Liver targeted presentation inhibition sequence combination and application thereof

Abstract

The invention provides a liver targeted presentation inhibition sequence combination, which comprises a small interfering RNA (Ribonucleic Acid) and a short hairpin RNA sequence as shown in SEQ ID NO.2, and the small interfering RNA comprises a small interfering RNA basic sequence as shown in SEQ ID NO.1. The invention also provides an application. The inhibition sequence combination is used for preparing a medicine for inhibiting FMO3 level and inhibiting TMA-FMO3-TMAO axis mediated bile acid metabolism and cholesterol transporter. The liver targeted presentation inhibition sequence combination has good specificity and high transfection efficiency, inhibits TMA-FMO3-TMAO axis mediated bile acid metabolism and cholesterol transporter by inhibiting the FMO3 level, has good potential for improving cholelithiasis and other related diseases, and is used for preparing cholelithiasis related drugs.

Description

Technical Field

[0001] This invention belongs to the field of liver-targeted presentation inhibitory sequence technology, specifically relating to a combination of liver-targeted presentation inhibitory sequences and their applications. Background Technology

[0002] The formation of cholelithiasis involves an imbalance in bile composition, leading to the deposition or aggregation of cholesterol, bile pigments, or mixed crystals in the gallbladder or biliary system, forming stones. The core pathology of cholesterol cholelithiasis is not simply cholesterol excess, but rather an imbalance in bile acid metabolism: hepatocytes convert cholesterol into bile acids via bile acid synthases and rely on cholesterol transporters to excrete them into the bile ducts; when this synthesis and transport axis leads to an imbalance in cholesterol metabolism in hepatocytes and an imbalance in the cholesterol efflux pump in the hepatic tubules, cholesterol in the bile becomes supersaturated, precipitating and crystallizing to form stones. Its global prevalence exceeds 10%, with a recurrence rate of nearly 30% within 5 years post-surgery. Currently, there is a lack of non-surgical drugs and precise intervention tools that can safely dissolve cholesterol stones. Therefore, developing usable liver-targeting novel molecules as a non-surgical approach for the treatment of cholelithiasis has translational value and market demand.

[0003] Flavin-containing monooxygenase 3 (FMO3) is a member of the flavin monooxygenase family. It can oxidize the food-derived precursor trimethylamine (TMA) to trimethylamine N-oxide (TMAO). The catalytic reaction formula is TMA + NADPH + H₂O. + +O2=TMAO+NADP + +H2O. It is most abundant in the liver, and also expressed to some extent in the kidneys. It participates in the TMA-TMAO metabolism of the gut microbiota-liver-kidney pathway. FMO3 gene deficiency leads to an autosomal recessive genetic disorder characterized by a fishy odor and is associated with metabolic diseases such as insulin resistance, diabetes, fatty liver, and atherosclerosis. Overexpression of FMO3 increases adipogenesis and gluconeogenesis, while FMO3 gene knockout has the opposite effect in mice. Furthermore, FMO3 and its metabolite TMAO regulate cholesterol absorption by affecting the expression of the hepatic tubular cholesterol transporter ATP-binding cassettes ABCG5 and ABCG8, and are associated with gallstone formation. All of the above studies indicate that intervention strategies targeting FMO3 are suitable for the development of therapeutic drugs related to glucose metabolism, lipid metabolism, and cholesterol metabolism, and have promising applications for patentability and clinical translation.

[0004] Small interfering RNA (siRNA) is a class of double-stranded RNA molecules, 19-25 base pairs in length, that operate within the RNA interference (RNAi) pathway. It interferes with the expression of specific genes with complementary nucleotide sequences by post-transcriptionally degrading mRNA, thus silencing genes by blocking translation. The U.S. Food and Drug Administration (FDA) and the European Commission have successively approved an RNAi-based oligonucleotide, ONPATTRO (patisiran, 2018), as a lipid-based injection for the treatment of peripheral neuropathy caused by hereditary transthyretin-mediated amyloidosis in adult patients. This marks the first time the FDA and the European Commission have approved an siRNA as a new drug, signifying a breakthrough in the technological barriers of RNAi therapy and providing the pharmaceutical industry with new options. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a combination of liver-targeted presentation inhibitory sequences and their applications, which have good specificity and high transfection efficiency. By inhibiting FMO3 levels, it inhibits bile acid metabolism and cholesterol transporters mediated by the TMA-FMO3-TMAO axis, and has good potential to improve cholelithiasis and related diseases. It can be used in the preparation of cholelithiasis-related drugs.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a combination of liver-targeted presentation inhibitory sequences, wherein the combination of inhibitory sequences includes FMO3 sequences designed with corresponding interfering RNA, specifically small interfering RNA and short hairpin RNA sequences; wherein the small interfering RNA includes a basic small interfering RNA sequence; The nucleotide sequence of the basic sequence of the small interfering RNA is shown in SEQ ID NO.1; The nucleotide sequence of the short hairpin RNA sequence is shown in SEQ ID NO.2.

[0007] Preferably, the small interfering RNA further includes one or more of the following: tail-modified small interfering RNA sequence, cholesterol-modified small interfering RNA sequence 1, cholesterol-modified small interfering RNA sequence 2, and GalNac-modified small interfering RNA sequence; The sequences of the positive and antisense strands of the tail-modified small interfering RNA sequence are as follows: 5'- CCAGACAGAUUACAUUGUUUAdTdT -3'; (SEQ ID NO.6) 5'- UAAACAAUGUAAUCUGUCUGGdTdT -3'; (SEQ ID NO.7) The sequences of the positive and antisense strands of the cholesterol-modified small interfering RNA sequence 1 (i.e., si-FMO3-Chol RNA sequence 1) are as follows: 5'- Cm Cm Am GACAGAUUACAUUGU Um Um Am dTdT-Chol-3'; (SEQ ID NO.8) 5'-phos-Um Am Am ​​AC Am Af Uf Gm Uf Am A Um C Um G Um Cm Um Gm GmdTdT-3'; (SEQ ID NO.9) The sequences of the positive and antisense strands of the cholesterol-modified small interfering RNA sequence 2 (i.e., si-FMO3-Chol RNA sequence 2) are as follows: 5'-Cm Cm Am GACAGAUUACAUUGU Um Um Am dTdT-Chol-3'; (SEQ ID NO.10) 5'-phos-Um Am Am ​​Af Cm Am Af Uf Gm Uf Am Af Um Cf Um Gf Um Cf Um GmGm dTdT-3'; (SEQ ID NO.11) The sequences of the positive and antisense strands of the GalNac-modified small interfering RNA sequence are as follows: 5'-CCAGACAGAUUACAUUGUUUAdTdT -3'-GalNac3; (SEQ ID NO. 12) 5'- UAAACAAUGUAAUCUGUCUGGdTdT -3'. (SEQ ID NO.7) The present invention also provides the application of the above-mentioned liver-targeted presentation inhibitory sequence combination, characterized in that the inhibitory sequence combination is used to prepare a drug that inhibits FMO3 (flavin monooxygenase 3) levels and inhibits TMA-FMO3-TMAO axis-mediated bile acid metabolism and cholesterol transporters.

[0008] Preferably, the inhibitory sequence combination is used to prepare a drug for improving cholelithiasis.

[0009] Compared with the prior art, the present invention has the following advantages: The liver-targeted presentation inhibitory sequence combination of the present invention has good specificity and high transfection efficiency. It has shown good performance in computer simulation, tool cell bioinformatics transfection verification, and in vitro experimental disease model verification. By inhibiting FMO3 level, it inhibits TMA-FMO3-TMAO axis-mediated bile acid metabolism and cholesterol transporter, which has good potential to improve cholelithiasis and related diseases, and can be used in the preparation of cholelithiasis-related drugs.

[0010] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. Attached Figure Description

[0011] Figure 1 This is a schematic diagram of the structure of cholesterol-modified small interfering RNA1 (SEQ ID NO. 8-11) and small interfering RNA2 (SEQ ID NO. 15-18) in Example 1 of the present invention.

[0012] Figure 2 This is a schematic diagram of the structure of GalNac-modified small interfering RNA 1 (SEQ ID NO.12 and SEQ ID NO.7) and GalNac-modified small interfering RNA 2 (SEQ ID NO.19 and SEQ ID NO.14) in Embodiment 1 of the present invention.

[0013] Figure 3 This is a graph showing the efficiency detection in vitro and in vivo experiments in Example 2 of this invention. In the graph, (A) represents the FMO3 level in hepatocytes after transfection with small interfering RNA; (B) represents the FMO3 level in mice with cholelithiasis; and (C) represents the TMAO level in mice with cholelithiasis. Sample size = 6 / group, **** indicates a significant difference of P < 0.0001, and ns indicates no significant difference.

[0014] Figure 4 This is an in vivo experimental diagram from Example 2 of the present invention. In the diagram, (A) represents the expression level of cholesterol transporter ABCG5 in a cholelithiasis model mouse; (B) represents the expression level of cholesterol transporter ABCG8; (C) represents the expression level of cholesterol transporter LDLR; (D) represents the expression level of cholesterol transporter SRB1; (E) represents the expression level of bile acid synthase CYP7A1; and (F) represents the expression level of bile acid synthase CYP27A1. Sample size = 6 / group, **** indicates P < 0.0001 significant difference.

[0015] Figure 5 This is a specific mechanism diagram in Embodiment 2 of the present invention. Detailed Implementation

[0016] Example 1 Design of S1, RNA1, and RNA2: S101, the design process of FMO3-related RNA1 and RNA2, the target mRNA used is Fmo3 mRNA number NM_008030.2 (SEQ ID NO.5) encoding FMO3 protein NP_032056.1.

[0017] S102. The computer designs multiple small interfering RNAs based on the target mRNAs mentioned above, as shown in Table 1: Table 1 Sequence listing of small interfering RNA design scheme 1 S103. Based on design scheme 1, the computer designed small interfering RNA design scheme 2, as shown in Table 2: Table 2 Sequence listing of small interfering RNA design scheme 2 Design scheme for S104 and GalNac modified small interfering RNA: GalNAc (N-acetylgalactosamine) is an amino sugar with the chemical formula C8H2O. 15 NO6 is characterized by the substitution of the hydroxyl group at the C-2 position of the galactose with an acetamino group (–NHCOCH3). GalNAc itself is a high-affinity natural ligand for the liver; therefore, its conjugation modification serves as a small nucleic acid drug delivery system, attaching GalNAc in a trivalent state to the 3′ end of the positive strand of small interfering RNAs of different sequences, forming a polysaccharide-small interfering RNA monoconjugate (specific structure shown in...). Figure 2 As shown in the diagram, GalNAc achieves specific delivery to hepatocytes and allows the drug to enter the cell and exert its function through endocytosis. GalNAc is a high-affinity targeting ligand for the desialyl glycoprotein receptor (ASGPR). ASGPR is highly specifically expressed on the surface of hepatocyte membranes, enabling rapid nanoscale affinity endocytosis and drug internalization. After subcutaneous injection, the small interfering RNA carrying GalNAc modification rapidly enters the liver through the circulatory system, is then rapidly internalized by hepatocytes via ASPGR-mediated endocytosis, accumulates in endosomes, and is slowly released, persistently loading into the RNA-induced silencing complex to achieve a long-lasting and stable inhibitory effect. The GalNAc delivery system consists of a three-touch GalNAc target, a linker arm, and a small interfering RNA molecule, as shown in the diagram. Figure 2 As shown in the figure. This method enables efficient, direct, and targeted delivery, avoiding the immunogenicity issues associated with lipids and increasing drug safety.

[0018] S105, short hairpin RNA design scheme: KHK gene ID: 14262, transcript number: NM_008030.2 (Fmo3), species: mouse, design region: coding sequence CDS region. Two different short hairpin or small interfering RNA sequences were designed for simultaneous validation to prevent off-target effects. The efficiency of the short hairpin RNA sequences was tested using computer-simulated BLAST. Specifically, the efficiency of the two different short hairpin RNA sequences was tested using BLAST. The results showed that the two short hairpin RNA sequence schemes could only simulate the FMO3 protein-related sequences with 100% similarity in BLAST calculations, indicating that the target sites were all located in homologous regions. In the maxscore calculation results of other encoded protein mRNA sequences, the maximum number of matching bases was 30.2, with 15 bases actually bound, resulting in 6 mismatches (>4). This indicates that the two different short hairpin RNA sequences have extremely high specificity, theoretically guaranteeing the specificity of the short hairpin RNA after entering the cell or organism.

[0019] Based on actual computer simulation screening, two sequences (Scheme 1 and Scheme 2) were finally selected as the design schemes with the highest transfection efficiency.

[0020] The two different small interfering RNAs include small interfering RNA1 and small interfering RNA2, and the two different short hairpins include short hairpin RNA1 and short hairpin RNA2.

[0021] The liver-targeted presentation inhibitory sequence combination 1 of scheme 1 includes: small interfering RNA 1 and short hairpin RNA 1 sequences; the small interfering RNA 1 includes the basic sequence of small interfering RNA 1; Small interfering RNA 1 may also include one or more of the following: tail-modified small interfering RNA 1 sequence, cholesterol-modified small interfering RNA 1 sequence 1 (si-FMO3-Chol RNA1 sequence 1), cholesterol-modified small interfering RNA 1 sequence 2 (si-FMO3-Chol RNA1 sequence 2), and GalNac-modified small interfering RNA 1 sequence.

[0022] The liver-targeted presentation inhibitory sequence combination 2 of scheme 2 includes: small interfering RNA 2 and short hairpin RNA 2 sequences; the small interfering RNA 2 includes the basic sequence of small interfering RNA 2; Small interfering RNA 2 also includes one or more of the following: tail-modified small interfering RNA 2 sequence, cholesterol-modified small interfering RNA 2 sequence 1 (si-FMO3-Chol RNA 2 sequence 1), cholesterol-modified small interfering RNA 2 sequence 2 (si-FMO3-Chol RNA 2 sequence 2), and GalNac-modified small interfering RNA 2 sequence.

[0023] (I) Scheme 1: The liver-targeted presentation inhibitory sequence combination 1 includes: small interfering RNA1 and short hairpin RNA1 sequences, which is the technical solution protected by this invention, and the specific sequences are as follows: 1. The liver-targeted presentation inhibitory sequence combination 1 includes: (1) The basic sequence of small interfering RNA 1 designed based on SEQ ID NO.5 (SEQ ID NO.1): 5'-CCAGACAGATTACATTGTTTA-3' (SEQ ID NO. 1); (2) The sequence of short hairpin RNA1 designed based on SEQ ID NO.1 (SEQ ID NO.2): .

[0024] 2. The liver-targeted presentation inhibitory sequence combination 1 also includes: (1) Tail-modified small interfering RNA 1 sequences designed based on SEQ ID NO.1 (SEQ ID NO.6-7): 5'- CCAGACAGAUUACAUUGUUUAdTdT -3' (SEQ ID NO. 6); 5'-UAAACAAUGUAAUCUGUCUGGdTdT-3' (SEQ ID NO. 7); (2) such as Figure 1 As shown, the cholesterol-modified small interfering RNA 1 sequence designed using SEQ ID NO.1 is as follows: ①si-FMO3-Chol RNA1 sequence 1 (SEQ ID NO. 8-9): 5'- Cm Cm Am GACAGAUUACAUUGU Um Um Am dTdT-Chol-3' (SEQ ID NO. 8); 5'-phos-Um Am Am ​​AC Am Af Uf Gm Uf Am A Um C Um G Um Cm Um Gm GmdTdT-3' (SEQ ID NO. 9); ②si-FMO3-Chol RNA1 sequence 2 (SEQ ID NO.10-11): 5'-Cm Cm Am GACAGAUUACAUUGU Um Um Am dTdT-Chol-3' (SEQ ID NO. 10); 5'-phos-Um Am Am ​​Af Cm Am Af Uf Gm Uf Am Af Um Cf Um Gf Um Cf Um GmGm dTdT-3' (SEQ ID NO. 11); (3) such as Figure 2 As shown, the GalNac-modified small interfering RNA1 sequences designed using SEQ ID NO.1 (SEQ ID NO.12, SEQ ID NO.7) are: Justice Chain 5'- CCAGACAGAUUACAUUGUUUAdTdT -3'-GalNac3 (SEQ ID NO.12); Antisense chain 5'- UAAACAAUGUAAUCUGUCUGGdTdT -3' (SEQ ID NO.7).

[0025] (ii) Option 2: The liver-targeted presentation inhibitory sequence combination 2 includes: small interfering RNA2 and short hairpin RNA2 sequences, the specific sequences of which are as follows: 1. The liver-targeted presentation inhibitory sequence combination 2 includes: (1) The basic sequence of small interfering RNA 2 designed based on SEQ ID NO.5 (SEQ ID NO.3): 5'-GCCTCTGTAAACGACATGAT-3' (SEQ ID NO. 3); (2) The sequence of short hairpin RNA2 designed based on SEQ ID NO.3 (SEQ ID NO.4): .

[0026] 2. The liver-targeted presentation inhibitory sequence combination 2 also includes: (1) The tail-modified small interfering RNA2 sequence designed based on SEQ ID NO.3 is (SEQ ID NO.13-14): 5'-GCCUUCUGUAAACGACAUGAUdTdT-3' (SEQ ID NO. 13); 5'-AUCAUGUCGUUUACAGAAGGCdTdT-3' (SEQ ID NO. 14); (2) such as Figure 1 As shown, the cholesterol-modified small interfering RNA 2 sequence designed in SEQ ID NO.3: ①si-FMO3-Chol RNA2 sequence 1 (SEQ ID NO.15-16): 5'-Gm Cm Cm UUCUGUAAACGACAU Gm Am Um dTdT-Chol-3' (SEQ ID NO. 15); 5'-phos-Am Um Cm AU Gm Uf Cf Gm Uf Uf U Am C Am Gm Am Am ​​Gm Gm CmdTdT-3' (SEQ ID NO. 16); ②si-FMO3-Chol RNA2 sequence 2 (SEQ ID NO.17-18): 5'-Gm Cm Cm UUCUGUAAACGACAU Gm Am Um dTdT-Chol-3' (SEQ ID NO. 17); 5'-phos-Am Um Cm Af Um Gm Uf Cf Gm Uf Um Uf Am Cf Am Gf Am Af Gm GmCm dTdT-3' (SEQ ID NO. 18); like Figure 2 As shown, the GalNac-modified small interfering RNA2 sequences designed using SEQ ID NO.3 (SEQ ID NO.19, SEQ ID NO.14) are: 5'-GCCUUCUGUAAACGACAUGAUdTdT-3'-GalNac3 (SEQ ID NO. 19); 5'-AUCAUGUCGUUUACAGAAGGCdTdT-3' (SEQ ID NO. 14).

[0027] Annotations for RNA1 and RNA2 sequences: The underlined GGGG and G indicate restriction enzyme sites; The underlined CTCGAG indicates a loop. TTTTT or dTdT: poly T terminates the 3' overhang of transcription; A: Adenine; U: uracil; G: Guanine; C: Cytosine; T: Thymine; dT: deoxythymine; m: 2'-O-methyl; f: 2'-Fluoro; phos-: 5'-phosphate; -Chol: 3'-Cholesterol; GalNac: N-acetylgalactosamine.

[0028] In this embodiment, the sequences such as SEQ ID NO.1-5 are all RNA sequences. When inputting into the WIPO Sequence software, the RNA sequence must only contain the symbols listed in Table 1 of Part 1 of ANNEX I in WIPO ST.26. The specific meaning of the symbol "t" is explained in the annotations of this RNA1 and RNA2 sequence.

[0029] S2. Efficiency of the designed small interference and short hairpin RNA sequences: S201, Efficiency detection of small interfering RNA design scheme 1: The sequence efficiency results obtained by this method are shown in Table 3: Table 3 Efficiency rating table for small interfering RNA design scheme 1 Therefore, the small interfering RNA of design scheme 1 has good stability and low off-target effect in theoretical calculations.

[0030] S202, Efficiency testing of small interfering RNA design scheme 2: The sequence efficiency test results obtained from this design scheme 2 are shown in Table 4: Table 4. Stability rating table for small interfering RNA design scheme 2 Therefore, the small interfering RNA of design scheme 2 has a good stability score in theoretical calculations.

[0031] S203. Efficiency detection of RNA sequence analysis using computer-simulated BLAST: This embodiment demonstrates the efficiency of computer-simulated BLAST testing for short hairpin RNA sequences. The specific method is as follows: The efficiency of RNA sequences was tested using computer-simulated BLAST. Specifically, BLAST was used to test the efficiency of two different short hairpin RNA sequences. The results showed that the two basic RNA sequence schemes could only simulate the FMO3 protein-related sequences with 100% similarity in the BLAST simulation, indicating that the target sites were located in homologous regions. In the max score calculation results for other protein-coding mRNA sequences, the maximum number of matching bases was 30.2, with 15 bases actually bound, resulting in 6 mismatches (>4). This demonstrates that the two different basic RNA sequences have extremely high specificity, theoretically guaranteeing the specificity of the RNA sequence after entering the cell or organism. Therefore, this embodiment demonstrates the specificity of the inhibitory sequence combination at the computer level.

[0032] S204. Real-time quantitative PCR detection of the knockdown efficiency of inhibitory sequence combinations: Small interfering RNA 1 (SMERNA1) and SMERNA2 (SMERNA2) were transfected into tool cells, and the transfection knockdown efficiency was detected by real-time quantitative PCR. The results showed that, compared with the control group, the knockdown efficiency of SMERNA1 was 88%, with a GC content of 33.33% and a length of 21 nt; the knockdown efficiency of SMERNA2 was 86%, with a GC content of 42.86% and a length of 21 nt. The knockdown efficiencies of both SMERNA1 and SMERNA2 in the in vitro tool cells were significantly greater than 50%, confirming at the cellular level that the combination of inhibitory sequences has high transfection efficiency.

[0033] Example 2 This embodiment is an application of the liver-targeted presentation inhibitory sequence combination in Example 1, used to prepare a drug that inhibits FMO3 (flavin monooxygenase 3) levels and inhibits TMA-FMO3-TMAO (trimethylamine-flavin monooxygenase 3-trimethylamine oxide) axis-mediated bile acid metabolism and cholesterol transporters, and is used to prepare a drug to improve cholelithiasis.

[0034] In the inhibitory sequence combination of this invention, small interfering RNA (SRNA) is used in in vitro experiments, short-term studies, or drug screening stages. Tail-modified SRNA improves stability for labeling and targeted delivery; cholesterol-modified SRNA enhances cell penetration and community efficiency; GalNac-modified SRNA enables efficient liver delivery, reducing hepatic dosage and toxicity. Short hairpin RNA sequences are used for in vivo gene therapy, in vitro experiments with difficult-to-transfect cells, long-term studies, and hereditary gene silencing. After selecting the required sequence types according to the research type, composition scheme 1 and composition scheme 2 are mixed in a 1:1 ratio and used in relevant experiments for in vitro and in vivo application of drugs to improve cholelithiasis. 1. In vivo and in vitro validation of the efficiency of inhibited sequence combination transfection (1) In vitro model construction and transfection efficiency verification Hepatocytes synthesize cholesterol and transport it into bile, determining the cholesterol concentration in bile components. This is closely related to cholelithiasis and is therefore selected as the target organ for the treatment of cholelithiasis in this invention. To explore the transfection efficiency of the inhibitory sequence combination of this invention, we used the hepatocyte AML12 cell line (Cell Number: CRL-2254, ATCC) as the target cells for transfection. Figure 3 (A) shows the in vitro FMO3 level of the hepatocyte line after transfection with the inhibitory sequence combination of the present invention. The results showed that the mRNA expression level of FMO3 was significantly reduced after transfection, and the average value of FMO3 mRNA expression level was 43.17%, with an inhibition rate of >50%, showing the effect of highly efficient transfection of target cells.

[0035] (2) In vivo model construction and axis-level efficiency verification To explore the transfection efficiency and therapeutic effect of the inhibitory sequence combination of the present invention, we used liver tissue from cholelithiasis model mice as the detection target. The cholelithiasis model mice were eight-week-old male C57BL / 6J mice, which were fed a high-cholesterol, high-fat diet (product number D12108C, ResearchDiets) containing 21.1% fat, 41% sucrose and 1.25% cholesterol for four consecutive weeks, and were injected intraperitoneally with TMAO (Sigma-Aldrich, product number T317594) for four consecutive weeks. The TMAO was dissolved in physiological saline to a final injection volume of 200 µL, and the dose was 20 mg / kg / day. Figure 3 In the middle (B) and (C), the levels of FMO3 (flavin monooxygenase 3) and TMAO (trimethylamine oxide) in the liver tissue of cholelithiasis model mice are respectively. The results showed that after injection of the inhibitory sequence combination of the present invention, the levels of FMO3 and TMAO in the liver tissue of cholelithiasis model mice were significantly reduced, and the therapeutic effect of the inhibitory sequence combination in liver tissue was verified at the TMA-FMO3-TMAO axis level.

[0036] 2. The effect of inhibitory sequence combination on the regulation of an in vivo cholelithiasis model Studies have shown that the TMA-FMO3-TMAO axis is associated with the incidence of gallstones. High plasma TMAO levels are positively correlated with the presence of gallstones in humans. Liver tissue FMO3 and TMAO levels in gallbladder-susceptible mice are significantly higher than in normal mice. Dietary TMAO supplementation also leads to an increase in hepatic tubular cholesterol transporters ABCG5 and ABCG8, increasing the incidence of gallstones in mice, indicating that FMO3 and its metabolite TMAO contribute to gallstone formation. TMAO causes cholesterol supersaturation by upregulating cholesterol transporters ABCG5 and ABCG8 and inhibiting bile acid synthase CYP7A1; inhibiting FMO3 can disrupt this pathway. Therefore, small interfering RNA targeting FMO3 in the liver may be a way to treat and block this pathophysiological pathway.

[0037] Cholesterol transporters ABCG5 and ABCG8 are located on the hepatic tubular membrane and mainly regulate cholesterol secretion and excretion into bile. Cholesterol transporter LDLR is located on the basement membrane of hepatocytes, hydrolyzing over 70% of systemic LDL-C into free cholesterol. Cholesterol transporter SRB1 is located on the hepatic tubular membrane and selectively takes up HDL-C, catalyzing its formation into cholesterol. Both provide substrates for bile acid synthesis. Bile acid synthases CYP7A1 and CYP27A1 are rate-limiting enzymes in the synthesis of bile acids from cholesterol in hepatocytes. The operation of these transporters and enzymes maintains the overall cholesterol-bile acid balance. To verify the efficacy of the inhibitory sequence combination therapy of this invention in in vivo, we examined the expression levels of the above transporters and enzymes in gallstone-inducing mice injected with this invention. The results are shown in [Figure number missing]. Figure 4 The results showed that ABCG5 and ABCG8 levels were significantly reduced in mice with gallstones induced by the injection of this invention, indicating a reduction in cholesterol excretion into bile. Figure 4 (A, B); LDLR and SRB1 were significantly reduced, indicating reduced cholesterol production in the liver ( Figure 4 (C, D); CYP7A1 and CYP27A1 expression were significantly elevated, indicating increased cholesterol conversion to bile acids (C, D). Figure 4 (E, F). Through the above pathways, the hypercholesterolemia state in cholelithiasis model mice was alleviated by the inhibitory combination sequence described in this invention, demonstrating the effect of the inhibitory sequence combination on regulating the in vivo cholelithiasis model from an in vivo experimental perspective.

[0038] The specific mechanism of the liver-targeted presentation inhibitory sequence combination of the present invention is described in [link to invention]. Figure 5A protein-rich diet is rich in amine complexes containing TMA. Gut microbiota produce trimethylamine lyase, which converts directly ingested or indirectly generated TMA precursors into fishy-smelling TMA. This TMA enters the liver via the portal circulation and is oxidized by FMO3 to produce odorless TMAO. TMAO, along with unmetabolized TMA, enters the bloodstream and is primarily excreted through urine via the kidneys, with some excretion through respiration, sweat, and other glands. Throughout this process, FMO3 is involved in metabolic diseases such as atherosclerosis, insulin resistance, diabetes, non-alcoholic fatty liver disease, gallstones, renal failure, and chronic kidney disease. Small interfering RNA and short hairpin RNA precisely target and present FMO3 inhibitor sequences in the liver, blocking this process.

[0039] The two embodiments above demonstrate that the liver-targeted presentation inhibitory sequence combination designed in this invention has high specificity and transfection efficacy, and shows good performance in computer simulation, tool cell bioinformatics transfection verification, and disease model verification. It can inhibit bile acid metabolism and cholesterol transporters mediated by the TMA-FMO3-TMAO axis by inhibiting FMO3 levels, and has good potential and application prospects for improving cholelithiasis, providing a possibility for the clinical application of liver-targeted presentation therapy for cholelithiasis.

[0040] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.

Claims

1. A combination of liver-targeted inhibitory sequences, characterized in that, The inhibitory sequence combination includes small interfering RNA and short hairpin RNA sequences; the small interfering RNA includes a basic small interfering RNA sequence. The nucleotide sequence of the basic sequence of the small interfering RNA is shown in SEQ ID NO.1; The nucleotide sequence of the short hairpin RNA sequence is shown in SEQ ID NO.

2.

2. The liver-targeted presentation inhibitory sequence combination according to claim 1, characterized in that, The small interfering RNA also includes one or more of the following: tail-modified small interfering RNA sequence, cholesterol-modified small interfering RNA sequence 1, cholesterol-modified small interfering RNA sequence 2, and GalNac-modified small interfering RNA sequence; The sequences of the positive and antisense strands of the tail-modified small interfering RNA sequence are as follows: 5'- CCAGACAGAUUACAUUGUUUAdTdT -3'; 5'- UAAACAAUGUAAUCUGUCUGGdTdT -3'; The sequences of the positive and antisense strands of the cholesterol-modified small interfering RNA sequence 1 are as follows: 5'- Cm Cm Am GACAGAUUACAUUGU Um Um Am dTdT-Chol-3'; 5'-phos-Um Am Am ​​AC Am Af Uf Gm Uf Am A Um C Um G Um Cm Um Gm Gm dTdT-3'; The sequences of the positive and antisense strands of the cholesterol-modified small interfering RNA sequence 2 are as follows: 5'-Cm Cm Am GACAGAUUACAUUGU Um Um Am dTdT-Chol-3'; 5'-phos-Um Am Am ​​Af Cm Am Af Uf Gm Uf Am Af Um Cf Um Gf Um Cf Um Gm GmdTdT-3'; The sequences of the positive and antisense strands of the GalNac-modified small interfering RNA sequence are as follows: 5'- CCAGACAGAUUACAUUGUUUAdTdT -3'-GalNac3; 5'- UAAACAAUGUAAUCUGUCUGGdTdT -3'.

3. The application of a liver-targeted presentation inhibitory sequence combination as described in claim 1 or 2, characterized in that, The combination of inhibitory sequences is used to prepare drugs that inhibit FMO3 levels and suppress TMA-FMO3-TMAO axis-mediated bile acid metabolism and cholesterol transporters.

4. The application according to claim 3, characterized in that, The combination of the inhibitory sequences is used to prepare a drug for improving gallstones.

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