RNA interference (RNAi) has unlimited potential for therapeutic applications in regulating disease-associated proteins. The versatility of RNAi allows for treating a wide variety of targets with high specificity, making it an ideal complement to small molecule drugs. Despite on-going clinical trials utilizing short interfering RNA (siRNA) therapeutics for eye diseases, cancer, kidney disorders, and antiviral defenses, none have yet been approved for clinical use. Also, in the majority of these trials, delivery is accomplished through topical administration, localized injections, or systemic delivery to the liver or kidneys (natural filtering organs). Difficulties in development can be partly attributed to complications in optimizing delivery and maximizing function, while minimizing cytotoxicity and immunogenicity. To meet these challenges, this research aims to improve siRNA therapeutics through (1) optimization of the siRNA molecules and (2) development of more efficient vehicles for delivery of active siRNAs into cells.When utilizing the RNAi pathway, merely selecting siRNA sequences complementary to the messenger RNA (mRNA) target does not guarantee target silencing. Factors such as 5' end stability are critical for ensuring incorporation of the correct strand into the RNA induced silencing complex (RISC). Two methods for determining this asymmetry, the relative likelihood of incorporating one strand compared to the other, are the terminal end sequence and relative terminal thermodynamic stability. To address the first aim, we developed an algorithm for predicting highly active siRNA sequences based on only these two parameters. The algorithm successfully predicts weakly and highly active sequences for two test proteins, enhanced green fluorescent protein (EGFP) and protein kinase R (PKR). Going forward, we plan to characterize these and additional mechanistic factors that are valuable in predicting siRNA function.For clinical applications, the primary roadblock in siRNA therapeutic development is delivery. Using cationic polymers to deliver siRNAs has the potential to address this issue but is limited by low delivery efficiencies. It is still not clear what properties are needed to ensure the formation of active polymer-siRNA complexes. To address part of the second aim, we sought to determine key characteristics of effective delivery vehicles by analyzing differences between siRNAs delivered using novel polymeric nanoparticles (NPs) and those delivered by linear polyethyleneimine (LPEI) and Lipofectamine 2000 (LF2K), two known effective delivery vehicles. Our results showed that for LF2K and LPEI, large quantities of siRNA were delivered rapidly, presumably overwhelming the basal levels of mRNA to initiate silencing. In contrast, our novel polymeric NPs showed delivery of siRNAs but at initial concentrations too low to achieve silencing. Nonetheless, the exceptionally low cytotoxicity of our NPs, and their ability for easy modification, makes them good candidates for further study and optimization.Finally, in addition to delivery to cells, delivery efficiency is hindered by our lack of knowledge on how siRNA-vehicle complexes traffic across the plasma membrane and into the cytoplasm. To address this challenge, we have developed novel dextran functionalized silica solid-core NPs with the ability to effectively deliver siRNA to human lung cancer (H1299) cells expressing EGFP. Through modifications to the particle size and amine content, we were able to relate the chemical and physical characteristics of the particles to changes in cellular uptake, endocytotic trafficking, and ultimately silencing efficiency. Taken together, this research provides new information to guide the continued development of effective siRNA therapeutics.
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