Application of siRNA compositions targeting LOC157273 in the preparation of drugs for treating non-alcoholic steatohepatitis and liver fibrosis

Abstract

This invention provides the application of a siRNA composition targeting LOC157273 in the preparation of drugs for treating non-alcoholic steatohepatitis (NASH) and liver fibrosis, belonging to the field of biomedical technology. The siRNA of this invention directly corrects the hyperlipidemic hepatic lipid synthesis, insulin resistance, and inflammatory activation caused by abnormal expression of PPP1R3B by specifically silencing the key fibrosis driver gene LOC157273. This fundamentally blocks the activation of hepatic stellate cells and excessive extracellular matrix deposition, achieving precise reversal of the fibrosis process and blocking the occurrence of NASH. The siRNA delivery system is optimized through liver-targeted modification, improving liver enrichment efficiency and reducing off-target effects caused by systemic distribution. It exhibits strong targeting, directly acting on upstream regulatory genes of fibrosis and NASH. Simultaneously, it silences multiple synergistic pathogenic gene sites, enhancing therapeutic efficacy and providing a highly efficient and safe novel intervention for liver fibrosis and NASH.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of a siRNA composition targeting LOC157273 in the preparation of a drug for treating non-alcoholic steatohepatitis and liver fibrosis. Background Technology

[0002] As the core regulatory organ for lipid metabolism, the liver is a key factor in the development of liver fibrosis due to the imbalance of lipid synthesis, breakdown, and storage homeostasis. When lipid intake or synthesis is excessive, triglycerides and free fatty acids accumulate abnormally in hepatocytes, leading to fatty liver. Excessive free fatty acids induce lipotoxicity through reactive oxygen species bursts and lipid peroxidation, resulting in hepatocyte damage and apoptosis. Signaling molecules released by damaged cells activate intrahepatic inflammatory responses, prompting Kupffer cells and other cells to release pro-inflammatory factors. The persistent inflammatory microenvironment further activates hepatic stellate cells, transforming them into myofibroblasts and synthesizing large amounts of extracellular matrix, ultimately leading to fibrosis due to excessive matrix deposition.

[0003] Nonalcoholic fatty liver disease (NAFLD) is a chronic liver disease characterized by fatty accumulation in the liver (hepatic steatosis), excluding alcohol and other secondary factors. Its typical course is progressive: from steatosis to nonalcoholic steatohepatitis (NASH) with inflammation, eventually leading to fibrosis or cirrhosis. End-stage patients die from liver failure, hepatocellular carcinoma (HCC), or require a liver transplant. Public data shows that NAFLD affects nearly 30% of adults worldwide, with approximately 25% progressing to NASH, resulting in a NASH prevalence of about 6% in the general population. The disease is highly prevalent in patients with abdominal obesity, insulin resistance / type 2 diabetes, hypertension, and dyslipidemia (i.e., "metabolic syndrome"), and increases cardiovascular risk.

[0004] Current treatments for NASH and liver fibrosis have significant limitations: First, they lack specific target targeting. Most drugs (such as some anti-inflammatory drugs and antioxidants) only target downstream pathways of inflammation or fibrosis, failing to precisely intervene in the core drivers of disease progression, resulting in limited efficacy and high relapse rates. Second, they lack specificity in addressing the underlying causes. Existing therapies focus on symptom relief (such as lowering transaminase levels and suppressing inflammation), lacking direct intervention in key triggers of NASH and fibrosis (such as liver metabolic disorders and abnormal regulation of specific genes), making it difficult to block the progression of the disease from hepatic steatosis to NASH and fibrosis. Third, there are safety and tolerability issues. Some drugs (such as certain immunosuppressants) have broad effects and may interfere with normal liver function or cause systemic adverse reactions, limiting long-term use. Fourth, they neglect the PPP1R3B-related mechanisms. Current technologies have not yet precisely regulated PPP1R3B, a key gene closely related to hepatic steatosis, inflammation, and fibrosis, failing to inhibit its abnormal expression and the resulting metabolic disorders and pathological processes at the source. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide an application of a siRNA composition targeting LOC157273 in the preparation of a drug for treating non-alcoholic steatohepatitis (NASH) and liver fibrosis. This composition specifically silences the key fibrosis driver gene LOC157273, directly correcting the hyperlipidemic hepatic lipid synthesis, insulin resistance, and inflammatory activation caused by abnormal PPP1R3B expression, thereby blocking the occurrence of NASH and the progression of fibrosis at the etiological level. Simultaneously, due to the high specificity of siRNA, interference with normal hepatocyte function is reduced, systemic side effects are decreased, and the safety and tolerability of the treatment are improved. Ultimately, this overcomes the shortcomings of existing technologies, such as vague target targeting and insufficient etiological intervention, providing a more efficient, precise, and safe treatment option for NASH and liver fibrosis.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides the use of siRNA compositions, chemically modified siRNA compositions, or pharmaceutical conjugates containing siRNA compositions in the preparation of medicaments for treating liver diseases, including non-alcoholic steatohepatitis or liver fibrosis.

[0008] Preferably, the siRNA composition comprises siRNA12, siRNA2+siRNA4, siRNA2+siRNA3+siRNA4 or siRNA2+siRNA3+siRNA4+siRNA8;

[0009] The siRNA2 comprises a sense strand as shown in SEQ ID No. 72 and an antisense strand as shown in SEQ ID No. 73.

[0010] The siRNA3 comprises a sense strand as shown in SEQ ID No. 74 and an antisense strand as shown in SEQ ID No. 75.

[0011] The siRNA4 comprises a sense strand as shown in SEQ ID No. 76 and an antisense strand as shown in SEQ ID No. 77.

[0012] The siRNA8 comprises a sense strand as shown in SEQ ID No. 84 and an antisense strand as shown in SEQ ID No. 85.

[0013] The siRNA12 includes a sense strand as shown in SEQ ID No. 92 and an antisense strand as shown in SEQ ID No. 93.

[0014] Preferably, the chemical modification method includes: changing the non-linked phosphate oxygen, replacing the non-linked phosphate oxygen, changing the linked phosphate oxygen, replacing the linked phosphate oxygen, replacing the phosphate moiety with a "dephosphorylated" linker, modifying the naturally occurring base, replacing the naturally occurring base, replacing the ribose phosphate backbone, and modifying one or more of the following:

[0015] Preferably, the chemical modification method includes, in the direction from the 5' end to the 3' end:

[0016] The nucleotides at positions 2 and 14 of the sense strand are modified with one or two of the following: 2'-fluoro modification, 4'-thio modification, 2'-fluoroarabinonucleotide, and a noncyclic nucleotide analog; and the antisense strand is modified with 2'-methoxyethoxy.

[0017] or

[0018] The sense strand is modified with 2'-methoxyethoxy, and the nucleotides at positions 2 and 14 of the antisense strand are modified with one or two of the following: 2'-fluoro, 4'-thio, 2'-fluoroarabinonucleotide, and a noncyclic nucleotide analog.

[0019] Preferably, the drug conjugate containing the siRNA combination further includes a ligand.

[0020] Preferably, the ligand is attached to the 3' end of the positive strand of the siRNA modifier via a linker group, and the ligand is GalNAc.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The siRNA of this invention directly corrects the hepatic lipogenesis, insulin resistance, and inflammation activation caused by abnormal expression of PPP1R3B by specifically silencing the key fibrosis driver gene LOC157273. This fundamentally blocks hepatic stellate cell activation and excessive extracellular matrix deposition, achieving precise reversal of the fibrosis process and blocking NASH. Simultaneously, by optimizing the siRNA delivery system (e.g., liver-targeted modification), its enrichment efficiency in the liver is improved, reducing off-target effects caused by systemic distribution. Its core features include: strong targeting, directly acting on upstream regulatory genes of fibrosis and NASH, avoiding the limitations of downstream molecular intervention; high local enrichment, reducing the impact on normal tissues; and the ability to simultaneously silence multiple synergistic pathogenic genes, enhancing therapeutic efficacy and providing a highly efficient and safe novel intervention for liver fibrosis and NASH. Attached Figure Description

[0023] Figure 1 Image showing the HE staining results of liver tissue sections from NC group cynomolgus monkeys;

[0024] Figure 2 Image showing the HE staining results of liver tissue sections from cynomolgus monkeys in the positive control group;

[0025] Figure 3 Image showing the HE staining results of liver tissue sections from experimental group 4 cynomolgus monkeys;

[0026] Figure 4 Image showing the HE staining results of liver tissue sections from experimental group 3 cynomolgus monkeys;

[0027] Figure 5 Image showing the HE staining results of liver tissue sections from experimental group 2 cynomolgus monkeys;

[0028] Figure 6 Image showing the HE staining results of liver tissue sections from experimental group 1 cynomolgus monkeys;

[0029] Figure 7 The changes in MRI-PDFF values ​​in different groups of cynomolgus monkeys. Detailed Implementation

[0030] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0031] Example 1

[0032] The innovative nucleic acid structure involved in this invention is a key tool for precisely inhibiting the expression of target genes within cells. This nucleic acid possesses at least one double-stranded region, which is composed of a partial fragment of the first strand and a partially complementary fragment of the second strand. The first strand is complementary to a partial fragment of RNA transcribed from the target gene, and the first strand contains modified or unmodified nucleotides at multiple locations. This specific arrangement of nucleotides effectively promotes the processing of nucleic acids by the RNA-induced silencing complex (RISC).

[0033] The target siRNA is selected based on GC content greater than 30% and less than 50%, Tm = 21.5℃, avoiding siRNAs with continuous GC and AT values ​​above 4, and silencing efficiency greater than 80%.

[0034] The expression levels of target genes after siRNA administration were detected in HepG2 cells to comprehensively evaluate the potential of siRNA as a therapeutic agent for liver fibrosis and to select the optimal target siRNA. Cells were seeded at a density of 150,000 cells per 6-well plate. After 24 hours, cells were transfected with 0.1 nM siRNA and 1 μg / ml lipo8000, and lysed after 48 hours. Total RNA was extracted, and gene expression levels were determined using TaqMan qRT-PCR. The associated genes and primers used for LOC157273 are shown in Table 1.

[0035] Table 1. Associated genes and primers used for LOC157273.

[0036]

[0037] Example 2

[0038] Synthesizing siRNA: After obtaining the gene sequence of LOC157273 from the Genebank database, 21bp siRNAs meeting the criteria were screened using R language. siRNAs with a GC content of 50% were further selected. The maximum Tm for seed double-strand stability was set at 21.51℃, and siRNAs meeting the criteria were further optimized. Simultaneously, siRNA sequences were chemically synthesized, and two dTdT dangling ends were added to the 3' end to enhance the stability of the siRNA.

[0039] Regarding the details of nucleotide modification, each group of siRNAs uses GalNAc as the delivery system. There are special requirements for the 2nd and 14th nucleotides at the 5′ end of the first strand: the 2nd and 14th nucleotides at the 5′ end of the first strand can be modified with one or two of the following: 2′F, 4′-S, 2′-FANA and UNA; while the 2nd and 14th nucleotides at the 5′ end of the second strand can be modified with 2′O-MOE.

[0040] Experiments were conducted in HepG2 cells to detect the silencing effect of a single siRNA on LOC157273 at the cellular level and to detect associated genes. Target siRNA combinations were further optimized. Detailed sequences of the siRNAs obtained from the cell experiments are shown in Table 2.

[0041] Table 2. Results of single siRNA cell screening experiments

[0042]

[0043]

[0044]

[0045]

[0046]

[0047]

[0048]

[0049] Example 3

[0050] At the cellular level, siRNAs were combined in pairs, groups of three, and groups of four to form combination drugs. The silencing efficiency of various combinations was verified using HepG2 cells. Some of the combinations are shown in Tables 3, 4, and 5.

[0051] Table 3. Results of cell experiments screening for pairwise combinations of siRNA

[0052]

[0053]

[0054]

[0055] Table 4. Results of siRNA Triple Combination Cell Screening Experiment

[0056]

[0057]

[0058] Table 5. Results of siRNA-based cell combination assays.

[0059]

[0060]

[0061]

[0062] Example 4

[0063] Based on the target silencing efficiency and the increased expression level of PPP1R3B, one combination from each approach was selected for in vivo large animal experiments. When selecting based on silencing efficiency, duplicate wells were used to ensure the reliability of results in cell experiments, and the final silencing efficiency data is expressed as the average of the duplicates. In the 3+3 combination, although the average silencing efficiency of 1+2+7 and 2+3+4 was the same, the data difference between each duplicate of 2+3+4 was smaller, indicating better data stability; therefore, 2+3+4 was selected. In the 4+4 combination, although the silencing efficiency of 2+3+4+12 and 3+4+5+18 was both 0.9, the data difference between duplicates of 3+4+5+18 was significant; therefore, 3+4+5+18 was excluded, and 2+3+4+8 was selected for subsequent experiments. The final selected combinations were siRNA12, siRNA2+siRNA4, siRNA2+siRNA3+siRNA4, or siRNA2+siRNA3+siRNA4+siRNA8.

[0064] The double-stranded RNAi agent used to reduce the expression of target genes in live large animals (cynomolgus monkey experiments) comprises an oligonucleotide duplex consisting of paired sense and antisense strands. The siRNA sequence is chemically synthesized, and the siRNA is linked to a GalNAc triplet (purchased from Guangzhou Ruibo Biotechnology Co., Ltd.). The 2nd and 14th nucleotides at the 5′ end of the sense strand are modified with 2′F; while the 2nd and 14th nucleotides at the 5′ end of the antisense strand are modified with 2′O-MOE to increase stability. The specific sequences of siRNA2, siRNA3, siRNA4, siRNA8, and siRNA12, as well as their modified sequences, are shown in Table 6 below.

[0065] Table 6 shows the specific sequences of siRNA2, siRNA3, siRNA4, siRNA8, and siRNA12, as well as their modified sequences.

[0066]

[0067]

[0068] Liver fibrosis is not a disease state independent of non-alcoholic steatohepatitis (NASH), but rather an important pathological marker of NASH progression to the intermediate or late stage. Therefore, this invention uses cynomolgus monkeys with pre-existing liver fibrosis (i.e., an intermediate or late-stage NASH model) as experimental subjects.

[0069] Using cynomolgus monkeys with a predisposition to obesity and fatty liver as the baseline model, a long-term high-calorie diet was induced in them for 6 to 24 months. The diet formulation included: high fat (providing 45%–60% of calories, mainly saturated fatty acids), high sugar (providing 20%–30% of calories, containing fructose, etc.), and high cholesterol (0.5%–2%). Feeding was done by daily fixed-amount feeding based on body weight (3%–5% of body weight), supplemented with fructose-containing drinking water. During the induction period, biochemical indicators such as body weight, blood glucose, blood lipid profile, and liver function were monitored regularly. The model was systematically validated through imaging examinations such as ultrasound and magnetic resonance proton density fat fraction (MRI-PDFF), combined with liver biopsy (to assess hepatic steatosis, inflammatory activity, and fibrosis).

[0070] 1. The above-mentioned combined small nucleic acid drug was administered to a cynomolgus monkey model via a single subcutaneous injection (10 mg / kg) on ​​Day 0, with no subsequent adjuvant administration. Liver tissue samples were obtained by liver biopsy on day 30 (D30). The samples were sectioned and stained with hematoxylin and eosin (HE) to assess the histopathological changes in the target indication (NASH with liver fibrosis).

[0071] 2. The above-mentioned combined small nucleic acid drug was administered to a cynomolgus monkey model via a single subcutaneous injection at a dose of 10 mg / kg. Day 0 was defined as the day of administration, with no subsequent booster doses. MRI-PDFF was performed on day 30. The MRI-PDFF detection method is as follows:

[0072] (1) Preoperative fasting: Fasting for 12 hours before the scan (you can drink water freely).

[0073] (2) Animal condition assessment: Check the weight and mental state of the crab-eating macaques to rule out acute diseases such as fever and diarrhea.

[0074] (3) Body surface preparation: Remove the hair on the monkey's body surface (especially the chest and abdomen, which can be gently treated with hair removal cream) to avoid hair interfering with radio frequency signals; check for any metallic foreign objects (such as implants or markers) on the body surface to prevent artifacts.

[0075] (4) Coil selection: Select a phased array coil for the body of the cynomolgus monkey or a flexible surface coil.

[0076] (5) Anesthesia: Intramuscular injection of ketamine (10mg / kg) + diazepam (0.5mg / kg), and after the animal is sedated, it is moved to the scanning bed.

[0077] (6) Maintenance anesthesia: Inhalation of isoflurane (wearing an animal-specific air anesthesia mask), maintained by an animal-specific anesthesia machine, with respiratory rate and blood oxygen saturation monitored throughout.

[0078] (7) Position fixation: Supine position, use a restraint strap to fix the limbs (to avoid limb movement), place a soft pillow under the abdomen (to keep the liver in a relatively horizontal position and reduce the influence of respiratory movements on the liver position); connect a respiratory gating sensor (attached to the chest and abdomen) for synchronous scanning to reduce respiratory artifacts.

[0079] (8) Parameter selection:

[0080] Sequence type: Multi-echo gradient echo, with at least 8 consecutive echoes;

[0081] Time: First echo time 1.2-2ms, interval echo time 1.0-1.5ms;

[0082] Flip angle: 5-10°.

[0083] (9) Calculation of change:

[0084] Absolute change: ΔPDFF = Post-test PDFF value - Pre-test PDFF value (negative value indicates fat reduction);

[0085] Relative change rate: (ΔPDFF / pre-tested PDFF value) × 100% (clinically, a relative reduction of ≥30% is often used as an indicator of effective treatment).

[0086] The specific groupings are shown in Table 7.

[0087] Table 7 Experimental Groups for Crab-Eating Macaques

[0088]

[0089] Experimental results: such as Figures 1-7 As shown, each siRNA combination has a certain therapeutic effect on NASH and liver fibrosis indicators, and multiple combination preparations can simultaneously have a long-term and stable inhibitory effect on various indicators.

[0090] As demonstrated by the above embodiments, the siRNA of this invention directly corrects the hepatic lipogenesis hypersynthesis, insulin resistance, and inflammation activation caused by abnormal expression of PPP1R3B by specifically silencing the key fibrosis driver gene LOC157273. This fundamentally blocks hepatic stellate cell activation and excessive extracellular matrix deposition, achieving precise reversal of the fibrosis process and blocking the occurrence of NASH. Simultaneously, by optimizing the siRNA delivery system (such as liver-targeted modification), its enrichment efficiency in the liver is improved, reducing off-target effects caused by systemic distribution. Its core features include: strong targeting, directly acting on upstream regulatory genes of fibrosis and NASH, avoiding the limitations of downstream molecular intervention; high local enrichment, reducing the impact on normal tissues; and the ability to simultaneously silence multiple synergistic pathogenic genes, enhancing therapeutic efficacy and providing a highly efficient and safe novel intervention strategy for liver fibrosis and NASH.

[0091] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The use of siRNA compositions or chemically modified siRNA compositions in the preparation of drugs for treating liver diseases, characterized in that, The liver disease described is non-alcoholic steatohepatitis; The siRNA composition includes siRNA12, siRNA2+siRNA4, siRNA2+siRNA3+siRNA4 or siRNA2+siRNA3+siRNA4+siRNA8; The siRNA2 consists of a sense strand as shown in SEQ ID No. 72 and an antisense strand as shown in SEQ ID No.

73. The siRNA3 consists of a sense strand as shown in SEQ ID No. 74 and an antisense strand as shown in SEQ ID No.

75. The siRNA4 consists of a sense strand as shown in SEQ ID No. 76 and an antisense strand as shown in SEQ ID No.

77. The siRNA8 consists of a sense strand as shown in SEQ ID No. 84 and an antisense strand as shown in SEQ ID No.

85. The siRNA12 consists of a sense strand as shown in SEQ ID No. 92 and an antisense strand as shown in SEQ ID No. 93; The chemical modification method is performed along the direction from the 5' end to the 3' end: The nucleotides at positions 2 and 14 of the sense strand are modified with one or two of 2'-fluoro, 4'-thio, and 2'-fluoroarabinonucleotide, and the antisense strand is modified with 2'-methoxyethoxy. or The sense strand is modified with 2'-methoxyethoxy, and the nucleotides at positions 2 and 14 of the antisense strand are modified with one or two of 2'-fluoro, 4'-thio, and 2'-fluoroarabinonucleotide.

2. The application according to claim 1, characterized in that, The non-alcoholic steatohepatitis mentioned refers to non-alcoholic steatohepatitis with liver fibrosis.

3. The application according to claim 1, characterized in that, The siRNA composition modified by the chemical modification method also includes GalNAc.

Images