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RNA inhibitor for inhibiting PCSK9 gene expression and application thereof

A technology of gene expression and inhibitors, applied in the field of RNA inhibitors that inhibit PCSK9 gene expression and its application, can solve the problems of statin drug intolerance, patients' maximum tolerated dose, etc.

Active Publication Date: 2021-08-10
KYLONOVA (XIAMEN) BIOPHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, statins are still the drug of choice for the treatment of atherosclerotic cardiovascular diseases, but there are still some patients who are intolerant to statins or the maximum tolerated dose still cannot reach the LDL-C therapeutic target (<1.8mmol / L( 70mg / dL) or <1.4mmol / L (55mg / dL))

Method used

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  • RNA inhibitor for inhibiting PCSK9 gene expression and application thereof
  • RNA inhibitor for inhibiting PCSK9 gene expression and application thereof
  • RNA inhibitor for inhibiting PCSK9 gene expression and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0298] The solid-phase phosphoramidite method synthesis of embodiment 1, RNA inhibitor

[0299] The RNA inhibitors of the present invention include but are not limited to Ky08-DS0103, Ky08-DS0105, Ky08-DS0107, Ky08-DS0109 and Ky08-DS0111 and Ky08-DS0113 are obtained by the solid-phase phosphoramidite method to obtain the respective sense strand and reverse The sense strand, the sense strand and the corresponding antisense strand are complementarily annealed to obtain the final product. The basic steps of the solid-phase phosphoramidite method include: 1) deprotection: remove the starting monomer Solid Support hydroxyl protecting group (DMTr); 2) coupling: add the first phosphoramidite monomer, pass 3' to Coupling reaction occurs in the 5' direction; 3) Oxidation: oxidize the resulting nucleoside phosphite to a more stable nucleoside phosphate (that is, trivalent phosphorus is oxidized to pentavalent phosphorus); 4) Blocking: the unreacted former One-step nucleotide monomer 5'...

Embodiment 2

[0397] Embodiment 2: Use Hep3B and Hela cell line to screen the sequence of RNA inhibitor

[0398] Description of the test process:

[0399] The corresponding RNA inhibitors Ky08-DS01-Ky08-DS15 were prepared using the mature phosphoramidite solid-phase synthesis method disclosed in the art. Prepare DMEM medium containing 10% fetal bovine serum. Cells (Hep3B and Hela cells) were cultured in 10 cm culture dishes to 80-90% confluence, and seeded into 6-well plates. The culture medium was poured off, and the cells were washed twice with 2 mL of PBS. Add 2mL Trypsin-EDTA solution, mix well, and place at 37°C for 3-5 minutes. Carefully suck off the trypsin solution, add 2 mL of DMEM culture solution containing 10% FBS, and pipette the cells to form a single-cell suspension. For hemocytometer counting, cells were diluted to 1.5 x 10 7 cells / mL. Press 1.5×10 6 Inoculate a 6-well plate at the concentration of cells / well, mix well and store at 37°C in 5% CO 2 Incubate for 24 hou...

Embodiment 3

[0401] Example 3: Exploration of the in vivo efficacy of RNA inhibitors in B6-hPCSK9 mouse model of hyperlipidemia 1

[0402] Description of test process: The corresponding RNA inhibitors Ky08-DS0101, Ky08-DS0401, Ky08-DS0601, Ky08-DS0701, Ky08-DS0801, and Ky08-DS1001 were prepared according to the method described in Example 1.

[0403] Thirty-five B6-hPCSK9 mice were randomly divided into control group and treatment group according to body weight after adaptive feeding, with 5 mice in each group. After feeding for 5 weeks, the mice were fasted for 4-5 hours, then blood was collected from the orbit (≤75 μL), and the level of low-density lipoprotein cholesterol (LDL-C) was detected after separating the plasma. When the average LDL-C level of mice in the WD feeding group reached 1.2mmol / L, it was determined that the model was established successfully. All mice recovered for one week after blood collection, and were randomly divided into 15 groups according to LDL-C levels, wit...

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Abstract

The invention belongs to the field of biochemistry, and particularly relates to an RNA inhibitor for inhibiting proprotein convertase subtilisin Kexin9 (PCSK9) gene expression in hepatocytes and application thereof. The RNA inhibitor is formed by a positive-sense strand and an antisense strand through base pairing, at least 85% of bases between the positive-sense strand and the antisense strand are complementary, -OH at the 2' site of partial or all nucleotide glycosyls is substituted by fluorine or methoxyl, and phosphate among at least three continuous nucleotides at the tail end is sulfated. The structure of the RNA inhibitor provided by the invention also contains 5' MVIP and 3' MVIP, so that the RNA inhibitor has specific liver targeting. In-vivo and in-vitro pharmacological experiments prove that: the RNA inhibitor directly acts on PCSK9 mRNA, continuously and efficiently inhibits PCSK9 gene expression and reduces the level of LDL-C in plasma, and is used for treating or / and preventing PCSK9 gene mediated diseases including but not limited to hyperlipidemia, atherosclerosis or PCSK9 gene mediated other diseases.

Description

technical field [0001] The invention belongs to the field of biochemistry, and specifically relates to an RNA inhibitor for inhibiting gene expression of proprotein convertase subtilisin Kexin9 (PCSK9) in liver cells and an application thereof, wherein the RNA inhibitor passes through a sense strand and an antisense strand Base pairing is formed, the sense strand and the antisense strand are at least 85% base complementary to each other, and part or all of the -OH at the 2' position of the nucleotide sugar group is replaced by fluorine or methoxy, and there are at least 3 consecutive Phosphates between nucleotides are thiolated. The structure of the RNA inhibitor provided by the present invention also contains the structures 5'MVIP and 3'MVIP that make the RNA inhibitor have liver targeting specificity, wherein the 5'MVIP is coupled to the sense strand of the RNA inhibitor and / or Or the 5' end of the antisense strand, the 3'MVIP is coupled to the 3' end of the antisense stran...

Claims

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Application Information

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IPC IPC(8): C12N15/113A61K31/713A61P3/06A61P9/10
CPCC12N15/1137A61K31/713A61P3/06A61P9/10C12N2310/14C12N2310/321C12N2310/322C12N2310/333C12N2310/334C12N2310/3341C12N2310/335C12N2310/336C12N2320/30
Inventor 崔坤元卢雪琴
Owner KYLONOVA (XIAMEN) BIOPHARMA CO LTD
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