Shrna specifically inhibiting expression of mlkl gene and application thereof

Abstract

The present application provides an shRNA for specifically inhibiting expression of an MLKL gene and application thereof. In particular, the present application provides an shRNA for specifically inhibiting expression of an MLKL gene, a recombinant vector, a recombinant lentivirus, an siRNA, a host cell, application of the shRNA or the recombinant vector or the recombinant lentivirus or the host cell or the siRNA in the preparation of a drug for specifically inhibiting expression of an MLKL gene, application in the preparation of a drug for preventing or treating liver fibrosis and / or liver injury, and a method for specifically inhibiting expression of an MLKL gene and a method for preventing or treating liver fibrosis and / or liver injury using the shRNA or the recombinant vector or the recombinant lentivirus or the host cell or the siRNA.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an shRNA that specifically inhibits the expression of the mixed-lineage kinase domain-like protein (MLKL) gene and its application. The shRNA targets MLKL and can be used to prevent or treat liver fibrosis and / or liver damage. Background Technology

[0002] The liver is a crucial hub for many physiological processes, including nutrient metabolism, immune defense, regulation of circulating blood volume, regulation of growth signaling pathways, lipid and cholesterol homeostasis, and the metabolism of exogenous compounds, including many existing drugs. Liver fibrosis is a leading cause of morbidity and mortality from chronic hepatitis and obesity-related fatty liver worldwide. Its causes are diverse, the most common including hepatotoxicity, viral hepatitis, autoimmune diseases, metabolic disorders, non-alcoholic steatohepatitis (NASH), and cholestatic liver disease. [1,2] wait.

[0003] Damage to hepatocytes triggers the infiltration of inflammatory cells, causing hepatic stellate cells (HSCs) to change from a resting state to an activated state. Activated HSCs can produce extracellular matrix (ECM), and long-term chronic damage leads to the gradual deposition of ECM, eventually causing liver fibrosis. [3] Studies in human diseases and animal models have revealed that liver fibrosis is a dynamic process. If damaging factors persist without effective intervention, chronic inflammation and endocrine disruption (ECM) accumulate, gradually replacing the liver parenchyma with scar tissue, leading to further progression of liver fibrosis into cirrhosis. Once chronic liver injury and fibrosis progress to cirrhosis, the chances of reversing this process decrease, potentially leading to a series of life-threatening complications such as portal hypertension, ascites, liver failure, and hepatic encephalopathy. [4] Reversing early-stage liver fibrosis in a timely manner is widely recognized as an effective means of preventing serious liver disease, but it also represents a significant challenge in the field of liver disease.

[0004] Hepatocellular death following injury is a crucial driver of liver fibrosis. Studies have reported that the effector protein MLKL in the programmed necrosis signaling pathway plays a vital role in hepatocellular death. [5] Other studies have found that knocking out MLKL affects autophagy in hepatocytes and inhibits hepatocyte damage and death, thereby alleviating diet-induced nonalcoholic steatohepatitis (NASH). [6] .

[0005] The development of new drugs for treating liver fibrosis is challenging due to the complex pathogenesis. Liver fibrosis results from the parallel interactions of multiple cell types and various tissues and organs, a common characteristic of chronic diseases. Increasing research demonstrates that the same gene often produces different, sometimes even completely opposite, effects in different tissues, organs, or cells. Therefore, targeted cell-specific regulation of gene expression is crucial when studying gene function. As a novel treatment approach, gene therapy has achieved significant success in treating specific diseases with well-defined targets. Summary of the Invention

[0006] MLKL plays a crucial role in hepatocyte damage and death. This invention designs multiple shRNAs that specifically knock down MLKL. Adeno-associated virus (AAV8) is used to deliver shRNAs specifically targeting mouse hepatocytes (shRNA expression driven by the human thyroid-binding globulin promoter TBG) to treat liver fibrosis. Simultaneously, multiple shRNAs specifically knocking down human MLKL are designed to alleviate chemically induced damage to HepG2 cells.

[0007] This invention has found that knocking out or downexpressing MLKL in hepatocytes inhibits hepatocyte damage and death induced by chemical toxins, thereby alleviating liver damage and liver fibrosis induced by carbon tetrachloride (CCl4), as well as liver damage and liver fibrosis induced by cholestasis (BDL).

[0008] One object of the present invention is to provide an shRNA that specifically inhibits the expression of the MLKL gene.

[0009] Another objective of this invention is to provide a recombinant vector that specifically inhibits MLKL gene expression.

[0010] Another objective of this invention is to provide a recombinant lentivirus that specifically inhibits MLKL gene expression.

[0011] Another object of the present invention is to provide an siRNA that specifically inhibits the expression of the MLKL gene.

[0012] Another object of the present invention is to provide a host cell.

[0013] Another object of the present invention is to provide the use of the above-mentioned shRNA or recombinant vector or recombinant lentivirus or host cell or siRNA in the preparation of a drug for specifically inhibiting MLKL gene expression.

[0014] Another object of the present invention is to provide the use of the above-mentioned shRNA or recombinant vector or recombinant lentivirus or host cell or siRNA in the preparation of a medicament for the prevention or treatment of liver fibrosis and / or liver injury.

[0015] Another object of the present invention is to provide a method for specifically inhibiting MLKL gene expression.

[0016] Another object of the present invention is to provide a method for preventing or treating liver fibrosis and / or liver injury.

[0017] On one hand, the present invention provides an shRNA that specifically inhibits the expression of the MLKL gene, wherein the shRNA targets a nucleotide sequence comprising a nucleotide sequence selected from the following:

[0018] 5'-AGGAGATTCCAAAGGAACATC-3' (SEQ ID NO: 1);

[0019] 5'-AGCTTGCAGGATTTGAGTTAA-3' (SEQ ID NO: 2);

[0020] 5'-AGCAGAGAGATCCAGTTCAAC-3' (SEQ ID NO: 3);

[0021] 5'-AGATCCAGTTCAACGATATAT-3' (SEQ ID NO: 4);

[0022] 5'-AGCCGGAGGCTACCAAGTAAA-3' (SEQ ID NO: 5);

[0023] 5'-CGGACAGCAAAGAGCACTAAA-3' (SEQ ID NO: 6);

[0024] 5'-TCCCAACATCTTGCGTATATT-3' (SEQ ID NO:7);

[0025] 5'-CCTGAAGCAATGCTCACTAAA-3' (SEQ ID NO:8);

[0026] 5'-CATTGGAATACCGTTTCAGAT-3' (SEQ ID NO:9);

[0027] 5'-GCGTATATTTGGGATTTGCAT-3' (SEQ ID NO: 10);

[0028] 5'-GCAGGATTTGAGTTAAGCAAA-3' (SEQ ID NO: 11);

[0029] 5'-CCCTGAGTTCTCCATTGTCAT-3' (SEQ ID NO: 12);

[0030] 5'-GGGACAGATCATCAAGTTA-3' (SEQ ID NO: 13);

[0031] 5'-CACAAGATCTCCAAATCAA-3' (SEQ ID NO: 14);

[0032] 5'-GGCCGAAAGTGTTGGAATA-3' (SEQ ID NO: 15);

[0033] 5'-CCCAACATCCTGCGTATATTT-3' (SEQ ID NO: 16);

[0034] 5'-CCTCTGTGGATGAAATCTTAA-3' (SEQ ID NO: 17);

[0035] 5'-TCCCGTTTCAAGGCTGTAATT-3' (SEQ ID NO: 18);

[0036] 5'-GAGTCAAATCTACAGCATATC-3' (SEQ ID NO: 19);

[0037] 5'-CCTTCGGCATTGGGTTATCTA-3' (SEQ ID NO: 20);

[0038] 5'-GAAGCTTCACTGAGACGATTA-3' (SEQ ID NO: 21);

[0039] 5'-GCCTCAATTCTCCATTGTCAT-3' (SEQ ID NO: 22);

[0040] 5'-GTGACTTGATTTGATCAATAG-3' (SEQ ID NO: 23);

[0041] 5'-AGAGATGAATACTGCAAGAA-3' (SEQ ID NO: 24);

[0042] 5'-CCTCTGACAGTAACTTTGATA-3' (SEQ ID NO: 25).

[0043] In some embodiments, the nucleotide sequence targeted by the shRNA comprises a nucleotide sequence selected from the following:

[0044] 5'-AGGAGATTCCAAAGGAACATC-3' (SEQ ID NO: 1);

[0045] 5'-AGCTTGCAGGATTTGAGTTAA-3' (SEQ ID NO: 2);

[0046] 5'-AGCAGAGAGATCCAGTTCAAC-3' (SEQ ID NO: 3);

[0047] 5'-CCCAACATCCTGCGTATATTT-3' (SEQ ID NO: 16);

[0048] 5'-CCTCTGTGGATGAAATCTTAA-3' (SEQ ID NO: 17).

[0049] In some embodiments, the shRNA has a length of approximately 44-61 bp and includes a sense strand, a loop, and an antisense strand, wherein the target sequence is approximately 21-23 bp in length.

[0050] In some embodiments, the nucleotide sequence encoding the shRNA comprises a nucleotide sequence selected from the following:

[0051] 5'-AGGAGATTCCAAAGGAACATCTAGTGAAGCCACAGATGTAGATGTTCCTTTGGAATCTCCT-3' (SEQ ID NO: 26);

[0052] 5'-AGCTTGCAGGATTTGAGTTAATAGTGAAGCCACAGATGTATTAACTCAAATCCTGCAAGCT-3' (SEQ ID NO: 27);

[0053] 5'-AGCAGAGAGATCCAGTTCAACTAGTGAAGCCACAGATGTAGTTGAACTGGATCTCTCTGCT-3' (SEQ ID NO: 28);

[0054] 5'-AGATCCAGTTCAACGATATATCTCGAGATATATCGTTGAACTGGATC-3' (SEQ ID NO: 29);

[0055] 5'-AGCCGGAGGCTACCAAGTAAAACTCGAGTTTACTTGGTAGCCTCCGGC-3' (SEQ ID NO: 30);

[0056] 5’-CGGACAGCAAAGAGCACTAAACTCGAGTTTAGTGCTCTTTGCTGTCCG-3’(SEQ ID NO:31);

[0057] 5’-TCCCAACATCTTGCGTATATTCTCGAGAATATACGCAAGATGTTGGGA-3’(SEQ ID NO:32);

[0058] 5’-CCTGAAGCAATGCTCACTAAACTCGAGTTTAGTGAGCATTGCTTCAGG-3’(SEQ ID NO:33);

[0059] 5’-CATTGGAATACCGTTTCAGATCTCGAGATCTGAAACGGTATTCCAATG-3’(SEQ ID NO:34);

[0060] 5’-GCGTATATTTGGGATTTGCATCTCGAGATGCAAATCCCAAATATACGC-3’(SEQ ID NO:35);

[0061] 5’-GCAGGATTTGAGTTAAGCAAACTCGAGTTTGCTTAACTCAAATCCTGC-3’(SEQ ID NO:36);

[0062] 5’-CCCTGAGTTCTCCATTGTCATCTCGAGATGACAATGGAGAACTCAGGG-3’(SEQ ID NO:37);

[0063] 5’-GGGACAGATCATCAAGTTACTCGAGTAACTTGATGATCTGTCCC-3’(SEQID NO:38);

[0064] 5’-CACAAGATCTCCAAATCAACTCGAGTTGATTTGGAGATCTTGTG-3’(SEQID NO:39);

[0065] 5’-GGCCGAAAGTGTTGGAATACTCGAGTATTCCAACACTTTCGGCC-3’(SEQID NO:40);

[0066] 5’-AGCTTGCAGGATTTGAGTTAACTCGAGTTAACTCAAATCCTGCAAGCT-3’(SEQ ID NO:41);

[0067] 5’-AGCAGAGAGATCCAGTTCAACCTCGAGGGTTGAACTGGATCTCTCTGCT-3’(SEQ ID NO:42);

[0068] 5’-CCCAACATCCTGCGTATATTTCTCGAGAAATATACGCAGGATGTTGGG-3’(SEQ ID NO:43);

[0069] 5’-CCTCTGTGGATGAAATCTTAACTCGAGTTAAGATTTCATCCACAGAGG-3’(SEQ ID NO:44);

[0070] 5’-TCCCGTTTCAAGGCTGTAATTCTCGAGAATTACAGCCTTGAAACGGGA-3’(SEQ ID NO:45);

[0071] 5’-GAGTCAAATCTACAGCATATCCTCGAGGATATGCTGTAGATTTGACTC-3’(SEQ ID NO:46);

[0072] 5’-CCTTCGGCATTGGGTTATCTACTCGAGTAGATAACCCAATGCCGAAGG-3’(SEQ ID NO:47);

[0073] 5’-GAAGCTTCACTGAGACGATTACTCGAGTAATCGTCTCAGTGAAGCTTC-3’(SEQ ID NO:48);

[0074] 5’-GCCTCAATTCTCCATTGTCATCTCGAGATGACAATGGAGAATTGAGGC-3’(SEQ ID NO:49);

[0075] 5’-GTGACTTGATTTGATCAATAGCTCGAGCTATTGATCAAATCAAGTCAC-3’(SEQ ID NO:50);

[0076] 5'-AGAGATGAATACTGCAAGAACTCGAGTTCTTGCAGTATTTCATCTC-3' (SEQ ID NO: 51);

[0077] 5'-CCTCTGACAGTAACTTTGATACTCGAGTATCAAAGTTACTGTCAGAGG-3' (SEQ ID NO: 52).

[0078] In a further embodiment, the nucleotide sequence encoding the shRNA comprises a nucleotide sequence selected from the following:

[0079] 5'-AGGAGATTCCAAAGGAACATCTAGTGAAGCCACAGATGTAGATGTTCCTTTGGAATCTCCT-3' (SEQ ID NO: 26);

[0080] 5'-AGCTTGCAGGATTTGAGTTAATAGTGAAGCCACAGATGTATTAACTCAAATCCTGCAAGCT-3' (SEQ ID NO: 27);

[0081] 5'-AGCAGAGAGATCCAGTTCAACTAGTGAAGCCACAGATGTAGTTGAACTGGATCTCTCTGCT-3' (SEQ ID NO: 28);

[0082] 5'-CCCAACATCCTGCGTATATTTCTCGAGAAATATACGCAGGATGTTGGG-3' (SEQ ID NO: 43);

[0083] 5'-CCTCTGTGGATGAAATCTTAACTCGAGTTAAGATTTCATCCACAGAGG-3' (SEQ ID NO: 44).

[0084] On the other hand, the present invention provides a recombinant vector that specifically inhibits MLKL gene expression, comprising adeno-associated virus 8 (AAV8) as a backbone vector and a nucleotide sequence encoding the shRNA of the present invention.

[0085] In some embodiments, the recombinant vector of the present invention introduces a hepatocyte-specific promoter (TBG) on AAV8 to control the expression of shRNA, thereby enabling specific knockdown of MLKL in hepatocytes.

[0086] In some embodiments, the recombinant vector of the present invention also contains a green fluorescent protein (GFP) sequence in the AAV8 virus, which can be used to indicate the targeting of AAV.

[0087] On the other hand, the present invention provides a recombinant lentivirus that specifically inhibits the expression of the MLKL gene, which is obtained by cloning the nucleotide sequence encoding the shRNA of the present invention into the lentiviral plasmid PLKO.1 to obtain a recombinant plasmid, and then co-transfecting the recombinant plasmid with the packaging plasmid psPAX2 and the envelope plasmid pMD2 into mammalian cells (such as HEK293T cells).

[0088] In another aspect, the present invention provides a siRNA that specifically inhibits MLKL gene expression, comprising an interference target sequence and a protective base sequence, wherein the interference target sequence comprises a nucleotide sequence selected from the following:

[0089] 5'-AGCUUGCAGGAUUUGAGUUAA-3' (SEQ ID NO: 53);

[0090] 5'-AGCAGAGAGAUCCAGUUCAAC-3' (SEQ ID NO: 54);

[0091] 5'-GGGACAGAUCAUCAAGUUA-3' (SEQ ID NO:55);

[0092] 5'-CACAAGAUCUCCAAAUCAA-3' (SEQ ID NO: 56);

[0093] 5'-GGCCGAAAGUGUUGGAAUA-3' (SEQ ID NO: 57);

[0094] 5'-AGGAGAUUCCAAAGGAACAUC-3' (SEQ ID NO: 58);

[0095] 5'-AGAUCCAGUUCAACGAUAUAU-3' (SEQ ID NO: 59);

[0096] 5'-AGCCGGAGGCUACCAAGUAAA-3' (SEQ ID NO: 60);

[0097] 5'-CGGACAGCAAAGAGCACUAAA-3' (SEQ ID NO: 61);

[0098] 5'-UCCCAACAUCUUGCGUAUAUU-3' (SEQ ID NO: 62);

[0099] 5'-CCUGAAGCAAUGCUCACUAAA-3'(SEQ ID NO:63);

[0100] 5'-CAUUGGAAUACCGUUUCAGAU-3'(SEQ ID NO:64);

[0101] 5'-GCGUAUAUUUGGGAUUUGCAU-3'(SEQ ID NO:65);

[0102] 5'-GCAGGAUUUGAGUUAAGCAAA-3'(SEQ ID NO:66);

[0103] 5'-CCCUGAGUUCUCCAUUGUCAU-3'(SEQ ID NO:67);

[0104] 5'-CCCAACAUCCUGCGUAUAUUU-3'(SEQ ID NO:68);

[0105] 5'-CCUCUGGGAUGAAAUCUUAA-3'(SEQ ID NO:69);

[0106] 5'-UCCCGUUUCAAGGCUGUAAUU-3'(SEQ ID NO:70);

[0107] 5'-GAGUCAAAUCUACAGCAUAUC-3'(SEQ ID NO:71);

[0108] 5'-CCUUCGGCAUUGGGUUAUCUA-3'(SEQ ID NO:72);

[0109] 5'-GAAGCUUCACUGAGACGAUUA-3'(SEQ ID NO:73);

[0110] 5'-GCCUCAAUUCUCCAUUGUCAU-3'(SEQ ID NO:74);

[0111] 5'-GUGACUUGAUUUGAUCAAUAG-3'(SEQ ID NO:75);

[0112] 5'-AGAGAUGAAAUACUGCAAGAA-3'(SEQ ID NO:76);

[0113] 5'-CCUCUGACAGUAACUUUGAUA-3' (SEQ ID NO:77).

[0114] In the siRNA of this invention, the protective base sequence can refer to the two nucleotide sequences at the 3' end of the siRNA: TT. The two TT bases are in a single-stranded suspended state, and their function is to enhance the stability of the siRNA double-stranded complex.

[0115] In some embodiments, the interference target sequence comprises a nucleotide sequence selected from the following:

[0116] 5'-AGCUUGCAGGAUUUGAGUUAA-3' (SEQ ID NO: 53);

[0117] 5'-AGCAGAGAGAUCCAGUUCAAC-3' (SEQ ID NO: 54);

[0118] 5'-GGGACAGAUCAUCAAGUUA-3' (SEQ ID NO:55);

[0119] 5'-CACAAGAUCUCCAAAUCAA-3' (SEQ ID NO: 56);

[0120] 5'-GGCCGAAAGUGUUGGAAUA-3' (SEQ ID NO: 57).

[0121] In some embodiments, the siRNA has a nucleotide sequence selected from the following sequences:

[0122] 5'-AGCUUGCAGGAUUUGAGUUAATT-3' (SEQ ID NO:78);

[0123] 5'-AGCAGAGAGAUCCAGUUCAACTT-3' (SEQ ID NO:79);

[0124] 5'-GGGACAGAUCAUCAAGUUATT-3' (SEQ ID NO:80);

[0125] 5'-CACAAGAUCUCCAAAUCAATT-3' (SEQ ID NO:81);

[0126] 5'-GGCCGAAAGUGUUGGAAUATT-3' (SEQ ID NO:82);

[0127] 5′-AGGAAUUCCAAAGGAACAUCTT-3′ (SEQ ID NO:83);

[0128] 5'-AGAUCCAGUUCAACGAUAUAUTT-3' (SEQ ID NO:84)

[0129] 5′-AGCCGGAGGCUACCAAGUAAATT-3′ (SEQ ID NO:85);

[0130] 5′-CGGACAGCAAAGAGCACUAAATT-3′ (SEQ ID NO:86);

[0131] 5′-UCCCAACAUCUUGCGUAUAUUTT-3′ (SEQ ID NO:87)

[0132] 5′-CCUGAAGCAAUGCUCACUAAATT-3′ (SEQ ID NO:88)

[0133] 5′-CAUUGGAAUACCGUUUCAGAUTT-3′ (SEQ ID NO:89)

[0134] 5′-GCGUAUAUUUGGAUUUGCAUTT-3′ (SEQ ID NO:90);

[0135] 5′-GCAGGAUUUGAGUUAAGCAAATT-3′ (SEQ ID NO:91)

[0136] 5′-CCCUGAGUUCUCCAUUGUCAUTT-3′ (SEQ ID NO:92)

[0137] 5′-CCCAACAUCCUGCGUAUAUUTT-3′ (SEQ ID NO:93);

[0138] 5′-CCUCUGUGGAUGAAAUCUUAATT-3′ (SEQ ID NO:94)

[0139] 5′-UCCCGUUUCAAGGCUGUAAUUTT-3′ (SEQ ID NO:95)

[0140] 5′-GAGUCAAAUCUACAGCAUAUCTT-3′ (SEQ ID NO:96)

[0141] 5'-CCUUCGGCAUUGGGUUAUCUATT-3' (SEQ ID NO:97);

[0142] 5'-GAAGCUUCACUGAGACGAUUATT-3' (SEQ ID NO:98);

[0143] 5'-GCCUCAAUUCUCCAUUGUCAUTT-3' (SEQ ID NO:99);

[0144] 5'-GUGACUUGAUUUGAUCAAUAAGTT-3' (SEQ ID NO: 100);

[0145] 5'-AGAGAUGAAAUACUGCAAGAATT-3' (SEQ ID NO: 101);

[0146] 5'-CCUCUGACAGUAACUUUGAUATT-3' (SEQ ID NO: 102).

[0147] In a further embodiment, the siRNA has a nucleotide sequence selected from the following sequences:

[0148] 5'-AGCUUGCAGGAUUUGAGUUAATT-3' (SEQ ID NO:78);

[0149] 5'-AGCAGAGAGAUCCAGUUCAACTT-3' (SEQ ID NO:79);

[0150] 5'-GGGACAGAUCAUCAAGUUATT-3' (SEQ ID NO:80);

[0151] 5'-CACAAGAUCUCCAAAUCAATT-3' (SEQ ID NO:81);

[0152] 5'-GGCCGAAAGUGUUGGAAUATT-3' (SEQ ID NO:82).

[0153] On the other hand, the present invention provides a host cell comprising at least one selected from the shRNA, siRNA, and their coding sequences of the present invention, the recombinant vector of the present invention, and the recombinant lentivirus of the present invention. The present invention does not particularly limit the type of host cell; for example, it may be the cell used to prepare the shRNA and other sequences of the present invention, such as the HEK293T cell described in the embodiments herein; or it may be a cell whose MLKL has been knocked down by the above-mentioned sequences of the present invention, such as the HepG2 cell and AML12 cell described in the embodiments herein, and includes, but is not limited to, hepatocyte lines of various species and primary hepatocytes of various species.

[0154] In another aspect, the present invention provides the use of the above-mentioned shRNA or recombinant vector or recombinant lentivirus or host cell or siRNA in the preparation of a drug for specifically inhibiting MLKL gene expression.

[0155] In another aspect, the present invention provides a method for specifically inhibiting MLKL gene expression, which includes the step of specifically inhibiting MLKL gene expression using the above-mentioned shRNA or recombinant vector or recombinant lentivirus or host cell or siRNA.

[0156] In another aspect, the present invention provides a method for treating liver fibrosis and / or liver injury, comprising administering a therapeutically effective amount of the above-mentioned shRNA or recombinant vector or recombinant lentivirus or host cell or siRNA to a subject in need.

[0157] In some implementations, the subject may be a mammal, including but not limited to humans, rats, dogs, pigs, cattle, sheep, horses, etc.

[0158] The beneficial effects of this invention are as follows:

[0159] 1. A gene therapy for knocking down MLKL in hepatocytes using AAV8-shRNA (also referred to as AAV8-shMLKL in this invention) was established, which can significantly treat CCl4-induced liver injury and liver fibrosis.

[0160] 2. A specific knockdown shRNA for HepG2 (also referred to as shMLKL in this invention) was identified, and lentivirus-shMLKL can alleviate CCl4-induced damage to HepG2 cells.

[0161] 3. A gene therapy using siRNA (also referred to as siMLKL in this invention) to specifically knock down MLKL in hepatocytes was established, which can significantly treat CCl4-induced liver injury.

[0162] The present invention has been described in detail above; however, the above embodiments are merely illustrative in nature and are not intended to limit the invention. Furthermore, this document is not limited to the foregoing prior art or the invention itself, or to any theory described in the following embodiments. Attached Figure Description

[0163] Figure 1 This is a graph showing the results of Western blot analysis of the efficiency of AAV8-shMLKL knockdown in liver tissue in Example 1.

[0164] Figure 2 This demonstrates how AAV8-shMLKL-1 specifically knocked down MLKL in hepatocytes of liver tissue in Example 1. Specifically: (A) Fluorescence images of frozen sections of mouse liver tissue 8 weeks after tail vein injection of AAV8-scramble (negative control) or AAV8-shMLKL-1, scale bar indicating 2 mm; (B) RT-qPCR analysis of MLKL expression in liver tissue (n=3); (C) Hepatocytes and hepatic immune cells (CD45+) isolated from liver tissue 8 weeks after AAV8-scramble or AAV8-shMLKL-1 injection in mice. + Hepatic stellate cells (HSCs) were analyzed by Western blotting to determine the knockdown efficiency of AAV8-shMLKL-1 in different types of liver cells.

[0165] Figure 3This demonstrates that AAV8-shMLKL-1 in Example 1 has a significant therapeutic effect on CCl4-induced liver injury and liver fibrosis. Specifically: (A) Flowchart of the experimental design for prophylactic administration (shMLKL-1-P) and therapeutic administration (shMLKL-1-T) of AAV8-shMLKL-1 in a CCl4-induced mouse fibrosis model; (B) Serum ALT and AST levels in mice in the scramble, shMLKL-1-P, and shMLKL-1-T groups after Oil and CCl4 induction (Oil group, n=5; CCl4 group, n=7); (C) Western blot analysis of MLKL, Vimentin, and α-SMA in liver samples from scramble, shMLKL-1-P, and shMLKL-1-T mice treated with Oil or CCl4; (D) Western blot analysis of MLKL, Vimentin, and α-SMA in samples from mice in both Oil and CCl4 groups; (D) Western blot analysis of MLKL, Vimentin, and α-SMA in samples from mice in both Oil and CCl4 groups. Quantitative analysis of the blotting results, in which all proteins were normalized to GAPDH in the same sample and then normalized to scramble-oil mice; (E) Representative images of α-SMA immunofluorescence staining of paraffin sections of livers from scramble, shMLKL-1-P, and shMLKL-1-T mice after Oil and CCl4 induction, with cell nuclei stained with Hoechst 33342; (F) Quantitative analysis of the staining results in (E), in which all data are shown as mean ± standard error (Means ± SEM), and statistical differences between the two groups were analyzed by unpaired t-test, *P<0.05, **P<0.01, ***P<0.001, scale bar indicates 100 μm.

[0166] Figure 4 This demonstrates the effect of shRNA (shMLKL-h1, shMLKL-h2) knockdown of MLKL in HepG2 cells in Example 2. Specifically, (A, B) RT-qPCR (A) and Western blotting (B) analyzed the knockdown efficiency of MLKL in scrambled, shMLKL-h1, and shMLKL-h2 cells, and in HepG2 cells 96 h after infection. All data are presented as mean ± standard error (Means ± SEM). Statistical differences between the two groups were assessed using unpaired t-tests, with ***P < 0.001.

[0167] Figure 5 This study demonstrates the effect of shMLKL-h1 and shMLKL-h2 in alleviating CCl4-induced HepG2 cell damage in Example 2. HepG2 cells were transfected with scramble, shMLKL-h1, and shMLKL-h2 for 96 hours, followed by CCl4 treatment. AST levels in the culture medium were measured at different time points. All data are presented as mean ± standard error (Means ± SEM). Statistical differences between the two groups were assessed using two-way ANOVA. ***P < 0.001.

[0168] Figure 6 A graph showing the knockdown efficiency of siMLKL in AML12 (mouse hepatocyte cell line) as analyzed by Western blot in Example 3.

[0169] Figure 7 This demonstrates that the lipid nanoparticles (LNPs) encapsulating siMLKL-1 (LNP-siMLKL-1) and siMLKL-2 (LNP-siMLKL-2) in Example 3 significantly treated CCl4-induced liver injury. (A) Representative images of hematoxylin and eosin (HE) staining of mouse liver paraffin sections after Oil and CCl4 induction. (B) Serum ALT and AST levels in LNP-scramble, LNP-siMLKL-1, and LNP-siMLKL-2 mice after Oil and CCl4 induction (Oil group, n=3; CCl4 group, n=5). All data are shown as mean ± standard error (Means ± SEM). Statistical differences between groups were assessed using unpaired t-tests. *P<0.05, **P<0.01, ***P<0.001. Scale bar represents 100 μm. Detailed Implementation

[0170] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0171] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0172]

[0173]

[0174] Example 1: shRNA targeting mouse MLKL

[0175] 1. shRNA coding sequence structure

[0176] shMLKL-1:

[0177]

[0178] shMLKL-2:

[0179]

[0180] shMLKL-3:

[0181]

[0182] shMLKL-4:

[0183]

[0184] shMLKL-5:

[0185]

[0186] shMLKL-6:

[0187]

[0188] shMLKL-7:

[0189]

[0190] shMLKL-8:

[0191]

[0192] shMLKL-9:

[0193]

[0194] shMLKL-10:

[0195]

[0196] shMLKL-11:

[0197]

[0198] shMLKL-12:

[0199]

[0200] shMLKL-13:

[0201]

[0202] shMLKL-14:

[0203]

[0204] shMLKL-15:

[0205]

[0206] shMLKL-16:

[0207]

[0208] shMLKL-17:

[0209]

[0210] Scramble (negative control):

[0211] Note: Each shRNA contains... Justice chain, circular sequence, antisense chain It consists of three parts. (The bold italic sequence represents the interference target sequence.)

[0212] The mouse MLKL gene is as follows:

[0213] >NM_029005.3|Mlkl[Mouse]|ORIGIN

[0214]

[0215] 2. Construction of AAV8 virus specifically targeting MLKL in hepatocytes:

[0216] The target genes (including scramble, shMLKL-1, shMLKL-2, and shMLKL-3) were obtained by PCR and constructed into the AAV8 vector (pAAV-TBG-sfGFP-3xFLAG-WPRE, Heyuan Biotechnology (Shanghai)) via ligation or homologous recombination / assembly. The vectors were then transformed into E. coli DH5α competent cells. Transformants were screened using colony PCR, and positive clones were sequenced. Plasmids were extracted from the validated clones.

[0217] The specific method is as follows:

[0218] 2.1 Obtaining the target gene fragment:

[0219] The target gene and its upstream and downstream sequences were retrieved from GenBank, and primers were designed using VectorNTI software.

[0220] PCR amplification of target genes (including scramble, shMLKL-1, shMLKL-2, shMLKL-3): The target genes were amplified using the high-fidelity PrimeSTAR enzyme.

[0221] The PCR products were subjected to agarose gel electrophoresis to detect the amplification effect. The target gene band was excised from the agarose gel after electrophoresis and recovered using the TaKaRa MiniBEST Agarose Gel DNAExtraction Kit Ver.3.0.

[0222] 2.2 Preparation of linearized expression vectors:

[0223] The expression vector was digested with restriction endonucleases (HindIII and XbaI). The digestion reaction system consisted of 2 μg plasmid, 5 μL 10x reaction buffer, 1 μL each restriction endonuclease, and water to a final volume of 50 μL. The mixture was incubated in a 37°C water bath for at least 2 hours. The digestion products were analyzed by agarose gel electrophoresis to assess the digestion efficiency. The target vector band was excised from the agarose gel and recovered using the TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver. 3.0.

[0224] 2.3 The target gene is constructed into a linearized expression vector:

[0225] Use a seamless cloning kit (suitable for single target gene inserts). Add the target gene fragment and the linearized vector to a centrifuge tube at a molar ratio of 2:1 to initiate the recombination reaction.

[0226] Insert fragment xμl Linearized carrier yμl Seamless cloning enzyme 2μl <![CDATA[ddH2O]]> Up to 20μl

[0227] Optimal insert size = [0.04 x number of insert bases] ng (0.03 pmol)

[0228] Optimal linearization vector usage = [0.02 x number of linearization vector bases] ng (0.03 pmol)

[0229] After mixing, incubate at 37°C for 30 minutes, then transfer to ice and place for 5 minutes. Conversion can be performed directly or stored at -20°C until thawed and converted as needed.

[0230] 2.4 Preparation and transformation of competent cells:

[0231] For details on the preparation and transformation of DH5 competent cells, please refer to "A Concise Guide to Molecular Biology Experiments" (FM Osber et al., translated by Ma Xuejun et al., A Concise Guide to Molecular Biology Experiments (4th Edition), Science Press, pp. 25-26).

[0232] 2.5 Colony PCR identification of positive transformants:

[0233] Transformants grown on the plate were picked and resuspended in 10 μL of LB medium. 1 μL of the resuspended transformant was used as a template for colony PCR identification.

[0234] 2.6 Positive clones were sent for sequencing:

[0235] Positive clones obtained from colony identification were sent to a sequencing company for sequencing verification. The sequencing results were compared and analyzed using software.

[0236] 2.7 Plasmid mini-prep:

[0237] For positive clones that pass sequencing, perform plasmid preparation according to the reagent instructions.

[0238] Finally, a three-plasmid system was used for AAV8 packaging in 293T cells. During AAV8 packaging, the packaging plasmid (the plasmid containing the target gene constructed above) encodes the target gene and two terminal inverted repeat sequences (ITRs, which play a crucial role in viral replication and packaging). The helper plasmid contains the cap (encoding the viral capsid protein) and rep (involved in viral replication) genes required for AAV packaging, as well as the adenovirus helper plasmid. After co-transfection of 293T cells with the three plasmids, AAV virus replication and packaging began. The same procedure was used to generate AAV8-scramble, AAV8-shMLKL-1, AAV8-shMLKL-2, and AAV8-shMLKL-3, respectively. The obtained viral particles were purified by ultracentrifugation, and the viral gene copy number was titrated by qPCR. Typically, the rAAV8 titer is around 10. 12 ~10 13 VG / ml is sufficient to meet usage requirements.

[0239] 3. Detect the targeting and knockdown efficiency of AAV8-shMLKL:

[0240] The AAV8 virus (pAAV-TBG-sfGFP-3xFLAG-WPRE) possesses a hepatocyte-specific promoter (TBG). Using this AAV8 virus, MLKL in hepatocytes can be specifically knocked down. Furthermore, this AAV8 virus contains a green fluorescent protein (GFP) sequence, which can be used to indicate the targeting specificity of the AAV8 virus. AAV8-scramble (negative control), AAV8-shMLKL-1, AAV8-shMLKL-2, and AAV8-shMLKL-3 were injected into mice via the tail vein (a single dose of AAV8 carrying shMLKL (10...)...) 12 (Genomic copy / animal)). Four weeks later, liver tissue was harvested. The knockdown efficiency of shRNA was determined by Western blotting of liver tissue proteins.

[0241] Experimental results showed that AAV8-shMLKL-1, AAV8-shMLKL-2, and AAV8-shMLKL-3 could all significantly knock down MLKL in the liver. Figure 1 The knockdown efficiency of all three shRNAs was relatively high.

[0242] Since all three shRNAs exhibited high knockdown efficiency, AAV8-shMLKL-1 was subsequently used to further verify its ability to specifically knock down MLKL in hepatocytes. A single dose of AAV8(10) 12(Genomic copies / animal) were injected via the tail vein, and samples were collected for testing 8 weeks later. Eight weeks after tail vein injection of AAV8-scramble and AAV8-shMLKL-1, AAV8 green fluorescence was observed distributed throughout the entire liver. Figure 2 (A). Simultaneously, 8 weeks later, hepatocytes and immune cells (CD45) were isolated from liver tissue. + Hepatic stellate cells were analyzed using Western blotting to determine the knockdown efficiency of AAV8-TBG-shMLKL-1. The results are as follows: Figure 2 As shown. RT-PCR analysis of liver tissue revealed that MLKL expression in the entire liver was significantly reduced in the AAV8-shMLKL-1 group. Figure 2 (B). Simultaneously, 8 weeks later, hepatocytes and immune cells (CD45) were isolated from liver tissue. + Hepatic stellate cells, and Western blot analysis of knockdown efficiency revealed that AAV8-shMLKL-1 specifically knocked down MLKL in hepatocytes (…). Figure 2 (C).

[0243] 4. The therapeutic effect of AAV8-shMLKL-1 on liver fibrosis:

[0244] Treatment was administered using AAV8-shMLKL-1. Two experimental protocols were designed: prophylactic administration and therapeutic administration. For the prophylactic administration group, a single dose of AAV8(10) was administered. 12 Genome copies / animal) were administered via tail vein injection, simultaneously with intraperitoneal injection of CCl4 (0.4 ml / kg), three times a week, for 8 weeks before sampling and testing. In the treatment group, after 4 weeks of CCl4 (0.4 ml / kg, three times a week) injection, a single dose of AAV8 (10) was administered via tail vein. 12 (Genomic copy / animal), then continuously injected with CCl4 (0.4 ml / kg, 3 times a week) for 4 weeks, for a total of 8 weeks of induction before sampling and testing.

[0245] The therapeutic effect of AAV8-shMLKL-1 on liver fibrosis was evaluated using the following methods:

[0246] Biochemical level detection: Aspartate aminotransferase (AST) and alanine aminotransferase (ALT).

[0247] Protein analysis: Expression analysis of liver fibrosis marker proteins, including α-SMA and Vimentin.

[0248] Detection of collagen fiber deposition in liver tissue: immunofluorescence staining of α-SMA in paraffin sections.

[0249] result:

[0250] AAV8-shMLKL-1 has a significant therapeutic effect on CCl4-induced liver injury and liver fibrosis.

[0251] Eight weeks after tail vein injection of AAV8-shMLKL-1, plasma AST and ALT levels in mice were measured to assess the degree of liver damage. Liver fibrosis-related proteins were detected, and immunofluorescence staining of tissue sections was performed to evaluate the degree of fibrosis. The therapeutic effect on liver fibrosis was assessed. Figure 3 As shown.

[0252] As described above, the AAV8-shMLKL-1 experimental design included two dosing protocols: prophylactic and therapeutic. For the prophylactic dosing group (shMLKL-1-P group), a single dose of AAV8 (10... 12 (Genomic copies / animal) were administered via tail vein injection, followed by intraperitoneal injection of CCl4 for 8 weeks to induce an immune response. For the treatment group (shMLKL-1-T group), after 4 weeks of CCl4 injection, a single dose of AAV8 was injected via tail vein, followed by continuous CCl4 injection for another 4 weeks. The control group (scramble) received an equal volume of olive oil via intraperitoneal injection. The administration procedure was as follows: Figure 3 A. First, the levels of AST and ALT in mouse serum were measured. The results showed that the serum ALT and AST levels were increased in the CCl4-treated group, but significantly decreased in the shMLKL-1-P and shMLKL-1-T groups. Figure 3 Western blot results showed that, compared with the scramble-oil group, CCl4 treatment significantly reduced MLKL levels in the livers of shMLKL-1-P and shMLKL-1-T mice, while fibrin levels decreased, including Vimentin and α-SMA (B). Figure 3 (C and D). Immunofluorescence staining results of α-SMA showed that, after CCl4 treatment, the fibrotic areas in the shMLKL-1-P and shMLKL-1-T groups were significantly reduced compared with the scramble-oil group. Figure 3 (E and F).

[0253] The above research results indicate that the AAV8-shMLKL gene therapy has a significant therapeutic effect on liver fibrosis.

[0254] Example 2: shRNA targeting human MLKL

[0255] 1. Structure of the shRNA coding sequence targeting human MLKL

[0256] shMLKL-h1:

[0257]

[0258] shMLKL-h2:

[0259]

[0260] shMLKL-h3:

[0261]

[0262] shMLKL-h4:

[0263]

[0264] shMLKL-h5:

[0265]

[0266] shMLKL-h6:(SEQ ID NO:47)

[0267]

[0268] (SEQ ID NO:48)

[0269] shMLKL-h7:

[0270]

[0271] shMLKL-h8:

[0272]

[0273] shMLKL-h9:

[0274]

[0275] shMLKL-h10: (SEQ ID NO:35)

[0276]

[0277] shMLKL-h11: (SEQ ID NO:51)

[0278]

[0279] Scramble (negative control):

[0280]

[0281] Note: Each shRNA contains... Justice chain, circular sequence, antisense chain It consists of three parts. (The bold italic sequence represents the interference target sequence.)

[0282] The human MLKL gene is as follows:

[0283] >NM_152649.4|MLKL[Homo sapiens]|ORIGIN

[0284]

[0285] 2. Construction of lentiviral viruses that specifically target human hepatocyte MLKL:

[0286] The DNA sequences encoding human shRNA (shMLKL-h1, shMLKL-h2) were cloned into the lentiviral plasmid PLKO.1 to obtain recombinant plasmids. The recombinant plasmid PLKO.1, along with the packaging plasmid psPAX2 and the envelope plasmid pMD2, were co-transfected into HEK293T cells to prepare lentiviruses producing shRNA. Viral supernatant was collected after 48 hours for transfection. HepG2 cells were cultured in 24-well dishes to 70%-80% confluence. The culture medium was then replaced with viral supernatant containing 8 μg / ml polystyrene, and the cells were centrifuged at 2500 rpm for 90 min to ensure viral infection. After 96 hours, the knockout efficiency was detected by RT-qPCR and Western blot, respectively. Results are as follows: Figure 4 As shown in A and B, both shRNAs (shMLKL-h1, shMLKL-h2) showed good knockdown efficiency.

[0287] 3. shRNA alleviates CCl4-induced HepG2 damage:

[0288] Two shRNAs (shMLKL-h1 and shMLKL-h2) were used simultaneously in the experiment. After HepG2 cells were infected with the virus for 96 h, the cells were treated with culture medium containing 20 mM CCl4. Cell supernatants were collected at 6 h, 12 h, and 24 h, and the AST content in the cell supernatants was detected.

[0289] The results showed that shRNA significantly alleviated CCl4-induced damage to HepG2 cells and increased AST secretion. Figure 5 ).

[0290] Example 3: siRNA targeting mouse MLKL

[0291] 1. Design of siRNA targeting mouse MLKL

[0292]

[0293] Note: Each siRNA consists of an interference target and a protective base (TT). (The underlined part indicates the interference target sequence.)

[0294] 2. siMLKL knockdown efficiency test:

[0295] 24-well plate, inoculate 5 x 10⁵ cells per well. 5AML12 cells (normal mouse hepatocytes) were transfected with Lipofectamine RNAiMAX Reagent when the cell confluence reached 80%. Samples were collected 48 hours post-transfection, and knockdown efficiency was assessed using Western blotting. Western blotting results showed that the knockdown efficiency of all five siMLKL lines (siMLKL-1, siMLKL-2, siMLKL-3, siMLKL-4, and siMLKL-5) was high. Figure 6 ).

[0296] 3. Treatment of liver injury using siMLKL:

[0297] The required components and ratios for preparing lipid nanoparticles (LNPs) are DlinMC3:PEG-DMG:DSPC:Cholesterol = 40:5:10:45. The molar ratio of DlinMC3 to siRNA is 7.6. The preparation steps are as follows: Each lipid component is dissolved in 90% ethanol to prepare an oil phase solution. siRNA is dissolved in pH 5 citrate buffer (ribozyme-free) to prepare an aqueous phase solution. pH 6 citrate buffer (ribozyme-free) is used as a diluent. All solutions are incubated at 40°C for 20 minutes. Using a syringe, the oil phase, aqueous phase, and diluent are drawn up in a volume ratio of 1:1:2 and inserted into the three inlets of a T-tube. The syringe is then pushed simultaneously to mix the three solutions, forming the LNP solution. This mixture is collected using a 100K ultrafiltration centrifuge tube and centrifuged at 4000 rpm for 20 minutes. Finally, the LNPs are replaced with phosphate-buffered saline (PBS) to obtain the LNPs.

[0298] Lipid nanoparticles (LNPs) carrying siMLKL (LNP-siMLKL-1 and LNP-siMLKL-2) were administered via tail vein injection at a dose of 1.5 mg / kg every other day. Simultaneously with the tail vein injection of LNP-siMLKL, intraperitoneal injection of CCl4 (0.4 ml / kg) was initiated, with three intraperitoneal injections per week for two weeks of induction. After two weeks, mice were anesthetized, and blood was collected. Liver tissue samples were prepared into paraffin sections and stained with hematoxylin and eosin (HE). The results showed that the LNP-siMLKL-1 and LNP-siMLKL-2 groups exhibited less immune cell infiltration and better liver tissue integrity. Figure 7 (A). AST and ALT test results showed that the LNP-siMLKL-1 and LNP-siMLKL-2 groups had milder liver damage. Figure 7 (B). These results consistently demonstrate that siMLKL has a significant therapeutic effect on CCl4-induced liver injury.

[0299] Example 4: siRNA targeting human MLKL

[0300] 1. Design of siRNA targeting human MLKL

[0301]

[0302] Note: Each siRNA consists of an interference target and a protective base (TT). (The underlined part indicates the interference target sequence.)

[0303] References

[0304] 1. Kisseleva, T. & Brenner, D. Molecular and cellular mechanisms of liverfibrosis and its regression. Nat Rev Gastroenterol Hepatol 18, 151-166 (2021).

[0305] 2. Hernandez-Gea, V. & Friedman, SLP Pathogenesis of Liver Fibrosis. Annu Rev Pathol-Mech 6, 425-456 (2011).

[0306] 3. Henderson, NC, Rieder, F. & Wynn, TA Fibrosis: from mechanisms to medicines. Nature 587, 555-566 (2020).

[0307] 4. Pellicoro, A., Ramachandran, P., Iredale, JP & Fallowfield, JALiverfibrosis and repair: immune regulation of wound healing in a solid organ. Nat Rev Immunol 14, 181-194 (2014).

[0308] 5. Dara, L., Liu, ZX & Kaplowitz, N. Amurder mystery in the liver: who done it and how? J Clin Invest 126,4068-4071(2016).

[0309] 6. Wu, SEQUENCE LISTING <110> Shanghai Institute of Materia Medica, Chinese Academy of Sciences <120> A shRNA that specifically inhibits MLKL gene expression and its application <130> DI22-0478-XC37 <160> 108 <170> PatentIn version 3.5 <210> 1 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 1 aggagattcc aaaggaacat c 21 <210> 2 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 2 agcttgcagg atttgagtta a 21 <210> 3 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 3 agcagagaga tccagttcaa c 21 <210> 4 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 4 agatccagtt caacgatata t 21 <210> 5 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 5 agccggaggc taccaagtaa a 21 <210> 6 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 6 cggacagcaa agagcactaa a 21 <210> 7 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 7 tcccaacatc ttgcgtatat t 21 <210> 8 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 8 cctgaagcaa tgctcactaa a 21 <210> 9 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 9 cattggaata ccgtttcaga t 21 <210> 10 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 10 gcgtatattt gggatttgca t 21 <210> 11 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 11 gcaggatttg agttaagcaa a 21 <210> 12 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 12 ccctgagttc tccattgtca t 21 <210> 13 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 13 gggacagatc atcaagtta 19 <210> 14 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 14 cacaagatct ccaaatcaa 19 <210> 15 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 15 ggccgaaagt gttggaata 19 <210> 16 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 16 cccaacatcc tgcgtatatt t 21 <210> 17 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 17 cctctgtgga tgaaatctta a 21 <210> 18 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 18 tcccgtttca aggctgtaat t 21 <210> 19 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 19 gagtcaaatc tacagcatat c 21 <210> 20 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 20 ccttcggcat tgggttatct a 21 <210> twenty one <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> twenty one gaagcttcac tgagacgatt a 21 <210> twenty two <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> twenty two gcctcaattc tccattgtca t 21 <210> twenty three <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> twenty three gtgacttgat ttgatcaata g 21 <210> twenty four <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> twenty four agagatgaaa tactgcaaga a 21 <210> 25 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 25 cctctgacag taactttgat a 21 <210> 26 <211> 61 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 26 aggagattcc aaaggaacat ctagtgaagc cacagatgta gatgttcctt tggaatctcc 60 t 61 <210> 27 <211> 61 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 27 agcttgcagg atttgagtta atagtgaagc cacagatgta ttaactcaaa tcctgcaagc 60 t 61 <210> 28 <211> 61 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 28 agcagagaga tccagttcaa ctagtgaagc cacagatgta gttgaactgg atctctctgc 60 t 61 <210> 29 <211> 47 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 29 agatccagtt caacgatata tctcgagata tatcgttgaa ctggatc 47 <210> 30 <211> 47 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 30 agccggaggc taccaagtaa actcgagttt acttggtagc ctccggc 47 <210> 31 <211> 48 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 31 cggacagcaa agagcactaa actcgagttt agtgctcttt gctgtccg 48 <210> 32 <211> 48 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 32 tcccaacatc ttgcgtatat tctcgagaat atacgcaaga tgttggga 48 <210> 33 <211> 48 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 33 cctgaagcaa tgctcactaa actcgagttt agtgagcatt gcttcagg 48 <210> 34 <211> 48 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 34 cattggaata ccgtttcaga tctcgagatc tgaaacggta ttccaatg 48 <210> 35 <211> 48 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 35 gcgtatattt gggatttgca tctcgagatg caaatcccaa atatacgc 48 <210> 36 <211> 48 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 36 gcaggatttg agttaagcaa actcgagttt gcttaactca aatcctgc 48 <210> 37 <211> 48 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 37 ccctgagttc tccattgtca tctcgagatg acaatggaga actcaggg 48 <210> 38 <211> 44 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 38 gggacagatc atcaagttac tcgagtaact tgatgatctg tccc 44 <210> 39 <211> 44 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 39 cacaagatct ccaaatcaac tcgagttgat ttggagatct tgtg 44 <210> 40 <211> 44 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 40 ggccgaaagt gttggaatac tcgagtattc caacactttc ggcc 44 <210> 41 <211> 48 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 41 agcttgcagg atttgagtta actcgagtta actcaaatcc tgcaagct 48 <210> 42 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 42 agcagagaga tccagttcaa cctcgagggt tgaactggat ctctctgct 49 <210> 43 <211> 48 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 43 cccaacatcc tgcgtatatt tctcgagaaa tatacgcagg atgttggg 48 <210> 44 <211> 48 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 44 cctctgtgga tgaaatctta actcgagtta agatttcatc cacagagg 48 <210> 45 <211> 48 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 45 tcccgtttca aggctgtaat tctcgagaat tacagccttg aaacggga 48 <210> 46 <211> 48 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 46 gagtcaaatc tacagcatat cctcgaggat atgctgtaga tttgactc 48 <210> 47 <211> 48 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 47 ccttcggcat tgggttatct actcgagtag ataacccaat gccgaagg 48 <210> 48 <211> 48 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 48 gaagcttcac tgagacgatt actcgagtaa tcgtctcagt gaagcttc 48 <210> 49 <211> 48 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 49 gcctcaattc tccattgtca tctcgagatg acaatggaga attgaggc 48 <210> 50 <211> 48 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 50 gtgacttgat ttgatcaata gctcgagcta ttgatcaaat caagtcac 48 <210> 51 <211> 47 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 51 agagatgaaa tactgcaaga actcgagttc ttgcagtatt tcatctc 47 <210> 52 <211> 48 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 52 cctctgacag taactttgat actcgagtat caaagttat gtcagagg 48 <210> 53 <211> twenty one <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 53 agcuugcagg auuugaguua a 21 <210> 54 <211> twenty one <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 54 agcagagaga uccaguucaa c 21 <210> 55 <211> 19 <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 55 gggacagauc aucaaguua 19 <210> 56 <211> 19 <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 56 cacaagaucu ccaaaucaa 19 <210> 57 <211> 19 <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 57 ggccgaaagu guuggaaua 19 <210> 58 <211> twenty one <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 58 aggagauucc aaaggaacau c 21 <210> 59 <211> twenty one <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 59 agauccaguu caacgauaua u 21 <210> 60 <211> twenty one <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 60 agccggaggc uaccaaguaa a 21 <210> 61 <211> twenty one <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 61 cggacagcaa agagcacuaa a 21 <210> 62 <211> twenty one <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 62 ucccaacauc uugcguauau u 21 <210> 63 <211> twenty one <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 63 ccugaagcaa ugcucacuaa a 21 <210> 64 <211> twenty one <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 64 cauuggaaua ccguuucaga u 21 <210> 65 <211> twenty one <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 65 gcguauauuu gggauuugca u 21 <210> 66 <211> twenty one <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 66 gcaggauuug aguuaagcaa a 21 <210> 67 <211> twenty one <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 67 cccugaguuc uccauuguca u 21 <210> 68 <211> twenty one <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 68 cccaacaucc ugcguauauu u 21 <210> 69 <211> twenty one <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 69 ccucugugga ugaaaucuua a 21 <210> 70 <211> twenty one <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 70 ucccguuuca aggcuguaau u 21 <210> 71 <211> twenty one <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 71 gagucaaauc uacagcauau c 21 <210> 72 <211> twenty one <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 72 ccuucggcau uggguuaucu a 21 <210> 73 <211> twenty one <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 73 gaagcuucac ugagacgauu a 21 <210> 74 <211> twenty one <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 74 gccucaauuc uccauuguca u 21 <210> 75 <211> twenty one <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 75 gugacuugau uugaucaaua g 21 <210> 76 <211> twenty one <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 76 agagaugaaa uacugcaaga a 21 <210> 77 <211> twenty one <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 77 ccucugacag uaacuuugau a 21 <210> 78 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 78 agcuugcagg auuugaguua att 23 <210> 79 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 79 agcagagaga uccaguucaa ctt 23 <210> 80 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 80 gggacagauc aucaaguuat t 21 <210> 81 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 81 cacaagaucu ccaaaucaat t 21 <210> 82 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 82 ggccgaaagu guuggaauat t 21 <210> 83 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 83 aggagauucc aaaggaacau ctt 23 <210> 84 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 84 agauccaguu caacgauaua utt 23 <210> 85 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 85 agccggaggc uaccaaguaa att 23 <210> 86 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 86 cggacagcaa agagcacuaa att 23 <210> 87 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 87 ucccaacauc uugcguauau utt 23 <210> 88 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 88 ccugaagcaa ugcucacuaa att 23 <210> 89 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 89 cauuggaaua ccguuucaga utt 23 <210> 90 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 90 gcguauauuu gggauuugca utt 23 <210> 91 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 91 gcaggauuug aguuaagcaa att 23 <210> 92 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 92 cccugaguuc uccauuguca utt 23 <210> 93 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 93 cccaacaucc ugcguauauu utt 23 <210> 94 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 94 ccucugugga ugaaaucuua att 23 <210> 95 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 95 ucccguuuca aggcuguaau utt 23 <210> 96 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 96 gagucaaauc uacagcauau ctt 23 <210> 97 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 97 ccuucggcau uggguuaucu att 23 <210> 98 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 98 gaagcuucac ugagacgauu att 23 <210> 99 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 99 gccucaauuc uccauuguca utt 23 <210> 100 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 100 gugacuugau uugaucaaua gtt 23 <210> 101 <211> 23 <212> DNA <213> artificial sequence <220> <223> Synthesis <400> 101 agagaugaaa uacugcaaga that 23 <210> 102 <211> 23 <212> DNA <213> artificial sequence <220> <223> Synthesis <400> 102 ccucugacag uaacuuugau that 23 <210> 103 <211> 61 <212> DNA <213> artificial sequence <220> <223> Synthesis <400> 103 ggaagtcgtg agaagtagaa ttagtgaagc cacagatgta attctacttc tcacgacttc 60 c 61 <210> 104 <211> 2007 <212> DNA <213> Mouse musculus <400> 104 cggagcagaa ctctggacag aaatagctgt cggacaggtt ggttttgagg actctgctgg 60 agcattgctg cgttggccca aatttgactt taggcgggaa ccgcggctgg tgcggagttc 120 ctccgagagg cgagagcttt aaggttgcac caagggaggg gactcaggac accgggggacc 180 tgctccgacc tgcgtgcat tgtcaacgag gcagagacc gcggtccggg agcggcggga 240 gactgatgat cctacaggaa ctgagcgcag gatagaccgg acgagcgccc ctcggagatc 300 acgtagaatc cagtgagctg tagcgcggca ggaggaagca ggagaatccc agaatcacag 360 attggacttc ctggaactta ggctatggat aaattgggac agatcatca gttaggccag 420 ctcatctatg aacagtgtga aagatgaaa tactgccgga aacaatgcca gcgtctagga 480 aaccgtgtgc acggcctgct acagccctc cagagactcc aggcccagg aaagagaac 540 ctgcccgatg acattactgc tgccctgggc cgtttgatg aagtcctgaa ggaggctaac 600 cagcagatag aaagttcag caagaagtcc catatttgga agtttgtgag tgtgggcaat 660 gataagatcc tcttccatga agtgaatgag aagctgagag acgtctggga ggagctgttg 720 ctgctgcttc aggtttaca ttggaatacc gtttcagatg tcagccagcc agcatcctgg 780 cagcaggaag atcgacagga tgcagaggaa gacggaatg aaatatgaa agttactcctg 840 atgcagttgc aaattagcgt ggaagaatc aaaaaaccc tgaagcaatg ctcactaaaa 900 cccacacagg agatcccaca agatctccaa atcaaggaga ttccaaagga acatcttgga 960 cctccgtgga ccaaactgaa gacaagtaaa atgagcacca tttatagagg agagtatcac 1020 agatctccag ttaccatcaa agtattcaac aacccccagg ccgaaagtgt tggaatagtg 1080 aggttcactt tcaatgacga gatcaaaacc atgaagaaat tcgattctcc caacatcttg 1140 cgtatatttg ggatttgcat tgatcaaaca gtgaagcccc ctgagttctc cattgtcatg 1200 gagtactgtg aacttggaac cctgagggaa ctgctggata gagaaaaga cctcacaatg 1260 agtgtgcgca gcctcctagt cctgagggca gccagaggct tatacaggct acaccattcg 1320 gaaacactcc acagaaacat cagcagctcc agttcctcg tagccggagg ctaccaagta 1380 aagcttgcag gatttgagtt aagcaaaaca cagaattcca tcagccggac agcaaagagc 1440 actaaagcag agagatccag ttcaacgata tatgtctccc ctgagagact gaaaaatcca 1500 ttttgccttt atgacataaa agctgaaata tatagctttg gaattgtact ctgggaaatt 1560 gccactggaa agatcccatt tgaaggctgt gattctaaga agatccggga gctggtggct 1620 gaggacaaga agcaggaacc agtgggtcag gattgccctg agttgttgcg ggaaatcatt 1680 aatgagtgtc gtgcccatga gccctcccaa cggccctctg tggacggaat cttggagaga 1740 ctgtctgcgg ttgaagaatc cacggacaag aaggtgtaag aggacaaagt ggacatttgt 1800 ccttcatatc tgttagcagc atcaacttat tgctgggagc aaatagcttc tttctccctt 1860 gcttgcttgc tttttttctt tcttttactt gagatgggat ctcaccatgt agcccctgaa 1920 aactgtgtag aacaggctgg ctcaaactca aggaaatctt ccagttctgc ctcctgaatg 1980 ctggaattaa aggcatagac caccaaa 2007 <210> 105 <211> 48 <212> DNA <213> Artificial sequence <220> <223> Synthesized <400> 105 cctaaggtta agtcgccctc gctcgagcga gggcgactta accttagg 48 <210> 106 <211> 2477 <212> DNA <213> Homo sapiens <400> 106 gtgagcctgg tggttggcag ctggagccac gtcggagggg gaagtgtcgc agcattctct 60 gcaggcatca cagacctgag gcagtggcct ccggaggggca ctggacagaa acagccatcc 120 aagtggctga gtggaggc cctgctcaag tgcagctgca gtggccgggg ttccctcag 180 gtagggatcg gggcgccttg tcgccgccag ccacgtgtgg cgtccggtac agtcagcaga 240 gtgcagggtg cgggcaccag gaaagggggc gcaggggaac tcccgggggc ctcgcgttg 300 caaacttctc gcctgggcag gaggcggtcg tgggaagaa ggtggaagg cgagcttttt 360 ggaactgtgc acgggacaga ttggacgcac acccctcgggg aggcgcgaag gcatggaaaa 420 tttgaagcat attatcaccc ttggccaggt catccacaaa cggtgtgaag agatgaaata 480 ctgcaagaaa cagtgccggc gcctgggcca ccgcgtccctc ggctgatca agcctctgga 540 gatgctccag gaccaaggaa agaggagcgt gccctctgag aagttaacca cagccatgaa 600 ccgcttcaag gctgccctgg aggaggctaa tgggata gaaagttca gcaatagatc 660 caatactctgc agttttctaa cagcaagcca ggacaaata ctctcagg acgtgaacag 720 gaagctgagt gagtctctga aggagctctc gctgttactt caggttgagc aacgcatgcc 780 tgtttcaccc ataagccaag gagcgtcctg ggcacaggaa gatcagcagg atgcagacga 840 agacaggcga gctttccaga tgctaagaag agataatgaa aaatagaag cttcactgag 900 acgattagaa atcaacatga aagaaatcaa ggaaactttg aggcagtatt taccaccaaa 960 atgcatgcag gagatcccgc aagagcaaat caaggagatc aagaaggagc agctttcagg 1020 atccccgtgg attctgctaa gggaaaatga agtcagcaca ctttataaag gagaatacca 1080 cagagctcca gtggccataa aagtattcaa aaaactccag gctggcagca ttgcaatagt 1140 gaggcagact ttcaataagg agatcaaaac catgaagaaa ttcgaatctc ccaacatcct 1200 gcgtattt gggatttgca ttgatgaaac agtgactccg cctcaattct ccattgtcat 1260 ggagtactgt gaactcggga ccctgaggga gctgttggat agggaaaaag acctcacact 1320 tggcaagcgc atggtcctag tcctgggggc agcccgaggc ctataccggc tacaccattc 1380 agaagcacct gaactccacg gaaaaatcag aagctcaaac ttcctggtaa ctcaaggcta 1440 ccaagtgaag cttgcaggat ttgagttgag gaaaacacag acttccatga gtttgggaac 1500 tacgagagaa aagacagaca gagtcaaatc tacagcatat ctctcacctc aggaactgga 1560 agatgtattt tatcaatatg atgtaaagtc tgaaatatac agctttggaa tcgtcctctg 1620 ggaaatcgcc actggagata tcccgtttca aggctgtaat tctgagaaga tccgcaagct 1680 ggtggctgtg aagcggcagc aggagccact gggtgaagac tgcccttcag agctgcggga 1740 gatcattgat gagtgccggg cccatgatcc ctctgtgcgg ccctctgtgg atgaaatctt 1800 aaagaaactc tccacctttt ctaagtagtg tatcaaaatc taaaccaagg agtctctgga 1860 caagaagctg ggagaggcac aaactggaca tctctctctc tcatatcctt cggcattggg 1920 ttatctatgg gtgcaaggag tgggcacgct tctctgttac aatagaaaa cgattccagt 1980 catacaggac acatcccact ccaaatgata tttccaaaaa catacctctg acagtaactt 2040 tgatagatgg tttgtcaaat gtatctttct gggtatccac acctcttggc aatgaaattt 2100 gcagctcctc ccttccataa atgaagtctc tttccccacc atttgaatct gggctggcac 2160 tgtgacttga tttgatcaat agaatgtgga agaagtgact gtatgccagt tccaagccta 2220 ggtttcaaga ggccttataa atgtctgttg gaaccttacc cagccatgaa catgttgagt 2280 gagcatgctg gagaatgaga gaccacatga agcagaaaca tgctttccta gctgaagtca 2340 tactagccca accaacatgg cagctaacac atgaatgagg ccaatcaaga ccagaagaac 2400 cactcaagca gatcccagcc caaattgccc attcacacaa tcaggagcta aataaattac 2460 tgttgtctta acactaa 2477 <210> 107 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 107 uucuccgaac gugucacgut t 21 <210> 108 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 108 ccuaagguua agucgcccuc gtt 23

Claims

1. A shRNA that specifically inhibits the expression of the MLKL gene, wherein the shRNA targets the nucleotide sequence of SEQ ID NO:

1.

2. The shRNA according to claim 1, wherein, The nucleotide sequence encoding the shRNA is the nucleotide sequence of SEQ ID NOS:

26.

3. A recombinant vector that specifically inhibits MLKL gene expression, comprising adeno-associated virus 8 (AAV8) as a backbone vector and a nucleotide sequence encoding the shRNA as described in claim 1 or 2.

4. The recombinant vector according to claim 3, in, Introduce the hepatocyte-specific promoter TBG onto AAV8 to control shRNA expression; and / or The AAV8 virus also contains the green fluorescent protein (GFP) sequence.

5. A recombinant lentivirus that specifically inhibits MLKL gene expression, obtained by cloning a nucleotide sequence encoding the shRNA as described in claim 1 or 2 into the lentiviral plasmid PLKO.1 to obtain a recombinant plasmid, and then co-transfecting the recombinant plasmid with the packaging plasmid psPAX2 and the envelope plasmid pMD2 into mammalian cells.

6. A siRNA that specifically inhibits MLKL gene expression, comprising an interference target sequence and a protective base sequence, wherein, The interference target sequence is the nucleotide sequence of SEQ ID NO:

58.

7. The siRNA according to claim 6, having a nucleotide sequence selected from SEQ ID NO:

83.

8. A host cell comprising at least one selected from the shRNA of claim 1 or 2, the siRNA of claim 6 or 7, the recombinant vector of claim 3 or 4, and the recombinant lentivirus of claim 5.

9. The use of the shRNA as described in claim 1 or 2, the recombinant vector as described in claim 3 or 4, the recombinant lentivirus as described in claim 5, the siRNA as described in claim 6 or 7, or the host cell as described in claim 8 in the preparation of a drug for specifically inhibiting MLKL gene expression.

10. A method for specifically inhibiting MLKL gene expression for non-diagnostic or therapeutic purposes, comprising the steps of using shRNA as described in claim 1 or 2, a recombinant vector as described in claim 3 or 4, a recombinant lentivirus as described in claim 5, siRNA as described in claim 6 or 7, or a host cell-specific inhibition of MLKL gene expression as described in claim 8.

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