Following the discovery of the RNA interference (RNAi) mechanism by Fire, Mello and colleagues in the late 1990s [1], there has been explosive interest in the application of short, interfering RNA (siRNA) in the treatment of genetic diseases. These duplex nucleic acids, about 21 base pairs in length, are capable of down regulating harmful genes in mammals with high sequence-specificity but they require a safe and effective delivery vehicle to carry them to the site of action. An ideal carrier would be able to protect the siRNA from enzymatic degradation in the blood stream, be selectively internalized by target cells and then release the siRNA only once it has reached the cytosol, where it can initiate the RNAi pathway. Polymers, particularly polycations, are very popular non-viral gene carriers because they can spontaneously associate with the negatively-charged phosphate backbone of nucleic acids to form “polyplexes”. However, among the drawbacks of these systems is that under physiological salt conditions, where repulsive forces between like-charged particles are screened, aggregation of cationic polyplexes can result [2]. Conjugation of poly(ethylene glycol), or PEG, to cationic polymers has been shown to confer several advantageous properties including colloidal stability and reduced aggregation of complexes due to steric shielding [2-4] and increased hydrophilicity/water solubility [4]. These advantages come at price, however, as “PEGylation” is also known to weaken the strength of binding between polycations and plasmid DNA [3-9], leaving the DNA susceptible to enzymatic degradation [4]. Fortunately, polymeric vehicles designed for delivery of DNA for gene therapy have been widely studied over the years, and knowledge from this field has been instrumental in identifying issues in siRNA delivery. In a recent study, we sought to evaluate the potential of three polycation-based gene carriers by studying their relative strengths of binding to siRNA, enzymatic protection and cytotoxic effects. This was illustrated with the use of two conventional gene-delivery approaches, (1) the polycationic homopolymer, poly(2-(dimethylamino)ethyl methacrylate) or PDMAEMA, and (2) a PEGylated polycation, PEG-PDMAEMA. Also, we introduce (3) a micelle derived from an A-B-C triblock copolymer, PEG-PnBA-PDMAEMA (PnBA denotes poly(n-butyl acrylate)). The micelle chemistry we present here has not been previously reported in the literature and differs from other micelle-based approaches because the polycation is indirectly connected to the PEG block through a hydrophobic segment. Contrary to what has been shown for DNA, our studies indicate that in the case of siRNA, PEGylation of polycations does not weaken electrostatic interactions nor promote enzymatic degradation when compared to their un-PEGylated counterparts. We hypothesize that because of molecular weight and/or chemistry, siRNA associates with polymers in a way such that the formed complexes are not adversely affected by PEGylation. Although the triblock micelles have demonstrated a performance comparable to the other formulations with respect to cytotoxicity and in their ability to bind and protect siRNA from enzymatic degradation, we expect that they will outperform the other formulations in the areas of in-vivo pharmacokinetics and cellular transfection. Because homopolymer and diblock complexes with siRNA are significantly smaller than micelle/siRNA complexes (<10 nm vs. ~70 nm), they have a higher probability of being eliminated from the bloodstream before eliciting their biological effects. Thus, micelle-based vehicles appear to be more promising candidates for efficient systemic delivery of gene silencing siRNA when compared with conventional polymer-based approaches. If time allows, we will also discuss our results from in vitro and in vivo gene silencing experiments. References 1. Fire, A., et al., Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature, 1998. 391(6669): p. 806-811. 2. Mishra, S., P. Webster, and M.E. Davis, PEGylation significantly affects cellular uptake and intracellular trafficking of non-viral gene delivery particles. European Journal of Cell Biology, 2004. 83(3): p. 97-111. 3. Glodde, M., S.R. Sirsi, and G.J. Lutz, Physiochemical properties of low and high molecular weight poly(ethylene glycol)-grafted poly(ethylene imine) copolymers and their complexes with oligonucleotides. Biomacromolecules, 2006. 7(1): p. 347-356. 4. Mullen, P.M., et al., Strength of conjugate binding to plasmid DNA affects degradation rate and expression level in vivo. Biochimica Et Biophysica Acta-General Subjects, 2000. 1523(1): p. 103-110. 5. Pun, S.H. and M.E. Davis, Development of a nonviral gene delivery vehicle for systemic application. Bioconjugate Chemistry, 2002. 13(3): p. 630-639. 6. Nguyen, H.K., et al., Evaluation of polyether-polyethyleneimine graft copolymers as gene transfer agents. Gene Therapy, 2000. 7(2): p. 126-138. 7. Gary, D.J., N. Puri, and Y.Y. Won, Polymer-Based siRNA Delivery: Perspectives on the Fundamental and Phenomenological Distinctions from Polymer-Based DNA Delivery. accepted in Journal of Controlled Release, 2007. 8. Garrett, S.W., et al., Synthesis and characterisation of polyamine-poly(ethylene glycol) constructs for DNA binding and gene delivery. Bioorganic & Medicinal Chemistry, 2000. 8(7): p. 1779-1797. 9. Oupicky, D., R.C. Carlisle, and L.W. Seymour, Triggered intracellular activation of disulfide crosslinked polyelectrolyte gene delivery complexes with extended systemic circulation in vivo. Gene Therapy, 2001. 8(9): p. 713-724.

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