SiRNA for inhibiting MGAT2 gene expression and application thereof

Abstract

The invention discloses siRNA for inhibiting MGAT2 gene expression and application thereof.The siRNA comprises a positive-sense strand and an antisense strand, the antisense strand has a nucleotide sequence composed of at least 15 continuous nucleotides, the difference between the nucleotide sequence shown in any one of SEQ ID NO.20-38 and the nucleotide sequence shown in any one of SEQ ID NO.20-38 and the nucleotide sequence shown in any one of SEQ ID NO.20-38 is not more than 3 nucleotides, and the nucleotide sequence shown in any one of SEQ ID NO.20-38 is a nucleotide sequence composed of at least 15 continuous nucleotides. The positive-sense strand and the antisense strand are at least partially complementary to form a double-strand region. The invention provides siRNA for inhibiting MGAT2 gene expression, the siRNA is modified to ensure the stability and inhibition activity of the siRNA, and the siRNA provided by the invention has great application prospects in preparation of drugs for preventing and / or treating diseases related to MGAT2 expression. A new treatment strategy is provided for treatment of glioblastoma, breast cancer, liver cancer and other cancers, metabolic diseases, neurodegenerative diseases and autoimmune diseases.

Description

Technical Field

[0001] This application belongs to the field of biomedical technology, specifically relating to an siRNA that inhibits MGAT2 gene expression and its application. Background Technology

[0002] The MGAT2 gene (mannoside acetylglucosaminyltransferase 2, N-acetylglucosamine transferase II) is located on human chromosome 14q21.3 and plays a crucial role in N-linked glycosylation of proteins. N-linked glycosylation is a widespread post-translational modification in eukaryotes, significantly influencing protein folding, stability, localization, and function. MGAT2 catalyzes the formation of complex N-glycans, playing a key role in cell recognition, immune responses, and signal transduction.

[0003] In recent years, studies have found that abnormal expression of MGAT2 is closely related to the occurrence and development of various diseases. For example, in some cancers, overexpression of MGAT2 is associated with increased tumor invasiveness, improved metastatic ability, and poor prognosis. Furthermore, abnormal MGAT2 expression is also associated with metabolic diseases, neurodegenerative diseases, and autoimmune diseases. Based on this, by reducing MGAT2 expression levels, abnormal glycosylation processes can be regulated, thereby affecting the progression of related diseases. Therefore, inhibiting MGAT2 gene expression has become a potential therapeutic strategy.

[0004] RNA interference (RNAi) technology, especially small interfering RNA (siRNA), is considered an effective gene silencing method. However, current technologies lack siRNAs that can effectively inhibit MGAT2 gene expression. The main challenge lies in ensuring the specificity, delivery efficiency, stability, and immunogenicity of siRNAs. Developing effective siRNAs targeting the MGAT2 gene could provide new insights for the treatment of related diseases. Summary of the Invention

[0005] The purpose of this application is to provide an siRNA that inhibits MGAT2 gene expression and its application, so as to provide a new treatment strategy for diseases related to abnormal MGAT2 gene expression.

[0006] To achieve the above objectives, the first aspect of this application provides an siRNA for inhibiting MGAT2 gene expression, comprising a sense strand and an antisense strand, wherein the antisense strand has a nucleotide sequence consisting of at least 15 consecutive nucleotides that differ from the nucleotide sequence shown in any one of SEQ ID NO. 20 to 38 by no more than 3 nucleotides, and the sense strand and the antisense strand are at least partially complementary to form a double-stranded region.

[0007] In one or more embodiments, the nucleotide sequence of the sense strand is shown in SEQ ID NO.1, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.20.

[0008] In one or more embodiments, the nucleotide sequence of the sense strand is shown in SEQ ID NO.2, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.21.

[0009] In one or more embodiments, the nucleotide sequence of the sense strand is shown in SEQ ID NO.3, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.22.

[0010] In one or more embodiments, the nucleotide sequence of the sense strand is shown in SEQ ID NO.4, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.23.

[0011] In one or more embodiments, the nucleotide sequence of the sense strand is shown in SEQ ID NO.5, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.24.

[0012] In one or more embodiments, the nucleotide sequence of the sense strand is shown in SEQ ID NO.6, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.25.

[0013] In one or more embodiments, the nucleotide sequence of the sense strand is shown in SEQ ID NO.7, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.26.

[0014] In one or more embodiments, the nucleotide sequence of the sense strand is shown in SEQ ID NO.8, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.27.

[0015] In one or more embodiments, the nucleotide sequence of the sense strand is shown in SEQ ID NO.9, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.28.

[0016] In one or more embodiments, the nucleotide sequence of the sense strand is shown in SEQ ID NO.10, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.29.

[0017] In one or more embodiments, the nucleotide sequence of the sense strand is shown in SEQ ID NO.11, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.30.

[0018] In one or more embodiments, the nucleotide sequence of the sense strand is shown in SEQ ID NO.12, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.31.

[0019] In one or more embodiments, the nucleotide sequence of the sense strand is shown in SEQ ID NO.13, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.32.

[0020] In one or more embodiments, the nucleotide sequence of the sense strand is shown in SEQ ID NO.14, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.33.

[0021] In one or more embodiments, the nucleotide sequence of the sense strand is shown in SEQ ID NO.15, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.34.

[0022] In one or more embodiments, the nucleotide sequence of the sense strand is shown in SEQ ID NO.16, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.35.

[0023] In one or more embodiments, the nucleotide sequence of the sense strand is shown in SEQ ID NO.17, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.36.

[0024] In one or more embodiments, the nucleotide sequence of the sense strand is shown in SEQ ID NO.18, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.37.

[0025] In one or more embodiments, the nucleotide sequence of the sense strand is shown in SEQ ID NO.19, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.38.

[0026] In one or more embodiments, at least one nucleotide in the sense strand and / or antisense strand is a modified nucleotide, the modification including one or more combinations of 2'-methoxy modification, 2'-fluoro modification, thiophosphate modification, vinyl phosphate modification and 2'-deoxynucleotide modification.

[0027] In one or more embodiments, the nucleotides at least at positions 1-6 and 10-19 of the positive strand are 2'-methoxy modified nucleotides, oriented from the 5' end to the 3' end.

[0028] In one or more embodiments, the nucleotide at least at positions 7 to 9 of the positive strand is a 2'-fluorinated nucleotide, in the direction from the 5' end to the 3' end.

[0029] In one or more embodiments, at least the nucleotides at positions 1 and 2 and at positions 2 and 3 of the sense strand are linked by thiophosphate groups along the 5' to 3' direction, and at least the nucleotides at positions 1 and 2, at positions 2 and 3, at positions 19 and 20, and at positions 20 and 21 of the antisense strand are linked by thiophosphate groups.

[0030] In one or more embodiments, the nucleotides at least at positions 2, 14, and 16 of the antisense strand are 2'-fluorinated nucleotides, arranged from the 5' end to the 3' end.

[0031] In one or more embodiments, the nucleotides at least at positions 2, 6, 14, and 16 of the antisense strand are 2'-fluoro-modified nucleotides, and the remaining positions are 2'-methoxy-modified nucleotides, in the direction from the 5' end to the 3' end.

[0032] In one or more embodiments, the nucleotide at least at position 1 of the antisense strand is a vinyl phosphate modified nucleotide, in the direction from the 5' end to the 3' end.

[0033] In one or more embodiments, the nucleotides at least at positions 2, 10, 12, 14, 16, 18, and 20 of the antisense strand are 2'-fluorinated nucleotides, arranged from the 5' end to the 3' end.

[0034] In one or more embodiments, the nucleotides at least at positions 1, 3, 4, 6, 8, 9, 11, 13, 15, 17, 19, and 21 of the antisense strand are 2'-methoxy modified nucleotides, in the direction from the 5' end to the 3' end.

[0035] In one or more embodiments, the nucleotides at least at positions 5 and 7 of the antisense strand are nucleotides modified with 2'-deoxynucleotides, in the direction from the 5' end to the 3' end.

[0036] To achieve the above objectives, a second aspect of this application provides an siRNA conjugate comprising the siRNA described in any of the above embodiments and a conjugate group coupled to the siRNA.

[0037] In one or more embodiments, the conjugation group is coupled to the 3' end of the positive strand of the siRNA.

[0038] In one or more embodiments, the conjugating group is N-acetylgalactosamine.

[0039] To achieve the above objectives, a third aspect of this application provides the use of the siRNA or siRNA conjugate described in any of the above embodiments in the preparation of a medicament for the prevention or treatment of diseases related to MGAT2 gene expression.

[0040] To achieve the above objectives, a fourth aspect of this application provides an MGAT2 inhibitor comprising the siRNA or the siRNA conjugate described in any of the above embodiments.

[0041] To achieve the above objectives, the fifth aspect of this application provides a medicament for the prevention or treatment of diseases related to MGAT2 gene expression, comprising the siRNA or siRNA conjugate described in any of the above embodiments; and a pharmaceutically acceptable carrier.

[0042] The advantages of this application, which differ from existing technologies, are: This application provides siRNA for inhibiting MGAT2 gene expression, and modifies the siRNA to ensure its stability and inhibitory activity. Experiments have shown that multiple siRNA conjugates of this application have significant inhibitory activity on MGAT2 gene expression in C57BL6 / J magnetic mice. Among them, MGAT2-54M38G inhibited MGAT2 gene expression in female C57BL6 / J mice by 81%. Therefore, the siRNA provided in this application has great application potential in the preparation of drugs for the prevention and / or treatment of diseases related to MGAT2 expression, and provides a new treatment strategy for the treatment of cancers such as glioblastoma, breast cancer and liver cancer, metabolic diseases, neurodegenerative diseases and autoimmune diseases. Attached Figure Description

[0043] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0044] Figure 1 This is a graph showing the relative expression levels of MGAT2 gene mRNA in each group in Example 3 of this application; Figure 2 This is a graph showing the relative expression levels of MGAT2 gene mRNA in each group in Example 4 of this application; Figure 3This is a graph showing the relative expression levels of the MGAT2 gene at different administration times in Example 5 of this application; Figure 4 This is a graph showing the relative expression levels of MGAT2 gene mRNA in each group in Example 6 of this application. Detailed Implementation

[0045] To enable those skilled in the art to better understand the technical solutions in this application, the technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without creative effort should fall within the scope of protection of this application.

[0046] It should be noted that, in this invention, "modified nucleotide" refers to a nucleotide or nucleotide analog formed by replacing the 2' hydroxyl group of the ribosome with another group, or a nucleotide whose base is a modified base, or a compound formed by modifying a nucleotide with a thiophosphate group. "Nucleotide analog" refers to a group that can replace a nucleotide in nucleic acids, but whose structure differs from adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, or thymine deoxyribonucleotide. Examples include isonucleotides, bridged nucleotides (BNA), or acyclic nucleotides.

[0047] In one embodiment of the present invention, "2'-fluorinated nucleotide" refers to a nucleotide formed by replacing the hydroxyl group at the 2' position of the ribosyl group with fluorine, which has the structure shown in formula (1).

[0048] In one embodiment of the present invention, "2'-methoxy modified nucleotide" refers to a nucleotide formed by replacing the 2'-hydroxyl group of the ribosome with a methoxy group, which has the structure shown in formula (2).

[0049] In one embodiment of the present invention, "2'-deoxynucleotide modified nucleotide" refers to a nucleotide formed by replacing the 2'-hydroxyl group of the ribosome with a hydrogen atom, which has the structure shown in the following formula (3).

[0050]

[0051] In one embodiment of the present invention, the thiophosphate group in the "thiophosphate-modified nucleotide" has a structure as shown in formula (4). In one embodiment of the present invention, the nucleotide linked to the thiophosphate group is shown in formula (5):

[0052] In one embodiment of the present invention, "vinyl phosphate modified nucleotide" refers to a nucleotide formed by replacing the oxygen atom in the phosphate group of a nucleotide with a vinyl group. Here, Base refers to the modified or unmodified nucleotide base A, U, G, C, T, or other nucleotide bases.

[0053] Aberrant expression of MGAT2 is closely related to the occurrence and development of various diseases. By reducing the expression level of MGAT2, abnormal glycosylation processes can be regulated, thereby affecting the progression of related diseases. Therefore, inhibiting the expression of the MGAT2 gene has become a potential therapeutic strategy. To this end, the applicant has developed a siRNA based on RNA interference technology that can knock down the expression level of the MGAT2 gene, which is described in detail below.

[0054] Example 1: Sequence Design

[0055] First, the applicant designed multiple siRNA sequences based on the NCBI database targeting the mRNA sequence encoding the MGAT2 gene. The specific siRNA sequences and the targeted human MGAT2 gene fragment (Homo MGAT2 position), mouse MGAT2 gene fragment (Mouse MGAT2 position), and Chinese mitten crab MGAT2 gene fragment (Crab position) are shown in Table 1 below.

[0056] Table 1. siRNA sequences, target sites, and their numbers that inhibit MGAT2.

[0057] Example 2: siRNA Modification

[0058] To improve the stability of siRNA and effectively suppress the expression of the target gene, the applicant further modified the siRNA sequences in Table 1 above.

[0059] Specifically, in one embodiment, the modification scheme of the above-mentioned siRNA is as follows: Following the direction from the 5' end to the 3' end, nucleotides located at positions 1-6 and 10-19 of the sense strand are modified with 2'-methoxy groups, and nucleotides located at positions 1, 3-5, 7-13, 15 and 17-21 of the antisense strand are modified with 2'-methoxy groups. Following the direction from the 5' end to the 3' end, the nucleotides located at positions 7 to 9 of the sense strand are 2'-fluorinated, and the nucleotides located at positions 2, 6, 14, and 16 of the antisense strand are 2'-fluorinated. Following the direction from the 5' end to the 3' end, the nucleotides located at positions 1 and 2 of the sense strand and at positions 2 and 3 are linked by thiophosphate groups, and the nucleotides located at positions 1 and 2 of the antisense strand, at positions 2 and 3 of the antisense strand, at positions 19 and 20 of the antisense strand, and at positions 20 and 21 of the antisense strand are linked by thiophosphate groups.

[0060] Based on the above modification scheme, the modified siRNAs were obtained, namely MGAT2-49M1, MGAT2-54M1, MGAT2-68M1, MGAT2-78M1, MGAT2-101M1, MGAT2-102M1, MGAT2-115M1, MGAT2-116M1, MGAT2-129M1, MGAT2-146M1, MGAT2-152M1, MGAT2-162M1, MGAT2-170M1, MGAT2-175M1, MGAT2-188M1, MGAT2-194M1, MGAT2-213M1, MGAT2-215M1, and MGAT2-240M1, as detailed in Table 2 below.

[0061] In addition, the applicant has adopted another modification scheme for MGAT2-54, MGAT2-146, and MGAT2-152. In another embodiment, the modification scheme specifically includes: Following the direction from the 5' end to the 3' end, nucleotides located at positions 1-6 and 10-19 of the sense strand are modified with 2'-methoxy groups, and nucleotides located at positions 1, 3, 4, 6, 8, 9, 11, 13, 15, 17, 19, and 21 of the antisense strand are modified with 2'-methoxy groups. Following the direction from the 5' end to the 3' end, the nucleotides located at positions 7 to 9 of the sense strand are 2'-fluorinated, and the nucleotides located at positions 2, 10, 12, 14, 16, 18, and 20 of the antisense strand are 2'-fluorinated. In the direction from the 5' end to the 3' end, the nucleotides located at the 1st and 2nd positions of the sense strand and the nucleotides located at the 2nd and 3rd positions are linked by thiophosphate groups, and the nucleotides located at the 1st and 2nd positions of the antisense strand, the nucleotides located at the 2nd and 3rd positions, the nucleotides located at the 19th and 20th positions, and the nucleotides located at the 20th and 21st positions are linked by thiophosphate groups. The nucleotides located at positions 5 and 7 of the antisense strand are modified with 2'-deoxynucleotides in the direction from the 5' end to the 3' end. Based on the above modification scheme, the modified siRNAs were obtained, namely MGAT2-54M38, MGAT2-146M38 and MGAT2-152M38.

[0062] In addition, the applicant further modified the MGAT2-54M1 to improve its stability and loading efficiency. The modifications include: The nucleotide at position 1 of the antisense strand of MGAT2-54M1 was modified with vinyl phosphate to obtain MGAT2-54M1VP.

[0063] In Table 2 below, m represents a nucleotide whose left adjacent nucleotide is 2'-methoxy modified; f represents a nucleotide whose left adjacent nucleotide is 2'-fluoro modified; s represents a nucleotide whose left and right adjacent nucleotides are modified with a thiophosphate group; (d) represents a nucleotide whose left adjacent nucleotide is 2'-deoxynucleotide modified; and VP represents a nucleotide whose right adjacent nucleotide is vinylphosphate modified.

[0064] Table 2 Modified siRNA sequences

[0065] Furthermore, the applicant constructed siRNA conjugates based on the aforementioned siRNAs to obtain a liver-targeting delivery system. The specific construction scheme for the siRNA conjugates is as follows: N-acetylgalactosamine (GalNAc) was coupled to the 3' end of the positive strand of each of the above siRNAs to obtain siRNA conjugates, which are designated as: MGAT2-49M1G, MGAT2-54M1G, MGAT2-68M1G, MGAT2-78M1G, MGAT2-101M1G, MGAT2-102M1G, MGAT2-115M1G, MGAT2-116M1G, MGAT2-129M1G, and MGAT2-146M1G. 1G, MGAT2-152M1G, MGAT2-162M1G, MGAT2-170M1G, MGAT2-175M1G, MGAT2-188M1G, MGAT2-194M1G, MGAT2- 213M1G, MGAT2-215M1G, MGAT2-240M1G, MGAT2-54M1GVP, MGAT2-54M38G, MGAT2-146M38G, MGAT2-152M38G.

[0066] It should be noted that this embodiment only shows conjugates coupled with the GalNAc group. In other embodiments, the conjugate groups coupled with the above-mentioned siRNAs can be adjusted according to actual needs.

[0067] Example 3: Detection of the inhibitory activity of siRNA conjugates on MGAT2 gene expression in female C57BL6 / J mice

[0068] Female C57BL6 / J mice (6-8 weeks old, provided by Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.) were divided into groups of 3 mice each. Each group of mice was given a single dose of 3 mg / kg of siRNA conjugate or saline (saline was used as the control group). On day 14 after administration, the mice were sacrificed, and liver samples were collected and flash-frozen in liquid nitrogen. mRNA was extracted from the livers of the mice, and the on-target activity of the siRNA conjugate was detected by RT-qPCR.

[0069] The specific experimental steps are as follows: Step 1: Extraction of liver mRNA S1: Take 20 mg of mouse liver tissue, add 1 mL of Trizol Lysis Buffer (purchased from Lifetechnology, catalog number 410701), and grind to lyse the tissue to obtain the grinding product; transfer the grinding product to an RNase-free 1.5 mL centrifuge tube, shake for 15 s to fully lyse the tissue cells, and let it stand at room temperature (25 ℃) for 5 min to obtain the lysate; S2: Open the cap of the centrifuge tube, add 200 μL of chloroform (purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd., catalog number 20140925), shake for 20 s, let stand at room temperature (25 ℃) for 3 min, and then centrifuge at 4 ℃ and 12000×g for 20 min. After centrifugation, transfer the supernatant to a new 2.0 mL centrifuge tube, and add 1.5 times the volume of anhydrous ethanol (purchased from Jiangsu Qiangsheng Functional Chemical Co., Ltd., catalog number 20210802) to the centrifuge tube. Invert and mix well to obtain the mixture. S3: Take a purification column with a collection tube (purchased from VWI, catalog number 11822AG0627), add 700 μL of the mixture obtained in step two to the purification column, let it stand for 2 min, and then centrifuge the purification column at 4 ℃ and 10000×g for 1 min. After centrifugation, discard the filtrate; repeat the above steps for the remaining mixture. S4: Add 700 μL of 80% (v / v) ethanol to the purification column, centrifuge the purification column at 4 ℃ and 10000×g for 1 min, and discard the filtrate after centrifugation. S5: Repeat S4; S6: Centrifuge the purification column at 4 ℃ and 10000×g for 2 min. After centrifugation, remove the purification column with the collection tube, discard the collection tube, and place the purification column into a new 1.5 mL centrifuge tube. Add 100 μL of DEPC water to the purification column, let it stand at room temperature (25 ℃) for 2 min, and then centrifuge at 4 ℃ and 10000×g for 1 min. After centrifugation, collect the RNA solution for subsequent experiments.

[0070] Step 2: RNA extraction Using HiScript III RT SuperMix for qPCR (purchased from Novizan, catalog number R323-01), a 20 μL reverse transcription reaction system was prepared according to the reverse transcription procedure in the kit instructions. Total RNA from the above tissues was reverse transcribed to obtain reverse transcription products (reverse transcription conditions: the reverse transcription reaction system was first incubated at 37 ℃ for 15 min, and then incubated at 85 ℃ for 5 s). 80 μL of DEPC water was added to each reverse transcription product to obtain a solution containing cDNA.

[0071] Step 3: Preparation of qPCR reaction system For each reverse transcription reaction system, take 4 μL of the above solution containing cDNA as a template, and use the reagents provided by the AceQ Universal SYBR qPCR Master Mix kit (purchased from Vazyme, catalog number Q511-02) to prepare a 20 μL qPCR reaction system on an ice box according to Table 3 (in the qPCR reaction system in Table 3, Primer1 and Primer2 are PCR primers used to amplify the target gene MGAT2 and the internal reference gene GAPDH, respectively, and the primer sequences are shown in Table 4). Each qPCR reaction system was placed on an ABI StepOnePlus Real-Time PCR instrument and amplified using a three-step method to obtain product W containing the amplified target gene MGAT2 and the internal reference gene GAPDH (the amplification program for the qPCR reaction was: 95℃ pre-denaturation for 10 min, then 95℃ denaturation for 30 s, 60℃ annealing for 30 s, and 72℃ extension for 30 s, and the above denaturation, annealing, and extension process was repeated for a total of 40 times). Product W was then incubated sequentially at 95 ℃ for 15 s, 60 ℃ for 1 min, and 95 ℃ for 15 s. The melting curves of the target gene MGAT2 and the internal reference gene GAPDH in product W were collected by real-time quantitative PCR instrument to obtain the Ct values ​​of the target gene MGAT2 and the internal reference gene GAPDH.

[0072] Table 3 RNA amplification reaction system

[0073] Table 4 qPCR primer information

[0074] The Ct values ​​of the target gene MGAT2 and the internal reference gene GAPDH were collected in each test group and control group. Then, the relative quantification of the target gene MGAT2 in each test group was calculated using the comparison Ct method (three replicates were set up for each group in the experiment).

[0075] The calculation method for the relative quantitative results is as follows: ΔCt(test group) = Ct(target gene in test group) – Ct(internal reference gene in test group); ΔCt(control group) = Ct(target gene in control group) – Ct(internal reference gene in control group); ΔCt(test group) = ΔCt(test group) - ΔCt(control group average); ΔCt(control group) = ΔCt(control group) - ΔCt(control group average); Wherein, ΔCt (control group mean) is the arithmetic mean of ΔCt (control group) of the three samples in the control group; thus, each sample in the test group and the control group corresponds to a ΔCt value; The MGAT2 mRNA expression level in the control group was defined as 100%. Using the control group as a baseline, the MGAT2 mRNA expression level in the test group was normalized. The relative expression level of MGAT2 mRNA in the test group = 2^(-ΔΔCt(test group)) × 100%; The MGAT2 mRNA level was compared with the internal reference gene GAPDH, and the value was normalized to the mean of the saline control group. The data were expressed as a percentage relative to the saline control group and presented as the mean plus the standard deviation.

[0076] Specifically, in this embodiment, MGAT2-49M1G, MGAT2-54M1G, MGAT2-68M1G, MGAT2-78M1G, MGAT2-101M1G, MGAT2-102M1G, MGAT2-115M1G, MGAT2-116M1G, MGAT2-129M1G, MGAT2-146M1G, MGAT2-152M1G, MGAT2-162M1G, MGAT2-170M1G, MGAT2-175M1G, MGAT2-188M1G, MGAT2-194M1G, MGAT2-213M1G, MGAT2-215M1G, and MGAT2-240M1G were used for experiments. The specific siRNA sequences can be found in Table 2. Figure 1The experimental results.

[0077] Please see Figure 1 , Figure 1 This is a graph showing the relative expression levels of MGAT2 gene mRNA in each group in Example 3 of this application.

[0078] like Figure 1 As shown, MGAT2-49M1G, MGAT2-54M1G, MGAT2-68M1G, MGAT2-78M1G, MGAT2-101M1G, MGAT2-102M1G, MGAT2-115M1G, MGA T2-116M1G, MGAT2-129M1G, MGAT2-146M1G, MGAT2-152M1G, MGAT2-162M1G, MGAT2-170M1G, MGAT2-175M1G, MG AT2-188M1G, MGAT2-194M1G, and MGAT2-215M1G all inhibited MGAT2 gene expression in female C57BL6 / J mice. Among them, compared with the saline control group, MGAT2-54M1G, MGAT2-146M1G, and MGAT2-152M1G inhibited MGAT2 gene expression in female C57BL6 / J mice by 74%, 64%, and 59%, respectively, demonstrating significant gene expression inhibition effects.

[0079] Example 4: Detection of the inhibitory activity of siRNA conjugates with different modification schemes on MGAT2 gene expression in female C57BL6 / J mice.

[0080] To verify the inhibitory activity of siRNA conjugates with different modification schemes on MGAT2 gene expression, the applicant selected the siRNAs with the best inhibitory effect in Example 3: MGAT2-54, MGAT2-146, and MGAT2-152. siRNA conjugates (MGAT2-54M1GVP, MGAT2-54M38G, MGAT2-146M38G, and MGAT2-152M38G) constructed based on other modification schemes were used in the experiment. The inhibitory activity of the siRNA conjugates on MGAT2 gene expression in female C57BL6 / J mice was tested using the same method as in Example 3.

[0081] The specific siRNA modification scheme can be found in Table 2. MGAT2-54M1G and negative control NC were also introduced. The negative control group was administered the same dose of NC to mice. The NC sequence is shown in Table 5.

[0082] Table 5 Negative control group sequences

[0083] Please see Figure 2 , Figure 2 This is a graph showing the relative expression levels of MGAT2 gene mRNA in each group in Example 4 of this application.

[0084] like Figure 2 As shown, MGAT2-54M1GVP, MGAT2-54M38G, MGAT2-146M38G, MGAT2-152M38G, and MGAT2-54M1G can all significantly inhibit the expression level of the MGAT2 gene in female C57BL6 / J mice. Among them, MGAT2-54M38G has an inhibition rate of 81% on the expression of the MGAT2 gene in female C57BL6 / J mice.

[0085] Example 5: Analysis of the long-term inhibitory effect of siRNA conjugates

[0086] To verify the long-lasting effect of the siRNA conjugate in vivo, and to investigate whether the inhibitory activity of the siRNA conjugate on MGAT2 gene expression in female C57BL6 / J mice was affected by administration time, the applicant selected MGAT2-54M1GVP for the experiment. The relative expression levels of the MGAT2 gene in female C57BL6 / J mice were tested 14 and 28 days after administration using the same method as in Example 3. Figure 3 .

[0087] Please see Figure 3 , Figure 3 This is a graph showing the relative expression levels of the MGAT2 gene at different administration times in Example 5 of this application.

[0088] like Figure 3 As shown, MGAT2-54M1GVP inhibited MGAT2 gene expression in female C57BL6 / J mice for 14 days by 76%, and also inhibited MGAT2 gene expression in female C57BL6 / J mice for 28 days by 69%, indicating that MGAT2-54M1GVP can inhibit MGAT2 expression for a long time.

[0089] Example 6: Detection of the inhibitory activity of different concentrations of siRNA on MGAT2 gene expression in Hep3B cells

[0090] To verify the inhibitory activity of siRNA conjugates with different modification schemes on MGAT2 gene expression, the applicant selected siRNAs MGAT2-54M1G and MGAT2-54M1GVP from Example 4 for the experiment. Quantitative Real-Time PCR (qPCR) was used to test the inhibitory activity of the siRNA conjugates on MGAT2 gene expression in Hep3B cells.

[0091] The specific experimental procedure is as follows: Hep3B cells (human hepatocellular carcinoma cells, purchased from the Cell Bank of the Chinese Academy of Sciences) were seeded into MEM medium (purchased from Gibco, catalog number 11095-080) containing 10% (v / v) fetal bovine serum (FBS, purchased from Hyclone) and 1% (v / v) penicillin-streptomycin mixture (Penicillin-Streptomycin, purchased from Gibco, catalog number 15140122) and cultured in a 5% (v / v) CO2, 37°C cell culture incubator for 48 h. After culture, the Hep3B cells were digested with trypsin (purchased from Gibco, catalog number 25200-072). After digestion, the cells were rinsed with PBS buffer and then resuspended in MEM medium to obtain a cell concentration of 3 × 10⁶ cells / year. 5 Cell suspension of cells / mL; Different siRNAs were diluted separately using opti-MEM (Gibco, catalog number 31985-070) to obtain siRNA dilutions containing different siRNAs. 25 μL of opti-MEM was mixed with 0.25 μL of Lipofectamine RNAiMAX transfection reagent (Thermo Fisher Scientific, catalog number 11668-019) to obtain transfection reagent dilutions. 25 μL of each siRNA dilution was then mixed with the transfection reagent dilutions and incubated at room temperature (25℃) for 15 min to obtain transfection solutions containing different siRNAs. Cell suspension was seeded into 96-well plates at a seeding rate of 50 μL / well. Two experimental groups, MGAT2-54M1G and MGAT2-54M1GVP, were set up in each well. A blank control group was also included, containing only the interfering reagent and no other sequence. 50 μL of transfection buffer containing different siRNAs was added to each well of the experimental groups. Specifically, the MGAT2-54M1G group received transfection buffer containing MGAT2-54M1G, and the MGAT2-54M1GVP group received transfection buffer containing MGAT2-54M1GVP. The final siRNA concentrations in each experimental group were 10 nM, 1 nM, 0.33 nM, 0.11 nM, 0.037 nM, 0.0123 nM, and 0.0041 nM. Seven experimental groups of nM were set up, with three replicates in each experimental group. The cells were then cultured in a 5% (v / v) CO2, 37°C cell culture incubator for 48 h before transfection. After transfection, discard the liquid in the wells, collect the cells, and lyse the cells in each well according to the method described in the FlysisAmp Cells Lysis Kit (purchased from Novizan, catalog number: CL101-01) to obtain cell lysate for subsequent qPCR detection.

[0092] For each reverse transcription reaction system, the cell lysate described above was used as a template, and the qPCR reaction system was prepared on an ice box according to Table 6. hMGAT2-F, hMGAT2-R, and hMGAT2-P are the primer and probe sequences for amplifying the target gene MGAT2, respectively, and HGAPDH-F, HGAPDH-R, and HGAPDH-PHGAPDH-P are the primer and probe sequences for amplifying the internal reference gene GAPDH, respectively. Please refer to Table 7 for the specific sequences.

[0093] The qPCR reaction system was amplified on a Roche 480 PCR instrument. The amplification program was 55℃ for 15 min, 95℃ for 30 s, followed by denaturation at 95℃ for 10 s, annealing at 58℃ for 30 s, extension at 72℃, and collection of fluorescence signals for 30 s. The denaturation, annealing, and extension process was repeated for a total of 40 times. The fluorescence signals of the target gene MGAT2 and the internal reference gene GAPDH were collected by a real-time quantitative PCR instrument to obtain the Ct values ​​of the target gene MGAT2 and the internal reference gene GAPDH.

[0094] Table 6 DNA Amplification Reaction System

[0095] Table 7 Primer Information

[0096] Ct values ​​of the target gene MGAT2 and the internal reference gene GAPDH were collected from each experimental group and control group. The data were then analyzed using the comparative Ct method, and the analysis was standardized for the blank control group. The specific analysis method was the same as in Example 3 and will not be repeated here. Figure 4 , Figure 4 This is a graph showing the relative expression levels of MGAT2 gene mRNA in each group in Example 6 of this application.

[0097] like Figure 4 As shown, both MGAT2-54M1G and MGAT2-54M1GVP exhibited significant inhibitory effects on MGAT2 gene expression in Hep3B cells. Moreover, the inhibition rate gradually increased with the increase of the final concentration of siRNA. When the final concentration of siRNA was 10 nM, the inhibition rate of MGAT2 gene expression by MGAT2-54M1G and MGAT2-54M1GVP reached about 80% compared with the blank control group.

[0098] Furthermore, after calculating the inhibition rate of the two groups of siRNAs on MGAT2 gene expression at different final concentrations, the applicant used GraphPad Prism software to fit and calculate the half-maximal inhibitory concentration (IC50) of the two groups of siRNAs, and obtained the data in Table 8 below.

[0099] Table 8 IC50 calculation results for Example 6

[0100] As shown in Table 8, the IC50 values ​​of MGAT2-54M1G and MGAT2-54M1GVP against the MGAT2 gene in Hep3B cells were both below 0.23 nM. Among them, the IC50 value of MGAT2-54M1GVP was as low as 0.1440, showing very outstanding inhibitory activity.

[0101] It will be apparent to those skilled in the art that this application is not limited to the details of the exemplary embodiments described above, and that this application can be implemented in other specific forms without departing from the spirit or essential characteristics of this application. Therefore, the embodiments should be considered illustrative and non-limiting in all respects, and the scope of this application is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within this application. No reference numerals in the claims should be construed as limiting the scope of the claims.

[0102] In addition, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. A siRNA that inhibits MGAT2 gene expression, characterized in that, It includes a sense strand and an antisense strand, wherein the antisense strand has a nucleotide sequence consisting of at least 15 consecutive nucleotides that differ from the nucleotide sequence shown in any one of SEQ ID NO. 20 to 38 by no more than 3 nucleotides, and the sense strand and the antisense strand are at least partially complementary to form a double-stranded region.

2. The siRNA according to claim 1, characterized in that, The nucleotide sequence of the sense strand is shown in SEQ ID NO.1, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.20; or, The nucleotide sequence of the sense strand is shown in SEQ ID NO.2, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.21; or, The nucleotide sequence of the sense strand is shown in SEQ ID NO.3, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.22; or, The nucleotide sequence of the sense strand is shown in SEQ ID NO.4, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.23; or, The nucleotide sequence of the sense strand is shown in SEQ ID NO. 5, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO. 24; or, The nucleotide sequence of the sense strand is shown in SEQ ID NO. 6, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO. 25; or, The nucleotide sequence of the sense strand is shown in SEQ ID NO. 7, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO. 26; or, The nucleotide sequence of the sense strand is shown in SEQ ID NO. 8, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO. 27; or, The nucleotide sequence of the sense strand is shown in SEQ ID NO. 9, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO. 28; or, The nucleotide sequence of the sense strand is shown in SEQ ID NO. 10, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO. 29; or, The nucleotide sequence of the sense strand is shown in SEQ ID NO. 11, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO. 30; or, The nucleotide sequence of the sense strand is shown in SEQ ID NO. 12, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO. 31; or, The nucleotide sequence of the sense strand is shown in SEQ ID NO. 13, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO. 32; or, The nucleotide sequence of the sense strand is shown in SEQ ID NO. 14, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO. 33; or, The nucleotide sequence of the sense strand is shown in SEQ ID NO. 15, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO. 34; or, The nucleotide sequence of the sense strand is shown in SEQ ID NO. 16, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO. 35; or, The nucleotide sequence of the sense strand is shown in SEQ ID NO. 17, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO. 36; or, The nucleotide sequence of the sense strand is shown in SEQ ID NO. 18, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO. 37; or, The nucleotide sequence of the sense strand is shown in SEQ ID NO.19, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.

38.

3. The siRNA according to claim 1 or 2, characterized in that, At least one nucleotide in the sense strand and / or antisense strand is a modified nucleotide, and the modification includes one or more combinations of 2'-methoxy modification, 2'-fluoro modification, thiophosphate modification, vinyl phosphate modification and 2'-deoxynucleotide modification.

4. The siRNA according to claim 3, characterized in that, In the direction from the 5' end to the 3' end, at least the nucleotides located at positions 1-6 and 10-19 of the positive strand are 2'-methoxy modified nucleotides; and / or, In the direction from the 5' end to the 3' end, at least the nucleotides located at positions 7 to 9 of the positive strand are 2'-fluorinated nucleotides; and / or, In the direction from the 5' end to the 3' end, at least the nucleotides at positions 1 and 2 and at positions 2 and 3 of the sense strand are linked by thiophosphate groups, and at least the nucleotides at positions 1 and 2, at positions 2 and 3, at positions 19 and 20, and at positions 20 and 21 of the antisense strand are linked by thiophosphate groups.

5. The siRNA according to claim 4, characterized in that, In the direction from the 5' end to the 3' end, at least the nucleotides located at positions 2, 14, and 16 of the antisense strand are 2'-fluorinated nucleotides.

6. The siRNA according to claim 5, characterized in that, Along the 5' to 3' direction, at least the nucleotides at positions 2, 6, 14 and 16 of the antisense strand are 2'-fluoro-modified nucleotides, and the remaining positions are 2'-methoxy-modified nucleotides.

7. The siRNA according to claim 6, characterized in that, In the direction from the 5' end to the 3' end, at least the nucleotide at the first position of the antisense strand is a vinyl phosphate modified nucleotide.

8. The siRNA according to claim 5, characterized in that, In the direction from the 5' end to the 3' end, at least the nucleotides located at positions 2, 10, 12, 14, 16, 18, and 20 of the antisense strand are 2'-fluorinated nucleotides; and / or, In the direction from the 5' end to the 3' end, at least the nucleotides located at positions 1, 3, 4, 6, 8, 9, 11, 13, 15, 17, 19, and 21 of the antisense strand are 2'-methoxy modified nucleotides; and / or, In the direction from the 5' end to the 3' end, at least the nucleotides at positions 5 and 7 of the antisense strand are nucleotides modified with 2'-deoxynucleotides.

9. A siRNA conjugate, characterized in that, It includes the siRNA as described in any one of claims 1 to 8 and the conjugate group coupled to the siRNA.

10. The siRNA conjugate according to claim 9, characterized in that, The conjugation group is coupled to the 3' end of the positive strand of the siRNA; and / or, The conjugating group is N-acetylgalactosamine.

11. Use of any siRNA according to claims 1 to 8 or any siRNA conjugate according to claims 9 or 10 in the preparation of a medicament for the prevention or treatment of diseases associated with MGAT2 gene expression.

12. An MGAT2 inhibitor, characterized in that, Includes the siRNA as described in any one of claims 1 to 8 or the siRNA conjugate as described in claim 9 or 10.

13. A medicament for the prevention or treatment of diseases associated with MGAT2 gene expression, characterized in that, Includes the siRNA as described in any one of claims 1 to 8 or the siRNA conjugate as described in claim 9 or 10; and a pharmaceutically acceptable carrier.

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