The Study On Application Of Cationic Liposomes Through Non-lysosome Route In Transfection Of Stem Cells And Gene Editing Of Tumor Cells

Objective:A novel cationic liposome,Pardaxin liposome(Par-lipo)through non-lysosome route is constructed with Pardaxin polypeptide modification.The vector is applied to sten cell transfection and gene editing of cancer cells,and studying the transport paths of Par-lipo in these two processes.Evaluate the in vivo&in vitro gene delivery capability of the liposome by analyzing the efficiency of transfection and gene editing.Methods:A novel cationic liposome,Par-lipo,was prepared by film dispersion-ultrasonic method.Par-lipo was characterized by Transmission Electron Microscopy(TEM)and Dynamic Light Scattering(DLS).The capacity of Par-lipo to bind with DNA through electrostatic adsorption was investigated by agarose gel electrophoresis(AGE)and measuring the particle size and zeta potential of the Par-lipo/DNA complexes at different mass ratios.EGFP was selected as the reporter gene,and the plasmid encoding the EGFP gene was used in transfection to investigate the gene delivery efficiency of Par-lipo on stem cells.Then,GFP was taken as the target gene,and the knockout plasmid was constructed by using CRISPR/Cas9 gene editing system.Par-lipo mediated knockout was analyzed to investigate the efficiency of its in vitro gene editing system delivery.The knockout results were verified by T7EI.The intracellular transport pathway of Par-lipo during gene editing was studied by fluorescence localization.CDC6 was selected as the endogenous target gene to perform in vivo knockout experiments on two tumor models with high expression level of it:breast cancer and liver cancer,to investigate the gene editing efficiency of Par-lipo in tumor cells and evaluate its anti-tumor effect.Results:The modification of Pardaxin not only endows the liposome with the transport mode of non-lysosome route,but also makes it easier to be uptake by stem cells,significantly improving the transfection efficiency;in the delivery of CRISPR/Cas9 gene editing system,Par-lipo can not only directly deliver the loaded plasmids to the endoplasmic reticulum,but also enrich free nucleic acids to the endoplasmic reticulum,protect the integrity of sgRNA,promote its binding to Cas9,and facilitate the return of Cas9/sgRNA editing complexes to the nucleus,so as to obtain efficient gene editing.Conclusions:Par-lipo can transport through the non-lysosome route in cells,significantly improving the efficiency of stem cell transfection and in vitro&in vivo gene editing.Therefore,Par-lipo has a great application prospect in gene therapy.