RNA interference (RNAi) provides a versatile therapeutic strategy via silencing of specific genes implicated in cancer and other diseases. Small interfering RNAs (siRNAs), which are double stranded RNAs typically 19-21 base pairs in length, have been extensively explored as RNAi drugs, but challenges in delivery to disease sites have hampered clinical translation, 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. As a result, excess amounts of cationic delivery materials are often required, leading to dose-limiting toxicities. Periodic short hairpin RNAs (p-shRNAs), consisting of repeating RNAi sequences linked together, can potentially address these delivery barriers by improving complexation with polycations. 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 silencing efficiencies at polymer 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 have found that PBAEs of low to intermediate molecular weight and increasing alkyl chain content deliver op-shRNA in vitro most efficiently, generating levels of gene knockdown comparable to those by commercial transfection reagents. Furthermore, we can enhance the stability of the complexes in physiological conditions by addition of a poly(ethylene glycol) (PEG)-PBAE copolymer. The complexes formed from combinations of our optimized PBAEs and a PEGylated PBAE, via electrostatic and hydrophobic interactions, possess an outer PEG layer that can provide stability in the bloodstream and thereby increase tumor accumulation. Thus, 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.