(227f) Optimization of Liposome-Encapsulated siRNA for Viral Gene Silencing

Inadequate nucleic acid delivery efficiency, caused by in vivo barriers such as nonspecific tissue interactions and extracellular and intracellular degradation, is the greatest obstacle to effective gene therapy.  In particular, an important gene therapy tool used for specific silencing of gene expression, siRNA, is especially sensitive to extracellular degradation.  Using a liposome formation method previously developed for plasmid DNA encapsulation, in which nucleic acids are first condensed into nanoparticles using a cationic polymer and then encapsulated in a lipid bilayer, siRNA was encapsulated for protective delivery in stealth liposomes.  It has been found that the size and charge of siRNA nanoparticles can be controlled by changing the siRNA:polymer ratio, and plasmid DNA encapsulation can be controlled by changing lipid film content.  Therefore, siRNA nanoparticles made with varying siRNA:polymer ratios were encapsulated in liposomes of varying lipid film content to optimize both siRNA yield and fraction of siRNA loaded liposomes.  siRNA designed to silence the expression of the E7 gene, a human papillomavirus gene associated with tumorigenesis in HeLa cells, was delivered to HeLa cells via stealth liposome-encapsulated siRNA. In order to understand the effect of the material properties of liposome-encapsulated siRNA on transfection efficiency, liposomes made with various siRNA:polymer ratios and load fractions were delivered to cells, and mRNA silencing and cancer cell death was measured.  Understanding how delivery vehicle assembly affects both vehicle properties and siRNA transfection efficiency is essential for design of effective in vivo gene delivery schemes.