(514a) A High-Throughput Method for Evaluating siRNA Delivery Materials In Vivo

RNA interference therapeutics represent a new class of gene-suppressing drugs which have marked potential to cure a wide variety of human diseases, including viral infections, heart disease, and cancer. The primary obstacle to the clinical application of siRNA is the development of safe and effective delivery system, and many laboratories are engaged in the design, synthesis, and evaluation of novel siRNA delivery materials. Typically, promising delivery vehicles are identified through a two-step screening process: first, materials are tested in vitro for transfection efficacy and any potential cytotoxicity. Then, leading candidates are formulated for in vivo use, and delivery to a target organ within an animal is evaluated. Although in vivo results are most relevant to clinical application, their cost along with their time-consuming, complicated nature limit the testing of all but the most promising materials.

Previously within our lab, several large libraries of lipid-like materials, termed ?lipidoids', were synthesized for use in siRNA delivery applications. These materials were evaluated using the methodology described above: lipidoids were initially screened for siRNA delivery efficacy and cytotoxicity in luciferase-expressing HeLa cells; then, select formulations were used to deliver anti-Factor VII siRNA to the liver in mice. A large number of our lipidoid materials have been tested both in vitro and in vivo, and through a comparison of the data sets, it has become apparent that in vitro transfection efficacy is often not indicative of in vivo transfection abilities. Interestingly, the most efficacious lipidoid identified to date, which is capable of silencing liver genes at doses as low as 0.01 mg/kg, exhibits minimal transfection ability in vitro.

This disconnect between cell culture and animal model experiments prompted us to re-examine our methodology for siRNA delivery material evaluation. In order to overcome the confusion caused by the translation of in vitro-in vivo experiments and to hasten the discovery of clinically relevant, suitable materials for RNA interference applications, a high-throughput in vivo method has been developed for delivery material evaluation. Traditionally, in vivo siRNA delivery experiments are performed by injecting one delivery vehicle with a specific siRNA, and gene silencing is assessed (either on the mRNA or protein level) only in the single organ which expresses that siRNA. Therefore, for every animal injection, traditional methods yield information for one delivery material in one organ. In contrast, we have developed a high-throughput method capable of testing up to 20 delivery materials in up to 5 organs with one injection. This method increases in vivo throughput by two orders of magnitude, and potentially offers the opportunity to test every delivery vehicle in vivo, significantly hastening the pace of viable delivery material discovery.