SiRNA of targeted FASN gene, carrier compound and application of siRNA and carrier compound

Abstract

The invention provides siRNA of a targeted FASN gene, a compound and application of the siRNA and the compound, and belongs to the technical field of gene therapy drugs. A sense strand and an antisense strand of the siRNA of the targeted FASN gene have sequences as shown in SEQ ID No.1 and SEQ ID No.2; the sequences are as shown in SEQ ID No.3 and SEQ ID No.4, or the sequences are as shown in SEQ ID No.3 and SEQ ID No.4; the sequences are shown as EQ ID No.5 and SEQ ID No.6, or the sequences are shown as EQ ID No.5 and SEQ ID No.6; the siRNA can inhibit expression of FASN genes and reduce protein abundance of FASN in the liver, so that deposition of liver fat is reduced, lipid toxicity and inflammatory response are reduced, and accurate treatment of MAFLD is achieved. The siRNA provided by the invention is chemically modified, so that the stability of the siRNA is improved.

Description

[0001] Cross-references

[0002] This application claims priority to Chinese patent application CN202410350602.3 filed on March 26, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present invention belongs to the technical field of gene therapy drugs, and in particular relates to a siRNA targeting the FASN gene, a complex and applications thereof. Background Art

[0004] Metabolic-associated fatty liver disease (MAFLD) is the most common form of chronic liver disease worldwide. It is a general term for a range of liver diseases, including steatosis, non-alcoholic steatohepatitis, cirrhosis, and ultimately hepatocellular carcinoma. MAFLD is a complex systemic metabolic disease influenced by multiple factors, including genetics and behavioral habits, and involving multiple pathways. Ectopic lipid deposition and insulin resistance are recognized as the two main causes, but the specific pathogenesis remains unclear.

[0005] Current treatments include lifestyle interventions, bariatric surgery, and medications. Targeting abnormal fatty acid metabolism to prevent liver fat accumulation and the development of a profibrotic environment may be a promising therapeutic strategy. However, the metabolic pathways involved in MAFLD are complex, and precise treatment is currently difficult to achieve given the unclear pathogenesis of MAFLD. Summary of the Invention

[0006] In view of this, the object of the present invention is to provide a siRNA targeting the FASN gene, a complex and its application. The siRNA targeting the FASN gene is chemically modified to improve the stability of the siRNA in vivo while maintaining the biological activity of the siRNA, thereby providing a new therapeutic approach for the treatment of MAFDL disease.

[0007] In a first aspect, the present invention provides an siRNA targeting the FASN gene, wherein the siRNA can inhibit the expression of the FASN gene.

[0008] In one embodiment, the sense strand and antisense strand of the siRNA have the sequences shown in SEQ ID No. 1 and SEQ ID No. 2; or have the sequences shown in SEQ ID No. 3 and SEQ ID No. 4; or have the sequences shown in SEQ ID No. 5 and SEQ ID No. 6.

[0009] Preferably, the sense strand and the antisense strand of the siRNA further have a tail of 2 to 3 nucleotides.

[0010] More preferably, the sense strand of the siRNA is modified as follows:

[0011] All nucleotides are PS modified, further, the 1st to 4th nucleotides at the 5' end are 2'ome modified, further, the 3rd to 6th nucleotides at the 3' end are 2'ome modified; further, two 2'deoxythymines are connected to the 3' end;

[0012] The antisense strand of the siRNA was modified as follows:

[0013] All nucleotides were PS modified, and further, the 6th and 7th nucleotides at the 5' end were GNA modified, and two 2' deoxythymines were connected to the 3' end.

[0014] The second aspect of the present invention provides a vector complex targeting the FASN gene, wherein the vector complex is a complex formed by connecting GalNAc to the 3' end of the sense strand of the siRNA.

[0015] The third aspect of the present invention provides use of the siRNA or carrier complex in preparing an agent for inhibiting FASN gene expression.

[0016] A fourth aspect of the present invention provides use of the siRNA or carrier complex in the preparation of a medicament for preventing and / or treating metabolism-related fatty liver disease.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] 1. The siRNA targeting the FASN gene provided by the present invention can inhibit the expression of the FASN gene and reduce the protein abundance of FASN in the liver, thereby reducing the deposition of liver fat, reducing lipotoxicity and inflammatory response, and thus achieving precise treatment for metabolic-associated fatty liver disease (MAFLD).

[0019] 2. The siRNA provided by the present invention is chemically modified, which improves the stability of the siRNA during delivery.

[0020] 3. The vector complex targeting the FASN gene provided by the present invention is different from traditional small molecule drugs. It has more precise targeting, can exert its effects continuously, and can achieve long-term lipid-lowering effects. It also has a single metabolic pathway and can be excreted through the kidneys, reducing the burden on the liver. Traditional small molecule drugs are mostly metabolized in the liver, which can cause severe liver damage. In addition, the metabolic dysfunction of MAFLD patients themselves will impose a certain burden on the disease. The vector complex provided by the present invention reduces the occurrence of the above-mentioned side effects.

[0021] 4. The carrier complex provided by the present invention is administered subcutaneously, which provides long-term inhibition and reduces the frequency of medication for patients, bringing patients a more convenient and effective treatment experience. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The effect of siRNA silencing the FASN gene in Example 1 is shown in the left figure, which shows human hepatocytes C3A and the right figure shows human hepatic stellate cells LX2. The upper parts of the left and right figures show the WB results, and the lower parts show the QPCR results.

[0023] Figure 2 To determine the effect of siRNA silencing the FASN gene and the IC50 value, the point where the two broken lines intersect with the red dotted line is the IC50 value;

[0024] siRNA-1 is the result of naked-strand FASN siRNA-289 transfection in C3A cells, and siRNA-2 is the result of naked-strand FASN siRNA-289 transfection in LX2 cells;

[0025] Figure 3 Schematic diagram of siRNA modification scheme;

[0026] Figure 4 To compare the IC50 values ​​of FASN gene silencing by modified and naked siRNA-289, C3A and LX2 cells were transfected with naked and modified siRNAs, respectively, and grouped into C3A+naked (siRNA-1), C3A+modified (siRNA-1P); LX2+naked (siRNA-2), LX2+modified (siRNA-2P). The modified siRNA significantly improved the silencing efficiency.

[0027] Figure 5 The CCK8 method was used to detect the effects of modified FASN siRNA-289 at different concentrations on cell viability. Cell refers to cells without any treatment, INTERFERin refers to cells only receiving transfection reagent, negative control refers to cells receiving siRNA that has no effect on the GADPH gene, and positive control refers to cells receiving siRNA targeting the GADPH gene. 0 nm, 1 nm, 10 nm, 100 nm, and 1 μM refer to cells receiving modified FASN siRNA-289 at different concentrations.

[0028] Figure 6To test the effect of FASN siRNA-289 on cell apoptosis, the figure shows no significant increase in the apoptosis rates of C3A and LX2 cells (C3A early apoptosis rate 7.12%, LX2 early apoptosis rate 5.24%), indicating that FASN siRNA-289 has no significant toxicity to cells and is relatively safe.

[0029] Figure 7 To introduce the naked sequence of FASN siRNA-289 into human liver cells (C3A) for FASN gene silencing, the results of Oil Red staining of C3A in different treatment groups;

[0030] Figure 8 Preparation and validation of NAFLD liver organoid model: Figure 8 A is a schematic diagram of IPS-induced differentiation of liver organoids. Figure 8 B shows the morphology of human liver organoids observed under bright field. Figure 8 C is the immunofluorescence examination of liver organoid-specific maker. Figure 8 D shows the construction of NAFLD liver organoid model and the lipid deposition in organoids after treatment. Figure 8 E shows the expression of FASN in the NAFLD liver organoid model. Figure 8 F represents the palmitic acid content in organoids of different groups. ***P<0.01.

[0031] Figure 9 Schematic diagram of the structure of the carrier complex;

[0032] Figure 10 The silencing efficiency of the FASN gene at different times after injection of different concentrations of vector complexes. DETAILED DESCRIPTION

[0033] The preferred embodiments of the present invention are described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0034] In the following description, the sequence name, number and sequence information of the siRNA are specifically shown in Table 1.

[0035] Table 1 siRNA sequences

[0036]

[0037] In the following description, ome represents 2'-O-methylation; * represents replacement of a sulfate bond with a phosphorothioate (PS) bond; GNA stands for Glycerol nucleic acid; and idT stands for 2'-deoxythymidine.

[0038] Example 1 siRNA sequence design

[0039] Based on the sequence of the FASN gene, multiple siRNAs were designed, and the following three siRNAs were selected. The specific sequences are as follows:

[0040] First: FASN siRNA-289 (FASN1):

[0041] UGUUCC UCGGAGU GAAUC U GG (antisense strand)

[0042] CCAGAU UCAC UCC GAGGAACA (sense strand)

[0043] Key points of this sequence design:

[0044] 1. Binding target is in the CDS region of FASN gene

[0045] 2. The first base at the 5' end of the SS chain is G or C

[0046] 3. The first base at the 5' end of the AS chain is A or U

[0047] 4. The SS chain base preference is close to the silence principle (A at position 19, C at position 10, and C at position 13)

[0048] 5. The sequence does not form a hairpin or palindrome structure

[0049] 6. Does not contain any form of three-base combination repeats

[0050] 7. The AS chain base preference meets the requirements (the 6th position is C)

[0051] 8. Bind to target positions 289...309.

[0052] Article 2: FASN siRNA-5309 (FASN2):

[0053] UCUGAGAAAG GUC GAAUUU GC (sense strand)

[0054] GCAAAU UCGACCU UUCUCAGA (antisense strand)

[0055] Key points of this sequence design:

[0056] 1. Binding target is in the CDS region of FASN gene

[0057] 2. The first base at the 5' end of the SS chain is G or C

[0058] 3. The first base at the 5' end of the AS chain is A or U

[0059] 4. The SS chain base preference is close to the silence principle (A at position 19, U at position 10, and U at position 13)

[0060] 5. The sequence does not form a hairpin or palindrome structure

[0061] 6. Repeats containing three base combinations

[0062] 7. The AS chain base preference meets the requirements (the 6th position is G)

[0063] 8. Binds to target positions 5309...5329 (closer to the 3' end).

[0064] Article 3: FASN siRNA-749 (FASN3):

[0065] GCACCAAUACAGAUGGCUU TT (sense strand)

[0066] AAGCCAUCUG UAU UGGUGC TT (antisense strand)

[0067] Key points of this sequence design:

[0068] 1. Binding target is in the CDS region of FASN gene

[0069] 2. The first base at the 5' end of the SS chain is G or C

[0070] 3. The first base at the 5' end of the AS chain is A or U

[0071] 4. Insufficient SS base preference (U at position 19, C at position 10, A at position 13)

[0072] 5. Sequence structure forms a hairpin

[0073] 6. Does not contain any form of three-base combination repeats

[0074] 7. Insufficient base preference of AS chain (A at position 6)

[0075] 8. Bind to target positions 749...767.

[0076] Comprehensive analysis revealed that the optimized FASN siRNA-289 sequence has the advantage of meeting the requirements for silencing efficiency and safety. However, compared with the optimized FASN siRNA-5309 sequence, its target location is further from the 3' end of the target gene. The optimized FASN siRNA-5309 sequence has the advantage of being closer to the 3' end of the target gene (potentially improving silencing efficiency), but the sequence contains three consecutive base repeats (AAA / UUU), which may terminate RNA Polymerase III-mediated transcription. Therefore, further parallel screening experiments were conducted on two siRNA pairs, FASN siRNA-289 and FASN-siRNA-749:

[0077] Sequence modification of FASN-siRNA-289

[0078] The modified sequence of FASN siRNA-289 is as follows:

[0079] Sense strand:

[0080] FASN siRNA-289-ss:

[0081] (ome-C)*(ome-C)*(ome-A)*(ome-G)*A*U*U*C*A*C*U*C*C*G*A*G*G*(ome-A)*(ome-A)*(ome-C)*(ome-A)* / idT / * / idT / *

[0082] FASN siRNA-289-as:

[0083] U*G*U*U*C* / GNA-C* / GNA-U*C*G*G*A*G*U*G*A*A*U*C*U*G*G* / id T / * / idT / *;

[0084] Among them, ome represents 2'-O-methyl modification; * represents the replacement of sulfate bond with phosphorothioate (PS) bond; GNA stands for Glycerol nucleic acid, and idT stands for 2' deoxythymidine.

[0085] Sequence modification of FASN-siRNA-749

[0086] The modified sequence of FASN siRNA-749 is as follows:

[0087] (ome-G)*(ome-C)*(ome-A)*(ome-C)*C*A*A*U*A*C*A*G*A*U*G*(ome-G)*(ome-C)*(ome-U)*(ome-U)*idT*idT*

[0088] A*A*G*C*C* / GNA-A / * / GNA-U / *C*U*G*U*A*U*U*G*G*U*G*C*idT*idT*

[0089] A hairpin structure was formed on the modified FASN siRNA-749 sequence, which in turn reduced the effective concentration and silencing efficiency of its siRNA. Therefore, the sequence with the best effect was finally determined to be FASN siRNA-289.

[0090] Example 2 Expression of siRNA in human liver cells and hepatic stellate cells

[0091] The three designed naked siRNA sequences were introduced into human liver cells C3A and human hepatic stellate cells LX2 to silence the FASN gene.

[0092] Human liver cells (C3A) and human hepatic stellate cells (LX2) were cultured at 37°C in 5% CO2. siRNA transfection was performed using INTERFERin transfection reagent according to the manufacturer's instructions. Six groups were set up: specific siRNA groups (siRNAs targeting C3A cells (1; 2; 3) and LX2 cells (1; 2; 3)) and a nonspecific siRNA group (negative control).

[0093] Prepare the transfection reagent: Add 400 μL of nuclease-free water to a tube and vortex for 10 seconds to dissolve the lipids. Store the reagent at -20°C after vortexing and vortex again before use. Select an appropriate mixing ratio (1:1-1:2 / liposome volume: siRNA mass) for transfection of cells. Add an appropriate volume of serum-free medium to a transfection tube. Add 10 nM / ml siRNA, vortex, then add 16 μL of transfection reagent and vortex again. Incubate the mixture at room temperature for 10-15 minutes. Aspirate the medium from the culture plate and wash once with PBS or serum-free medium. Add the mixture and return C3A and LX2 cells to 70-80% confluency in the incubator for one hour. Then, add complete medium and continue culturing for 24-48 hours. RNA extraction and quantitative real-time PCR (qPCR): 48 hours after transfection, extract total RNA using an RNA extraction kit and reverse transcribe the RNA into cDNA using a reverse transcription reagent. Specific primers were used to quantitatively analyze the mRNA expression level of the target gene by qPCR technology. This was done to verify the protein-level changes in the siRNA silencing effect. In addition, in order to confirm the siRNA silencing efficiency, a gradient of siRNA concentrations (1e-8, 1e-7, 1e-6, 1e-5, 1e-4) was set to calculate the IC50 to select the optimal concentration for intervention of C3A and LX2. Figure 2 , Figure 2siRNA1 is FASN siRNA-289, and siRNA2 is FASN siRNA-749. The point where the two broken lines intersect with the red dotted line is the IC50 value. According to the experimental requirements, a concentration of 1e-4 was selected for intervention.

[0094] The concentration gradients of FASN siRNA-289 and FASN siRNA-749 were compared, with 1×10 -8 , 1×10 -7 , 1×10 -6 , 1×10 -5 , 1×10 -4 The concentration of moles / well was used to observe the silencing efficiency of the two siRNAs under different concentration gradients.

[0095] The results are as follows Figure 4 As shown, siRNA-1 shows the results of naked FASN siRNA-289 transfection in C3A cells, and siRNA-1p shows the results of modified FASN siRNA-289 transfection in C3A cells; siRNA-2 shows the results of naked FASN siRNA-289 transfection in LX2 cells, and siRNA-2P shows the results of modified FASN siRNA-289 transfection in LX2 cells. As the concentration gradually increased, the silencing efficiency of FASN siRNA-289 and FASN siRNA-749 differed significantly, with FASN siRNA-289 having a higher silencing efficiency. The trends before and after modification were consistent, with the modified siRNA having a higher silencing efficiency.

[0096] Furthermore, we studied the effect of siRNA on lipid deposition in liver cells by using a high glucose environment. The naked sequence of FASN siRNA-289 was introduced into human liver cells (C3A), and a high glucose group and a high glucose + FASN-siRNA-289 group ( Figure 7 The high glucose + FASN1 group was compared with the C3A cells, and the cells were divided into groups and stained with Oil Red O. The fat deposition was then observed under a microscope. The results are as follows Figure 7 As shown, transfection of FASN siRNA-289 into C3A cells cultured in high glucose reduced lipid deposition.

[0097] To evaluate the safety of FASN siRNA-289, we used CCK-8 to detect the effect of modified FASN siRNA-289 on cell viability ( Figure 5 ). The cells are grouped as follows:

[0098] cell is a cell that does not undergo any treatment.

[0099] INTERFERin is to add transfection reagent only to cells.

[0100] Negative control (negative control) is to add universal negative siRNA (NC-SUUCUCCGAACGUGUCACGUTT (SEQ ID NO.7); NC-AACGUGACACGUUCGGAGAATT (SEQ ID NO.8)) to the cells.

[0101] Positive control: siRNA targeting the endogenous gene GADPH (human-GADPH-S: GUAUGACAACAGCCUCAAGTT (SEQ ID NO. 9); human-GADPH-A: CUUGAGGCUGUUGUCAUACTT (SEQ ID NO. 10)) was added to the cells.

[0102] 0 nm, 1 nm, 10 nm, 100 nm, and 1 μm refer to the addition of different concentrations of modified FASN siRNA-289 to cells.

[0103] The results showed that the modified FASN siRNA-289 had good safety in both C3A and LX2 cells.

[0104] Flow cytometry was used to detect the effect of FASN siRNA-289 on cell apoptosis. Figure 6 As shown, the apoptosis rates of C3A and LX2 cells did not increase significantly (the early apoptosis rate of C3A was 7.12%, and the early apoptosis rate of LX2 was 5.24%), indicating that FASN siRNA-289 had no obvious toxicity to cells.

[0105] Example 3 Verification of the therapeutic effect of FASN siRNA-289 using an organoid model

[0106] Liver organoids were constructed using induced pluripotent stem cells (iPSCs). iPSCs were provided by the Research Institute Co., Ltd. and cultured in cell culture medium under standard conditions. The induced differentiation of liver organoids was carried out using a reported differentiation protocol, which specifically involves inducing the differentiation of iPSCs into liver progenitor cells by adding specific growth factors (such as HGF, FGF4, Oncostatin M, etc.), and further maturation into liver organoids. Cell morphology, proliferation capacity, and expression of liver function markers were regularly evaluated during the differentiation process to confirm the successful construction of organoids.

[0107] The induced differentiation of human liver organoids first starts with the cultivation of iPSCs, using mTeSR1 culture medium to maintain their self-renewal ability. After the cells reach a confluence of 60-80%, inhibitors such as LIF are removed, and the cells are transferred to a flat culture dish. Differentiation medium containing liver induction factors, such as BMP4, Wnt3a and HGF, is added to begin inducing differentiation. At this time, iPSCs differentiate into the germ layer to form liver precursor cells. After 2-3 days of germ layer induction, factors such as HGF, FGF and BMP4 are continued to be added to promote the maturation of liver precursors and stimulate the formation of liver-like cells. In the next 6-7 days, these liver precursor cells are transferred to a three-dimensional culture system (polymer hydrogel) and the formation and development of organoids are further promoted by culturing growth factors such as HGF and EGF. Through this process, organoids with liver characteristics can be successfully induced, such as Figure 8 AC shows that it provides a good model for further liver research or drug screening.

[0108] The experiment used 5mg / mL glucose + 10mM Glucosamine for 4 days to obtain a fatty liver organoid model. During the model induction process, different concentrations of FASN siRNA-289 were administered to observe lipid deposition. The results showed that ( Figure 8 D), by staining the model organoids with oil red, it was found that 10 micromolar of the drug significantly reduced lipid deposition in the liver organoids. In addition, 20 micromolar also showed a reduction in liver lipid accumulation. However, the low dose of 5 micromolar did not show a significant effect. In addition, by detecting the expression of FASN in the induced fatty liver organoid model, it was found that ( Figure 8 E), FASN expression increased significantly and was significantly associated with high sugar. Palmitic acid was detected and found ( Figure 8 F), Palmitic acid in the model group was significantly higher than that in the normal group, while the expression of palmitic acid in the drug-treated group was significantly improved.

[0109] Example 4: Vector complex targeting the FASN gene

[0110] The carrier complex targeting the FASN gene was prepared by conjugating GalNAc to the 3' end of the modified FASN siRNA-289 sense chain in Example 1 ( Figure 9 ).

[0111] A total of 240 6-week-old male C57BL6 mice were divided into 24 groups of 10 mice each. The vector complex was subcutaneously injected into the C57BL6 mice at concentrations of 1 mg / kg (low dose), 5 mg / kg (medium dose), and 10 mg / kg (high dose). FASN silencing efficiency was measured by QPCR every four weeks (10 mice were randomly selected from each group each time) until week 24. Mice that did not receive the vector complex served as controls and were designated as NC.

[0112] The results are as follows Figure 10 As shown, wherein Week represents the week number, for example, W4 represents the fourth week; it was found that the vector complex can silence FASN for 24 weeks.

[0113] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A siRNA targeting the FASN gene, characterized in that: The siRNA can inhibit the expression of FASN gene.

2. The siRNA according to claim 1, wherein The sense strand and antisense strand of the siRNA have the sequences shown in SEQ ID No. 1 and SEQ ID No. 2; or having the sequences shown in SEQ ID No. 3 and SEQ ID No. 4; Or have the sequence shown as SEQ ID No.5 and SEQ ID No.

6.

3. The siRNA according to claim 1, wherein The sense strand and antisense strand of the siRNA further have a 2-3 nucleotide tail.

4. The siRNA according to claim 2, wherein The sense strand of the siRNA was modified as follows; All nucleotides were PS modified. The 1st to 4th nucleotides at the 5' end are modified with 2'ome, and the 3rd to 6th nucleotides at the 3' end are modified with 2'ome; two 2'deoxythymines are connected to the 3' end; The antisense strand of the siRNA was modified as follows: All nucleotides were PS modified. The 6th and 7th nucleotides at the 5' end are modified with GNA, and two 2' deoxythymines are connected to the 3' end.

5. A vector complex targeting the FASN gene, characterized in that: GalNAc is linked to the 3' end of the sense strand of the siRNA according to any one of claims 1 to 3.

6. Use of the siRNA according to any one of claims 1 to 4 and the carrier complex according to claim 5 in the preparation of an agent for inhibiting FASN gene expression.

7. Use of the siRNA according to any one of claims 1 to 4 and the carrier complex according to claim 5 in the preparation of a medicament for preventing and / or treating metabolism-related fatty liver disease.

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