Nucleic acid interference drug for treating liver injury

Abstract

A nucleic acid interference drug for treating liver injury. The nucleic acid interference drug comprises an siRNA capable of inhibiting and silencing COL1A1 gene expression and a pharmaceutically acceptable delivery system for delivering the siRNA. Designing and screening more small interfering nucleic acids that can effectively knock down the COL1A1 target in vivo will be of great significance for the effective treatment and prevention of liver injury-related diseases. At the same time, selecting a suitable delivery vector can improve the in vivo delivery effect of small interfering nucleic acids and the efficient knockdown of the COL1A1 target in vivo under the premise of ensuring safety, thereby promoting the development progress of clinical drugs for treating liver injury.

Description

A nucleic acid interference drug for treating liver damage

[0001] This invention claims priority to the Chinese patent application filed with the China Patent Office on December 26, 2023, with application number 202311802721X and invention name “A Nucleic Acid Interference Drug for Treating Liver Injury”, the entire contents of which are incorporated by reference into the application. Technical Field

[0002] The present invention relates to the field of biomedicine technology, and in particular to a nucleic acid interference drug for treating liver damage. Background Art

[0003] Numerous studies have shown that the pathogenesis of liver fibrosis is that pathogenic, toxic, metabolic or viral diseases lead to liver cell damage and immune cell infiltration, activating hepatic stellate cells (HSCs) to transdifferentiate into myofibroblasts that produce collagen (mainly type I collagen), resulting in excessive deposition of extracellular matrix (ECM), thereby destroying the normal liver structure.

[0004] Hepatic stellate cells (HSCs) are the primary cell type producing type I collagen in both normal and cirrhotic livers. Activation of HSCs plays a key role in the development of liver fibrosis. When the liver is damaged (including by HBV or HCV infection, NAFLD, alcohol, chemicals, autoimmune diseases, cholestasis, and tumors), hepatocytes undergo necrosis and apoptosis, producing damage-associated molecules (DAMPs) and cytokines. These molecules further activate endothelial cells and Kupffer cells (phagocytes located on the inner surface of the sinusoids) lining the liver, which continue to secrete cytokines, including TGF-β1 and PDGF. All of these factors act as activators of HSCs, which differentiate into proliferative, fibrogenic, and contractile myofibroblasts. HSCs are the primary cellular source for the production and secretion of ECM components. The ECM components of liver fibrosis primarily include collagen (primarily COL1A1) and several glycoproteins. Data show that COL1A1 is strongly upregulated in the livers of CCL4-induced fibrosis mice and in human liver fibrosis patients. Liver fibrosis can further develop into cirrhosis, leading to portal hypertension or liver cancer, and ultimately liver failure.

[0005] In terms of liver fibrosis treatment, there are three main ways commonly used clinically to intervene in the liver fibrosis process, including reducing oxidative stress by inhibiting hepatocyte apoptosis, reducing the progression of fibrous scars by inhibiting HSC activation, and reducing fibrosis through immune regulation. However, there are currently no effective and recognized anti-liver fibrosis drugs approved for marketing. Although some patients can hope to reverse the early stages of liver fibrosis through some causal treatments (such as controlling viral infections, controlling weight, stopping alcohol consumption, relieving cholestasis, etc.), for patients who cannot eliminate the cause or have entered the late stage of cirrhosis, there is still an urgent need for effective anti-liver fibrosis drugs.

[0006] In addition, the extracellular matrix (ECM) can act as a non-cellular component in the tumor microenvironment to provide structural support and bioactive molecules to tumor cells. In some tumors, such as breast cancer (BRCA) and pancreatic ductal adenocarcinoma (PDAC), fibrostromal proliferation accounts for 90% of the tumor mass. The abnormal presence of ECM proteins or the enrichment of ECM expression signatures are associated with poor prognosis in various cancer types. The ECM regulates almost all characteristics of cancer; for example, increased density of collagen I promotes the occurrence, growth, and invasion of breast tumors. Therefore, targeting the synthesis and components of the ECM, such as COL1A1, is expected to become a potential strategy to limit the progression of connective tissue proliferation tumors. Although the development of small nucleic acid drugs has become the research and development focus of current pharmaceutical companies, due to the complexity and uncertainty of the in vivo action environment, the in vivo effects of small nucleic acids screened in vitro are not completely consistent, and there may be problems such as off-target effects. As a result, no nucleic acid interference drugs for the treatment of liver damage have entered the clinic or been marketed. Summary of the Invention

[0007] The purpose of the present invention is to provide a nucleic acid interference pharmaceutical composition for treating liver damage.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is:

[0009] A nucleic acid interference drug for treating liver damage, comprising siRNA capable of inhibiting and silencing COL1A1 gene expression, wherein the siRNA comprises any one or more of the siRNAs listed in Table 1.

[0010] Preferably, the 5' end and / or 3' end of the sense strand and / or antisense strand of the siRNA is truncated or extended by 1-6 bases, preferably, the truncation or extension forms 1-3 unpaired single bases at the 3' end of the antisense strand of the siRNA.

[0011] Still more preferably, the 3' end of the antisense strand of the siRNA forms 1-3 unpaired single bases during truncation or extension, and the number of bases in the sequence of the antisense strand of the siRNA is 1-3 more than that in the sequence of the sense strand.

[0012] In the present invention, unique siRNA modification can be used to ensure the knockdown effect of siRNA on the COL1A1 gene while improving the stability of siRNA in the body, making siRNA less susceptible to enzyme degradation, so that it can continue to play a role in the body.

[0013] Preferably, the siRNA molecule is chemically modified, and the chemical modification includes one or more of base 2'-OME modification, base 2'-F modification, and phosphorothioate backbone modification.

[0014] Further preferably, the siRNA is any one or more of the siRNAs in Table 4, preferably C1a1-049# and / or C1a1-052#.

[0015] Preferably, the nucleic acid interference drug further comprises a pharmaceutically acceptable delivery system for delivering the siRNA.

[0016] Further preferably, the delivery system is a small peptide lipid nano-delivery system and / or an amino acid lipid nano-delivery system.

[0017] Further preferably, the delivery system is a small peptide lipid nano-delivery system, and the small peptide lipid is preferably Ser-Glu-(C17)2, Ser-Asp-(C17)2, Ser-N-Ser-Asp-(C17)2, D-Ser-D-Glu-(C17)2, Ser-Glu-N(C17)2, Ser-Glu-(Lin)2, Ser-Glu-(C25)2, Ser-Asp-(C25)2, Thr-Asp-(C25)2, Met-Asp-(C2 5)2, Ser-Lys-(C17)(NC16), Ser-Glu-[Glu-(C17)2]2, Ser-Glu-[Glu-(C17)2]2, Ser-Glu-αC17(NC17), Ser-Glu-C17(αNC17), Gly-Glu-(C17)2, Leu-Glu-(C17)2, His-Glu-(C17)2, Ser-AAD-(C17)2, Ser-Glu-[C17(=)2]2.

[0018] Still further preferably, the delivery system further selectively includes one or more of auxiliary lipids, cholesterol and its derivatives, and PEGylated lipids (PEG-Lipid).

[0019] More preferably, the molar ratio of the small peptide lipid to the auxiliary lipid, cholesterol and its derivatives and PEG-Lipid is (40-99.5):(0-15):(0-50):(0.5-3).

[0020] Further preferably, the delivery system is an amino acid lipid nanodelivery system, and the amino acid lipid is preferably one or more of Met-Tris-3MOA, Gly-Tris-3MOA, Gly-Tris-3MA, Gly-Tris-3POA, Gly-Tris-3DOA, Gly-Tris-3OA, Gly-Tris-3Lin, Gly-Tris-3PA, Met-Tris-3MA, Met-Tris-3LA, Met-Tris-3C10, and Met-PEL-3MOA.

[0021] Still further preferably, the delivery system also selectively includes one or more of auxiliary lipids, cholesterol and its derivatives, and PEGylated lipids (PEG-Lipid).

[0022] More preferably, the molar ratio of the amino acid lipid to the auxiliary lipid, cholesterol and its derivatives and PEG-Lipid is (40-99.5):(0-15):(0-50):(0.5-3).

[0023] The helper lipids described in the present invention are selected from phospholipids and their derivatives, preferably one or more of PC, DPPC, DOPC, DSPC, DOPE, and DPPG.

[0024] The PEG-Lipid described in the present invention is selected from one or more of PEG-DMG, PEG-C-DMG, and PEG-DSPE.

[0025] Further preferably, the N / P molar ratio of the drug delivery system and the siRNA is 2 / 1-6 / 1, for example, 2 / 1, 2.5 / 1, 3 / 1, 3.5 / 1, 4 / 1, 4.5 / 1, 5 / 1, 5.5 / 1 or 6 / 1.

[0026] More preferably, the nucleic acid interfering drug is a nanoparticle.

[0027] Still further preferably, the size of the nanoparticles is 50-300 nm, for example, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, 155 nm, 160 nm, 165 nm m, 170nm, 175nm, 180nm, 185nm, 190nm, 195nm, 200nm, 205nm, 210nm, 215nm, 220nm, 225nm, 230nm, 23 5nm, 240nm, 245nm, 250nm, 255nm, 260nm, 265nm, 270nm, 275nm, 280nm, 285nm, 290nm, 295nm, 300nm.

[0028] More preferably, the dosage form of the nucleic acid interfering drug is a lyophilized agent.

[0029] More preferably, the nucleic acid interference drug is administered by subcutaneous injection and / or intravenous injection.

[0030] More preferably, the subject of administration of the nucleic acid interference drug is a mammal.

[0031] Further preferably, the nucleic acid interference drug is obtained by mixing the siRNA solution and the delivery system stock solution by microfluidics, followed by dialysis against a neutral buffer solution and ultrafiltration concentration.

[0032] In the present invention, the liver damage includes one or more of liver fibrosis, liver cirrhosis, and liver cancer.

[0033] In the present invention, the nucleic acid interference drug also includes siRNA that can inhibit and silence the expression of other liver damage-related genes.

[0034] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art:

[0035] The present invention designs and screens more small interfering nucleic acids that can effectively knock down the COL1A1 target in vivo, which will be of great significance for the effective treatment and prevention of liver injury-related diseases. At the same time, the selection of appropriate delivery vectors can improve the in vivo delivery effect of small interfering nucleic acids and the efficient knockdown of the COL1A1 target in vivo while ensuring safety, greatly advancing the development of clinical drugs for the treatment of liver injury and hopefully providing more options for patients with liver injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] FIG1 is a schematic diagram of the administration of the test drug siCOL1A1 to mice with CCl4-induced liver injury;

[0037] FIG2 is a graph showing the effect of the test drug siCOL1A1 on the expression of COL1A1 in liver tissue of mice with CCl4-induced liver injury;

[0038] FIG3 shows the effects of different doses of the test drug siCOL1A1 on the expression of COL1A1 in liver tissue of mice with CCl4-induced liver injury;

[0039] FIG4 shows the effects of the test drug siCOL1A1 in different carriers on the expression of COL1A1 in liver tissue of mice with CCl4-induced liver injury;

[0040] FIG5 shows the effects of the test drug siCOL1A1 in different carriers on the expression of α-SMA in the liver tissue of mice with CCl4-induced liver injury;

[0041] FIG6 shows the residual amount detection results of the test drug siCOL1A1 in different carriers in the liver tissue of mice with CCl4-induced liver injury 48 hours after the last administration;

[0042] FIG7 is a graph showing the effects of the test drug siCOL1A1 containing COL1A1 siRNA in different optimized forms on the expression of COL1A1 in liver tissue of mice with CCl4-induced liver injury;

[0043] FIG8 shows the residual amount of the test drug siCOL1A1 with optimized sequences in different vectors in the liver tissue of mice with CCl4-induced liver injury 48 hours after the last administration;

[0044] FIG9 is a schematic diagram of the administration of the test drug siCOL1A1 to mice with TAA-induced liver injury;

[0045] FIG10 is a graph showing the effects of the test drug siCOL1A1 containing COL1A1 siRNA in different optimized forms on the expression of COL1A1 in liver tissue of mice with TAA-induced liver injury;

[0046] FIG11 shows the residual amount of the test drug siCOL1A1 with optimized sequences in different vectors in the liver tissue of mice with TAA-induced liver injury 48 hours after the last administration;

[0047] FIG12 is a graph showing the effects of different delivery vectors on mouse body weight.

[0048] Implementation Method

[0049] The present invention is further described below with reference to the following examples. However, the present invention is not limited to the following examples. The implementation conditions used in the examples may be further adjusted according to the specific requirements of the application. Unspecified implementation conditions are conventional conditions in the industry. The technical features involved in the various embodiments of the present invention may be combined with each other as long as they do not conflict with each other.

[0050] In the following examples and comparative examples, unless otherwise specified, all experimental materials used are commercially available, and all experimental methods used are conventional methods in the art.

[0051] Example 1

[0052] Design siRNA sequence for target (COL1A1)

[0053] The design process used human COL1A1 mRNA NM_000088.4 and mouse COL1A1 mRNA NM_007742.4. Using a specific algorithm and programming with several parameter conditions, a series of siRNA sequences, including 25- and 21-base-pair siRNA sequences, were designed against the target gene COL1A1. These sequences were characterized by, but not limited to, targeting both coding and non-coding sequences, reasonable thermodynamic stability, and low expected toxic side effects. siRNA sequences targeting the COL1A1 gene are theoretically capable of binding to and degrading COL1A1 mRNA in target cells through the RNAi mechanism, thereby blocking protein translation and inhibiting COL1A1 protein expression.

[0054] Table 1 siRNA sequences targeting COL1A1

[0055] Example 2

[0056] In vitro screening of siRNA sequences targeting the COL1A1 gene (cellular level)

[0057] In this example, the cell lines selected for screening effective small nucleic acids are cell lines that can highly express target genes, including human cervical cancer cells (Hela), human pancreatic cancer cells (PANC-1), and human bile duct cancer cells (HUCCT), and in vitro screening of target siRNA sequences is performed at the cellular level.

[0058] Cell culture and transfection

[0059] The cells were cultured in a 37°C, 5% CO2 incubator. When the cells were in good condition and the cell density reached 80%-90%, a screening experiment was performed. The cells were first transferred to a clean bench, the stock solution was discarded, PBS was added and shaken slightly to wash the residual culture medium, and 1-2 mL of trypsin (Biosharp, BL512A) was added. When the trypsin was evenly covered on the cells, the excess trypsin was carefully removed with a pipette, leaving a small amount of trypsin in the culture bottle. The culture bottle was placed in an incubator at 37°C for digestion for 1-5 minutes. The culture bottle was removed and observed under a fluorescent light. When the cells were observed to fall off, the cells were observed to become round and discrete under a microscope. 10 mL of complete culture medium containing 10% fetal bovine serum was immediately added to terminate the digestion. The digested cells were repeatedly blown to detach and disperse them to prepare a cell suspension. A small amount of the cell suspension was placed in an EP tube for cell counting.

[0060] Hela (MEM complete medium, Procell), PANC-1 (DMEM complete medium, Procell) and HUCCT (RPMI1640 complete medium, Procell) were inoculated into 48-well cell culture plates (0.7×10 4 / well), the cell culture plate was placed in a cell culture incubator and cultured for 24 hours. The cells were observed under an inverted microscope and the transfection experiment was performed under the premise that the cells were in good condition and the density was 70%-80%.

[0061] Cells were transfected with the commercial transfection reagent Lipo2000 (Invitrogen, 1734988) carrying the siRNA sequences listed in Table 1 or a negative control (NC siRNA: sense strand 5'-UUCUCCGAACGUGUCACGUdTdT-3', antisense strand 5'-ACGUGACACGUUCGGAGAAdTdT-3'). The siRNA transfection concentration was 20 nM. Untreated cells served as a blank control. Lipo2000 was diluted with serum-free medium to prepare a Lipo2000 dilution solution at a volume ratio of 1:200. 150 μL of the Lipo2000 dilution solution was added to 150 μL of the siRNA dilution solution, mixed, and incubated at room temperature for 20 minutes to form a transfection complex. After incubation, remove the cell culture plate to be used, discard the original culture medium, wash the plate with serum-free medium, add 200 μL (48-well plate) transfection complex to each well, transfect in the incubator for 4 hours, and then add 500 μL of fresh complete culture medium to each well and continue to culture for 24 hours.

[0062] RNA extraction

[0063] Total RNA was extracted from cells 24 hours after transfection. Cell lysis buffer (20 μL β-mercaptoethanol per 1 mL TRK Lysis Buffer) was prepared based on the sample number. The culture medium in the cell culture plate was aspirated with a pipette. 250 μL of cell lysis buffer was added to each well. After gentle shaking, 250 μL of 70% ethanol was added. The sample was pipetted evenly throughout the well and transferred to an RNA extraction column. The column was centrifuged at 10,000 rpm for 1 minute at room temperature. The waste liquid in the bottom tube was discarded. 500 μL of RNA Wash Buffer I was added to the RNA extraction column. The column was centrifuged at 10,000 rpm for 1 minute at room temperature. The bottom tube and waste liquid were discarded. The RNA extraction column was placed in a new 2 mL tube. 110 μL of DNase working solution (Sangon Biotech (Shanghai) Co., Ltd., I117KA3326) was added to the RNA extraction column. The reaction was allowed to incubate at room temperature for 15 minutes. After digestion, add 500 μL RNA wash buffer I to each RNA extraction column and centrifuge at 10,000 rpm for 1 min at room temperature. Discard the waste liquid in the bottom tube. Add 600 μL RNA wash buffer II (diluted with ethanol) to each RNA extraction column and centrifuge at 10,000 rpm for 1 min at room temperature. Discard the waste liquid in the bottom tube. Add another 600 μL RNA wash buffer II (diluted with ethanol) to each RNA extraction column and centrifuge at 10,000 rpm for 1 min at room temperature. Discard the waste liquid in the bottom tube. For each sample, centrifuge at 10,000 rpm for 2 min at room temperature without adding buffer. Discard the bottom tube and transfer the RNA extraction column to a 1.5 mL centrifuge tube. Open the RNA extraction column cap and evaporate at room temperature for 5-10 min to remove residual ethanol. Add 30 μL RNase-free water to the RNA extraction column for elution. Centrifuge at 10,000 rpm for 1 min at room temperature and detect the concentration under UV light.

[0064] cDNA synthesis and real-time PCR

[0065] The Evo-M-MLV RT Kit (ACCURATE BIOLOGY, AG11707) was used according to the manufacturer's instructions for the relevant experimental procedures. Reverse transcription was performed using a standard PCR instrument to obtain cDNA. 10 μL of the cDNA stock solution was diluted 5-fold to a final volume of 50 μL. A 20 μL qPCR reaction system was prepared according to the instructions for Power Green PCR Master Mix (2X) (Applied Biosystems, 01059548). 15 μL of the qPCR reaction mix was added to each well, followed by 5 μL of the cDNA template. The relative expression level of the target gene COL1A1 mRNA was determined by real-time PCR and normalized to the housekeeping gene β-actin. The effectiveness of gene knockdown is expressed as a percentage of the blank control. The results are shown in Table 2.

[0066] Table 2. Preliminary screening results of siRNA sequences targeting COL1A1 at the cellular level Note: % is mRNA expression level%; KD% = 100% - mRNA expression level%.

[0067] The results are shown in Table 2. The knockdown effects of COL1A1 siRNA sequences in human Hela and PANC-1 cells were significant, while some sequences showed knockdown effects in HUCCT but the knockdown effects were not significant. In Hela cells, the knockdown effects of sequences C1a1-003#, C1a1-006#, C1a1-007#, C1a1-008#, C1a1-009#, C1a1-010#, C1a1-016#, C1a1-018#, C1a1-019#, C1a1-021#, C1a1-022#, C1a1-023#, C1a1-030#, and C1a1-032# all reached more than 85%; in PANC-1 cells, the knockdown effects of sequences C1a1-007#, C1a1-008#, The knockdown effect of C1a1-010#, C1a1-018#, C1a1-019#, C1a1-021#, C1a1-030#, C1a1-032#, and C1a1-039# was about 75%; in HUCCT cells, the knockdown effect of sequences C1a1-007#, C1a1-022#, and C1a1-030# was about 70%, and the knockdown effect of sequences C1a1-001#, C1a1-018#, C1a1-021#, C1a1-023#, and C1a1-032# was about 60%. Based on the preliminary screening results in Hela, PANC-1 and HUCCT cells, C1a1-007#, C1a1-010#, C1a1-018#, C1a1-021#, C1a1-030#, C1a1-032# and C1a1-039# were selected as candidate sequences for subsequent EC50 data detection and analysis in Hela and PANC-1; C1a1-007#, C1a1-018#, C1a1-021#, C1a1-022#, C1a1-023#, C1a1-030# and C1a1-032# were selected as candidate sequences for subsequent EC50 data detection and analysis in HUCCT.

[0068] Example 3

[0069] EC50 data analysis and detection of the candidate sequences selected in Example 2 in the corresponding cell lines

[0070] First, HeLa, PANC-1, and HUCCT cells were seeded into 48-well cell plates (0.7×104 / well) and transfected with the siRNA sequences shown in Table 1 or a negative control (NC siRNA) using the commercial transfection reagent Lipo2000. The siRNA concentration was 100 nM and diluted fivefold to a concentration gradient of 100 nM, 20 nM, 4 nM, 0.8 nM, 0.16 nM, 0.032 nM, and 0.0064 nM. Untreated cells served as blank controls. Cell culture, plating, transfection, total RNA extraction, reverse transcription, and Q-PCR were performed using the same procedures as in Example 2. Finally, data curves were plotted using GraphPad Prism 9 software and EC50 values ​​were calculated.

[0071] Table 3 EC50 results of COL1A1 siRNA in PANC-1, Hela and HUCCT

[0072] The results are shown in Table 3. EC50 analysis was performed on the candidate sequences identified in the initial screening in PANC-1, Hela, and HUCCT. An EC50 value greater than 10 nM was considered to indicate no knockdown effect, an EC50 greater than 1 nM but less than 10 nM was considered to indicate a mild knockdown effect, and an EC50 less than 1 nM was considered to indicate a significant knockdown effect. In PANC-1 cells, the EC50 values ​​of sequences C1a1-007#, C1a1-010#, C1a1-021#, C1a1-030#, C1a1-032# and C1a1-039# were all less than 1nM, showing significant knockdown effects; in Hela cells, the EC50 values ​​of sequences C1a1-010#, C1a1-021#, C1a1-030# and C1a1-032# were all less than 1nM, showing significant knockdown effects; in HUCCT cells, the EC50 values ​​of sequences C1a1-021#, C1a1-023#, C1a1-030# and C1a1-032# were all less than 1nM, showing significant knockdown effects.

[0073] In summary, after EC50 screening, C1a1-021#, C1a1-030# and C1a1-032# were preliminarily selected as candidate sequences for in vivo experiments.

[0074] Example 4

[0075] The candidate sequences C1a1-021#, C1a1-030#, and C1a1-032# selected in Example 3 were optimized and modified (Table 4), and the modified sequences were analyzed and tested for EC50 data. First, Hela, PANC-1, and HUCCT cells were inoculated into 48-well cell plates (0.7×10 4Cells were transfected with the commercial transfection reagent Lipo2000 (Table 4) carrying the siRNA sequences or negative control (NC siRNA). The highest siRNA concentration was 100 nM, and the siRNA was diluted fivefold to a concentration gradient of 100 nM, 20 nM, 4 nM, 0.8 nM, 0.16 nM, 0.032 nM, and 0.0064 nM. Untreated cells served as blank controls. The cell culture, plating, transfection, total RNA extraction, reverse transcription, and Q-PCR procedures were identical to those in Example 2. GraphPad Prism 9 software was used to plot data and calculate EC50 values.

[0076] Table 4 Note: m indicates base 2'-OME modification, f indicates base 2'-F modification

[0077] Table 5. EC50 results of optimized and modified COL1A1 siRNA sequences in Hela, PANC-1, and HUCCT

[0078] The results are shown in Table 5. C1a1-021#, C1a1-030#, and C1a1-032# were modified in different ways, and the EC50 values ​​of the modified sequences were analyzed and tested. An EC50 value greater than 10 nM was considered to indicate no knockdown effect, an EC50 greater than 1 nM and less than 10 nM was considered to indicate a mild knockdown effect, and an EC50 less than 1 nM was considered to indicate a significant knockdown effect. In PANC-1 cells, the EC50 values ​​of sequences C1a1-045#, C1a1-046#, C1a1-047#, C1a1-048#, C1a1-049#, C1a1-050#, C1a1-052# and C1a1-055# were all less than 1nM, showing significant knockdown effects; in Hela cells, the EC50 values ​​of sequences C1a1-048#, C1a1-050# and C1a1-052# were all less than 1nM, showing significant knockdown effects; in HUCCT cells, the EC50 values ​​of sequences C1a1-045#, C1a1-048#, C1a1-049#, C1a1-050#, C1a1-051#, C1a1-052# were all less than 1nM, showing significant knockdown effects.

[0079] In summary, after screening, C1a1-021#, C1a1-030#, C1a1-032#, C1a1-045#, C1a1-046#, C1a1-048#, C1a1-049#, C1a1-050# and C1a1-052# will be used as candidate sequences for in vivo experiments.

[0080] Example 5

[0081] Preparation and identification of nanopharmaceutical preparations

[0082] The delivery system used in this patent is described in patents 202211209610.3 and 202310228776.8, which are hereby incorporated by reference.

[0083] The preparation process for the peptide-lipid LANP delivery system was as follows: the peptides listed in Table 6 were mixed with DSPC, cholesterol, and PEG-C-DMG at a molar ratio of 50 / 10 / 38.5 / 1.5, respectively, and dissolved in anhydrous ethanol to obtain lipid solution (a). At a molar ratio of 5:1, siRNA was dissolved in a citric acid buffer at a pH of 3-4 to obtain siRNA solution (b). Lipid solution (a) and siRNA solution (b) were mixed using a microfluidic system at a flow rate ratio of 3:1 to obtain product (c). Product (c) was dialyzed against PBS buffer at pH 7.2-7.4 for 24 hours and then concentrated by ultrafiltration at 4°C to obtain siRNA-encapsulated peptide-lipid nanoparticles as shown in Table 6. The sample encapsulation efficiency was greater than 90%, and its particle size potential is shown in Table 7.

[0084] The preparation process for the amino acid lipid ATLNP delivery system was as follows: the amino acid lipids listed in Table 6 were mixed with DSPC, cholesterol, and PEG-C-DMG at a molar ratio of 50 / 10 / 38.5 / 1.5, respectively, and dissolved in anhydrous ethanol to obtain lipid solution (a). At a molar ratio of 5:1 (N / P) between the amino acid lipids and siRNA, siRNA was dissolved in a citric acid buffer at a pH of 3-4 to obtain siRNA solution (b). The amino acid lipid solution (a) and siRNA solution (b) were mixed using microfluidics at a flow rate ratio of 3:1 to obtain product (c). Product (c) was dialyzed against PBS buffer at pH 7.2-7.4 for 24 hours and then concentrated by ultrafiltration at 4°C to obtain siRNA-loaded amino acid lipid nanoparticles as shown in Table 6. The particle size potential is shown in Table 7.

[0085] Table 6. Small nucleic acids and vector information used in in vivo pharmacodynamics validation Note: The sense strand of PC(5354) is 5'-GUCUAGACAUGUUCAGCUUdTdT-3', and the antisense strand is 5'-AAGCUGAACAUGUCUAGACdTdT-3' (US9944671B2).

[0086] Table 7. Key parameters of small nucleic acid drug formulations used in in vivo pharmacodynamics validation

[0087] Example 6

[0088] In vivo testing of COL1A1 siRNA in a CCl4-induced liver injury mouse model

[0089] The experimental mouse model used in this example is a liver injury model induced by the chemical reagent CCl4. This mouse model is a well-studied chemical-induced liver injury model. To evaluate the knockdown of COL1A1 by siCOL1A1 in the CCl4 mouse model, liver injury was induced by oral gavage of CCl4 (final dose of 1.75 ml / kg) on ​​days 1 and 8 in a BALB / c liver injury mouse model. The blank control group received no treatment. On day 9, the drug group received an intravenous injection of LANP-loaded siRNA, the model group received PBS, and the blank group received no drug treatment. The siCOL1A1 (SE-(Lin)2 / C1a1-021, SE-(Lin)2 / C1a1-030, and SE-(Lin)2 / C1a1-032) dose was 3 mg / kg, respectively. Approximately 48 hours after siRNA injection on day 11, mice were sacrificed, and livers were harvested and cryopreserved for future use (as shown in Figure 1). Taqman-QPCR was used to detect the mRNA levels of COL1A1 and β-actin, with 6 animals in each group.

[0090] The results in Figure 2 show that after two oral administrations of CCl4, COL1A1 mRNA expression in the Model group increased significantly compared to the Blank group, indicating successful model establishment. SE-(Lin)2 / C1a1-032# demonstrated a greater knockdown effect on COL1A1 mRNA than SE-(Lin)2 / C1a1-021# and SE-(Lin)2 / C1a1-030#. Compared to the Model group, SE-(Lin)2 / C1a1-032# significantly reduced COL1A1 mRNA expression by 56% (P < 0.01).

[0091] Example 7

[0092] In vivo testing of different doses of COL1A1 siRNA in a CCl4-induced liver injury mouse model

[0093] To evaluate the dose-response effect of LANP-based delivery of siCOL1A1 in a CCl4-induced liver injury model, BALB / c mice were orally gavaged with CCl4 at a final dose of 1.75 ml / kg on days 1 and 8. Control animals were not treated. On day 9, mice were intravenously injected with different doses of LANP-loaded siRNA. The Model group was given PBS, while the Blank group was not treated with the drug. The mice were then killed approximately 48 hours after siRNA injection on day 11, and the livers were collected and frozen for later use (as shown in Figure 1). The siRNAs used were SE-(Lin)2 / C1a1-021, SE-(Lin)2 / C1a1-032, SE-(Lin)2 / C1a1-050, and SE-(Lin)2 / C1a1-052, and three doses were tested, including 0.5 mpk, 1 mpk, and 3 mpk. Taqman-QPCR was used to analyze COL1A1 and β-actin mRNA levels in the liver. Six animals were used per group for each dose.

[0094] The results in Figure 3 show that after two oral administrations of CCl4, COL1A1 mRNA expression in the Model group increased significantly compared to the Blank group, indicating successful model establishment. Using LANP (SE-(Lin)2) as a delivery vehicle, a 3 mpk dose of C1a1-021# siRNA significantly knocked down liver COL1A1 expression by 34% (P < 0.01 vs. Model group). SE-(Lin)2 / C1a1-032 and SE-(Lin)2 / C1a1-050 (modified siRNA sequences) did not significantly knock down the target gene. SE-(Lin)2 / C1a1-052 (modified siRNA sequence) effectively knocked down the expression of COL1A1, and this knockdown was dose-dependent. The three doses of 0.5mpk, 1mpk and 3mpk knocked down the COL1A1 mRNA level by 28%, 50% and 60%, respectively, among which 1mpk and 3mpk were extremely significant (P<0.0001vs Model group).

[0095] Example 8

[0096] In vivo testing of COL1A1 siRNA delivered by different vectors in a CCl4-induced liver injury mouse model

[0097] The knockdown of COL1A1 and the residual status in the liver of siCOL1A1 delivered by different vectors in the CCl4 mouse model were studied. 45 BALB / c mice were orally gavaged with CCl4 at a final dose of 1.75 ml / kg on days 1 and 8. The control animals were not treated. On day 9, the mice were intravenously injected with siRNA formulated with different delivery vectors. The Model group was given PBS, and the Blank group was not treated with the drug. Then, about 48 hours after the siRNA injection on day 11, the mice were killed and the livers were collected and frozen (as shown in Figure 1). The siRNAs used were Met-Tris-3LA / C1a1-052, Gly-Tris-3MOA / C1a1-052, SE-(Lin)2 / C1a1-052, GE-ρ2 / C1a1-052, and MC3 / C1a1-052, and the dosage was 3 mpk, respectively. Taqman-QPCR was used to analyze COL1A1 and α-SMAm RNA levels in the liver. SL-RT-QPCR (stem loop RT-QPCR) was used to quantify the antisense strand of the siRNA. Six animals were used per group.

[0098] The results in Figure 4 show that after two oral administrations of CCl4, COL1A1 mRNA expression in the Model group increased significantly compared to the Blank group, indicating successful model establishment. In the Met-Tris-3LA-delivered siCOL1A1 (C1a1-052#) group, liver COL1A1 expression was equivalent to 36% of that in the Model group, a significant difference compared to the Model group (P < 0.05). In vivo knockdown of siCOL1A1 delivered by GE-ρ2 was comparable to that of MC3, with liver COL1A1 expression in both groups equivalent to 22% of that in the Model group, a highly significant difference compared to the Model group (P < 0.001).

[0099] The results in Figure 5 show that compared with the Blank group, the expression level of α-SMA increased after CCl4 injury, and the Met-Tris-3LA-delivered siCOL1A1 group and the GE-ρ2-delivered siCOL1A1 group could significantly inhibit the increase in α-SMA expression (P<0.05 vs Model group).

[0100] The residual amount detection results (Figure 6) showed that siCOL1A1 (C1a1-052#) was detectable in all drug-delivered groups 48 hours after administration, among which the residual amount in the GE-ρ2-delivered siCOL1A1 group was the highest.

[0101] Example 9

[0102] In vivo testing of COL1A1 siRNAs with different optimization methods in a CCl4-induced liver injury mouse model

[0103] To evaluate the knockdown of COL1A1 by siCOL1A1 with different optimization methods in the CCl4 mouse model, liver injury was induced by oral gavage of CCl4 (final dose of 1.75 ml / kg) on ​​days 1 and 8 in a BALB / c liver injury mouse model. The blank control group received no treatment. On day 9, the drug group received an intravenous injection of GE-ρ2-loaded siRNA, the model group received PBS, and the blank group received no drug treatment. The dose of siCOL1A1 (GE-ρ2 / C1a1-032, GE-ρ2 / C1a1-046, GE-ρ2 / C1a1-049, and GE-ρ2 / PC(5354)) was 3 mg / kg, respectively. PC(5354) is a sequence validated in vivo (US9944671B2). The sense strand is 5'-GUCUAGACAUGUUCAGCUUdTdT-3', and the antisense strand is 5'-AAGCUGAACAUGUCUAGACdTdT-3'. Mice were then sacrificed on day 11 (approximately 48 hours after siRNA injection), and livers were harvested. Taqman-QPCR was used to measure COL1A1 and β-actin mRNA levels, and the COL1A1 siRNA content in liver tissue was also analyzed using Taqman-QPCR. Six animals were included in each group.

[0104] The results in Figure 7 show that under the same GE-ρ2 vector conditions, GE-ρ2 / C1a1-032# and GE-ρ2 / C1a1-046# sequences did not show knockdown, while GE-ρ2 / C1a1-049# and GE-ρ2 / PC(5354) sequences had comparable effects, with a knockdown of approximately 40%.

[0105] The results in Figure 8 show that significant amounts of siRNA residue were detected in the liver in all treatment groups. Using the same GE-ρ2 vector, the C1a1-032# sequence showed less liver residue than the C1a1-046#, C1a1-049#, and PC(5354) sequences. Furthermore, under the same PC(5354) conditions, the GE-ρ2 vector produced more siRNA residue than the MC3 vector.

[0106] Example 10

[0107] In vivo testing of COL1A1 siRNAs with different optimized sequences in a TAA-induced liver injury mouse model

[0108] Thioacetamide, abbreviated as TAA, acts slower than CCl4. The TAA model can cause uniform liver damage and is therefore considered to be closer to simulating liver damage in the human population. This example evaluates the knockdown of COL1A1 in a TAA mouse model using siCOL1A1 with different optimization methods. In the BALB / c TAA liver injury mouse model, liver damage was induced by intraperitoneal injection of TAA (final dose of 200 mg / kg) on ​​days 0, 3, and 7. The Blank control group was not treated. Subsequently, on day 8, the drug-treated group was injected with GE-ρ2-loaded siRNA via the tail vein, the Model group was given PBS, and the Blank group was not treated with the drug. The dosage of siCOL1A1 (GE-ρ2 / C1a1-032, GE-ρ2 / C1a1-046, GE-ρ2 / C1a1-049, and GE-ρ2 / PC(5354)) was 3 mg / kg, respectively. On day 9, approximately 24 hours after siRNA injection, mice were sacrificed and livers were harvested (as shown in Figure 9 ). Taqman-QPCR was used to measure COL1A1 and β-actin mRNA levels, and Taqman-QPCR was used to analyze COL1A1 siRNA levels in liver tissue. Six animals were included in each group.

[0109] The results in Figure 10 show that compared with the Model group, the GE-ρ2 / C1a1-032# group knocked down 48% (P<0.05), the GE-ρ2 / C1a1-046# group knocked down 29%, the GE-ρ2 / C1a1-048# group knocked down 74% (P<0.001), the GE-ρ2 / PC(5354) group knocked down 72% (P<0.01), and the MC3 / PC(5354) group knocked down 43% (P<0.05). Among the different sequences delivered by the GE-ρ2 vector, the C1a1-049# sequence had the best knockdown effect (74%, P<0.001 vs Model group), slightly better than PC(5354) (72%, P<0.01 vs Model group).

[0110] The residue results in Figure 11 show that there is little difference in the residue amounts of each group. Under the same sequence conditions of PC(5354), the residue amount of GE-ρ2 vector is higher than that of MC3 vector (P<0.05).

[0111] Example 11

[0112] Safety testing of different delivery vehicles in mice

[0113] To evaluate the safety of different delivery vectors in mice, 90 ICR mice were divided into 9 groups, with 10 mice in each group. The drug-treated group was intravenously injected with siRNA prepared with GE-ρ2 and MC3. At the same time, the empty control group was intravenously injected with GE-ρ2 and MC3, and the blank group was given PBS. The doses of siTF1 (GE-ρ2 / TF1 and MC3 / TF1) and the empty group were 4 mg / kg and 8 mg / kg. The TF1 positive chain is 5'-CCCAAGGGCUACCAUGCCAACUUCU-3', and the antisense chain is 5'-AGAAGUUGGCAUGGUAGCCCUUGGG-3'. The body weight was measured before and one day after administration, and the weight change rate of the mice was analyzed 24 hours after administration.

[0114] As shown in Figure 12, the 8-mpk GE-ρ2 / siTF1 and MC3 / siTF1 groups showed a significant decrease in body weight, while the 4-mpk group showed relatively less toxicity. The toxicity of both the 4-mpk and 8-mpk empty GE-ρ2 and MC3 groups was relatively low. At the same dose, the body weight loss rate of GE-ρ2 / siTF1 was slightly lower than that of MC3 / siTF1, and similarly, the body weight loss rate of empty GE-ρ2 was slightly lower than that of empty MC3. This suggests that GE-ρ2 has a lesser effect on mouse body weight than MC3.

[0115] The above embodiments are intended only to illustrate the technical concepts and features of the present invention. Their purpose is to enable those skilled in the art to understand the contents of the present invention and implement them accordingly. They are not intended to limit the scope of protection of the present invention. Any equivalent changes or modifications made in accordance with the spirit of the present invention are intended to be covered by the scope of protection of the present invention.

Claims

1. A nucleic acid interference drug for treating liver damage, characterized in that: The nucleic acid interference drug includes siRNA capable of inhibiting and silencing COL1A1 gene expression, and the siRNA includes any one or more of the siRNAs listed in Table 1.

2. The nucleic acid interference drug for treating liver damage according to claim 1, characterized in that: The 5' end and / or 3' end of the sense strand and / or antisense strand of the siRNA is truncated or extended by 1 to 6 bases. Preferably, the truncation or extension is performed so that the 3' end of the antisense strand of the siRNA forms 1 to 3 unpaired single bases.

3. The nucleic acid interference drug for treating liver damage according to claim 1, characterized in that: The siRNA molecule is chemically modified, and the chemical modification includes one or more of base 2'-OME modification, base 2'-F modification, and phosphorothioate backbone modification.

4. The nucleic acid interference drug for treating liver damage according to claim 2, characterized in that: The siRNA is any one or more of the siRNAs listed in Table 4, preferably C1a1-049# and / or C1a1-052#.

5. The nucleic acid interference drug for treating liver damage according to claim 1, characterized in that: The nucleic acid interference drug also includes a pharmaceutically acceptable delivery system for delivering the siRNA.

6. The nucleic acid interference drug for treating liver damage according to claim 5, characterized in that: The delivery system is a small peptide lipid nano delivery system and / or an amino acid lipid nano delivery system.

7. The nucleic acid interference drug for treating liver damage according to claim 6, characterized in that: The delivery system comprises a small peptide lipid or an amino acid lipid, wherein the small peptide lipid is preferably Ser-Glu-(C17)2, Ser-Asp-(C17)2, Ser-N-Ser-Asp-(C17)2, D-Ser-D-Glu-(C17)2, Ser-Glu-N(C17)2, Ser-Glu-(Lin)2, Ser-Glu-(C25)2, Ser-Asp-(C25)2, Thr-Asp-(C25)2, Met-Asp-(C25)2, Ser-Lys-(C17)(NC16), Ser-Glu-[Glu-(C17)2]2, Ser-Glu-[Glu-(C17)2]2, Ser-Glu-αC17(NC17), Ser-Glu-C17(αNC17), Gly-Glu-(C17) 2, Leu-Glu-(C17)2, His-Glu-(C17)2, Ser-AAD-(C17)2, Ser-Glu-[C17(=)2]2, the amino acid lipid is preferably one or more of Met-Tris-3MOA, Gly-Tris-3MOA, Gly-Tris-3MA, Gly-Tris-3POA, Gly-Tris-3DOA, Gly-Tris-3OA, Gly-Tris-3Lin, Gly-Tris-3PA, Met-Tris-3MA, Met-Tris-3LA, Met-Tris-3C10, Met-PEL-3MOA, and the delivery system further selectively includes one or more of auxiliary lipids, cholesterol and its derivatives, and PEGylated lipids (PEG-Lipid).

8. The nucleic acid interference drug for treating liver damage according to claim 7, characterized in that: The auxiliary lipid is selected from phospholipids and their derivatives, preferably one or more of PC, DPPC, DOPC, DSPC, DOPE, and DPPG; the PEG-Lipid is selected from one or more of PEG-DMG, PEG-C-DMG, and PEG-DSPE; and the molar ratio of the small peptide lipid or amino acid lipid to the auxiliary lipid, cholesterol and its derivatives, and PEG-Lipid is (40-99.5):(0-15):(0-50):(0.5-3).

9. The nucleic acid interference drug for treating liver damage according to claim 5, characterized in that: The N / P molar ratio of the drug delivery system and the siRNA is 2 / 1 to 6 / 1; And / or, the nucleic acid interfering drug is a nanoparticle; And / or, the nucleic acid interference drug is administered by subcutaneous injection and / or intravenous injection; And / or, the subject to which the nucleic acid interfering drug is administered is a mammal; And / or, the nucleic acid interference drug is obtained by mixing the siRNA solution and the delivery system stock solution through microfluidics, and then dialyzing against a neutral buffer solution and concentrating through ultrafiltration.

10. The nucleic acid interference drug for treating liver damage according to any one of claims 1 to 9, characterized in that: The liver damage includes one or more of liver fibrosis, liver cirrhosis, and liver cancer; And / or, the nucleic acid interference drug also includes siRNA that can inhibit and silence the expression of other liver damage-related genes.

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