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. Furthermore, p-shRNA exhibits immunostimulatory properties that can be combined with RNAi to dramatically enhance therapeutic efficacy. To develop an optimal delivery vehicle for op-shRNA, we used factorial design to synthesize a library of poly(beta-amino ester)s (PBAEs). Screening of this library showed that p-shRNA silencing efficiency increases with increasing alkyl side chain percentage and decreasing molecular weight. Our PBAEs 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. We further modified the complexes with a poly(ethylene glycol) (PEG)-PBAE copolymer containing alkyl side chains, introducing an outer PEG layer that can enhance colloidal stability and decrease cytotoxicity. By directing op-shRNA against signal transducer and activator of transcription 3 (STAT3), which promotes an immunosuppressive tumor microenvironment, we can significantly reduce tumor progression and prolong survival in a B16F10 melanoma model. 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 enable higher therapeutic efficacy in vivo through RNA interference.

Biodegradable periodic shRNA systems for enhanced gene silencing

256th National Meeting (2018)

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