Role of SNP Characteristics on the Endocytosis and Intracellular Trafficking of siRNA

Short interfering RNA (siRNA) therapeutics, have garnered considerable attention for their potential to treat a wide variety of disease-associated proteins through the use of RNA interference (RNAi). siRNA therapeutics have been developed for cancers, genetic disorders, and infectious diseases, but their progress through clinical pipelines has been limited by delivery vehicles that are toxic, immunogenic, or ineffective. Despite the variety of delivery vehicles that have been developed, the essential chemical and physical characteristics of effective delivery vehicles remain unknown. It is also unclear if RNAi is initiated more potently following endocytosis by particular mechanisms and if this variability is specific to a certain cell type or delivery vehicle chemistry.

Our goal is to define nanoparticle characteristics that are essential for potent siRNA delivery. Using silica nanoparticles (SNPs) for their tunable synthesis, we are investigating delivery criteria in four main categories: sequence specific mRNA degradation (silencing), membrane translocation, endosomal escape, and association with RNAi proteins. Using SNPs, vehicle size, structure, charge, and surface functionalization can be varied to determine optimal vehicle design criteria in a variety of cell types.

The presentation will discuss our novel live cell assay that allows independent intracellular trafficking of delivery vehicle, antisense, and sense siRNA. Using confocal microscopy, we track vehicle-siRNA complexes during endocytosis (whether by Clathrin, Caveolin, ARF6, GRAF1, Flotillin, and Macropinocytosis), endosomal trafficking (Early, Late, Recycling, and Lysosome), trafficking to the endoplasmic reticulum or golgi, and exocytosis. Our results to date demonstrate that multiple vehicle characteristics affect siRNA delivery and demonstrate the critical role of intracellular trafficking in maximizing siRNA potency.