(20c) Engineering Periodic shRNA Delivery Systems with High Silencing Efficacy

Challenges in the delivery of small interfering RNAs (siRNAs) to disease sites have hampered clinical translation of RNA interference, particularly in cancer therapeutics. Due to its low valency and high rigidity, siRNA is not readily condensed into stable nanoparticles that can successfully protect it from nuclease degradation and deliver it into target cells. Periodic short hairpin RNAs (p-shRNAs), consisting of repeating RNAi sequences linked together, can potentially address these delivery barriers by improving complexation with cationic delivery materials. Ranging from under 200 to over 5000 nucleotides in length, p-shRNA can be generated in large quantities through rolling circle transcription, in which an RNA polymerase repeatedly traverses a small circular DNA template. We have engineered these p-shRNA molecules via template design and selective enzymatic digestion of their hairpin loops to generate an open-ended p-shRNA (op-shRNA) that induces greater gene silencing than p-shRNA and siRNA in multiple cancer cell lines up to nine days.

The high valency and flexibility of op-shRNA dramatically improves complexation with low molecular weight polycations compared to siRNA. To develop a delivery vehicle optimized for the biophysical properties of op-shRNA, we have synthesized a library of biodegradable poly(beta-amino ester)s (PBAEs) via factorial design. Our designed PBAEs condense op-shRNA into compact nanoparticles below 100 nm in diameter, and demonstrate high transfection efficiencies at polymer and RNA concentrations much lower than typically required for PBAE-mediated gene delivery. By varying the molecular weight and incorporation of alkyl chains in our PBAE library, we were able to develop correlations between these parameters and multiple parameters influencing op-shRNA delivery, including complex assembly, stability, intracellular disassembly, cellular uptake, and cytotoxicity. In particular, PBAEs of low to intermediate molecular weight and increasing alkyl chain content delivered op-shRNA in vitro most efficiently, generating levels of gene knockdown comparable to those by commercial transfection reagents. Through nucleic acid engineering and rational carrier design, we have successfully developed a stable, potent RNAi delivery platform that can trigger significant gene silencing at low doses, and potentially enable higher therapeutic efficacy in vivo.