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Delivery system and biological preparation of PCSK9 (Proprotein Convertase subtilisin/kexin type 9) inhibitor hypolipidemic drug

A technology of lipid nanoparticles and dioleoyl phosphatidylethanolamine, which is applied in the field of medicine, can solve the problems of adenovirus safety problems, toxic and side effects, and achieves the effect of avoiding safety problems and toxic and side effects

Active Publication Date: 2018-07-31
谭旭
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The existing CRISPR-Cas9 delivery in vivo is through the use of adenovirus expression system to express Cas9 and guideRNA, but the safety of adenovirus is still problematic
Moreover, the long-term stable expression of Cas9, a DNA enzyme, in cells by adenoviruses may also cause long-term toxic side effects

Method used

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  • Delivery system and biological preparation of PCSK9 (Proprotein Convertase subtilisin/kexin type 9) inhibitor hypolipidemic drug
  • Delivery system and biological preparation of PCSK9 (Proprotein Convertase subtilisin/kexin type 9) inhibitor hypolipidemic drug
  • Delivery system and biological preparation of PCSK9 (Proprotein Convertase subtilisin/kexin type 9) inhibitor hypolipidemic drug

Examples

Experimental program
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Effect test

Embodiment 1

[0094] Example 1: Cas9mRNA and sgRNA-mE3, hC1, hC4 delivered by transient transfection method, hC5 in NIH-3T3 and Targeted cleavage of PCSK9 gene sequence in 293T cell line

[0095] As mentioned above, the PX330-PCSK9gRNA (mE3, hC1-hC5) we constructed can express Cas9 protein and corresponding sgRNA simultaneously in cells, so we transiently transfected PX330-PCSK9gRNA (mE3, hC1, 4, 5) After expressing the vector, evaluate the targeted cutting effect of the Cas9 protein and the corresponding sgRNA on the PCSK9 gene sequence.

[0096] Figure 5 It shows that Cas9mRNA and sgRNA-mE3, hC1, hC4, hC5 delivered by transient transfection method have targeted cleavage effect on PCSK9 gene sequence in NIH-3T3 and 293T cell lines. Figure 5 A shows a schematic diagram of the experimental process; Figure 5 A shows the T7E1 mismatch enzyme assay to detect the effect of Cas9 / sgRNA (mE3) in NIH-3T3 cells to target DNA double-strand shearing, and the target band after digestion is mark...

Embodiment 2

[0098] Example 2: Cas9mRNA and sgRNA-mE3 delivered by TT3-O3 formulation lipid nanoparticles in wild-type C57 / B6 Inhibition of PCSK9 protein in a mouse model

[0099] After demonstrating the targeted cleavage of the PCSK9 gene sequence by expressing Cas9 mRNA and sgPsck9-mE3 in the NIH-3T3 cell line based on a transient transfection method, we further evaluated Cas9 mRNA and Effect of sgPsck9-mE3 on inhibiting PCSK9 protein in wild-type C57 / B6 mouse model.

[0100] Figure 7 and Figure 8 Shown is the effect of Cas9 mRNA and sgPsck9-mE3 delivered by TT3-O3 formulated lipid nanoparticles to inhibit PCSK9 protein in a wild-type C57 / B6 mouse model. Figure 7 , 8A shows a schematic diagram of the mouse experiment process; Figure 7 , 8B shows the expression level of PCSK9 protein mRNA in mouse liver detected by real-time fluorescent quantitative PCR; Figure 7 , 8C shows that the T7E1 mismatch enzyme assay detects that sgPsck9-mE3 causes specific gene splicing in the Psck9...

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Abstract

The present invention discloses a lipid nanoparticle comprising a lipopeptide molecule, dioleoyl phosphoethanolamine, cholesterol, and polyethylene glycol, and the lipid nanoparticle encapsulates Cas9mRNA and a sgRNA molecule in a gene conserved region of target mouse and human Proprotein Convertase subtilisin / kexin type 9 ( PCSK9). The present invention also discloses a preparation method and use of the lipid nanoparticle, and a pharmaceutical composition and formulation comprising the lipid nanoparticle. The lipid nanoparticle and the biological preparation can be used as a novel delivery system for a PCSK9 inhibitor hypolipidemic drug.

Description

technical field [0001] The invention relates to the field of medicine, in particular to the field of PCSK9-inhibiting hypolipidemic drugs, and more specifically to a novel delivery system and biological preparation of a novel PCSK9-inhibiting hypolipidemic drug. Background technique [0002] Cardiovascular disease is currently the leading cause of death in developed countries. With the improvement of living standards in our country and the change of related environmental factors, the incidence of cardiovascular disease has also been increasing year by year, gradually becoming a serious medical and social problem. Plasma low-density lipoprotein cholesterol (LDL-C) levels are closely related to the risk of cardiovascular disease. Although statins are currently the first choice for lowering LDL-C levels in clinical practice, some patients receive maximum doses Statins are still unable to effectively reduce blood lipid levels, and there are serious drug side effects in a small ...

Claims

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Application Information

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IPC IPC(8): C12N15/113A61K9/51A61K47/10A61K31/7088A61P3/06
CPCA61K9/5146C12N15/1137C12N2310/10
Inventor 谭旭董一洲蒋超梅淼祖文红李斌
Owner 谭旭
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