'-alkoxy modified nucleotides, 2'-substituted alkoxy modified nucleotides, 2'-alkyl modified nucleotides, 2'-substituted alkyl modified nucleotides, 2'-amino modified nucleotides, 2'-substituted amino modified nucleotides, and 2'-deoxynucleotides.

[0262] In some embodiments, the modified double-stranded RNA molecule is arranged in a 5' to 3' direction, with the nucleotides at positions 1-6 and 10-19 of the sense strand being 2'-methoxy modified nucleotides, the nucleotides at positions 7-9 being 2'-fluoro modified nucleotides, and the nucleotides at positions 1 and 2, and 2 and 3 linked by thiophosphate; the nucleotides at positions 1, 3-5, 7-13, 15, and 17-21 of the antisense strand being 2'-methoxy modified nucleotides, the nucleotides at positions 2, 6, 14, and 16 being 2'-fluoro modified nucleotides, and the nucleotides at positions 1 and 2, 2 and 3, 19 and 20, and 20 and 21 linked by thiophosphate.

[0263] The double-stranded RNA molecules used in this application can all be prepared using methods known in the art. For example, phosphoramide solid-phase synthesis is a method known to those skilled in the art for the synthesis of siRNA. All nucleoside monomers used in this application are commercially available.

[0264] The unmodified or modified double-stranded RNA molecules of this application can be further coupled with ligands.

[0265] The ligand is known in the art and can be selected from small molecule compounds, polypeptides, short peptides, proteins, antibodies, etc.

[0266] The ligand can be coupled to any nucleotide at the 3'-terminus, 5'-terminus, or middle of the sense strand. In some embodiments, the ligand is coupled to the 3'-terminus of the sense strand.

[0267] Those skilled in the art will understand that the coupling of the ligand to the sense strand can be direct coupling or indirect coupling via a linker. In some embodiments, the ligand is coupled to the 3'-terminus of the sense strand via a linker.

[0268] Pharmaceutical Composition

[0269] This application also provides a pharmaceutical composition comprising the above-described unmodified double-stranded RNA molecule or modified double-stranded RNA molecule, and a pharmaceutically acceptable carrier.

[0270] The pharmaceutically acceptable carrier may be an excipient, stabilizer, suspending agent, or diluent, as is well known to those skilled in the art.

[0271] The pharmaceutical composition of this application can be administered at a dose sufficient to inhibit gene expression. Typically, a suitable dose of the double-stranded RNA molecule of this application is about 0.001 to about 200.0 mg per kilogram of body weight per day, usually about 1 to 50 mg per kilogram of body weight per day. Typically, a suitable dose of the double-stranded RNA molecule of this application is about 0.1 mg / kg to about 10 mg / kg, for example, about 1 mg / kg to about 5 mg / kg, about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg.

[0272] Repeated dosing regimens may include the administration of therapeutic doses of nucleic acid at regular intervals (e.g., every other day or once a year). In some implementations, the frequency of administration of nucleic acid (e.g., siRNA) is from about once a month to about once a year, such as about once every three months, about once every six months, or about once every nine months.

[0273] In some embodiments, the double-stranded RNA molecule of this application is administered at a dose of about 0.1 mg / kg to about 5.0 mg / kg, approximately once a week, once a month, once every two months, once a quarter (i.e., once every three months), once every six months, once every nine months, or once a year. In some embodiments, the double-stranded RNA molecule of this application is administered to the subject once a week. In some embodiments, the double-stranded RNA molecule of this application is administered to the subject once a month. In some embodiments, the double-stranded RNA molecule of this application is administered once a quarter (i.e., once every three months). In some embodiments, the double-stranded RNA molecule of this application is administered once every six months.

[0274] After the initial treatment regimen, the frequency of treatment can be reduced. For example, after applying once a week or every two weeks for three months, it can be repeated once a month for six months or a year; or longer.

[0275] The pharmaceutical composition of this application can be administered in a variety of ways, depending on whether local or systemic treatment is required and depending on the area to be treated. Administration can be local (e.g., via a transdermal patch), pulmonary (e.g., by inhalation or blowing of powder or aerosol, including via a nebulizer), intratracheal, intranasal, epidermal and transdermal, oral, or parenteral. Parenteral administration includes intravenous, intra-arterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; subcutaneous, for example, via an implanted device; or intracranial, for example, via intraparenchymal, intrathecal, or intraventricular administration. In some preferred embodiments, the composition is administered by intravenous infusion or injection. In some embodiments, the composition is administered by subcutaneous injection. In some embodiments, the double-stranded RNA molecule of this application is administered subcutaneously to the subject.

[0276] Treatment methods and therapeutic uses

[0277] This application also provides the use of the above-mentioned unmodified double-stranded RNA molecules or modified double-stranded RNA molecules in any of the following:

[0278] D1) Application in the preparation of drugs that inhibit KRAS gene expression;

[0279] Application of D2 in inhibiting KRAS gene expression;

[0280] Application of D3 in the treatment of diseases related to KRAS gene targets;

[0281] Application of D4 in the preparation of drugs for treating diseases related to KRAS gene targets.

[0282] Inhibition of KRAS gene expression can refer to inhibition of at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 100%.

[0283] KRAS gene target-related diseases can encompass diseases known in the art caused by abnormal KRAS gene expression.

[0284] In some embodiments, the disease caused by abnormal KRAS gene expression is a disease caused by upregulation of KRAS gene expression.

[0285] In some embodiments, the KRAS gene target-related diseases are selected from one or more of the following groups:

[0286] Metastatic non-small cell lung cancer, metastatic pancreatic ductal adenocarcinoma, KRAS G12C+ non-small cell lung cancer, diabetic retinopathy, leukemia, cholangiocarcinoma, metastatic colorectal cancer, gastric cancer, advanced non-small cell lung cancer, stage IV non-small cell lung cancer, gastric adenocarcinoma, sepsis, KRAS G12C-mutated advanced non-small cell lung cancer, gallbladder cancer, age-related macular degeneration, malignant solid tumors, metastatic pancreatic cancer, non-squamous non-small cell lung cancer, pancreatic cancer, fibrosis, metastatic solid tumors, KRAS G12C-mutated metastatic non-small cell lung cancer, bone metastases, EGFR-mutated advanced non-small cell lung cancer, non-small cell lung cancer, brain metastases, advanced solid tumors, lung cancer, gastroesophageal junction adenocarcinoma, esophageal cancer, KRAS G12D-mutated advanced solid tumors, KRAS / G12C-mutated advanced malignant solid tumors, scleroderma, colorectal cancer, pancreatic ductal adenocarcinoma, colorectal cancer, ovarian cancer, esophageal adenocarcinoma, KRAS G12C mutation metastatic solid tumors.

[0287] In some embodiments, the KRAS gene target-related diseases are selected from one or more of the following groups:

[0288] Bile duct cancer, cervical cancer, bladder cancer, liver cancer, breast cancer.

[0289] This application also provides a method for inhibiting KRAS gene expression in cells, the method comprising:

[0290] The cells are brought into contact with the above-mentioned unmodified double-stranded RNA molecule or modified double-stranded RNA molecule, or the above-mentioned pharmaceutical composition;

[0291] Maintaining the cell for a sufficient period of time to allow for the degradation of the KRAS gene mRNA transcript can suppress KRAS gene expression in the cell.

[0292] In some embodiments, the cells are located inside the subject. In some embodiments, the cells are located outside the subject. The subject can be a mammal, including primates (e.g., humans or non-human primates, such as monkeys or chimpanzees) and non-primates (e.g., cattle, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, mice, horses, or whales). In some embodiments, the subject is a human.

[0293] In some embodiments, the subject suffers from a KRAS gene target-related disease. The KRAS gene target-related disease is as described above.

[0294] Example

[0295] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0296] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0297] The experimental reagents, consumables, and instruments used in the following examples are as follows:

[0298] Table 2 Experimental Reagents and Consumables

[0299] For any experimental steps or conditions not specified in the following examples, the procedures or conditions described in the literature in this field can be followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0300] Example 1: Preparation and synthesis of siRNA to inhibit KRAS gene expression

[0301] 1. KRAS mRNA

[0302] KRAS mRNA refers to mRNAs with GeneBank registration numbers NM_004985.5, NM_033360.4, NM_033360.4, NM_001369787.1, XM_005570378.3, XM_005570379.3, XM_005570381.3, NM_021284.7, NM_001403240.1, and NM_00140324. 1.1. mRNAs containing the sequences shown in NM_001403242.1, NM_001403243.1, NM_001403244.1, NM_001403245.1, NM_001403246.1, NM_005343.4, NM_176795.5, NM_001130442.3, NM_001318054.2, or NM_002524.5.

[0303] 2. Design and synthesis of KRAS siRNA

[0304] siRNAs were designed based on the NM_002524.5 transcript of KRAS mRNA (submission date: 01-JUL-2024). The siRNA sequences used in the experiments were synthesized by GenePharma using a solid-phase synthesis method known in the art and stored at -20°C. The sequences of the unmodified KRAS siRNAs are shown in Table 3.

[0305] Table 3 Unmodified KRAS siRNA sequences

[0306] In Table 3 above, column 1 or 6 is the name of the KRAS siRNA, column 2 or 7 is the KRAS siRNA sense strand, column 4 or 9 is the KRAS siRNA antisense strand, column 3 is the sequence number of the KRAS siRNA sense strand in column 2, column 5 is the sequence number of the KRAS siRNA antisense strand in column 4, column 8 is the sequence number of the KRAS siRNA sense strand in column 7, and column 10 is the sequence number of the KRAS siRNA antisense strand in column 9.

[0307] The KRAS siRNA sequences shown in Table 3 were modified according to the M1 modification pattern, and the modified siRNA was obtained using the solid-phase synthesis method described above.

[0308] M1 Modification Mode:

[0309] Chain of Justice 5'-3' ms-ms-mmmmfffmmmmmmmmmm

[0310] That is, the nucleotides at positions 1-6 and 10-19 of the positive chain are 2'-methoxy modified nucleotides, the nucleotides at positions 7-9 are 2'-fluorinated modified nucleotides, and the nucleotides at positions 1 and 2, as well as positions 2 and 3, are linked by thiophosphates.

[0311] antisense chain 5'-3' ms-fs-mmmfmmmmmmmfmfmm-ms-ms-m

[0312] That is, the nucleotides at positions 1, 3-5, 7-13, 15, and 17-21 of the antisense strand are 2'-methoxy modified nucleotides, the nucleotides at positions 2, 6, 14, and 16 are 2'-fluorinated modified nucleotides, and the nucleotides at positions 1 and 2, 2 and 3, 19 and 20, and 20 and 21 are linked by thiophosphates.

[0313] In the M1 modification pattern described above, m indicates that the nucleotide is 2'-methoxy modified, f indicates that the nucleotide is 2'-F modified, and s indicates that the two nucleotides are linked by a phosphate thioester.

[0314] According to the M1 modification pattern, the unmodified siRNAs in Table 3 are modified to obtain M1-modified siRNAs. The name of the modified siRNA is the corresponding naked sequence name followed by M1. For example, the unmodified siRNA named Kras-1 is modified by M1 to obtain the modified siRNA named Kras-1M1, and the unmodified siRNA named Kras-85 is modified by M1 to obtain the modified siRNA named Kras-85M1.

[0315] An example of an M1-modified siRNA is as follows: For instance, the siRNA modified with M1 pattern according to Kras-85 is designated Kras-85M1, and its corresponding positive strand is: UmsCmsAmCmCmAmUfUfAfUmAmGmAmGmAmAmCmAmAm(SEQ ID NO.303),

[0316] The ansense chain is: UmsUfsGmUmUmCfUmCmUmAmUmAmAmUfGmGfUmGmAmsAmsUm(SEQ ID NO.304).

[0317] Example 2: Screening of siRNAs that inhibit KRAS gene expression

[0318] I. Preliminary screening of highly active siRNAs by measuring the inhibitory level of KRAS mRNA in cells through single-concentration transfection with modified KRAS siRNA.

[0319] 1. Obtaining cell culture plates

[0320] Human pancreatic cancer cell line PANC-1 was purchased from Wuhan Pronosei Biotechnology Co., Ltd.; human non-small cell lung cancer cell line A549 was purchased from the Chinese Academy of Sciences Cell Bank. Cells were cultured according to the manufacturer's instructions. PANC-1 / A549 cells cultured in 10cm culture dishes were passaged for 48 hours, then digested with trypsin, and resuspended in complete culture medium to prepare 6×10⁶ cells / mL. 5 Cell suspension per mL. 50 μL was seeded into each well of a 96-well plate and incubated at 37°C in a 5% CO2, 95% aerosol atmosphere.

[0321] 2. Transfection

[0322] Divided into the following groups:

[0323] Kras siRNA group: 150 modified KRAS siRNAs were transfected (siRNAs in Table 3 were siRNAs modified according to the M1 modification pattern);

[0324] NC group: transfected with negative control siRNA (NC) as a negative control;

[0325] MOCK group: Only transfection reagent is added, without any siRNA sequence.

[0326] BLANK group: Normal cell culture group, without any other components added.

[0327] Systemic control group: Candidate sequence KRAS-104M1, used as the systemic reference sequence.

[0328] siRNA transfection was performed using the Lipofectamine RNAiMAX transfection reagent. The transfection complex was prepared according to the manufacturer's instructions. For example, to prepare a single-well transfection complex: siRNA was diluted with 25 μL of Opti-MEM and gently mixed to prepare the siRNA dilution buffer; 25 μL of Opti-MEM and 0.25 μL of Lipofectamine RNAiMAX transfection reagent were gently mixed to prepare the transfection reagent dilution buffer. The two dilution buffers were mixed and gently stirred, incubated at room temperature for 10-20 minutes, and then added to the corresponding wells of the cell plate, resulting in a final siRNA concentration of 10 nM. After 48 hours of transfection, the cell culture medium in each well was discarded, and 50 μL of cell lysis buffer (Germage) was added to each well. After incubation for 5 minutes for complete lysis, 5 μL of stop solution was added to each well to obtain the final lysis product.

[0329] 3. RT-qPCR detection

[0330] The above cell lysis products were treated with DNase I to remove genomic DNA, and then an RNA template was obtained.

[0331] Prepare the RT-qPCR reaction system as shown in Table 4, and perform the above RNA on an LC480 using the reaction system shown in Table 4, the primers shown in Table 5, and the procedure shown in Table 6.

[0332] Table 4 RT-qPCR probe method reaction system

[0333] Table 5 Primer Information

[0334] Table 6 RT-qPCR reaction procedure

[0335] The relative quantification of the target gene KRAS in each test group was performed using the comparative Ct(ΔΔCt) method, as follows: ΔCt(test group) = Ct(target gene in test group) – Ct(internal reference gene in test group) ΔCt(control group) = Ct(target gene in control group) – Ct(internal reference gene in control group) ΔΔCt(test group) = ΔCt(test group) - ΔCt(average in control group) ΔΔCt(control group) = ΔCt(control group) - ΔCt(average in control group)

[0336] Wherein, ΔCt (control group mean) is the arithmetic mean of ΔCt (control group) for each sample in the control group; thus, each sample in the test group and the control group corresponds to a ΔCt value.

[0337] Using the control group as a baseline, the expression level of KRAS mRNA in the test group was normalized, and the expression level of KRAS mRNA in the control group was defined as 100%.

[0338] The relative expression level of KRAS mRNA in the test group was 2. ^-ΔΔCt (Test group) × 100%

[0339] The above test groups are Kras-1M1 to Kras-150M1 groups;

[0340] The above-mentioned internal reference genes are all GAPDH;

[0341] The control group mentioned above was the MOCK group.

[0342] The KRAS mRNA level was compared with the internal reference gene GAPDH, and the value was normalized to the mean of the Mock control group. The data were expressed as a percentage relative to the Mock control group and presented as the mean plus the standard deviation.

[0343] The KRAS mRNA silencing effects after transfecting PANC-1 and A549 cell lines with 150 modified KRAS siRNAs are shown in Table 7. It can be seen that in PANC-1 cells, 58 sequences of 10 nM modified KRAS siRNAs exhibited an inhibition rate greater than 85% (residual activity below 0.15) against KRAS mRNA (siRNA names shown in Table 8), and 44 sequences exhibited an inhibition rate greater than 85% in PANC-1 cells (siRNA names shown in Table 9).

[0344] Table 7 summarizes the inhibition rates of modified KRAS siRNA on KRAS mRNA in the PANC-1 / A549 cell line.

[0345] II. Further screening of the initially screened siRNAs was conducted by measuring the inhibitory levels of KRAS mRNA in cells through high, medium, and low concentrations of KRAS siRNA transfection.

[0346] The above experimental method was used to test the inhibitory effect of siRNA on PANC-1 or A549 cell lines. The final concentrations of siRNA were 10 nM, 1 nM and 0.1 nM.

[0347] The KRAS mRNA silencing effect after transfection of the PANC-1 cell line with 58 modified KRAS siRNAs is shown in Table 8. The KRAS mRNA silencing effect after transfection of the A549 cell line with 44 modified KRAS siRNAs is shown in Table 9.

[0348] The 17 lines that showed the best efficacy in PANC-1 cells were: KRAS-76M1, KRAS-10M1, KRAS-85M1, KRAS-13M1, KRAS-113M1, KRAS-96M1, KRAS-12M1, KRAS-109M1, KRAS-81M1, KRAS-21M1, KRAS-25M1, KRAS-14M1, KRAS-27M1, KRAS-94M1, KRAS-39M1, KRAS-17M1, and KRAS-74M1.

[0349] The 19 lines that showed the best results in A549 cells were: KRAS-85M1, KRAS-74M1, KRAS-94M1, KRAS-10M1, KRAS-76M1, KRAS-113M1, KRAS-103M1, KRAS-42M1, KRAS-96M1, KRAS-31M1, KRAS-5M1, KRAS-89M1, KRAS-32M1, KRAS-34M1, KRAS-39M1, KRAS-21M1, KRAS-13M1, KRAS-38M1, and KRAS-12M1.

[0350] Table 8. Summary of the inhibition rates of 58 modified KRAS siRNAs on KRAS mRNA in the PANC-1 cell line.

[0351] Table 9. Summary of the inhibition rates of 44 modified KRAS siRNAs on KRAS mRNA in the A549 cell line.

[0352] III. IC50 of siRNA-modified sequences silencing KRAS mRNA in the PANC-1 / A549 cell line 50

[0353] The above experimental method was used to test the inhibitory effects of the 17 siRNAs that showed the best effect in PANC-1 cells and the 19 siRNAs that showed the best effect in A549 cells in PANC-1 or A549 cell lines. The final concentrations of siRNAs were 10 nM, 1 nM, 0.333 nM, 0.111 nM, 0.037 nM, 0.012 nM, 0.004 nM, and 0.001 nM.

[0354] IC50 was performed in PANC-1 and A549 cells. 50 Experiment, IC detection 50 value.

[0355] Plotting the log value of siRNA concentration on the X-axis and the percentage inhibition rate on the Y-axis, dose-response curves were fitted using the analysis software GraphPadPrism to derive the IC50 of each siRNA. 50 value.

[0356] The fitting formula is: Y = Bottom + (Top – Bottom) / (1 + 10^((LogIC50 – X) × HillSlope))

[0357] Where: Top represents the percentage inhibition rate at the top plateau, and the standard for the Top of the curve is generally between 80% and 120%; Bottom represents the percentage inhibition rate at the bottom plateau, and the Bottom of the curve is generally between -20% and 20%; HillSlope represents the slope of the percentage inhibition rate curve.

[0358] PANC-1 cell line KRAS mRNA silencing effect IC 50 The experimental results are shown in Table 10. The IC50 value of KRAS mRNA silencing in the A549 cell line is shown in Table 10. 50 The experimental results are shown in Table 14.

[0359] Table 10

[0360] Table 14

[0361] The present application has been described in detail above. Those skilled in the art will recognize that the present application can be implemented in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments are given in this application, it should be understood that further modifications can be made to the present application. In summary, in accordance with the principles of this application, this application is intended to include any changes, uses, or improvements to the present application, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A double-stranded RNA molecule, wherein the double-stranded RNA molecule comprises a sense strand and an antisense strand that at least partially form a double-stranded region, the antisense strand comprising 17-21 consecutive nucleotides of any even-numbered nucleotide sequence in SEQ ID NO. 1-300, and the sense strand comprising 17-19 consecutive nucleotides of any odd-numbered nucleotide sequence in SEQ ID NO. 1-300.

2. The double-stranded RNA molecule according to claim 1, wherein the length of the sense strand is no more than 30 nucleotides, and / or the length of the antisense strand is no more than 30 nucleotides, preferably, the length of the sense strand is no more than 21 nucleotides, and / or the length of the antisense strand is no more than 23 nucleotides, more preferably, the length of the sense strand is no more than 19 nucleotides, and / or the length of the antisense strand is no more than 21 nucleotides.

3. The double-stranded RNA molecule according to claim 1 or 2, wherein the sense strand and antisense strand of the double-stranded RNA molecule comprise or are selected from any combination of the following: a sense strand with nucleotide sequence number n and an antisense strand with nucleotide sequence number n+1, wherein n is an odd number from 1 to 300.

4. The double-stranded RNA molecule according to claim 3, wherein the sense strand and antisense strand of the double-stranded RNA molecule comprise or are selected from any combination of the following: The positive chain shown in SEQ ID NO.1 and the negative chain shown in SEQ ID NO.2; The positive chain shown in SEQ ID NO.7 and the negative chain shown in SEQ ID NO.8; The positive chain shown in SEQ ID NO.9 and the negative chain shown in SEQ ID NO.10; The positive chain shown in SEQ ID NO.15 and the negative chain shown in SEQ ID NO.16; The positive chain shown in SEQ ID NO.17 and the negative chain shown in SEQ ID NO.18; The positive chain shown in SEQ ID NO.19 and the negative chain shown in SEQ ID NO.20; The positive chain shown in SEQ ID NO.21 and the negative chain shown in SEQ ID NO.22; The positive chain shown in SEQ ID NO.23 and the negative chain shown in SEQ ID NO.24; The positive chain shown in SEQ ID NO.25 and the negative chain shown in SEQ ID NO.26; The positive chain shown in SEQ ID NO.27 and the negative chain shown in SEQ ID NO.28; The positive chain shown in SEQ ID NO.29 and the negative chain shown in SEQ ID NO.30; The positive chain shown in SEQ ID NO.33 and the negative chain shown in SEQ ID NO.34; The positive chain shown in SEQ ID NO.41 and the negative chain shown in SEQ ID NO.42; The positive chain shown in SEQ ID NO.45 and the negative chain shown in SEQ ID NO.46; The positive chain shown in SEQ ID NO.49 and the negative chain shown in SEQ ID NO.50; The positive chain shown in SEQ ID NO. 53 and the negative chain shown in SEQ ID NO. 54; The positive chain shown in SEQ ID NO. 57 and the negative chain shown in SEQ ID NO. 58; The positive chain shown in SEQ ID NO. 59 and the negative chain shown in SEQ ID NO. 60; The positive chain shown in SEQ ID NO. 61 and the negative chain shown in SEQ ID NO. 62; The positive chain shown in SEQ ID NO. 65 and the negative chain shown in SEQ ID NO. 66; The positive chain shown in SEQ ID NO. 67 and the negative chain shown in SEQ ID NO. 68; The positive chain shown in SEQ ID NO. 69 and the negative chain shown in SEQ ID NO. 70; The positive chain shown in SEQ ID NO. 71 and the negative chain shown in SEQ ID NO. 72; The positive chain shown in SEQ ID NO.75 and the negative chain shown in SEQ ID NO.76; The positive chain shown in SEQ ID NO.77 and the negative chain shown in SEQ ID NO.78; The positive chain shown in SEQ ID NO. 83 and the negative chain shown in SEQ ID NO. 84; The positive chain shown in SEQ ID NO. 85 and the negative chain shown in SEQ ID NO. 86; The positive chain shown in SEQ ID NO. 99 and the negative chain shown in SEQ ID NO. 100; The positive chain shown in SEQ ID NO. 105 and the negative chain shown in SEQ ID NO. 106; The positive chain shown in SEQ ID NO. 107 and the negative chain shown in SEQ ID NO. 108; The positive chain shown in SEQ ID NO. 109 and the negative chain shown in SEQ ID NO. 110; The positive chain shown in SEQ ID NO. 113 and the negative chain shown in SEQ ID NO. 114; The positive chain shown in SEQ ID NO. 117 and the negative chain shown in SEQ ID NO. 118; The positive chain shown in SEQ ID NO. 123 and the negative chain shown in SEQ ID NO. 124; The positive chain shown in SEQ ID NO. 125 and the negative chain shown in SEQ ID NO. 126; The positive chain shown in SEQ ID NO. 143 and the negative chain shown in SEQ ID NO. 144; The positive chain shown in SEQ ID NO. 147 and the negative chain shown in SEQ ID NO. 148; The positive chain shown in SEQ ID NO. 151 and the negative chain shown in SEQ ID NO. 152; The positive chain shown in SEQ ID NO.161 and the negative chain shown in SEQ ID NO.162; The positive chain shown in SEQ ID NO. 167 and the negative chain shown in SEQ ID NO. 168; The positive chain shown in SEQ ID NO. 169 and the negative chain shown in SEQ ID NO. 170; The positive chain shown in SEQ ID NO. 177 and the negative chain shown in SEQ ID NO. 178; The positive chain shown in SEQ ID NO. 187 and the negative chain shown in SEQ ID NO. 188; The positive chain shown in SEQ ID NO.191 and the negative chain shown in SEQ ID NO.192; The positive chain shown in SEQ ID NO. 205 and the negative chain shown in SEQ ID NO. 206; The positive chain shown in SEQ ID NO. 211 and the negative chain shown in SEQ ID NO. 212; The positive chain shown in SEQ ID NO. 213 and the negative chain shown in SEQ ID NO. 214; The positive chain shown in SEQ ID NO. 217 and the negative chain shown in SEQ ID NO. 218; The positive chain shown in SEQ ID NO. 225 and the negative chain shown in SEQ ID NO. 226; The positive chain shown in SEQ ID NO. 227 and the negative chain shown in SEQ ID NO. 228; The positive chain shown in SEQ ID NO. 235 and the negative chain shown in SEQ ID NO. 236; The positive chain shown in SEQ ID NO. 245 and the negative chain shown in SEQ ID NO. 246; The positive chain shown in SEQ ID NO. 247 and the negative chain shown in SEQ ID NO. 248; The positive chain shown in SEQ ID NO. 251 and the negative chain shown in SEQ ID NO. 252; The positive chain shown in SEQ ID NO. 255 and the negative chain shown in SEQ ID NO. 256; The positive chain shown in SEQ ID NO. 257 and the negative chain shown in SEQ ID NO. 258; The positive chain shown in SEQ ID NO.267 and the negative chain shown in SEQ ID NO.268; The positive chain shown in SEQ ID NO. 281 and the negative chain shown in SEQ ID NO. 282; The positive chain shown in SEQ ID NO. 63 and the negative chain shown in SEQ ID NO. 64; The positive chain shown in SEQ ID NO.97 and the negative chain shown in SEQ ID NO.98; The positive chain shown in SEQ ID NO. 171 and the negative chain shown in SEQ ID NO. 172; The positive chain shown in SEQ ID NO. 189 and the negative chain shown in SEQ ID NO. 190; The positive chain shown in SEQ ID NO.259 and the negative chain shown in SEQ ID NO.

260.

5. A modified double-stranded RNA molecule comprising any one of claims 1-4, wherein at least one nucleotide is chemically modified, preferably, the chemical modification comprising replacing the 2' hydroxyl group of the nucleotide's ribosyl group with another group, and / or modifying a base on the nucleotide, and / or replacing the 5' hydroxyl group of the nucleotide's ribosyl group with another group, more preferably, the chemically modified nucleotide is selected from at least one of the following: 2'-methoxy modified nucleotides, 2'-fluoro modified nucleotides, 5'-(E)-vinylphosphonate modified nucleotides, 2'-methyl modified nucleotides, deoxy-nucleotides, 2'-deoxy-nucleotides, 3'-terminal deoxy-thymidine (dT) nucleotides, 2'-deoxy-modified nucleotides, locked nucleotides, non-locked nucleotides, configuration-restricted nucleotides, and restricted ethyl nucleosides. Acids, non-basic nucleotides, 2'-amino-modified nucleotides, 2'-O-allyl-modified nucleotides, 2'-C-alkyl-modified nucleotides, 2'-hydroxy-modified nucleotides, 2'-methoxyethyl-modified nucleotides, 2'-O-alkyl-modified nucleotides, morpholinyl nucleotides, nucleotides containing non-natural bases, tetrahydropyran-modified nucleotides, 1,5-dehydrohexyl alcohol-modified nucleotides, cyclohexenyl-modified nucleotides, glycerol nucleotides (GNA), open-ring nucleotides (UNA), nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphate esters, and nucleotides containing 5'-phosphate ester mimics, more preferably, the chemically modified nucleotides are selected from at least one of the following: 2'-methoxy-modified nucleotides, 2'-fluoro-modified nucleotides, and 5'-(E)-vinylphosphonate-modified nucleotides.

6. The modified double-stranded RNA molecule according to claim 5, further comprising modification of the phosphodiester between nucleotides, preferably, the modification of the phosphodiester is a thiophosphate diester modification.

7. The modified double-stranded RNA molecule according to claim 5 or 6, wherein the modified double-stranded RNA molecule has the following modification pattern: Following the direction from the 5' end to the 3' end, the nucleotides at positions 1-6 and 10-19 of the sense strand are 2'-methoxy modified nucleotides, and the nucleotides at positions 7-9 are 2'-fluorinated modified nucleotides. The nucleotides at positions 1 and 2, and positions 2 and 3 are linked by thiophosphate. The nucleotides at positions 1, 3-5, 7-13, 15, and 17-21 of the antisense strand are 2'-methoxy modified nucleotides, and the nucleotides at positions 2, 6, 14, and 16 are 2'-fluorinated modified nucleotides. The nucleotides at positions 1 and 2, 2 and 3, 19 and 20, and 20 and 21 are linked by thiophosphate.

8. The double-stranded RNA molecule according to any one of claims 1-4 or the modified double-stranded RNA molecule according to any one of claims 5-7, further coupled with a ligand, preferably, the ligand is selected from one or more small molecule compounds, polypeptides, short peptides, proteins, and antibodies.

9. A pharmaceutical composition comprising a double-stranded RNA molecule as described in any one of claims 1-4 or a modified double-stranded RNA molecule as described in any one of claims 5-7, and a pharmaceutically acceptable carrier.

10. The use of the double-stranded RNA molecule according to any one of claims 1-4, or the modified double-stranded RNA molecule according to any one of claims 5-7, or the pharmaceutical composition according to claim 11, in any of the following: D1) Application in the preparation of drugs that inhibit KRAS gene expression; Application of D2 in inhibiting KRAS gene expression; Application of D3 in the treatment of diseases related to KRAS gene targets; Application of D4 in the preparation of drugs for treating diseases related to KRAS gene targets.

11. A method for treating a disease related to a KRAS gene target, comprising administering to a subject a therapeutically effective amount of a double-stranded RNA molecule as described in any one of claims 1-4, or a modified double-stranded RNA molecule as described in any one of claims 5-7, or a pharmaceutical composition as described in claim 9.

12. A method for inhibiting KRAS gene expression in cells, the method comprising: The cells are brought into contact with the double-stranded RNA molecule of any one of claims 1-4, or the modified double-stranded RNA molecule of any one of claims 5-7, or the pharmaceutical composition of claim 9; Maintaining the cell for a sufficient period of time to allow for the degradation of the KRAS gene mRNA transcript, thereby inhibiting KRAS gene expression in the cell; Preferably, the cells are located inside or outside the subject's body; More preferably, the subject suffers from a KRAS gene target-related disease.

13. The application according to claim 10, or the method according to claim 11 or 12, wherein the KRAS gene target-related disease is selected from one or more of the following group: Metastatic non-small cell lung cancer, metastatic pancreatic ductal adenocarcinoma, KRAS G12C+ non-small cell lung cancer, diabetic retinopathy, leukemia, cholangiocarcinoma, metastatic colorectal cancer, gastric cancer, advanced non-small cell lung cancer, stage IV non-small cell lung cancer, gastric adenocarcinoma, sepsis, KRAS G12C-mutated advanced non-small cell lung cancer, gallbladder cancer, age-related macular degeneration, malignant solid tumors, metastatic pancreatic cancer, non-squamous non-small cell lung cancer, pancreatic cancer, fibrosis, metastatic solid tumors, KRAS G12C-mutated metastatic non-small cell lung cancer, bone metastases, EGFR-mutated advanced non-small cell lung cancer, non-small cell lung cancer, brain metastases, advanced solid tumors, lung cancer, gastroesophageal junction adenocarcinoma, esophageal cancer, KRAS G12D-mutated advanced solid tumors, KRAS / G12C-mutated advanced malignant solid tumors, scleroderma, colorectal cancer, pancreatic ductal adenocarcinoma, colorectal cancer, ovarian cancer, esophageal adenocarcinoma, KRAS G12C mutation metastatic solid tumors.

Images