Targeted Gene Delivery System Using Ionizable Cationic Lipids

Ionizable cationic lipids were designed to have headgroups with p Ka from 6.0 to 7.0.The lipids would have different net charges at different p Hs.At p H4.0,the headgroups are positively charged to enable efficient binding to nucleic acids,while at p H7.4,they would deprotonate and maintain a close to neutral charge density.Such characteristics were suggested to also help nucleic acids release from endosomes or lysosomes.Several ionizable lipids have been developed successfully for si RNA delivery and were shown to be able to achieve high efficiency and good safety in vivo.We would like to use DLin-MC2-MPZ,an ionizable lipid developed in our lab,to construct a DNA delivery system for use in cancer gene therapy.The R-spondin1 protein was used as the targeting ligand for specific interactions with the LGR family proteins,which are frequently up-regulated in cancer cells,especially cancer stem cells.Two different ligand conjugation methods were used: the direct-coupling method and post-insertion method.The resulted RSPO1-lipoplexes were examined for their binding and uptake by LGR+ cells,using FACS and confocal microscopy.These targeted lipoplexes showed very efficient binding and rapid uptake by Lo Vo cells and A549 cells,however the reporter gene transfection efficiency was still low.We then further evaluated the different processes involved during RSPO1-lipoplex preparation and our data suggested the RSPO1 conjugation method may be critical to DNA release and transfection in cell culture..In summary,we had developed a highly reproducible method to formulated DNA delivery systems and demonstrated their efficacies in specific cell experiments.Based on our studies,it may be possible to construct gene delivery systems carrying therapeutic genes to cancer cells and other diseased tissues.Further studies are ongoing to evaluating the gene delivery efficiencies and specificities in vivo using these systems.