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SiRNA composition for inhibiting angiogenesis

An angiogenesis and composition technology, applied in the fields of molecular biology and biomedicine, can solve the problems of single target and insufficient curative effect, and achieve the effects of small side effects, reliable conclusions, and great clinical transformation prospects

Inactive Publication Date: 2022-07-29
WUHAN ZEZHI BIOLOGICAL PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In order to solve the defects of current anti-angiogenic drugs with a single target (only for VEGFA or its receptor extracellular domain) and insufficient curative effect, the present invention targets the key targets of neovascularization regulation, such as VEGFA, VEGFR1, VEGFR2 and VEGFR3 et al., designed angiogenesis-inhibiting siRNA compositions that specifically silence these cytokines

Method used

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  • SiRNA composition for inhibiting angiogenesis
  • SiRNA composition for inhibiting angiogenesis
  • SiRNA composition for inhibiting angiogenesis

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Experimental program
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Effect test

specific Embodiment 1

[0071] RT-qPCR was used to detect the silencing efficiency of the above siRNA sequences on target genes in primary human umbilical vein endothelial cells (HUVECs).

[0072] (1) siRNA transfection:

[0073] HUVECs by 1×10 5The density of cells / well was inoculated into 12-well plates, and the number of cells in each well was kept the same. After culturing for 12 hours, washed with PBS, replaced with ECM serum-free medium and starved for 6 hours, and recovered ECM complete medium (cell fusion). After culturing for 6 hours, the transfection group was transfected with 100 nM siRNA with Lipofectamine 2000: prepare two 1.5ml RNase-free EP tubes, add 120µL OPTI and 5µL Lipofectamine to a 1.5ml RNase-free EP tube 2000, add 120 µL OPTI and 5 µL siRNA to another 1.5 ml RNase-free EP tube, wait for 5 minutes, mix the two EP tubes, and wait for 15 minutes to form a transfection solution. Wash the cells twice with PBS, add 750µL OPTI to the transfection group, add 1mL OPTI to the blank gr...

specific Embodiment 2

[0085] siRNA inhibits angiogenesis in HUVEC cells.

[0086] (1) siRNA and combined siRNA samples:

[0087] The concentrations of siVEGFA, siVEGFR1, siVEGFR2, siVEGFR3 and other stock solutions were all 20 μmol·L / L, and equal volumes of siVEGFA stock solutions were mixed with equal volumes of siVEGFR1 stock solutions to obtain siVEGFA / siVEGFR1 containing siVEGFA / siVEGFR1 with a total siRNAs concentration of 20 mol·L / L. The combined siRNA solution, equal volume of siVEGFA stock solution and equal volume of siVEGFR2 stock solution were mixed to obtain a solution containing siVEGFA / siVEGFR2 with a total siRNAs concentration of 20 mol·L / L, and equal volume of siVEGFA stock solution was mixed with equal volume of siVEGFR3 and other stock solutions , to obtain a siVEGFA / siVEGFR3-containing solution with a total siRNAs concentration of 20 mol·L / L, and mix an equal volume of siVEGFR1 stock solution with an equal volume of siVEGFR2 and other stock solutions to obtain a siVEGFR1 / siVEGFR2...

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Abstract

The invention discloses a siRNA composition for inhibiting angiogenesis, which is a composition of a nucleic acid molecule for down-regulating VEGFA expression and a nucleic acid molecule for down-regulating VEGFR3 expression, or a composition of a nucleic acid molecule for down-regulating VEGFR1 expression and a nucleic acid molecule for down-regulating VEGFR2 expression. Or a composition of a nucleic acid molecule for down-regulating the expression of VEGFR1, a nucleic acid molecule for down-regulating the expression of VEGFR2 and a nucleic acid molecule for down-regulating the expression of VEGFR3. The invention can efficiently inhibit the generation of new vessels, including the total length of new vessels, the growth of branch vessels and the generation of vascular intersections; the application prospect of treating the vascular proliferation caused by the gene mutation and the diseases caused by the vascular proliferation is realized.

Description

technical field [0001] The invention belongs to the siRNA double-strand molecular structure and its combination technology in the technical field of molecular biology and biomedicine. Background technique [0002] The growth and maturation of new blood vessels is a highly complex and coordinated process requiring sequential activation of a series of ligand-receptor complexes, of which VEGF signaling is often a key rate-limiting step in physiological angiogenesis, VEGF Signaling is also important during pathological angiogenesis, such as the growth of blood vessels in solid tumors. The VEGF family includes members such as VEGFA, VEGFB, VEGFC, VEGFD, and placental growth factor PLGF, among which VEGFA is a key regulator of pathological angiogenesis, inducing tumor growth and angiogenesis, while VEGFC and VEGFD only regulate lymphangiogenesis. VEGF receptors are tyrosine kinase receptors, VEGFR1 and VEGFR2 are expressed in most vascularized tissues, while VEGFR3 is largely res...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/87C12N5/071A61K31/713A61P35/00A61P27/02
CPCC12N15/1136C12N15/1138C12N15/87C12N5/069A61K31/713A61P35/00A61P27/02C12N2310/141C12N2501/165C12N2501/65A61K2300/00
Inventor 季爱民岑柏宏陈佳扬黎国仙陈漳浦杨伶
Owner WUHAN ZEZHI BIOLOGICAL PHARMA
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