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siRNA molecules that enhance the silencing efficiency of target genes and their applications

A gene-targeting and silencing technology, applied in DNA/RNA fragments, medical preparations containing active ingredients, genetic engineering, etc., can solve the problems of low potency, complicated preparation process and high preparation cost of siRNA molecule delivery system

Active Publication Date: 2021-02-09
WUHAN ZEZHI BIOLOGICAL PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Viral delivery systems are not suitable for clinical application due to their immunogenicity and tumorigenicity; non-viral delivery systems such as cationic polymer nanoparticles and nanoliposome preparations have complex preparation processes, high preparation costs, and poor formulations. Defects such as stability and high toxicity in vivo application directly restrict the clinical transformation of siRNA molecular nanoparticle drug delivery system (Lee SH, et al. Advanced Drug Delivery Reviews. 2016;104:78-92.)
Not only that, the key and bottleneck problem commonly faced by siRNA molecule delivery systems is that even though siRNA molecules are taken up by cells through the drug delivery system, they are easily encapsulated into endosomes and lysosomes, and only a small part (<0.01 %) successfully released into the cytoplasm to exert gene silencing effect, thus resulting in low titer of siRNA molecule delivery system, which directly leads to limited gene silencing activity

Method used

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  • siRNA molecules that enhance the silencing efficiency of target genes and their applications
  • siRNA molecules that enhance the silencing efficiency of target genes and their applications
  • siRNA molecules that enhance the silencing efficiency of target genes and their applications

Examples

Experimental program
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Effect test

Embodiment 1

[0056] Embodiment 1, siRNA preparation:

[0057] The present invention refers to the methods provided by Elbashir and Reynolds et al. (Elbashir SM, et al.Methods.2002; 26:199-213. and Reynolds A, et al.Nature Biotechnology. 2004; 22:326-330), designed and synthesized Multiple candidate siRNA molecular sequences. Blast comparative analysis shows that the siRNA sequence designed in the present invention has no homology with other genes other than the target gene.

[0058] The target genes selected for the preparation of siRNA in the present invention are Vascular Endothelial Growth Factor Receptor 2 (VEGFR2), Phosphoinositide 3-Kinase Catalytic Beta Polypeptide (PIK3CB), and epidermal Growth factor receptor (Epidermal Growth Factor Receptor, EGFR). These cytokines are closely related to cell growth and survival. VEGFR2 is expressed at a high level on the surface of neovascular endothelial cells, and angiogenesis runs through the whole process of malignant tumors, affecting tu...

Embodiment 2

[0062] Example 2, modifying the stability of the siRNA molecular structure to increase its gene silencing activity:

[0063] siRNA molecules have nucleic acid-like structural properties. When used as drug molecules in the body, they will be degraded by nucleoside enzymes in body fluids and cells. The stability modification of each base of siRNA nucleotides can effectively prevent nucleosides in the body. Enzymatic degradation prolongs the action time in vivo. However, the modification of nucleotides may reduce the gene silencing activity of the entire siRNA molecule, increase the clinical dosage, and produce new adverse reactions, which is not conducive to clinical transformation. The modification rule found in the present invention has the following characteristics: 1) In the double strand, except for the 3' terminal base of the positive sense strand, the 2'-OH position on the pentose sugar structure in all nucleotides is replaced by 2'-fluoro (2' -F) or 2'-methoxy (2'-OMe) ...

Embodiment 3

[0064] Example 3, tissue targeting molecules modify siRNA molecules to increase their gene silencing activity:

[0065]siRNA molecules have the structural characteristics of nucleic acid molecules. Even if the structure is modified by the above-mentioned modification method, it cannot actively enter the cell to exert gene silencing activity. It must be assisted by a drug carrier to enter through receptor-mediated or cell membrane fusion. biological activity in cells. The present invention provides tissue-targeting structural molecules to modify siRNA molecules at one or both ends of the sense strand, which can enhance the gene silencing efficiency of the siRNA molecular structure, and has the following properties: 1) biRGD-siRNA can target a high level of integrin αvβ3 receptor The expressed neovascular endothelial cell membrane and part of the tumor cell membrane endocytose the siRNA carried into the cell through a receptor-mediated manner, thereby exerting the gene silencing...

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Abstract

The invention discloses an siRNA molecule that enhances the silencing efficiency of a target gene. Except for the 3' terminal base of the sense chain, the 2'-OH positions on the pentose sugar structures in all nucleotides are replaced by 2'-fluorine or 2 '-methoxyl substitution; the bases at the 5' end of the antisense strand are modified by phosphorylation, the odd-numbered bases after the start are modified with 2'-methoxy, the even-numbered bases are modified with 2'-fluorine, and the antisense The first three bases from the 3' end of the sense strand that are complementary to the target gene are all modified with 2'-methoxy groups; the 3' of the antisense strand of the siRNA molecule can be linked with 1-10 deoxy groups by phosphate bonds or phosphorothioate bonds Thymine, and ensure that it contains 2 phosphorothioate bond modified nucleotide backbones. The present invention can be used to silence the gene expression of cytokines VEGFR2, PIK3CB or EGFR, modify siRNA molecules with tissue-targeting molecules to improve transfection efficiency, inhibit the function of cytokines related to endosomal plasma membrane circulation, and increase their gene silencing efficiency; at the same time, these siRNA molecules Used alone or in combination, they can be used to treat diseases caused by gene mutation or overexpression.

Description

technical field [0001] The present invention relates to the nucleotide sequence of the small interfering RNA molecule and its technology for enhancing gene silencing. Background technique [0002] Small interfering RNA (Small interfering RNA, siRNA) is the effector molecule of the RNA interference theory (2006 Nobel Prize in Physiology and Medicine), which can specifically silence overexpressed and mutated genes that cause diseases in the cytoplasm or nucleus, and has the potential to treat diseases Application prospects (Sullenger B A, et al. Science. 2016; 352(6292):1417-1420.). The siRNA molecule is a completely symmetrical double-stranded structure or an asymmetric double-stranded structure formed by two single-stranded RNA molecules due to the complete complementarity or partial complementarity of the sequence, one of which can be partially or partially compatible with the nucleotide sequence of the target RNA in the cell Completely complementary, known as the guide st...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113A61K31/713A61P35/00
CPCA61K31/713A61P35/00C12N15/113C12N2310/14C12N2310/321C12N2310/322
Inventor 季爱民岑柏宏黄文黎权辉
Owner WUHAN ZEZHI BIOLOGICAL PHARMA