Sirna for inhibiting MGMT gene expression, pharmaceutical composition comprising same, and use thereof

Abstract

Provided are an siRNA for inhibiting MGMT gene expression and a conjugate thereof, and a pharmaceutical composition comprising the siRNA or the conjugate. The siRNA and the conjugate thereof can specifically degrade MGMT mRNA by means of base pairing to inhibit MGMT protein production, thereby reducing the dose of an alkylating agent such as TMZ, achieving the effect of reducing the toxic and side effects of the alkylating agent, and having a significant clinical value and good application prospects for tumor treatment.

Description

siRNA inhibiting expression of MGMT gene, pharmaceutical composition containing the siRNA and use thereof

[0001] Cross-reference to Related Applications

[0002] This application claims priority to Chinese Patent Application No. 202411073930.X, filed on August 6, 2024, the entire contents of which are incorporated herein by reference in their entirety. TECHNICAL FIELD

[0003] The present application relates to siRNA and conjugates thereof capable of inhibiting the expression of O6-methylguanine-DNA-methyltransferase (MGMT), pharmaceutical compositions containing the siRNA or conjugates thereof, and uses thereof. BACKGROUND

[0004] O6-methylguanine-DNA-methyltransferase (MGMT) is a DNA repair enzyme that specifically targets DNA damage caused by alkylating agents, and it can transfer the methyl group on O6-methylguanine to its 145th amino acid, thereby restoring the base to normal. When temozolomide (TMZ) is used to treat glioma, the expression of MGMT in glioma cells increases, leading to the development of TMZ resistance. Patients with high MGMT DNA methylation levels have low MGMT expression levels and are more sensitive to TMZ, while patients with low methylation levels have high MGMT expression levels and are more likely to develop resistance to TMZ. TMZ is currently the first-line drug for brain glioma, and its toxic side effects mainly include myelosuppression, hematotoxicity, leukopenia, thrombocytopenia, neutropenia, infection, urinary incontinence, etc. The small molecule inhibitor O6-BG (O6-benzylguanine) can significantly reduce the resistance of tumor cells to TMZ and reduce the dosage of TMZ, but because it can inhibit MGMT throughout the body, it does not improve the toxicity of TMZ, so it has been discontinued (Phase III). In addition, O6-BG is a small molecule drug with a short half-life.

[0005] Therefore, it is an urgent problem to be solved to develop an MGMT inhibitor that can effectively improve the toxicity of TMZ. Small interfering RNA (siRNA) is a new class of therapeutic agents that mainly bind to target mRNA in the cytoplasm to down-regulate gene expression at the post-transcriptional level via the RNA interference (RNAi) mechanism, thereby achieving the purpose of treating diseases. There is a need in the art to develop siRNA inhibitors for inhibiting or reducing MGMT expression to meet the above needs. SUMMARY

[0006] The present application provides siRNA and conjugates thereof capable of effectively inhibiting the expression of MGMT gene, and their applications in tumor treatment, etc.

[0007] In one aspect, the application provides an siRNA for inhibiting expression of a MGMT gene, the siRNA comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 17 contiguous nucleotides differing by no more than 4 (e.g., 0, 1, 2, 3, or 4) nucleotides from the nucleotide sequence set forth in any one of SEQ ID NOs: 1-9, 60, 62, 64, 66, 68, 70, 103, 105, 108, 127, 139, 154, 168, and the sense strand comprises a nucleotide sequence that is at least partially complementary to the antisense strand.

[0008] In some embodiments, the antisense strand comprises at least 17 contiguous nucleotides differing by no more than 2 (e.g., 0, 1, or 2) nucleotides from the nucleotide sequence set forth in any one of SEQ ID NOs: 1-9, 60, 62, 64, 66, 68, 70, 103, 105, 108, 127, 139, 154, 168.

[0009] In some embodiments, the sense strand has no more than 6 (e.g., 0, 1, 2, 3, 4, 5, or 6) nucleotide mismatches with the antisense strand; preferably, the sense strand has no more than 2 (e.g., 0, 1, or 2) nucleotide mismatches with the antisense strand.

[0010] In some embodiments, the mismatch is at any one of the 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, or 19th nucleotide of the sense strand, further preferably, the mismatch is at any one of the 16th, 17th, 18th, or 19th nucleotide of the sense strand.

[0011] In some embodiments, the sense strand comprises at least 17 contiguous nucleotides differing by no more than 4 (e.g., 0, 1, 2, 3, or 4) nucleotides from the nucleotide sequence set forth in any one of SEQ ID NOs: 10-18, 59, 61, 63, 65, 67, 69, 71-80, 102, 104, 106, 107, 109-115, 122-126, 134-138, 152, 153, 155-163, 169; preferably, the sense strand comprises at least 17 contiguous nucleotides differing by no more than 2 (e.g., 0, 1, or 2) nucleotides from the nucleotide sequence set forth in any one of SEQ ID NOs: 10-18, 59, 61, 63, 65, 67, 69, 71-80, 102, 104, 106, 107, 109-115, 122-126, 134-138, 152, 153, 155-163, 169.

[0012] In some embodiments, the siRNA comprises blunt ends and / or 1-4 nucleotides overhangs; preferably, the siRNA comprises 1 or 2 nucleotides overhangs; preferably, the overhangs are present at the 5' end and / or the 3' end of the antisense strand and / or the sense strand; preferably, the 3' end of the antisense strand of the siRNA comprises a 2 nucleotides overhang, the 3' end of the sense strand of the siRNA is blunt; preferably, the 3' end of the antisense strand and the 3' end of the sense strand of the siRNA respectively comprise a 2 nucleotides overhang.

[0013] In some embodiments, the antisense strand and the sense strand are each independently 17-30 nucleotides in length; preferably, the antisense strand is 19-27 nucleotides in length; preferably, the sense strand is 17-25 nucleotides in length.

[0014] In some embodiments, the antisense strand is 21-23 nucleotides in length, and the sense strand is 19-21 nucleotides in length.

[0015] In some embodiments, the sequence of the sense strand and the antisense strand of the siRNA is selected from the corresponding sense strand and antisense strand sequence of any one of siRNA 1 to siRNA 9 and siRNA 21 described in Table 1-1, siRNA 8-1 to siRNA 8-16 described in Table 2-2, siRNA 4-1 to siRNA 4-11 described in Table 3-2, siRNA 2-1 to siRNA 2-5 described in Table 4-2, siRNA 3-1 to siRNA 3-5 described in Table 5-2, and siRNA 7-1 to siRNA 7-11 described in Table 6-2; preferably, the sequence of the sense strand and the antisense strand of the siRNA is selected from the corresponding sense strand and antisense strand sequence of any one of siRNA 1 to siRNA 9 and siRNA 21 described in Table 1-1, siRNA 8-5, siRNA 8-7, siRNA 8-16, siRNA 8-8 described in Table 2-2, siRNA 4-10, siRNA 4-11, siRNA 4-8, siRNA 4-7, or siRNA 4-9 described in Table 3-2, siRNA 3-3 described in Table 5-2, and siRNA 7-1 described in Table 6-2.

[0016] In some embodiments, the siRNA comprises at least one modified nucleotide.

[0017] In some embodiments, all the nucleotides in the sense strand and / or the antisense strand of the siRNA are modified nucleotides or nucleotide analogs.

[0018] In some embodiments, the modified nucleotide or nucleotide analog is selected from a 2'-methoxy nucleotide, a 2'-fluoro nucleotide, a 2'-deoxy nucleotide, a 2',3'-seco nucleotide analog, a 2'-fluoroarabinonucleotide, a 2'-methoxyethyl nucleotide, a 2'-amino modified nucleotide, a 2'-alkyl modified nucleotide, a 3'-methoxy nucleotide, a 2'-allyl modified nucleotide, a glycol modified nucleotide, an abasic nucleotide, a morpholino nucleotide, a locked nucleotide, an unlocked nucleotide, or a glycerol nucleotide.

[0019] In some embodiments, all of the nucleotides in the sense strand and / or the antisense strand of the siRNA are modified nucleotides selected from a 2'-methoxy nucleotide, a 2'-fluoro nucleotide.

[0020] In some embodiments, the 5' end of the antisense strand of the siRNA comprises a phosphorous-containing group; preferably, the phosphorous-containing group is selected from a 5' phosphate (5'-P), a 5' phosphorothioate (5'-PS), a 5' phosphorodithioate (5'-PS2), a 5' vinylphosphonate (5'-VP), a 5' methylphosphonate (MePhos), or a 5'-deoxy-5'-C-malonato; preferably, the 5' end of the antisense strand of the siRNA comprises a 5' vinylphosphonate (5'-VP).

[0021] In some embodiments, the sense strand and / or the antisense strand of the siRNA comprises a modified internucleoside linkage; preferably, the internucleoside linkage between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides, at the 5' end and / or the 3' end of the sense strand is a phosphorothioate linkage; preferably, the internucleoside linkage between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides, at the 5' end and / or the 3' end of the antisense strand is a phosphorothioate linkage.

[0022] In some embodiments, the sequence of the sense strand and the antisense strand of the siRNA is selected from the corresponding sense strand and antisense strand sequence of any one of siRNA 10 to siRNA 18 and siRNA 20 described in Table 1-2, siRNA 17-1 to siRNA 17-16 described in Table 2-1, siRNA 17-17 to siRNA 17-29 described in Table 2-3, siRNA 13-1 to siRNA 13-11 described in Table 3-1, siRNA 11-1 to siRNA 11-5 described in Table 4-1, siRNA 12-1 to siRNA 12-5 described in Table 5-1, and siRNA 16-1 to siRNA 16-11 described in Table 6-1; preferably, the sequence of the sense strand and the antisense strand of the siRNA is selected from the corresponding sense strand and antisense strand sequence of any one of siRNA 10 to siRNA 18 and siRNA 20 described in Table 1-2, siRNA 17-5, siRNA 17-7, siRNA 17-16, siRNA 17-8, siRNA 17-17, siRNA 17-18, siRNA 17-19, siRNA 17-20, siRNA 17-21 or siRNA 17-25 described in Table 2-1, siRNA 13-10, siRNA 13-11, siRNA 13-8, siRNA 13-7 or siRNA 13-9 described in Table 3-1, siRNA 12-3 described in Table 5-1, and siRNA 16-1 described in Table 6-1.

[0023] In another aspect, the present application provides a conjugate comprising the siRNA described in any one of the preceding embodiments and a conjugating moiety linked thereto. In some embodiments, the conjugating moiety comprises a targeting molecule.

[0024] In some embodiments, the conjugating moiety is 1 or 2 or 3 or 4, when the conjugating moiety is more than 1, the conjugating moieties are the same or different.

[0025] In some embodiments, the targeting molecule is a GalNAc derivative, a lipophilic moiety or a peptide chain.

[0026] In some embodiments, the GalNAc derivative is L96.

[0027] In some embodiments, the lipophilic moiety is selected from aliphatic, alicyclic or polyalicyclic compounds, preferably C4-C30 hydrocarbyl (e.g. alkyl or alkenyl), more preferably C6-C18 hydrocarbyl (e.g. alkyl or alkenyl), further preferably C16 hydrocarbyl (e.g. alkyl or alkenyl).

[0028] In some embodiments, the peptide chain is a peptide chain having 5-50 amino acids, preferably, the peptide chain is a peptide chain having 7-18 amino acids, further preferably, the peptide chain is CHRPYIAH (SEQ ID NO: 346), HAIYPRHC (SEQ ID NO: 347), PWVPSWMPPRHT (SEQ ID NO: 348) or THRPPMWSPVWP (SEQ ID NO: 349).

[0029] In some embodiments, the coupling moiety is linked via a linker containing an ether bond, a thioether bond, a urea, a carbonate, an amine, an amide, a maleimide-thioether, a disulfide, a phosphate, a phosphodiester, a sulfonamide or a carbamate, a hydrazone group, an ester group, an acetal group or a ketal group, a peptide bond, or the linker is a biologically cleavable linker selected from the group consisting of DNA, RNA, disulfide, amide, functionalized monosaccharide or oligosaccharide of galactosamine, glucosamine, glucose, galactose, mannose, and combinations thereof.

[0030] In some embodiments, the coupling moiety is linked at a position selected from the group consisting of a nucleotide at the 5' end of the sense strand, a nucleotide at a middle position other than the two positions at the 5' and 3' ends of the sense strand, a nucleotide at the 3' end of the sense strand; more preferably, the lipophilic moiety is conjugated at the 2 position of the nucleotide sugar ring at the above-mentioned position.

[0031] In another aspect, the present application provides a pharmaceutical composition, wherein the pharmaceutical composition comprises the siRNA or the conjugate according to any one of the preceding embodiments, and a pharmaceutically acceptable carrier and / or excipient. In some embodiments, the pharmaceutically acceptable carrier and / or excipient is a delivery vehicle; preferably, the siRNA or conjugate is encapsulated by the delivery vehicle.

[0032] In another aspect, the present application provides use of the siRNA, conjugate or pharmaceutical composition according to any one of the preceding embodiments in the manufacture of a medicament for reducing or preventing resistance of a tumor to an alkylating agent. Also provided is a method for reducing or preventing resistance of a tumor to an alkylating agent, comprising administering to a subject in need thereof an effective amount of the siRNA, conjugate or pharmaceutical composition according to any one of the preceding embodiments.

[0033] In another aspect, the present application provides the use of the siRNA, conjugate or pharmaceutical composition described in any of the preceding embodiments and an alkylating agent in the manufacture of a medicament for treating a tumor. Also provided is a method for treating a tumor comprising administering to a subject in need thereof an effective amount of an alkylating agent and an effective amount of the siRNA, conjugate or pharmaceutical composition described in any of the preceding embodiments. In some embodiments, the tumor is resistant to an alkylating agent. In some embodiments, the alkylating agent is temozolomide (TMZ), Lomustine or Carmustine. In some embodiments, the tumor is a glioma. In some embodiments, the alkylating agent is administered in any order, e.g., simultaneously, before or after, with the siRNA, conjugate or pharmaceutical composition.

[0034] In another aspect, the present application provides the use of the siRNA, conjugate or pharmaceutical composition described in any of the preceding embodiments in the manufacture of a medicament for treating and / or preventing a pathological condition or disease associated with MGMT. In some embodiments, the pathological condition or disease associated with MGMT involves MGMT overexpression or would benefit from a reduction or inhibition of MGMT levels.

[0035] In another aspect, the present application provides a method of preventing and / or treating a pathological condition or disease associated with MGMT in a subject, the method comprising administering to a subject in need thereof an effective amount of the siRNA, conjugate or pharmaceutical composition described in any of the preceding embodiments.

[0036] In some embodiments, the pathological condition or disease associated with MGMT involves MGMT overexpression or would benefit from a reduction or inhibition of MGMT levels.

[0037] In another aspect, the present application provides the siRNA, conjugate or pharmaceutical composition described in any of the preceding embodiments for use in preventing and / or treating a pathological condition or disease associated with MGMT.

[0038] In some embodiments, the pathological condition or disease associated with MGMT involves MGMT overexpression or would benefit from a reduction or inhibition of MGMT levels.

[0039] In another aspect, the present application provides a method of inhibiting MGMT expression in a cell, the method comprising introducing into the cell the siRNA, conjugate or pharmaceutical composition described in any of the preceding embodiments. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 shows exemplary structures of MGMT siRNA molecules.

[0041] Figure 2A shows the IC50 of MGMT siRNA molecules for in vitro inhibition activity. 50 Results.

[0042] Figure 2B shows the results of MGMT siRNA molecules for in vivo inhibition activity.

[0043] Figures 3A-3B show the results of in vitro inhibition activity assays for siRNA 17 structure-activity relationship variants.

[0044] Figure 4 shows the IC50 of siRNA 17 structure-activity relationship variants for in vitro inhibition activity. 50 Results.

[0045] Figures 5A-5D show the results of in vivo inhibition activity assays for siRNA 17 structure-activity relationship variants and conjugates with L96.

[0046] Figures 6A-6B show the results of in vitro inhibition activity assays for siRNA 13 structure-activity relationship variants.

[0047] Figure 7 shows the IC50 of siRNA 13 structure-activity relationship variants for in vitro inhibition activity. 50 Results.

[0048] Figure 8 shows the results of in vitro inhibition activity assays for siRNA 11 structure-activity relationship variants.

[0049] Figure 9 shows the IC50 of siRNA 11 structure-activity relationship variants for in vitro inhibition activity. 50 Results.

[0050] Figure 10 shows the results of in vitro inhibition activity assays for siRNA 12 structure-activity relationship variants.

[0051] Figure 11 shows the IC50 of siRNA 12 structure-activity relationship variants for in vitro inhibition activity. 50 Results.

[0052] Figures 12A-12B show the results of in vitro inhibition activity assays for siRNA 16 structure-activity relationship variants.

[0053] Figure 13 shows the IC50 of siRNA 16 structure-activity relationship variants for in vitro inhibition activity. 50 Results.

[0054] Figure 14A shows MGMT expression in U118MG after transfection with siRNA.

[0055] Figure 14B shows the IC50 of U118MG for TMZ after transfection with siRNA. 50 Results.

[0056] Figure 14C shows the MGMT expression of T98G after transfection of siRNA.

[0057] Figure 15 shows the KD activity assay results of siRNA 17-L96 in C57 mice liver.

[0058] Figure 16 shows the knockdown results of MGMT mRNA of siRNA 17-C1 after free uptake of tumor cells.

[0059] Figure 17 shows the efficacy of siRNA 17-C1 in mouse GL261 subcutaneous tumor.

[0060] Figure 18 shows the efficacy of siRNA 17-C2 in mouse U251 subcutaneous tumor.

[0061] Figure 19A shows the off-target cytotoxicity of siRNA in vitro using CCK8 detection method.

[0062] Figure 19B shows the off-target cytotoxicity of siRNA in vitro using PI detection method.

[0063] Figure 20A is the structure of siRNA 17-C1.

[0064] Figure 20B is the structure of siRNA 17-C2. Beneficial effects

[0065] The siRNA of the present application can effectively inhibit the expression of MGMT gene in vitro and / or in vivo, reduce the resistance of tumor cells to alkylating agents (such as TMZ), thereby reducing the amount of alkylating agents (such as TMZ) used, reducing the toxic side effects of alkylating agents (such as TMZ), and having important clinical value for the treatment of tumors. DETAILED DESCRIPTION

[0066] The specific embodiments of the present application are described in detail below. It should be understood that the specific embodiments described herein are merely intended to illustrate and explain the present application, and are not intended to limit the present application.

[0067] Definitions

[0068] Unless specific definitions are provided, the nomenclature utilized in connection with, and the procedures and techniques of, molecular biology, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques can be used for chemical synthesis and chemical analysis. Certain such techniques and methods can be found, for example, in "Carbohydrate Modifications in Antisense Research" ed. Sangvi and Cook American Chemical Society, Washington, 1994; "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa., 18th edition, 1990; and "Antisense Drug Technology, Principles, Strategies, and Applications" ed. Stanley T. Crooke, CRC Press, Boca Raton, Fla.; in Sambrook et al., "Molecular Cloning, A laboratory Manual," 2nd edition, Cold Spring Harbor Laboratory Press, 1989, which are incorporated by reference for any purpose. All patents, applications, published applications and other publications, and other data cited herein are fully incorporated by reference in their entirety where permitted.

[0069] In the present context, the term "target gene", "target sequence" or "target mRNA" refers to any nucleic acid molecule whose expression or activity can be modulated by an siRNA compound; including but not limited to RNA (including but not limited to mRNA precursors and mRNA or parts thereof) resulting from transcription of DNA encoding a target protein, as well as cDNA derived from such RNA, and miRNA. As used herein, the term "target gene", "target mRNA" refers to MGMT mRNA, the sequence of which is well known to the person skilled in the art, see various public databases. MGMT mRNA refers to the mRNA shown under Genbank accession number NM_002412.5. As used herein, the term "inhibiting the MGMT gene" refers to inhibiting the expression of MGMT mRNA and / or protein, unless otherwise indicated.

[0070] In this context, capital letters C, G, U, A represent the base composition of a nucleotide; lower case letter m represents that the nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide; lower case letter f represents that the nucleotide adjacent to the left of the letter f is a fluoro-modified nucleotide; lower case letter s represents that the two nucleotides adjacent to the left and right of the letter s are connected by phosphorothioate group; and letter combination VP represents that the nucleotide adjacent to the right of the letter combination VP is a 5'-(E)-vinyl phosphonate (E-VP) modified nucleotide.

[0071] In this context, the term "modified nucleotide" refers to a nucleotide independently having a modified ribose moiety, a modified internucleoside linkage, or a modified base. Thus, the term "modified nucleotide" encompasses substitution, addition, or removal (e.g., using a functional group or atom) to the internucleoside linkage, ribose moiety, or base. Modifications suitable for use in the present application include all types of modifications disclosed herein or known in the art. "Methoxy-modified nucleotide" refers to a nucleotide in which the hydroxyl group at the 2' position of the ribosyl group of the nucleotide is replaced by a methoxy group. "Fluoro-modified nucleotide" refers to a nucleotide in which the hydroxyl group at the 2' position of the ribosyl group of the nucleotide is replaced by a fluorine. "Nucleotide analog" refers to a group that can replace a nucleotide in a nucleic acid, but is structurally different from an adenine ribonucleotide, a guanine ribonucleotide, a cytosine ribonucleotide, a uracil ribonucleotide, or a thymine deoxyribonucleotide, such as an iso-nucleotide, a bridged nucleic acid (BNA), or an acyclic nucleotide, etc.

[0072] In the present context, the expression "complementary" has the meaning well known to the person skilled in the art, i.e. in a double-stranded nucleic acid molecule, the bases of one strand each pair with the bases on the other strand in a complementary manner. In DNA, the purine base adenine (A) always pairs with the pyrimidine base thymine (T) (or in RNA with uracil (U)); the purine base guanine (G) always pairs with the pyrimidine base cytosine (C). Each base pair comprises one purine and one pyrimidine. When the adenine on one strand always pairs with the thymine (or uracil) on the other strand and the guanine always pairs with the cytosine, the two strands are considered to be complementary to each other and the sequence of one strand can be deduced from the sequence of its complementary strand. Correspondingly, "mismatch" means in the art that the bases at the corresponding positions in a double-stranded nucleic acid do not pair in a complementary manner. The person skilled in the art is able to determine the conditions most suitable for testing the complementarity of two sequences depending on the end application of the hybridized nucleotides. Such conditions can for example be stringent conditions, e.g. 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°C or 70°C for 12-16 hours, followed by washing. Other conditions, e.g. physiologically relevant conditions that can be encountered in an organism, can also be applied.

[0073] In the present context, "substantially complementary" means that there are no more than 3 base mismatches between the two nucleotide sequences concerned; "essentially complementary" means that there are no more than 1 base mismatch between the two nucleotide sequences; "completely complementary" means that there are no base mismatches between the two nucleotide sequences.

[0074] In the present context, "nucleotide sequence difference" means that the base type of the nucleotide at the same or corresponding position has changed compared to the original nucleotide sequence. For example, if the nucleotide base at a position in the original nucleotide sequence is A, a nucleotide sequence difference at this position is considered to exist if the nucleotide base at the same or corresponding position is changed to U, C, G or dT, dC, dG, etc. It is noted that a nucleotide sequence difference at a position is not considered to exist if the nucleotide at the same or corresponding position differs only in the presence or type of modification compared to the original nucleotide sequence.

[0075] In the present context, the term "overhang" or "nucleotide overhang" refers to at least one unpaired nucleotide projecting from the duplex structure of the siRNA. For example, there is an overhang when the 3' end of one strand of the siRNA extends beyond the 5' end of the other strand (or vice versa). The siRNA can comprise an overhang of at least one nucleotide, or the overhang can comprise at least 2 nt, at least 3 nt, at least 4 nt, at least 5 nt, or more. The overhang can comprise or consist of nucleotides / nucleoside analogs, including deoxynucleotides / nucleosides. The overhang can be on the sense strand, the antisense strand, or any combination thereof. The nucleotides of the overhang can be present on the 5' end, the 3' end, or both ends of the antisense strand or the sense strand of the siRNA. Accordingly, the term "blunt end" refers to the absence of a nucleotide overhang. In addition, where two oligonucleotides are designed to hybridize with the formation of one or more single-stranded overhangs, such overhangs should not be considered mismatches for purposes of determining complementarity.

[0076] In the present context, the term "pharmaceutically acceptable carriers and / or excipients" refers to carriers, delivery vehicles, diluents, adjuvants, and / or salts / esters / hydrates thereof, etc. that are generally chemically and / or physically compatible with the other ingredients constituting a certain pharmaceutical dosage form, such as the siRNA of the present application, and physiologically compatible with the subject. The "pharmaceutically acceptable carriers and / or excipients" do not exert or are not intended to exert a therapeutic effect at the intended dosage. Such ingredients can serve a function of: a) aiding in the processing of the drug delivery system during manufacturing, b) protecting, supporting or enhancing the stability, bioavailability or patient acceptability of the active ingredients, c) aiding in product identification, and / or d) enhancing the overall safety, effectiveness, delivery, etc. of any other attributes of the active ingredients during storage and use. For example, the siRNA of the present application can be encapsulated by a delivery vehicle. The "pharmaceutically acceptable carriers and / or excipients" include, but are not limited to: viruses, liposomes, nanoparticles, bacteria, lipid nanoparticles (LNP), neutral liposomes (NL), polymeric nanoparticles, double-stranded RNA binding motifs (dsRBMs), pH modulators, surfactants, adjuvants, ionic strength enhancers, diluents, agents to maintain osmotic pressure, agents to delay absorption, preservatives. For example, viruses include, but are not limited to, retroviruses, adenoviruses, lentiviruses, baculoviruses, AAVs. Liposomes include, but are not limited to, Lipofectamine, cationic DOTAP, neutral DOPC. Nanoparticles include, but are not limited to, cationic polymers, PEI. Bacteria include, but are not limited to, tkRNAi. Polymeric nanoparticles include, but are not limited to, low molecular weight polymers or high molecular weight polymers. pH modulators include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic or non-ionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. Agents to maintain osmotic pressure include, but are not limited to, sugars, NaCl and the like. Agents to delay absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), etc.

[0077] As used herein, the terms "prevent" or "preventing" refer to an approach undertaken to stop or slow the development of a disease or condition or symptom in a subject; the terms "treat" or "treating" refer to an approach undertaken to obtain a beneficial or desired clinical result. For purposes of this application, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0078] As used herein, the term "effective amount" means an amount that is sufficient to achieve or at least partially achieve a desired effect. For example, an effective amount for preventing a disease means an amount that is sufficient to prevent, stop, or slow the development of the disease; an effective amount for treating a disease means an amount that is sufficient to cure or at least partially arrest the disease and its complications in a patient already having the disease. Determining such effective amounts is well within the capabilities of those skilled in the art. For example, an amount effective for therapeutic purposes will depend on the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient such as age, body weight, and sex, the mode of administration of a drug, and other therapies being administered concurrently, etc.

[0079] siRNA

[0080] The present application provides siRNAs for inhibiting expression of the MGMT gene. As used herein, the term "siRNA" means an RNA molecule capable of inducing the RNAi phenomenon in a sequence-specific manner, consisting of a sense strand and an antisense strand, and having a double-stranded structure with at least partial complementarity. By at least partial complementarity is meant that the two sequences can be substantially complementary, essentially complementary, or completely complementary, or not more than 6, 5, 4, 3, 2, or 1 mismatched base pairs overall, while retaining the ability to hybridize under the relevant conditions. The term "antisense strand" refers to a strand that includes a region of complementarity to a target sequence. The term "sense strand" refers to a strand that includes a region of complementarity to the antisense strand. When the region of complementarity is not completely complementary to the target sequence or the antisense strand, the mismatches can be internal or in the terminal regions of the molecule.

[0081] In some embodiments, the sense and antisense strands can have the same or different lengths. In some embodiments, the sense and antisense strands can each be 16 to 49 nucleotides in length. In some embodiments, the antisense strand is 17 to 30 (e.g., 17 to 29, 17 to 28, 17 to 27, 19 to 30, 19 to 29, 19 to 28, 19 to 27, 19 to 25, 19 to 23, 21 to 25, 21 to 23) nucleotides in length. In some embodiments, the sense strand is 17 to 30 (e.g., 17 to 29, 17 to 28, 17 to 27, 17 to 26, 17 to 25, 17 to 21, 19 to 21) nucleotides in length. In some embodiments, the antisense strand is 19 to 27 nucleotides in length; the sense strand is 17 to 25 nucleotides in length. In some embodiments, the antisense strand is 21 to 23 nucleotides in length; the sense strand is 19 to 21 nucleotides in length. In some embodiments, the antisense strand is 23 nucleotides in length; the sense strand is 21 nucleotides in length. In some embodiments, the antisense strand is 21 nucleotides in length; the sense strand is 19 nucleotides in length. In some embodiments, the antisense strand is 21 nucleotides in length; the sense strand is 21 nucleotides in length. In some embodiments, the antisense strand is 23 nucleotides in length; the sense strand is 23 nucleotides in length.

[0082] In some embodiments, the sense strand forms a duplex region with the antisense strand, the duplex region being 15 to 30 nucleotide pairs in length, e.g., 15 to 25 nucleotide pairs, 15 to 23 nucleotide pairs, 15 to 21 nucleotide pairs, e.g., 15, 16, 17, 18, 19, 20, or 21 nucleotide pairs.

[0083] In some embodiments, the sense strand has no more than 6 nucleotide mismatches with the antisense strand. In some embodiments, the sense strand has no more than 5 nucleotide mismatches with the antisense strand. In some embodiments, the sense strand has no more than 4 nucleotide mismatches with the antisense strand. In some embodiments, the sense strand has no more than 3 nucleotide mismatches with the antisense strand. In some embodiments, the sense strand has no more than 2 nucleotide mismatches with the antisense strand. In some embodiments, the sense strand has no more than 1 nucleotide mismatch with the antisense strand. In some embodiments, the sense strand is completely complementary to the antisense strand.

[0084] In some embodiments, the mismatch is at any one of positions 11, 12, 13, 14, 15, 16, 17, 18, or 19 of the sense strand, further preferably, the mismatch is at any one of positions 16, 17, 18, or 19 of the sense strand.

[0085] In some embodiments, the siRNA comprises a blunt end and / or an overhang.

[0086] In some embodiments, the siRNA comprises one or more single-stranded nucleotide overhangs. For example, 1, 2, 3, or 4 nucleotide overhangs. In some embodiments, the overhangs can be on the sense strand, the antisense strand, or any combination thereof. In some embodiments, the overhangs are present on the 5' end, the 3' end, or both ends of the antisense strand or the sense strand of the siRNA.

[0087] In some embodiments, the 3' end of the antisense strand of the siRNA comprises a 2 nucleotide overhang, and the 3' end of the sense strand of the siRNA is blunt.

[0088] In some embodiments, the 3' end of the antisense strand and the 3' end of the sense strand of the siRNA each comprise a 2 nucleotide overhang.

[0089] In some embodiments, the antisense strand of an siRNA of the application comprises at least 17 contiguous nucleotides (e.g., 17, 18, 19, 20, or 21 contiguous nucleotides) differing by no more than 4 (e.g., 0, 1, 2, 3, or 4) nucleotides from the nucleotide sequence set forth in any one of SEQ ID NOs: 1-9, 60, 62, 64, 66, 68, 70, 103, 105, 108, 127, 139, 154, 168. In some embodiments, the antisense strand comprises at least 17 contiguous nucleotides (e.g., 17, 18, 19, 20, or 21 contiguous nucleotides) differing by no more than 3 (e.g., 0, 1, 2, or 3) nucleotides from the nucleotide sequence set forth in any one of SEQ ID NOs: 1-9, 60, 62, 64, 66, 68, 70, 103, 105, 108, 127, 139, 154, 168. In some embodiments, the antisense strand comprises at least 17 contiguous nucleotides (e.g., 17, 18, 19, 20, or 21 contiguous nucleotides) differing by no more than 2 (e.g., 0, 1, or 2) nucleotides from the nucleotide sequence set forth in any one of SEQ ID NOs: 1-9, 60, 62, 64, 66, 68, 70, 103, 105, 108, 127, 139, 154, 168. In some embodiments, the antisense strand comprises at least 17 contiguous nucleotides (e.g., 17, 18, 19, 20, or 21 contiguous nucleotides) differing by no more than 1 (e.g., 0 or 1) nucleotide from the nucleotide sequence set forth in any one of SEQ ID NOs: 1-9, 60, 62, 64, 66, 68, 70, 103, 105, 108, 127, 139, 154, 168. In some embodiments, the antisense strand comprises at least 17 contiguous nucleotides (e.g., 17, 18, 19, 20, or 21 contiguous nucleotides) of the nucleotide sequence set forth in any one of SEQ ID NOs: 1-9, 60, 62, 64, 66, 68, 70, 103, 105, 108, 127, 139, 154, 168. In some embodiments, the antisense strand is a nucleotide sequence differing by no more than 2 (e.g., 0, 1, or 2) nucleotides from the nucleotide sequence set forth in any one of SEQ ID NOs: 1-9, 60, 62, 64, 66, 68, 70, 103, 105, 108, 127, 139, 154, 168. In some embodiments, the antisense strand is the nucleotide sequence set forth in any one of SEQ ID NOs: 1-9, 60, 62, 64, 66, 68, 70, 103, 105, 108, 127, 139, 154, 168.

[0090] In some embodiments, the sense strand of the siRNA of the application is substantially complementary, substantially complementary, or fully complementary to the antisense strand over at least 17 contiguous nucleotides. In some embodiments, the sense strand is substantially complementary, substantially complementary, or fully complementary to the antisense strand over at least 18 contiguous nucleotides. In some embodiments, the sense strand is substantially complementary, substantially complementary, or fully complementary to the antisense strand over at least 19 contiguous nucleotides.

[0091] In some embodiments, the sense strand of the siRNA comprises at least 17 contiguous nucleotides (e.g., 17, 18, or 19 contiguous nucleotides) differing by no more than 4 (e.g., 0, 1, 2, 3, or 4) nucleotides from the nucleotide sequence set forth in any one of SEQ ID NOs: 10-18, 59, 61, 63, 65, 67, 69, 71-80, 102, 104, 106, 107, 109-115, 122-126, 134-138, 152, 153, 155-163, 169. The at least 17 contiguous nucleotides of the sense strand and the antisense strand of the siRNA comprise a duplex region. In some embodiments, the sense strand comprises at least 17 contiguous nucleotides (e.g., 17, 18, or 19 contiguous nucleotides) differing by no more than 3 (e.g., 0, 1, 2, or 3) nucleotides from the nucleotide sequence set forth in any one of SEQ ID NOs: 10-18, 59, 61, 63, 65, 67, 69, 71-80, 102, 104, 106, 107, 109-115, 122-126, 134-138, 152, 153, 155-163, 169. In some embodiments, the sense strand comprises at least 17 contiguous nucleotides (e.g., 17, 18, or 19 contiguous nucleotides) differing by no more than 2 (e.g., 0, 1, or 2) nucleotides from the nucleotide sequence set forth in any one of SEQ ID NOs: 10-18, 59, 61, 63, 65, 67, 69, 71-80, 102, 104, 106, 107, 109-115, 122-126, 134-138, 152, 153, 155-163, 169. In some embodiments, the sense strand comprises at least 17 contiguous nucleotides (e.g., 17, 18, or 19 contiguous nucleotides) differing by no more than 1 (e.g., 0 or 1) nucleotide from the nucleotide sequence set forth in any one of SEQ ID NOs: 10-18, 59, 61, 63, 65, 67, 69, 71-80, 102, 104, 106, 107, 109-115, 122-126, 134-138, 152, 153, 155-163, 169. In some embodiments, the sense strand comprises at least 17 contiguous nucleotides (e.g., 17, 18, or 19 contiguous nucleotides) of the nucleotide sequence set forth in any one of SEQ ID NOs: 10-18, 59, 61, 63, 65, 67, 69, 71-80, 102, 104, 106, 107, 109-115, 122-126, 134-138, 152, 153, 155-163, 169.In some embodiments, the sense strand comprises a nucleotide sequence differing by no more than 2 (e.g., 0, 1, or 2) nucleotides from the nucleotide sequence set forth in any one of SEQ ID NOs: 10-18, 59, 61, 63, 65, 67, 69, 71-80, 102, 104, 106, 107, 109-115, 122-126, 134-138, 152, 153, 155-163, 169. In some embodiments, the sense strand is the nucleotide sequence set forth in any one of SEQ ID NOs: 10-18, 59, 61, 63, 65, 67, 69, 71-80, 102, 104, 106, 107, 109-115, 122-126, 134-138, 152, 153, 155-163, 169.

[0092] In some embodiments, the antisense strand is the nucleotide sequence set forth in SEQ ID NO: 1, and the sense strand comprises a nucleotide sequence differing by no more than 2 (e.g., 0, 1, 2) nucleotides from SEQ ID NO: 10. In some embodiments, the antisense strand is the nucleotide sequence set forth in SEQ ID NO: 2, and the sense strand comprises a nucleotide sequence differing by no more than 2 (e.g., 0, 1, 2) nucleotides from SEQ ID NO: 11. In some embodiments, the antisense strand is the nucleotide sequence set forth in SEQ ID NO: 3, and the sense strand comprises a nucleotide sequence differing by no more than 2 (e.g., 0, 1, 2) nucleotides from SEQ ID NO: 12. In some embodiments, the antisense strand is the nucleotide sequence set forth in SEQ ID NO: 4, and the sense strand comprises a nucleotide sequence differing by no more than 2 (e.g., 0, 1, 2) nucleotides from SEQ ID NO: 13. In some embodiments, the antisense strand is the nucleotide sequence set forth in SEQ ID NO: 5, and the sense strand comprises a nucleotide sequence differing by no more than 2 (e.g., 0, 1, 2) nucleotides from SEQ ID NO: 14. In some embodiments, the antisense strand is the nucleotide sequence set forth in SEQ ID NO: 6, and the sense strand comprises a nucleotide sequence differing by no more than 2 (e.g., 0, 1, 2) nucleotides from SEQ ID NO: 15. In some embodiments, the antisense strand is the nucleotide sequence set forth in SEQ ID NO: 7, and the sense strand comprises a nucleotide sequence differing by no more than 2 (e.g., 0, 1, 2) nucleotides from SEQ ID NO: 16. In some embodiments, the antisense strand is the nucleotide sequence set forth in SEQ ID NO: 8, and the sense strand comprises a nucleotide sequence differing by no more than 2 (e.g., 0, 1, 2) nucleotides from SEQ ID NO: 17. In some embodiments, the antisense strand is the nucleotide sequence set forth in SEQ ID NO: 9, and the sense strand comprises a nucleotide sequence differing by no more than 2 (e.g., 0, 1, 2) nucleotides from SEQ ID NO: 18. In some embodiments, the antisense strand is the nucleotide sequence set forth in SEQ ID NO: 168, and the sense strand comprises a nucleotide sequence differing by no more than 2 (e.g., 0, 1, 2) nucleotides from SEQ ID NO: 169. In some embodiments, the antisense strand is the nucleotide sequence set forth in SEQ ID NO: 68, and the sense strand comprises a nucleotide sequence differing by no more than 2 (e.g., 0, 1, 2) nucleotides from SEQ ID NO: 67.

[0093] In some embodiments, the sequence of the sense strand and the antisense strand of the siRNA is selected from the corresponding sense strand and antisense strand sequences of any one of siRNA 1 to siRNA 9 and siRNA 21 described in Table 1-1, siRNA 8-1 to siRNA 8-16 described in Table 2-2, siRNA 4-1 to siRNA 4-11 described in Table 3-2, siRNA 2-1 to siRNA 2-5 described in Table 4-2, siRNA 3-1 to siRNA 3-5 described in Table 5-2, or siRNA 7-1 to siRNA 7-11 described in Table 6-2.

[0094] In some embodiments, the sequence of the sense strand and the antisense strand of the siRNA is selected from the corresponding sense strand and antisense strand sequences of any one of siRNA 1 to siRNA 9 and siRNA 21 described in Table 1-1, siRNA 8-5, siRNA 8-7, siRNA 8-16, siRNA 8-8 described in Table 2-2, siRNA 4-10, siRNA 4-11, siRNA 4-8, siRNA 4-7, or siRNA 4-9 described in Table 3-2, siRNA 3-3 described in Table 5-2, or siRNA 7-1 described in Table 6-2.

[0095] In some embodiments, the sequence of the antisense strand of the siRNA is selected from the corresponding antisense strand sequence of any one of siRNA 8 described in Table 1-1, siRNA 8-5, siRNA 8-7, siRNA 8-16, siRNA 8-8 described in Table 2-2. The sense strand of the siRNA is substantially complementary, essentially complementary, or fully complementary to the antisense strand over at least 17, at least 18, or at least 19 contiguous nucleotides, for example, with no more than 2 (e.g., 0, 1, or 2) nucleotide mismatches. In some embodiments, the sequence of the sense strand and the antisense strand of the siRNA is selected from the corresponding sense strand and antisense strand sequences of any one of siRNA 8 described in Table 1-1, siRNA 8-5, siRNA 8-7, siRNA 8-16, siRNA 8-8 described in Table 2-2.

[0096] In some embodiments, the sequence of the antisense strand of the siRNA is selected from the corresponding antisense strand sequence of siRNA 21 described in Table 1-1. The sense strand of the siRNA is substantially complementary, essentially complementary, or fully complementary to the antisense strand over at least 17, at least 18, or at least 19 contiguous nucleotides, for example, with no more than 2 (e.g., 0, 1, or 2) nucleotide mismatches. In some embodiments, the sequence of the sense strand and the antisense strand of the siRNA is selected from the corresponding sense strand and antisense strand sequences of siRNA 21 described in Table 1-1.

[0097] Modifications

[0098] The siRNAs of the application can be modified in the nucleobase structure or in the ribose-phosphate backbone structure to reduce off-target effects, and / or to increase the biological stability of the molecule, or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids. Thus, siRNA sequences comprising any modifications are also encompassed within the scope of the application. The siRNA molecules comprising ribonucleoside analogs or derivatives must retain the ability to form a duplex and allow or mediate specific degradation of the target RNA via the RISC pathway.

[0099] In some embodiments, the siRNA comprises at least one modified nucleotide. The modification need not be the same for each of the plurality of modified ribonucleosides in the siRNA.

[0100] In some embodiments, all of the nucleotides in the sense strand and / or the antisense strand of the siRNA are modified nucleotides or nucleotide analogs.

[0101] In some embodiments, the modified nucleotides include, but are not limited to, 2'-methoxy nucleotides, 2'-fluoro nucleotides, 2'-deoxy nucleotides, 2',3'-seco nucleotide analogs, 2'-fluoroarabinonucleotides, 2'-methoxyethyl nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, 3'-methoxy nucleotides, 2'-allyl modified nucleotides, glycol modified nucleotides, abasic nucleotides, morpholino nucleotides, locked nucleotides (LNA), unlocked nucleotides (UNA), or glycerol nucleotides (GNA).

[0102] In some embodiments, all of the nucleotides in the sense strand and / or the antisense strand of the siRNA are modified nucleotides selected from the group consisting of: 2'-methoxy nucleotides, 2'-fluoro nucleotides.

[0103] In some embodiments, the siRNAs of the application can comprise a phosphorous-containing group at the 5' end of the sense strand or the antisense strand. The 5' end phosphorous-containing group can be a 5' phosphate (5'-P), a 5' phosphorothioate (5'-PS), a 5' phosphorodithioate (5'-PS2), a 5' vinylphosphonate (5'-VP), a 5' methylphosphonate (MePhos), or a 5'-deoxy-5'-C-malonate.

[0104] In some embodiments, the 5' terminal nucleotide of the antisense strand of the siRNA is a 5'-phosphate nucleotide or a 5'-phosphate analog modified nucleotide. The 5'-phosphate nucleotide or 5'-phosphate analog modified nucleotide commonly used is well known to one skilled in the art, such as the 5'-phosphate nucleotide can have the following structure:

[0105] For example, Anastasia Khvorova and Jonathan K. Watts, The chemical evolution of oligonucleotide therapies of clinical utility. Nature Biotechnology, 2017, 35(3): 238-48 discloses the following 4 5'-phosphate analog modified nucleotides:

[0106] wherein R a is selected from H, OH, methoxy, fluoro; Base represents a base selected from A, U, C, G, or T.

[0107] In some embodiments, the 5' end of the antisense strand of the siRNA comprises a 5' end vinylphosphonate (5'-VP) represented by Formula (2).

[0108] In some embodiments, the siRNA of the present application comprises modified internucleoside linkages or modified backbones. The modified internucleoside linkages or backbones include, but are not limited to, phosphorothioate, alkylphosphonate, phosphodiester, phosphodi thioate, alkylphosphonothioate, phosphoramidate, carbamate, carbonate, phosphate triester, acetamidate, carboxymethyl ester, and combinations thereof.

[0109] In some embodiments, the 5' and 3' ends of the sense strand independently comprise 1 or 2 phosphorothioate linkages, respectively. In some embodiments, the internucleotidic linkage between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides, of the 5' and / or 3' end of the sense strand is a phosphorothioate linkage. In some embodiments, the internucleotidic linkage between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides, of the 5' end of the sense strand is a phosphorothioate linkage.

[0110] In some embodiments, the 5' and 3' ends of the antisense strand independently comprise 1 or 2 phosphorothioate linkages, respectively. In some embodiments, the internucleotidic linkage between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides, of the 5' and / or 3' end of the antisense strand is a phosphorothioate linkage. In some embodiments, the internucleotidic linkage between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides, of the 5' end of the antisense strand is a phosphorothioate linkage. In some embodiments, the internucleotidic linkage between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides, of the 3' end of the antisense strand is a phosphorothioate linkage.

[0111] In some embodiments, at least one of the linkages between the nucleotides at positions 1 and 2 of the 5' end of the sense strand, between the nucleotides at positions 2 and 3 of the 5' end of the sense strand, between the nucleotides at positions 1 and 2 of the 3' end of the antisense strand, between the nucleotides at positions 2 and 3 of the 3' end of the antisense strand, between the nucleotides at positions 1 and 2 of the 5' end of the antisense strand, and between the nucleotides at positions 2 and 3 of the 5' end of the antisense strand is a phosphorothioate linkage; preferably, at least four of the linkages are phosphorothioate linkages; more preferably, all six of the linkages are phosphorothioate linkages.

[0112] In some embodiments, the sequences of the sense strand and the antisense strand of the siRNA are selected from the corresponding sense strand and antisense strand sequences of any one of siRNA 10 to siRNA 18 and siRNA 20 described in Table 1-2, siRNA 17-1 to siRNA 17-16 described in Table 2-1, siRNA 17-17 to siRNA 17-29 described in Table 2-3, siRNA 13-1 to siRNA 13-11 described in Table 3-1, siRNA 11-1 to siRNA 11-5 described in Table 4-1, siRNA 12-1 to siRNA 12-5 described in Table 5-1, and siRNA 16-1 to siRNA 16-11 described in Table 6-1.

[0113] In some embodiments, the sequences of the sense strand and the antisense strand of the siRNA are selected from the corresponding sense strand and antisense strand sequences of any one of siRNA 10 to siRNA 18 and siRNA 20 described in Table 1-2, siRNA 17-5, siRNA 17-7, siRNA 17-16, siRNA 17-8 described in Table 2-1, siRNA 17-17, siRNA 17-18, siRNA 17-19, siRNA 17-20, siRNA 17-21 or siRNA 17-25 described in Table 2-3, siRNA 13-10, siRNA 13-11, siRNA 13-8, siRNA 13-7 or siRNA 13-9 described in Table 3-1, siRNA 12-3 described in Table 5-1, and siRNA 16-1 described in Table 6-1.

[0114] In some embodiments, the sequences of the sense strand and the antisense strand of the siRNA are selected from the corresponding sense strand and antisense strand sequences of any one of siRNA 17 described in Table 1-2, siRNA 17-5, siRNA 17-7, siRNA 17-16, siRNA 17-8 described in Table 2-1.

[0115] In some embodiments, the sequences of the sense strand and the antisense strand of the siRNA are selected from the corresponding sense strand and antisense strand sequences of siRNA 20 described in Table 1-2.

[0116] delivery

[0117] The siRNAs of the application can be delivered or introduced (e.g., in vitro to cells, or in vivo to a patient) by any means known in the art. For example, for in vivo delivery, the siRNAs can be injected into a tissue site, or administered systemically. In vivo delivery can also be performed by a beta-glucan delivery system. In vitro introduction to cells includes methods known in the art, such as electroporation and lipofection.

[0118] In some embodiments, the delivery methods include, but are not limited to, viral delivery (retrovirus, adenovirus, lentivirus, baculovirus, AAV); liposomes (Lipofectamine, cationic DOTAP, neutral DOPC); nanoparticles (cationic polymers, PEI); bacterial delivery (tkRNAi); chemical modification of siRNA (such as LNA) to increase stability; lipid nanoparticles (LNP); neutral liposomes (NL); polymeric nanoparticles (low molecular weight polymers or high molecular weight polymers); double-stranded RNA binding motifs (dsRBMs); and other delivery systems known in the art that can be suitable for nucleic acid or oligonucleotide delivery.

[0119] conjugates

[0120] The siRNAs of the application can be further linked to one or more non-nucleotide groups to form a conjugate. The non-nucleotide groups can alter the targeting, delivery, pharmacokinetic, or biodistribution properties of the siRNA, among others.

[0121] In some embodiments, the conjugates of the application comprise an siRNA of the application and a conjugating moiety linked thereto, the conjugating moiety comprising a targeting molecule.

[0122] In some embodiments, the conjugating moiety is optionally linked by a linker.

[0123] In some embodiments, the siRNA can be non-covalently or covalently conjugated to the conjugating moiety.

[0124] In some embodiments, the conjugating moiety is a targeting molecule. The targeting molecule can be a targeting molecule conventionally used in the art of siRNA drug delivery, which typically enhances the pharmacokinetic or biodistribution properties of the siRNA to which it is attached, improves the cell-specific (or organ-specific) distribution and cell-specific (or organ-specific) uptake of the siRNA. Representative targeting molecules include, but are not limited to, a compound having affinity for a cell surface molecule, a cell receptor ligand, a hapten, an antibody or antibody fragment, an antibody mimetic, and the like. In some embodiments, the targeting molecule includes, but is not limited to, one or more of the following targeting molecules or derivatives thereof: an integrin; a lipophilic molecule, such as cholesterol, bile acid, a vitamin (e.g., vitamin E), a lipid molecule of varying chain length; a polymer, such as polyethylene glycol; a polypeptide, such as a transmembrane peptide; an aptamer; an antibody; a quantum dot; a saccharide, such as lactose, polylactose, mannose, galactose, N-acetylgalactosamine (GalNAc); a folate; or a receptor ligand expressed by a liver parenchymal cell, such as an asialoglycoprotein, an asialoglycoside residue, a lipoprotein (e.g., high-density lipoprotein, low-density lipoprotein, etc.), a glucagon, a neurotransmitter (e.g., epinephrine), a growth factor, a transferrin, a polypeptide that binds to a transferrin receptor, and the like.

[0125] In some embodiments, the conjugating moiety is 1 or 2 or 3 or 4, and when the conjugating moiety is more than 1, the conjugating moieties are the same or different.

[0126] In some embodiments, the targeting molecule is a GalNAc derivative, a lipophilic moiety, or a peptide chain.

[0127] In some embodiments, the N-acetylgalactosamine (GalNAc) is an L96 structure, which is a conjugating molecule having a structure as shown below:

[0128] In some embodiments, the lipophilic moiety is selected from an aliphatic, alicyclic, or polyalicyclic compound, such as a C4-C30 hydrocarbon group (e.g., an alkyl or alkenyl group), such as a C6-C18 hydrocarbon group (e.g., an alkyl or alkenyl group), and such as a C16 hydrocarbon group (e.g., an alkyl or alkenyl group).

[0129] In some embodiments, the peptide chain is a peptide chain having 5-50 amino acids, for example, the peptide chain is a peptide chain having 7-18 amino acids, for example, the peptide chain is CHRPYIAH (DRI-T8, SEQ ID NO: 346), HAIYPRHC (T8, SEQ ID NO: 347), PWVPSWMPPRHT (RP12, SEQ ID NO: 348), or THRPPMWSPVWP (P12, SEQ ID NO: 349).

[0130] In some embodiments, the conjugate comprises an siRNA described in any of the preceding embodiments, a lipophilic moiety (e.g., C16) linked to the 5’ end of the sense strand thereof, and a peptide chain (e.g., DRI-T8) linked to the 3’ end thereof. In some embodiments, the siRNA in the conjugate comprises a 5’-terminal vinylphosphonate (5’-VP) at the 5’ end of the antisense strand. In some embodiments, the siRNA in the conjugate comprises the sense and antisense strand sequences of siRNA 8 in Table 1-1, for example, siRNA 17 in Table 1-2. In some embodiments, the siRNA in the conjugate comprises the sense and antisense strand sequences of siRNA 21 in Table 1-1, for example, siRNA 20 in Table 1-2.

[0131] In some embodiments, the conjugate moiety is linked via a linker containing an ether linkage, a thioether linkage, a urea, a carbonate, an amine, an amide, a maleimide-thioether, a disulfide, a phosphonate, a phosphodiester, a sulfonamide, or a carbamate, a hydrazone group, an ester group, an acetal group or a ketal group, a peptide bond, or the linker is a biologically cleavable linker selected from the group consisting of DNA, RNA, disulfide, amide, functionalized monosaccharides or oligosaccharides of galactosamine, glucosamine, glucose, galactose, mannose, and combinations thereof; the linker can be one or more of the following linker moieties or derivatives thereof commonly used in the art of siRNA drug delivery, including but not limited to, amide linker moieties, amino linker moieties, carbonyl linker moieties, carbamate linker moieties, urea linker moieties, ether linker moieties, disulfide linker moieties, succinamido linker moieties, and the like.

[0132] In some embodiments, the targeting molecule can be linked to the siRNA of the present application directly or indirectly via a linker / attachment group. In some embodiments, the targeting molecule is linked to the siRNA via a labile, cleavable, or reversible bond or linker. In some embodiments, the targeting molecule is linked to at least one terminus of the sense strand and / or the antisense strand of the siRNA. In some embodiments, the targeting molecule is linked to the 5’ end and / or the 3’ end of the sense strand. In some embodiments, the targeting molecule is linked to the 5’ end and / or the 3’ end of the antisense strand.

[0133] In some embodiments, the targeting molecule is attached at a position selected from the group consisting of: a nucleotide at the 5' end of the sense strand, a nucleotide at the 3' end of the sense strand; more preferably, the targeting molecule is conjugated to the 2' position of the nucleotide sugar ring at the above-mentioned positions.

[0134] Pharmaceutical compositions

[0135] The siRNAs of the present application can be prepared as pharmaceutical compositions suitable for inhibiting the expression of a target mRNA in a target cell, cell population, tissue, or organism.

[0136] In some embodiments, the pharmaceutical composition of the present application comprises at least one siRNA of the present application. In some embodiments, the pharmaceutical composition contains one siRNA as described above as an active ingredient. In other embodiments, the pharmaceutical composition contains at least two siRNAs (for example, but not limited to, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) as described above as active ingredients. Preferably, the at least two siRNAs each target a different target sequence in the MGMT gene, whereby a synergistic effect can be expected from simultaneous action on different target sequences. Here, "different target sequences" means that there is no overlap between the target sequences, or the number of consecutive nucleotides that overlap between the target sequences is less than 5 (for example, the number of consecutive nucleotides that overlap is 4, 3, 2, 1, 0). In this case, the at least two siRNAs as described above can be present in any different ratio. Preferably, the at least two siRNAs as described above can be present in a molar ratio of 1:100 to 100:1 with respect to each other; more preferably, the at least two siRNAs as described above can be present in a molar ratio of 1:10 to 10:1, 1:5 to 5:1, or 1:2 to 2:1 with respect to each other. In some embodiments, the at least two siRNAs as described above are present in the same molar ratio.

[0137] In some embodiments, the pharmaceutical composition comprises an effective amount of siRNA. An "effective amount" means the amount of siRNA that is effective for producing a desired pharmacological, therapeutic, or prophylactic result. For example, a therapeutically effective amount of a drug for treating a disease or condition is the amount that achieves at least a 10% reduction in a measurable parameter associated with the disease or condition, if a given clinical treatment is considered effective when there is at least a 10% reduction in the parameter. For example, a therapeutically effective amount of an siRNA targeting MGMT can reduce the level of MGMT mRNA by at least 10%.

[0138] In some embodiments, the pharmaceutical composition of any of the above embodiments, the siRNA can be linked to a targeting molecule to form a conjugate. Thus, in some embodiments, the pharmaceutical composition comprises a conjugate of the present application.

[0139] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

[0140] In some embodiments, the pharmaceutically acceptable carrier and / or excipient is a delivery vehicle. A delivery vehicle is a substance that improves the delivery of a nucleic acid or oligonucleotide to a cell or tissue. Such substances can be any delivery vehicle known in the art to be suitable for nucleic acid or oligonucleotide delivery, including but not limited to: viruses (retrovirus, adenovirus, lentivirus, baculovirus, AAV); liposomes (Lipofectamine, cationic DOTAP, neutral DOPC); nanoparticles (cationic polymers, PEI); bacteria (tkRNAi); lipid nanoparticles (LNP); neutral liposomes (NL); polymeric nanoparticles (low molecular weight polymers or high molecular weight polymers); double-stranded RNA binding motifs (dsRBMs), and the like.

[0141] In some embodiments, the siRNA can be encapsulated by the delivery vehicle.

[0142] In some embodiments, the siRNA can be directly or indirectly linked to the delivery vehicle via a linker / linker group. In some embodiments, the delivery vehicle is linked to the siRNA via a labile, cleavable, or reversible bond or linker. In some embodiments, the delivery vehicle is linked to at least one end of the sense strand and / or the antisense strand of the siRNA. In some embodiments, the delivery vehicle is linked to the 5’ end and / or the 3’ end of the sense strand. In some embodiments, the delivery vehicle is linked to the 5’ end and / or the 3’ end of the antisense strand.

[0143] In some embodiments, the pharmaceutical composition of the present application is formulated in a dosage form compatible with its intended route of administration, such as local administration (e.g., direct injection or implantation), systemic administration, or subcutaneous, intravenous, intraperitoneal, or parenteral routes of administration, including intracranial (e.g., intraventricular, intramembranous, or intrathecal), intramuscular, transdermal, airway (aerosol), nasal, oral, rectal, or topical (including buccal and sublingual) administration.

[0144] In some embodiments, the pharmaceutical composition is administered by inhalation, intranasally, intratracheally, or oropharyngeal inhalation. Formulations suitable for administration by inhalation are prepared by incorporating the active ingredient into appropriate formulations to be nebulized. Generally, formulations for administration by inhalation are sterile solutions at physiological pH and have low viscosity. Salts can be added to the formulation to balance tonicity. In some cases, a surfactant or cosolvent can be added to increase active ingredient solubility and improve aerosol properties. In some cases, excipients can be added to control viscosity in order to ensure droplet size and distribution of the aerosolized droplets.

[0145] In other embodiments, the pharmaceutical composition can be administered by injection, such as intravenous, intramuscular, subcutaneous, intradermal, intraarticular, intraocular, intraperitoneal, or topical administration. Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, or phosphate buffered saline (PBS), among others. The pharmaceutical composition should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms such as bacteria and fungi. For example, sterile injectable solutions can be prepared by incorporating the active ingredient into an appropriate solvent with one or more of the other ingredients enumerated above, as required, followed by filtered sterilization. In addition, sterile solutions can be prepared as sterile powders (e.g., by vacuum drying or freeze-drying) for reconstitution with a suitable solvent prior to use.

[0146] The siRNAs of the present application can be formulated in dosage unit form for ease of administration. Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[0147] In the present application, dosing schedules can be adjusted to achieve the best response for the purpose (e.g., therapeutic or prophylactic response). For example, dosing can be single, can be multiple over a period of time, or can be reduced or increased proportionally to the urgency of the therapeutic situation.

[0148] Inhibition of expression of MGMT

[0149] The siRNAs of the application can be used to inhibit the expression of MGMT in vitro and / or in vivo. In the present context, the term "inhibit" refers to a situation where the expression of a target gene is down-regulated as a result of siRNA-mediated degradation of the mRNA of the target gene. By "down-regulation" is meant a decrease in the level of expression of the target gene of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or even 100% relative to the absence of siRNA treatment. A decrease in the level of expression of the target gene of 100% means no detectable level of expression of the target gene.

[0150] In one aspect, the application provides a method of inhibiting the expression of MGMT in a cell, the method comprising introducing into the cell an siRNA, a conjugate or a pharmaceutical composition of the application. In some embodiments, the method is performed in vitro. The siRNAs of the application can be introduced by any nucleic acid delivery means known in the art, such as electroporation or lipofection.

[0151] In some embodiments, the inhibition of the expression of MGMT can be manifested by a decrease in the amount of detectable MGMT mRNA. In some embodiments, the degree of inhibition is expressed as: (mRNA in control cells) - (mRNA in treated cells) / (mRNA in control cells) * 100%. In other embodiments, the degree of inhibition can be given as a decrease in a parameter functionally linked to the expression of the MGMT gene, such as the amount of protein encoded by the MGMT gene. In principle, MGMT gene silencing can be measured in any cell expressing MGMT (constitutively or by genetic engineering), and by any suitable assay. The measurement can be performed at various time points, before, during and after the administration of the siRNA, to measure the effect of the siRNA. The level or expression of MGMT can be measured by evaluation of the mRNA (e.g. by Northern blot or PCR) or of the protein (e.g. Western blot). The effect of the siRNA on the expression of MGMT can be measured, for example, by measuring the rate of transcription of the MGMT gene (e.g. by RT-PCR).

[0152] In some embodiments, the expression of the MGMT gene is suppressed by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by administration of the siRNAs of the application. In some embodiments, the expression of the MGMT gene is suppressed by at least about 60%, 70%, or 80% by administration of the siRNAs of the application. In some embodiments, the expression of the MGMT gene is suppressed by at least about 85%, 90%, or 95% by administration of the siRNAs of the application. In some embodiments, the expression of the MGMT gene is suppressed by at least about 96%, 97%, 98%, 99%, or 100% by administration of the siRNAs of the application.

[0153] In some embodiments, the siRNA, the conjugate, or the pharmaceutical composition is used alone or in combination with another pharmaceutically active agent (e.g., an siRNA targeting a different target sequence in the MGMT gene or an siRNA targeting another target).

[0154] In some embodiments, one siRNA provided herein is used. In other embodiments, at least two siRNAs provided herein are used (e.g., but not limited to, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more), preferably the at least two siRNAs each target a different target sequence in the MGMT gene.

[0155] Tumor treatment

[0156] As a DNA repair enzyme, once DNA alkylation occurs, a large amount of MGMT rushes into the nucleus of the cell to facilitate DNA repair. During the repair process, MGMT binds in the minor groove of the damaged DNA, and the O6-methylguanine flips out of the DNA helix and binds to MGMT. The methyl group of O6-methylguanine is transferred to the cysteine at position 145 of MGMT. Then, due to the change in the DNA binding region conformation of MGMT, the alkylated MGMT is separated from the repaired DNA and degraded by the ubiquitin-proteasome system. Thus, MGMT actually performs a suicidal repair on the DNA, in which the DNA is repaired while MGMT is degraded.

[0157] The siRNAs of the application inhibit the production of MGMT protein by degrading MGMT mRNA, thereby improving the sensitivity of tumors to alkylating agents and increasing the efficacy of alkylating agents on tumors.

[0158] In one aspect, the present application provides a method for reducing or preventing resistance of a tumor to an alkylating agent, comprising administering to a subject in need thereof an effective amount of the siRNA, conjugate or pharmaceutical composition of the present application. Also provided is the use of the siRNA, conjugate or pharmaceutical composition of the present application in the manufacture of a medicament for reducing or preventing resistance of a tumor to an alkylating agent. In some embodiments, the alkylating agent is temozolomide (TMZ), Lomustine or Carmustine. In some embodiments, the tumor is a glioma, such as glioblastoma, anaplastic astrocytoma. In some embodiments, the subject has a tumor. In some embodiments, the subject is a tumor patient who has been, is being or plans to be treated with an alkylating agent.

[0159] In one aspect, the present application provides a method for treating a tumor, comprising administering to a subject in need thereof an effective amount of an alkylating agent and an effective amount of the siRNA, conjugate or pharmaceutical composition of the present application. Also provided is the use of the siRNA, conjugate or pharmaceutical composition of the present application and an alkylating agent in the manufacture of a medicament for treating a tumor. In some embodiments, the tumor is resistant to an alkylating agent. In some embodiments, the alkylating agent is temozolomide (TMZ), Lomustine or Carmustine. In some embodiments, the tumor is a glioma, such as glioblastoma, anaplastic astrocytoma.

[0160] It is understood by one skilled in the art that both the alkylating agent and the siRNA, conjugate or pharmaceutical composition of the present application can be administered together in a single formulation, or separately in different formulations. The alkylating agent and the siRNA, conjugate or pharmaceutical composition of the present application can be administered in any order, such as simultaneously, before or after.

[0161] In some embodiments of the methods or uses as described above, the subject is a mammal, such as a human.

[0162] In some embodiments of the methods or uses as described above, the siRNA, the conjugate or the pharmaceutical composition is used alone, or in combination with another pharmaceutically active agent (such as an siRNA targeting a different target sequence in the MGMT gene or an siRNA targeting another target), such as simultaneous or sequential administration.

[0163] In some embodiments of the method or use as described above, one siRNA provided herein is used. In other embodiments, at least two siRNAs provided herein are used (e.g., but not limited to, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more), preferably said at least two siRNAs each target a different target sequence in the MGMT gene.

[0164] Treatment of MGMT-related diseases

[0165] The siRNAs of the present application can be used to treat a disease or condition that would benefit from a reduction or inhibition of MGMT levels.

[0166] In one aspect, the present application provides a method of preventing and / or treating a pathological condition or disease associated with MGMT in a subject, said method comprising administering to a subject in need thereof an effective amount of a siRNA, a conjugate or a pharmaceutical composition of the present application. The present application also relates to the use of a siRNA, a conjugate or a pharmaceutical composition of the present application for the manufacture of a medicament for the treatment and / or prevention of a pathological condition or disease associated with MGMT.

[0167] In some embodiments, said pathological condition or disease associated with MGMT involves MGMT overexpression. MGMT overexpression refers to MGMT levels (e.g., MGMT levels present in the plasma of a subject or in a tissue and preferably in a damaged tissue) that are higher than normal MGMT levels (e.g., corresponding levels in a healthy control).

[0168] In some embodiments, said pathological condition or disease associated with MGMT would benefit from a reduction or inhibition of MGMT levels.

[0169] In some embodiments, said subject is a mammal, e.g., a human.

[0170] In some embodiments, said siRNA, said conjugate or said pharmaceutical composition is used alone or in combination with (e.g., simultaneously or sequentially) another pharmaceutically active agent, e.g., a siRNA targeting a different target sequence in the MGMT gene or a siRNA targeting another target.

[0171] In some embodiments, one siRNA provided herein is used. In other embodiments, at least two siRNAs provided herein are used (e.g., but not limited to, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more), preferably said at least two siRNAs each target a different target sequence in the MGMT gene.

[0172] Examples

[0173] The present application will now be described with reference to the following examples which are intended to be illustrative only and not limiting of the present application.

[0174] It is known to those skilled in the art that the examples describe the present application by way of example only and are not intended to limit the scope of the application as claimed. The experimental methods in the examples are conventional methods unless specifically stated otherwise. Where specific conditions are not specified in the examples, they are carried out under conventional conditions or under conditions recommended by the manufacturer. Where the manufacturer of a reagent or instrument is not indicated, it is a conventional product that can be obtained commercially.

[0175] It is known to those skilled in the art that the siRNAs described in the present application can be obtained by conventional siRNA preparation methods in the art, such as solid phase synthesis and liquid phase synthesis, both of which have commercial custom service. It is also known to those skilled in the art that modified nucleotide groups can be introduced into the siRNAs described in the present application by using nucleotide monomers with corresponding modifications. Methods for preparing nucleotide monomers with corresponding modifications are well known to those skilled in the art, and commercial monomers are available on the market.

[0176] Example 1: siRNA synthesis

[0177] The siRNA sequences listed in Table 1-1 and Table 1-2 were synthesized by solid phase synthesis method, respectively, and the siRNAs provided in the present application were obtained by dissolving equal moles of the sense and antisense strands complementary to each other in Table 1-1 and Table 1-2, respectively, followed by annealing using DEPC water.

[0178] The naked sequences of MGMT siRNA molecules are shown in Table 1-1, and the modified sequences are shown in Table 1-2. Figure 1 shows an exemplary structure of the modified sequence, where the dark circles represent methoxy-modified nucleotides, the light circles represent fluorine-modified nucleotides, and the dark curved lines represent phosphorothioate group connections.

[0179] Table 1-1. MGMT siRNA sequence (naked sequence) information

[0180] Table 1-2. MGMT siRNA sequence (modified sequence) information

[0181] In this context, uppercase letters C, G, U, and A represent the base composition of nucleotides; lowercase letter m indicates that the nucleotide adjacent to the left of letter m is a methoxy-modified nucleotide; lowercase letter f indicates that the nucleotide adjacent to the left of letter f is a fluorinated nucleotide; and lowercase letter s indicates that the two nucleotides adjacent to the left and right of letter s are linked by thiophosphate groups.

[0182] Experimental Example 1: Activity Detection

[0183] 1.1 Detection of in vitro inhibitory activity of siRNA

[0184] According to 3*10 4 A549 cells (obtained from Beina Chuanglian Biotechnology Co., Ltd.) were seeded into 24-well plates with 500 μL of complete culture medium (DMEM + 10% FBS + 1% P / S) per well, with two replicates per group. Transfection was performed 24 h after cell seeding. Pre-diluted siRNA and transfection reagent (CALNP) were added to each well. TM RNAiin vitro transfection reagent (DN001-10, purchased from Beijing Dona Pharmaceutical Technology Co., Ltd.) was mixed well and the procedure was performed according to standard transfection reagent methods. After culturing in a cell culture incubator at 37℃ and 5% CO2 for 24 hours, the culture medium was aspirated, and 450 μl of lysis buffer was added. Lysis was performed at room temperature for 10 minutes. RNA extraction reagent (…) was then used… RNA was extracted using a Magnetic Tissue / Cell Total RNA Kit, 18600ES60, purchased from Nanjing Novizan Biotechnology Co., Ltd. 1 μg of total RNA and 5 μL of reverse mixing buffer were added to an RNase-free EP tube, followed by RNase-free ddH2O to a final volume of 20 μL. The mixture was incubated at 50°C for 20 min, then reacted at 85°C for 5 s, and finally stored at -20°C overnight. An RT-PCR kit was used. The qRT-PCR detection was performed using the IIIOne Step RT-qPCR SYBR Green Kit (11143ES80, Novizan). Results analysis was based on the Blank group, and the ΔCT value between the target gene and the internal reference gene (GAPDH) was calculated. The average ΔCT value of the Blank group was then set as 1 according to the relative quantification principle. The final expression level was calculated by summing the ΔΔCT of each group and 2^(-ΔΔCT).

[0185] The inhibition activity of the naked sequences was detected by the above method. The siRNA1-siRNA6, siRNA8-siRNA9 and siRNA21 were 9 siRNA molecules, and the in vitro KD results were shown in Tables 1-3, which indicated that they had stronger inhibition activity on human MGMT gene (i.e. residual MGMT mRNA in A549 at 1 nM concentration).

[0186] Table 1-3 KD activity of naked sequence at 1 nM concentration

[0187] The inhibition activity of the modified sequences was detected by the above method. According to the 0.1 nM screening results, the siRNAs with higher activity were selected for IC 50 study, and finally 10 siRNA molecules of siRNA10-siRNA18 and siRNA20 were obtained, and the IC 50 results were shown in Figure 2A, which indicated that they had stronger inhibition activity on human MGMT gene.

[0188] 1.2 siRNA in vivo activity detection

[0189] After the above siRNA was conjugated with L96 structure, the in vivo inhibition activity was evaluated in mice. The specific method was as follows: C57BL / 6J mice (Sibeifu (Beijing) Biotechnology Co., Ltd.) were subcutaneously administered at a dose of 1 mpk. One week after administration, the mice were sacrificed, the liver was taken, and the RNA Later was stored at -80℃. The total RNA was extracted by magnetic bead method using Magnetic Tissue / Cell Total RNA Kit (18600ES60), and the OD value was determined. The cDNA was obtained by reverse transcription using AdvanceFast One-step RT-gDNA Digestion SuperMix for qPCR (Nanjing Novozyme Biotech Co., Ltd., Cat: R333-01, Lot: 7E731J3), and was stored at -20℃. The target gene expression was detected by qPCR using Taq Pro Universal SYBR qPCR Master Mix (Nanjing Novozyme Biotech Co., Ltd., Cat: Q712-03, Lot: 7E760D3) kit.

[0190] The inhibition activity of the modified sequences was detected by the above method, and the results were shown in Figure 2B, which indicated that they had stronger inhibition activity on human MGMT gene.

[0191] Experimental Example 2: Variants based on siRNA17 and activity detection thereof

[0192] 2.1 Design of structure-activity relationship variants and detection of in vitro inhibitory activity

[0193] Based on the structure-activity relationship of siRNA17, the following variants were further designed, the modified sequences and the corresponding naked sequences are shown in Table 2-1 ~ Table 2-2, and these variant molecules were prepared by the method described in Example 1.

[0194] The in vitro inhibitory activity of siRNA17 structure-activity relationship variants was detected according to the method described in Experimental Example 1, and the inhibitory activity results at a concentration of 1 nM are shown in Figures 3A-3B, and the IC 50 The detection results are shown in Figure 4. These variants showed good inhibitory activity, among which the inhibitory activity of the SS19 mismatch variant siRNA17-16 was significantly improved compared with siRNA17.

[0195] Table 2-1: siRNA17 structure-activity relationship variant sequences

[0196] Table 2-2: Naked sequence of siRNA17 structure-activity relationship variant

[0197] 2.2 Design of modified variants

[0198] Based on siRNA17, variants with different modification modes were further designed, and the sequences are shown in Table 2-3.

[0199] Table 2-3: siRNA17 modified variant sequences

[0200] Among them, (VP) represents that the right adjacent nucleotide is an (E)-vinyl phosphate modified nucleotide, and the meanings of the remaining letters are the same as above.

[0201] 2.3 Evaluation of in vivo inhibitory activity

[0202] After the above siRNA-17 variants were conjugated with L96 structure, in vivo inhibitory activity evaluation was carried out in mice. The specific method is: C57BL / 6J mice (Sibeifang (Beijing) Biotechnology Co., Ltd.) were subcutaneously administered at a dose of 1 mpk; one week after administration, the mice were sacrificed, the liver was taken out and stored in RNA Later, and stored at -80℃. The total RNA was extracted by magnetic bead method using Magnetic Tissue / Cell Total RNA Kit (18600ES60), and the OD value was determined, and the AdvanceFast One-step RT-gDNA Digestion SuperMix for qPCR (Nanjing Novogene BioTech Co., Ltd., Cat: R333-01, Lot: 7E731J3) was used to obtain cDNA by reverse transcription, and stored at -20°C. Taq Pro Universal SYBR qPCR Master Mix (Nanjing Novogene BioTech Co., Ltd., Cat: Q712-03, Lot: 7E760D3) kit was used for qPCR detection of target gene expression.

[0203] The results are shown in Figures 5A-5D. In the structure-activity relationship variants, the SS11 mismatch variant siRNA17-8 showed better in vivo inhibitory activity. In the modification variants, the in vivo activity of different modification mode variants was improved compared with siRNA-17, and the activity of siRNA17-25 carrying VP modification was the most prominent.

[0204] Experimental Example 3: Variants based on siRNA13 and detection of their activity

[0205] The following variants were further designed based on the structure-activity relationship of siRNA13, and their modification sequences and corresponding naked sequences are shown in Tables 3-1-3-2. The in vitro inhibitory activity of the above siRNA13 structure-activity relationship variants was detected according to the method described in Experimental Example 1, and the inhibitory activity results at a concentration of 1 nM are shown in Figures 6A-6B, and IC 50 The detection results are shown in Figure 7. These variants showed good inhibitory activity, among which the SS19 mismatch variant siRNA13-10 and siRNA13-11, the SS16 mismatch variant siRNA13-7, the SS17 mismatch variant siRNA13-8, and the SS18 mismatch variant siRNA13-9 all had improved activity compared with siRNA13, and the inhibitory activity of the SS17 mismatch variant siRNA13-8 was the best.

[0206] Table 3-1: siRNA13 structure-activity relationship variant sequences

[0207] Table 3-2: Naked sequence of siRNA13 structure-activity relationship variant

[0208] Experimental Example 4: Variants based on siRNA11 and detection of their activity

[0209] Based on the structure-activity relationship of siRNA 11, the following variants were further designed, the modified sequences and the corresponding naked sequences are shown in Table 4-1~Table 4-2 respectively. The in vitro inhibitory activity of the above siRNA 11 structure-activity relationship variants was detected according to the method described in Experimental Example 1, and the inhibitory activity results at a concentration of 1 nM are shown in Figure 8, IC 50 The detection results are shown in Figure 9. These variants show good inhibitory activity.

[0210] Table 4-1: siRNA 11 structure-activity relationship variant sequences

[0211] Table 4-2: Naked sequence of siRNA 13 structure-activity relationship variant

[0212] Experimental Example 5: Variants based on siRNA 12 and detection of their activity

[0213] Based on the structure-activity relationship of siRNA 12, the following variants were further designed, the modified sequences and the corresponding naked sequences are shown in Table 5-1~Table 5-2 respectively. The in vitro inhibitory activity of the above siRNA 11 structure-activity relationship variants was detected according to the method described in Experimental Example 1, and the inhibitory activity results at a concentration of 1 nM are shown in Figure 10, IC 50 The detection results are shown in Figure 11. These variants show good inhibitory activity.

[0214] Table 5-1: siRNA 12 structure-activity relationship variant sequences

[0215] Table 5-2: Naked sequence of siRNA 12 structure-activity relationship variant

[0216] Experimental Example 6: Variants based on siRNA 16 and detection of their activity

[0217] Based on the structure-activity relationship of siRNA 16, the following variants were further designed, the modified sequences and the corresponding naked sequences are shown in Table 6-1~Table 6-2 respectively. The in vitro inhibitory activity of the above siRNA 16 structure-activity relationship variants was detected according to the method described in Experimental Example 1, and the inhibitory activity results at a concentration of 1 nM are shown in Figure 12A-12B, IC 50 The detection results are shown in Figure 13. These variants show good inhibitory activity.

[0218] Table 6-1: siRNA 16 structure-activity relationship variant sequences

[0219] Table 6-2: Naked sequence of siRNA 16 structure-activity relationship variant

[0220] Experimental Example 7: Effect of siRNA on Sensitivity of Tumor Cells to TMZ

[0221] Recover U118MG cells (human glioblastoma cells, obtained from Shangcheng Beinaikai Biotechnology Co., Ltd.), inoculate into 96-well plates, 5 x 10 3 cells per well, 100 μL of complete culture medium, and incubate at 37°C in a 5% CO2 incubator. After the cells adhere, use CALNP TM mRNA in vitro transfection reagent (Dona, DN001-10) to dilute siRNA (siRNA17 or siRNA19) to 2 μM with PBS, incubate for 5 min according to 2 uL siRNA + 14 uL A + 4 uL B, then add 20 uL of culture medium, take 10 uL of transfection complex and add it to the 96-well plate, and after 24 h, replace the culture medium containing TMZ (the concentration of TMZ is set to 6.25, 12.5, 25, 50, 100, 200, and 400 μg / mL, respectively), and after another 72 h, use 10% CCK8 to detect the OD450 cell viability.

[0222] The expression of MGMT after transfection of siRNA is shown in Figure 14A, and the siRNA has a good knockdown efficiency, which can be stably achieved above 90% and has a long duration. The IC 50 The results are shown in Figure 14B, and after knocking down MGMT, the sensitivity of U118MG to TMZ is significantly improved, and the IC 50 is reduced to about 1 / 3 of the original.

[0223] Recover T98G cells (human malignant glioblastoma cells, obtained from Shangcheng Beinaikai Biotechnology Co., Ltd.), inoculate into 96-well plates, 5 x 10 3 cells per well, 100 μL of complete culture medium, and incubate at 37°C in a 5% CO2 incubator. After the cells adhere, use CALNP TM mRNA in vitro transfection reagent (Dona, DN001-10) to dilute siRNA (siRNA20) to 2 μM with PBS, incubate for 5 min according to 2 uL siRNA + 14 uL A + 4 uL B, then add 20 uL of culture medium, take 10 uL of transfection complex and add it to the 96-well plate, and after 24 h, replace the culture medium containing TMZ (the concentration of TMZ is set to 6.25, 12.5, 25, 50, 100, 200, and 400 μg / mL, respectively), and after another 72 h, use 10% CCK8 to detect the OD450 cell viability.

[0224] The MGMT expression after transfection of siRNA is shown in Figure 14C. The siRNA has good knockdown efficiency, which can be stably achieved at about 90%, and has a long duration.

[0225] Experimental Example 8: KD activity of siRNA in C57 mouse liver

[0226] siRNA 17 and siRNA 19 were conjugated with L96 to obtain siRNA 17-L96 and siRNA 19-L96, respectively. C57 mice were administered a single dose subcutaneously, and the dose was 1 mpk. One week after administration, the mice were sacrificed, the liver was removed, and the target gene expression was detected by qPCR. The results are shown in Figure 15. siRNA 17 showed significantly better in vivo inhibition activity than siRNA 19.

[0227] Example 9: Knockdown of MGMT mRNA by siRNA after free uptake of tumor cells

[0228] The U118MG cells (obtained from Nanjing Kebai Biotechnology Co., Ltd.) were recovered and inoculated into a 96-well plate at 5x10 3 cells per well in a volume of 90 μL, 6 replicates, 100 μL complete culture medium, and incubated in a 37°C, 5% CO2 incubator. After the cells adhered, the siRNA (siRNA 17 conjugated with C16 and DRI-T8 carrier to obtain siRNA 17-C1, structure shown in Figure 20A) lyophilized powder was diluted to 1 μM with 1x PBS, and 10 uL was added to the 96-well plate. After 24 h, the medium was replaced with 200 μL per well, and after 72 h of culture, 3 replicates were added with lysis solution to extract RNA. The subsequent RNA extraction, quantification, reverse transcription, and qRT-PCR were used to detect the KD efficiency at the RNA level.

[0229] The results are shown in Figure 16. The siRNA 17 conjugated with the carrier had a good knockdown efficiency of 78% on MGMT mRNA at a free uptake of 100 nM in U118MG cells. However, the sequence without the carrier had no KD on MGMT mRNA at a free uptake of 100 nM in U118MG cells.

[0230] Example 10: Efficacy of siRNA in C57 mouse GL261 subcutaneous tumor

[0231] The mouse-derived glioma GL261 cells (obtained from Shangcheng Beinaeliang Biotechnology Co., Ltd.) were subcutaneously inoculated into nude mice (obtained from Saiye (Suzhou) Biotechnology Co., Ltd.), and the tumor volume was allowed to grow to 500-1000 mm 3When the tumor volume reached 500-1000 mm3, the tumor was collected and separated, other tissues were removed under sterile conditions, and the tumor mass was cut into small pieces of about 3 mm x 3 mm x 3 mm, which was inoculated into the right axillary of nude mice using a trocar, and when the average tumor volume of tumor-bearing mice reached 150-250 mm3, the tumor was collected and separated, other tissues were removed under sterile conditions, and the tumor mass was cut into small pieces of about 3 mm x 3 mm x 3 mm, which was inoculated into the right axillary of nude mice using a trocar, and when the average tumor volume of tumor-bearing mice reached 150-250 mm 3 When the tumor volume reached 500-1000 mm3, the tumor was collected and separated, other tissues were removed under sterile conditions, and the tumor mass was cut into small pieces of about 3 mm x 3 mm x 3 mm, which was inoculated into the right axillary of nude mice using a trocar, and when the average tumor volume of tumor-bearing mice reached 150-250 mm

[0232] The sequence siRNA17-C1 with carrier was administered one day before TMZ, and was administered intratumorally twice a week; TMZ was administered by gavage once a day, continuously for 5 days, and stopped for 2 days. The tumor mass volume was measured every week, and the animal weight was measured once a week.

[0233] Table 7: Grouping and administration

[0234] The efficacy of the subcutaneous tumor of nude mice is shown in FIG. 17, 50 mpk TMZ has an inhibitory effect on GL261 subcutaneous tumor, the sequence siRNA17-C1 with carrier has no obvious inhibitory effect on GL261 subcutaneous tumor, and the combination of the sequence siRNA17-C1 with carrier and TMZ can significantly increase the tumor inhibition effect of TMZ.

[0235] Example 11: Efficacy of siRNA in U251 subcutaneous tumor of mice

[0236] Human glioma U251 cells (obtained from Wuhan Ponsay Life Science Co., Ltd.) were subcutaneously inoculated into NKG mice (obtained from Saiye (Suzhou) Biological Technology Co., Ltd.), and when the tumor volume reached 500-1000 mm 3 When the tumor volume reached 500-1000 mm3, the tumor was collected and separated, other tissues were removed under sterile conditions, and the tumor mass was cut into small pieces of about 3 mm x 3 mm x 3 mm, which was inoculated into the right axillary of nude mice using a trocar, and when the average tumor volume of tumor-bearing mice reached 150-250 mm 3 When the tumor volume reached 500-1000 mm3, the tumor was collected and separated, other tissues were removed under sterile conditions, and the tumor mass was cut into small pieces of about 3 mm x 3 mm x 3 mm, which was inoculated into the right axillary of nude mice using a trocar, and when the average tumor volume of tumor-bearing mice reached 150-250 mm

[0237] The sequence siRNA17-C2 (SS 5 end conjugated with C16, SS 3 end conjugated with DRI-T8, AS modified with VP, and the structure of siRNA17-C2 is shown in FIG. 20B) was administered one day before TMZ, and was administered intratumorally once a week; TMZ was administered by gavage once a day, continuously for 5 days, and stopped for 2 days. The tumor mass volume was measured twice a week, and the animal weight was measured once a week.

[0238] Table 8: Grouping and administration

[0239] The pharmacodynamics of nude mice subcutaneous tumor is shown in Figure 18. 10mpk TMZ has inhibitory effect on U251 subcutaneous tumor. The sequence siRNA17-C2 with carrier has no obvious inhibitory effect on U251 subcutaneous tumor. After the combination of the sequence siRNA17-C2 with carrier and TMZ, the inhibitory effect of TMZ on tumor can be obviously increased.

[0240] Example 12: In vitro off-target cytotoxicity

[0241] Four siRNA sequences, siRNA13, siRNA14, siRNA17 and siRNA20, were selected to verify whether there is toxicity in the cell line caused by off-target. Different concentrations of siRNA were transfected in A549 (obtained from North China Nankai Link Biotechnology Co., Ltd.) and KYSE-150 cells (obtained from North China Nankai Link Biotechnology Co., Ltd.) using RNAiMAX, and the changes in cell viability and cell death levels were detected by CCK8 (Cell Counting Kit-8) and PI (propidium iodide) staining methods, respectively.

[0242] The results shown in Figures 19A and 19B indicate that the four sequences have no obvious cytotoxicity at low / high dose levels.

Claims

1. An siRNA for inhibiting the expression of a MGMT gene, the siRNA comprising a sense strand and an antisense strand, wherein, the antisense strand comprises at least 17 contiguous nucleotides differing by no more than 4 (e.g. 0, 1, 2, 3 or 4) nucleotides from the nucleotide sequence of any one of SEQ ID NOs: 1-9, 60, 62, 64, 66, 68, 70, 103, 105, 108, 127, 139, 154, 168 and the sense strand comprises a nucleotide sequence that is at least partially complementary to the antisense strand; Preferably, the antisense strand comprises at least 17 contiguous nucleotides differing by no more than 2 (e.g. 0, 1 or 2) nucleotides from the nucleotide sequence of any one of SEQ ID NOs: 1-9, 60, 62, 64, 66, 68, 70, 103, 105, 108, 127, 139, 154, 168.

2. The siRNA of claim 1, wherein, the sense strand has no more than 6 (e.g. 0, 1, 2, 3, 4, 5 or 6) nucleotide mismatches with the antisense strand; Preferably, the sense strand has no more than 2 (e.g. 0, 1 or 2) nucleotide mismatches with the antisense strand; Preferably, the mismatch is at any one of positions 11, 12, 13, 14, 15, 16, 17, 18 or 19 of the sense strand, further preferably, the mismatch is at any one of positions 16, 17, 18 or 19 of the sense strand.

3. The siRNA of claim 1 or 2, wherein, the sense strand comprises at least 17 contiguous nucleotides differing by no more than 4 (e.g. 0, 1, 2, 3 or 4) nucleotides from the nucleotide sequence of any one of SEQ ID NOs: 10-18, 59, 61, 63, 65, 67, 69, 71-80, 102, 104, 106, 107, 109-115, 122-126, 134-138, 152, 153, 155-163, 169; Preferably, the sense strand comprises at least 17 contiguous nucleotides differing by no more than 2 (e.g. 0, 1 or 2) nucleotides from the nucleotide sequence of any one of SEQ ID NOs: 10-18, 59, 61, 63, 65, 67, 69, 71-80, 102, 104, 106, 107, 109-115, 122-126, 134-138, 152, 153, 155-163, 169.

4. The siRNA of any one of claims 1-3, wherein, the siRNA comprises a blunt end and / or 1-4 nucleotides overhang; Preferably, the siRNA comprises a 1 or 2 nucleotide overhang; Preferably, the overhang is present at the 5' end and / or the 3' end of the antisense strand and / or the sense strand; Preferably, the 3' end of the antisense strand of the siRNA comprises a 2 nucleotide overhang and the 3' end of the sense strand of the siRNA is blunt; Preferably, the 3' end of the antisense strand and the 3' end of the sense strand of the siRNA each comprise a 2 nucleotide overhang.

5. The siRNA of any one of claims 1-4, wherein, The length of the antisense strand and the length of the sense strand are each independently 17-30 nucleotides; preferably, the length of the antisense strand is 19-27 nucleotides; preferably, the length of the sense strand is 17-25 nucleotides.

6. The siRNA of any one of claims 1-4, wherein, The length of the antisense strand is 21-23 nucleotides, and the length of the sense strand is 19-21 nucleotides.

7. The siRNA of any one of claims 1-6, wherein, The sequence of the sense strand and the sequence of the antisense strand of the siRNA are selected from the corresponding sense strand and antisense strand sequences of any one of siRNA1 to siRNA9 and siRNA21 described in Table 1-1, siRNA8-1 to siRNA8-16 described in Table 2-2, siRNA4-1 to siRNA4-11 described in Table 3-2, siRNA2-1 to siRNA2-5 described in Table 4-2, siRNA3-1 to siRNA3-5 described in Table 5-2, and siRNA7-1 to siRNA7-11 described in Table 6-2. Preferably, the sequence of the sense strand and the sequence of the antisense strand of the siRNA are selected from the corresponding sense strand and antisense strand sequences of any one of siRNA1 to siRNA9 and siRNA21 described in Table 1-1, siRNA8-5, siRNA8-7, siRNA8-16, siRNA8-8 described in Table 2-2, siRNA4-10, siRNA4-11, siRNA4-8, siRNA4-7, or siRNA4-9 described in Table 3-2, siRNA3-3 described in Table 5-2, and siRNA7-1 described in Table 6-2.

8. The siRNA of any one of claims 1-7, wherein, The siRNA contains at least one modified nucleotide.

9. The siRNA of claim 8, wherein, All the nucleotides in the sense strand and / or the antisense strand of the siRNA are modified nucleotides or nucleotide analogs.

10. The siRNA of claim 9, wherein, The modified nucleotide or nucleotide analog is selected from 2'-methoxy nucleotide, 2'-fluoro nucleotide, 2'-deoxy nucleotide, 2',3'-seco nucleotide analog, 2'-fluoro arabinonucleotide, 2'-methoxy ethyl nucleotide, 2'-amino modified nucleotide, 2'-alkyl modified nucleotide, 3'-methoxy nucleotide, 2'-allyl modified nucleotide, glycol modified nucleotide, abasic nucleotide, morpholino nucleotide, locked nucleotide, unlocked nucleotide, or glycerol nucleotide.

11. The siRNA of any one of claims 8-10, wherein, All the nucleotides in the sense strand and / or the antisense strand of the siRNA are modified nucleotides, and the modified nucleotide is selected from 2'-methoxy nucleotide and 2'-fluoro nucleotide.

12. The siRNA of any one of claims 8-11, wherein, The 5' end of the antisense strand of the siRNA comprises a phosphorus-containing group; Preferably, the phosphorus-containing group is selected from 5' phosphate (5'-P), 5' thiophosphate (5'-PS), 5' dithiophosphate (5'-PS2), 5' vinylphosphate (5'-VP), 5' methylphosphate (MePhos), or 5'-deoxy-5'-C-malonate; Preferably, the 5' end of the antisense strand of the siRNA comprises 5' vinylphosphate (5'-VP).

13. The siRNA of any one of claims 1-12, wherein, The sense strand and / or the antisense strand of the siRNA comprises a modified internucleoside linkage. Preferably, the nucleotides between the 1st and 2nd position and between the 2nd and 3rd position of the 5' end and / or 3' end of the sense strand are linked by phosphorothioate group; Preferably, the nucleotides between the 1st and 2nd position and between the 2nd and 3rd position of the 5' end and / or 3' end of the antisense strand are linked by phosphorothioate group.

14. The siRNA of any one of claims 8-13, wherein, Preferably, the sequence of the sense strand and the antisense strand of the siRNA is selected from the corresponding sense strand and antisense strand sequence of any one of siRNA10 to siRNA18 and siRNA20 described in Table 1-2, siRNA17-1 to siRNA17-16 described in Table 2-1, siRNA17-17 to siRNA17-29 described in Table 2-3, siRNA13-1 to siRNA13-11 described in Table 3-1, siRNA11-1 to siRNA11-5 described in Table 4-1, siRNA12-1 to siRNA12-5 described in Table 5-1, and siRNA16-1 to siRNA16-11 described in Table 6-1. Preferably, the sequence of the sense strand and the antisense strand of the siRNA is selected from the corresponding sense strand and antisense strand sequence of any one of siRNA10 to siRNA18 and siRNA20 described in Table 1-2, siRNA17-5, siRNA17-7, siRNA17-16, siRNA17-8 described in Table 2-1, siRNA17-17, siRNA17-18, siRNA17-19, siRNA17-20, siRNA17-21 or siRNA17-25 described in Table 2-3, siRNA13-10, siRNA13-11, siRNA13-8, siRNA13-7 or siRNA13-9 described in Table 3-1, siRNA12-3 described in Table 5-1, and siRNA16-1 described in Table 6-1.

15. A conjugate comprising the siRNA of any one of claims 1-14 and a conjugating moiety linked thereto; preferably, the conjugating moiety comprises a targeting molecule; Preferably, the conjugating moiety is 1 or 2 or 3 or 4, and when the conjugating moiety is more than 1, the conjugating moieties are the same or different; Preferably, the targeting molecule is a GalNAc derivative, a lipophilic moiety or a peptide chain; preferably wherein, the GalNAc derivative is L96; the lipophilic moiety is selected from aliphatic, alicyclic or polyalicyclic compounds, preferably C4-C30 hydrocarbyl (e.g. alkyl or alkenyl), more preferably C6-C18 hydrocarbyl (e.g. alkyl or alkenyl), further preferably C16 hydrocarbyl (e.g. alkyl or alkenyl); the GalNAc derivative is L96; the peptide chain is a peptide chain having 5-50 amino acids, preferably, the peptide chain is a peptide chain having 7-18 amino acids, further preferably, the peptide chain is CHRPYIAH (SEQ ID NO: 346), HAIYPRHC (SEQ ID NO: 347), PWVPSWMPPRHT (SEQ ID NO: 348) or THRPPMWSPVWP (SEQ ID NO: 349); preferably, the coupling moiety is linked via a linker, the linker containing an ether bond, a thioether bond, a urea, a carbonate, an amine, an amide, a maleimide-thioether, a disulfide, a phosphate, a phosphodiester, a sulfonamide or a carbamate, a hydrazone group, an ester group, an acetal group or a ketal group, a peptide bond, or the linker is a biologically cleavable linker selected from the group consisting of DNA, RNA, disulfide, amide, functionalized monosaccharide or oligosaccharide of galactosamine, glucosamine, glucose, galactose, mannose, and combinations thereof; preferably, the coupling moiety is linked at a position selected from the group consisting of a nucleotide at the 5' end of the sense strand, a nucleotide at a middle position between the two positions of the 5' end and the 3' end of the sense strand, a nucleotide at the 3' end of the sense strand; more preferably, the lipophilic moiety is conjugated at the 2 position of the nucleotide sugar ring at the above-mentioned position.

16. A pharmaceutical composition, wherein, the pharmaceutical composition comprises the siRNA of any one of claims 1-14 or the conjugate of claim 15, and a pharmaceutically acceptable carrier and / or excipient; preferably, the pharmaceutically acceptable carrier and / or excipient is a delivery vehicle; preferably, the siRNA or conjugate is encapsulated by the delivery vehicle.

17. Use of the siRNA of any one of claims 1-14, the conjugate of claim 15 or the pharmaceutical composition of claim 16 in the manufacture of a medicament for reducing or preventing resistance of a tumor to an alkylating agent.

18. Use of the siRNA of any one of claims 1-14, the conjugate of claim 15 or the pharmaceutical composition of claim 16 and an alkylating agent in the manufacture of a medicament for treating a tumor; preferably, the tumor is resistant to an alkylating agent.

19. A method for reducing or preventing resistance of a tumor to an alkylating agent, comprising administering to a subject in need thereof an effective amount of the siRNA of any one of claims 1-14, the conjugate of claim 15 or the pharmaceutical composition of claim 16.

20. A method for treating a tumor, comprising administering to a subject in need thereof an effective amount of an alkylating agent and an effective amount of the siRNA of any one of claims 1-14, the conjugate of claim 15 or the pharmaceutical composition of claim 16; preferably, the alkylating agent and the siRNA, conjugate or pharmaceutical composition are administered in any order, e.g., simultaneously, before or after.

21. The use of claim 17 or 18 or the method of claim 19 or 20, wherein, the alkylating agent is temozolomide (TMZ), Lomustine or Carmustine.

22. The use of claim 17 or 18 or the method of claim 19 or 20, wherein, the tumor is a glioma.

23. Use of the siRNA according to any one of claims 1 to 14, of the conjugate according to claim 15 or of the pharmaceutical composition according to claim 16 for the manufacture of a medicament for the treatment and / or prevention of a pathological condition or disease associated with MGMT; Preferably, the pathological condition or disease associated with MGMT involves MGMT overexpression or would benefit from a decrease or inhibition of MGMT levels.

24. A method for the prevention and / or treatment of a pathological condition or disease associated with MGMT in a subject, said method comprising administering to a subject in need thereof an effective amount of the siRNA according to any one of claims 1 to 14, of the conjugate according to claim 15 or of the pharmaceutical composition according to claim 16; Preferably, the pathological condition or disease associated with MGMT involves MGMT overexpression or would benefit from a decrease or inhibition of MGMT levels.

25. The siRNA according to any one of claims 1 to 14, the conjugate according to claim 15 or the pharmaceutical composition according to claim 16 for use in the prevention and / or treatment of a pathological condition or disease associated with MGMT; Preferably, the pathological condition or disease associated with MGMT involves MGMT overexpression or would benefit from a decrease or inhibition of MGMT levels.

26. A method of inhibiting MGMT expression in a cell, the method comprising: introducing into a cell the siRNA according to any one of claims 1 to 14, the conjugate according to claim 15 or the pharmaceutical composition according to claim 16.

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