Biomolecular engineering of siRNA therapeutics

RNA interference (RNAi) provides a powerful means for regulating gene expression. Although it is a relatively recent discovery, it has already proven useful in identification of gene/protein functions and is in the process of being utilized for disease treatment in humans. Continued use of this pathway for a variety of applications would benefit from a more thorough understanding of the role of the initiator molecules of RNAi, small interfering RNA (siRNA). As the efficacy of siRNAs that mediate RNAi is known to vary greatly, the aim of this thesis was to study three steps of the pathway that pose barriers to identifying and developing highly active siRNA molecules for use as therapeutic agents.;The first of these factors investigated in this research was the effect of secondary structure within the mRNA site targeted by siRNA. The results obtained from both experimental and computational studies show that sites with extensive mRNA secondary structure were less susceptible to silencing, while those containing unpaired nucleotides at either the 5'- or 3'-end were generally more amenable. Similar observations were made when taking into account siRNA guide strand structure. Taken together, there is a correlation between RNA structure and silencing efficiency that ultimately can be included in existing and future siRNA selection algorithms for the improved identification of active siRNAs, thus reducing the number of sequences to be tested before selecting one capable of significantly silencing the target gene.;The second aspect of RNAi explored here was the influence of siRNA sequence and hybridization stability on recognition by the TAR RNA binding protein (TRBP). This protein is part of the RNA induced silencing complex (RISC) responsible for targeting and cleaving mRNAs to achieve silencing. It was found that TRBP can detect the overall and relative stability of the two ends of the siRNA by interacting primarily with the more stable end, and that the interaction is similarly reflected when binding to single-stranded RNAs alone. Additionally, TRBP interactions can be altered through the inclusion of mismatches within the siRNA sequence or DNA substitutions. These observations suggest a role for TRBP in the mechanism of RISC formation as well as a means to improve siRNA functionality through modification of the RNA or its sequence.;The third focus was to develop a biocompatible and biodegradable cationic polymer capable of delivering siRNA into cells grown in vitro, with potential use later for in vivo applications. These nanoparticles (NPs) were generated with "click" chemistry, allowing for a systematic study of a range of variables contributing to siRNA binding. Strong siRNA binding required a NP containing a combination of primary and secondary amine groups to facilitate electrostatic interactions. Inclusion of alkyl chains enhanced binding, perhaps by causing vesicle-like formation, while polyethylene glycol (PEG) reduced overall binding affinity, consistent with its known charge shielding effects. The NPs were capable of delivering siRNAs to cells, but were unable to release them to initiate RNAi. Collectively, the work presented here provides a framework for identifying and engineering more active siRNA molecules by taking into account mRNA target structure and TRBP binding preferences, as well as achieving efficient cellular uptake of such engineered molecules with a novel type of polymeric nanoparticle.