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siRNA for inhibiting expression of human PCSK9 gene and application thereof

A gene expression and target gene technology, applied in the field of biomedicine, can solve difficult gene silencing and other problems

Pending Publication Date: 2020-05-15
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although siRNA can specifically inhibit the expression of target genes, siRNA in cells is easily degraded, making it difficult to achieve stable gene silencing

Method used

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  • siRNA for inhibiting expression of human PCSK9 gene and application thereof
  • siRNA for inhibiting expression of human PCSK9 gene and application thereof
  • siRNA for inhibiting expression of human PCSK9 gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Design and synthesis of siRNA targeting human PCSK9

[0040] The PCSK9 mRNA sequences of human, golden hamster and monkey were obtained from Genebank by bioinformatics method (RefSeq ID numbers are NM_174936.3, XM_013114871.1 and NM_001112660.1), and the conserved regions of mRNA were found by DNA man (V6) software sequence. Use RNA structure analysis software to predict the secondary structure of PCSK mRNA sequence, and comprehensively analyze its GC content, secondary structure of mRNA action site, and base preference requirements of siRNA according to the design principles of siRNA. On the basis of siRNA online design software DSIR design, we further searched for suitable siRNA target sequences, and preliminarily screened out 3 pairs of siRNA sequences. Then use the BLAST software provided by NCBI GeneBank to select the Transcript Reference Sequences database (Transcript Reference Sequences), and perform homology analysis on the above siRNA sequences to ex...

Embodiment 2

[0050] Example 2 Western blot detection of the impact of siRNA targeting human PCSK9 transfected HepG2 and LO2 cells on the expression of PCSK9 and LDLR

[0051] HepG2 and LO2 cells (both purchased from the Basic Medical Cell Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) were adjusted to 1 × 10 5 Each well was inoculated in a 12-well plate, 1ml / well, cultured at 37°C for 12 hours, the medium was aspirated, and replaced with 500 μl MEM medium without serum, penicillin and streptomycin. The siRNA sequence of the PCSK9 siRNA drug Inclisiran was set as the positive control (PC), and a blank control group (NC) was set up separately. Dilute siRNA (negative control NC, positive control PC, si1309, si1318, si1432) and liposome lipofectamine with serum-free Opti-MEM medium TM 3000 (Invitrogen Company), after incubating at room temperature for 5 minutes, mix siRNA with liposomes, and after incubating at room temperature for 15 minutes, add dropwise t...

Embodiment 3

[0052] Example 3 Flow cytometry detection of the impact of transfected human PCSK9 siRNA on the expression of LDLR on the surface of HepG2 and LO2 cells

[0053] Transfect HepG2 and LO2 cells according to the method described in Example 2. After 48 hours of transfection, each well was replaced with serum-free Opti-MEM medium for cultivation. LDLR levels were detected by flow cytometry. The specific process is as follows: The cells were digested with trypsin and washed with PBS, and the cells were collected by centrifugation. Fix with 80% methanol for 5 min, centrifuge to discard the supernatant, add blocking solution (PBS buffer containing 10% goat serum, 0.3M glycine) to resuspend the pellet, and let stand at room temperature for 30 min. Discard the supernatant by centrifugation, resuspend the pellet with Anti-LDLR Receptor antibody (Abcam, Cat. No. ab52818) diluted 1:100 in PBS containing 1% BSA, and let stand at room temperature for 60 min; Alexa diluted 1:300 in PBS 48...

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Abstract

The invention discloses an siRNA sequence for specifically inhibiting the expression of a human protein convertase subtilisin 9 (PCSK9) target gene and an application thereof. The siRNA can be chemically synthesized, and can also be constructed into corresponding recombinant lentivirus or adeno-associated virus and the like according to the sequence of the siRNA. After human hepatoma carcinoma cell HepG2 and human normal hepatoma carcinoma cell LO2 are transfected by the siRNA or the virus, the expression of PCSK9 can be effectively inhibited; the protein level of a low-density lipoprotein receptor (LDLR) is increased; and the activity of the hepatoma carcinoma cell for taking in low-density lipoprotein (LDL) is remarkably enhanced. After the recombinant lentivirus is subjected to high-pressure caudal vein injection administration, the levels of total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) in plasma of a hypercholesterolemia model mouse can be remarkably reduced. The siRNA or the recombinant virus or a modifier thereof can be used for preventing and / or treating diseases like hyperlipidemia, atherosclerosis, cardiovascular and cerebrovascular diseases, obesity, diabetes and nephropathy.

Description

technical field [0001] The invention belongs to the field of biomedicine, in particular to a siRNA that inhibits the expression of human protein convertase subtilisin 9 (PCSK9) gene and its preparation and treatment of related diseases mediated by PCSK9, such as hyperlipidemia, atherosclerosis, heart and brain Application in drugs for vascular diseases, obesity, diabetes, kidney disease and other diseases. Background technique [0002] Hypercholesterolemia can be divided into familial and non-familial, clinically manifested as high plasma low-density lipoprotein cholesterol (LDL-C), which is an important factor in the occurrence of cardiovascular diseases. At present, statins are the main lipid-lowering drugs widely used clinically, but a considerable number of patients will develop tolerance to them (Eckel (2010) J Clin Endocrinol Metab 95:2015-22), and LDL cannot be reduced after receiving statins. The -C level is reduced to the expected value (Toutouzas et al. (2010) Exp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/867A61K31/713A61P3/04A61P3/06A61P3/10A61P9/10A61P9/00A61P13/12
CPCC12N15/1137C12N15/86A61P3/04A61P3/06A61P3/10A61P9/10A61P9/00A61P13/12C12N2310/14C12Y304/21061C12N2740/15043C12N2800/107
Inventor 谭树华王维陈佳利陈雪梅
Owner CHINA PHARM UNIV
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