(16d) Peg-Poly(beta-amino ester) Delivery Systems for Periodic shRNA

RNA interference (RNAi) provides a versatile therapeutic strategy via silencing of specific genes implicated in cancer and other diseases. However, clinical translation of RNAi for cancer therapeutics remains unrealized, due to challenges in delivery of small interfering RNA (siRNA) to tumors. The low valency and high rigidity of siRNA often requires high excesses of cationic delivery materials to condense into stable nanoparticles, leading to dose-limiting toxicities. To address this challenge, we adopt an RNAi platform based on periodic short hairpin RNAs (p-shRNAs). Consisting of siRNA sequences linked together, these polymeric RNAi molecules are generated by the repeated action of an RNA polymerase around a small circular DNA template. Through template design and selective enzymatic digestion, we can design p-shRNA structures that are efficiently processed inside cells into siRNAs and induce significant gene silencing.

With its much higher valency and flexibility compared to siRNA, p-shRNA requires far less polycationic material to be condensed, and forms more stable complexes. Poly(beta-amino ester)s (PBAEs) in particular have shown promise as gene delivery vehicles, due to their biodegradability and design versatility. To develop an optimal PBAE structure for p-shRNA delivery, we used factorial design to synthesize a library of PBAE variants of a base structure, poly-1. Screening of this library showed that p-shRNA silencing efficiency increases with increasing alkyl side chain percentage and decreasing molecular weight. Our designed poly-1 structures are able to fully condense p-shRNA into sub-100 nm complexes with high silencing efficiency, at much lower polymer-to-RNA ratios than those typically required for PBAE gene delivery. To enhance the colloidal stability of the complexes in physiological conditions, we added a poly(ethylene glycol) (PEG)-poly-1 copolymer containing alkyl side chains to the core complex. The resulting complexes, assembled via electrostatic and hydrophobic interactions, possess an outer PEG layer that can provide stability in the bloodstream and thereby increase tumor accumulation. We further introduced active tumor targeting by conjugating folate to the PEG terminus. Expanding our library to screen combinations of the core poly-1 and PEG-poly-1 structures revealed an optimal blend of hydrophobicities and molecular weights in the two polymer components. Thus, through nucleic acid engineering and rational carrier design, we have successfully developed a stable, potent RNAi delivery system that can trigger significant gene silencing at low doses, and potentially enable higher therapeutic efficacy in vivo.