(235a) Relationships between Molecular Properties of Polymer-Oligonucleotide Complexes and Cellular Antisense Activity

Antisense oligonucleotides present a powerful means to inhibit expression of specific genes. They are short, single-stranded oligonucleotides (ODNs) that bind to complementary mRNA via Watson-Crick base pairing. However, poor cellular delivery impedes their wide spread utilization in therapy and biotechnological research. Numerous delivery agents have already been developed to enhance their cellular uptake while also protecting them from degradation. Further improvements in the design of carriers for enhanced ODN delivery demand a better understanding of the role of the vector on the extent and time course of the antisense effect. The objective of this work is to obtain structure-property relationships that will aid in the rational design of new vectors, by correlating vector properties with the observed antisense effects. For this study, we chose a series of molecular weights (MWs) of the branched and linear forms of the widely studied cationic polymer, polyethylenimine (PEI). We also utilize various ODN backbone chemistries: phosphodiester, phosphorothioate and a mixed backbone ODN (a phosphodiester sequence modified with two phosphorothioate linkages at each end). Using a range of biophysical techniques, we studied the polymer-ODN molecular interactions and further evaluated their efficacy in delivering ODNs to cells. We accomplish this by measuring simultaneously the dynamics of both ODN uptake and antisense inhibition using a cellular assay based on single cell fluorescence measurements. We found that the extent and time scale of observed antisense effects was distinctly dependent on the particular combinations of the ODN chemistry and polymer properties. The trends in delivery and antisense activity with respect to polymer branching and molecular weight correlate in most cases with those of a biophysical assay involving heparin challenge of polymer-ODN complexes. The results from our work highlight several key factors that affect PEI mediated delivery of AS ODNs. In particular, our systematic variation of polymer and ODN properties enables us to perturb the dynamics of intracellular ODN availability and observe its effect on the appearance of the antisense effect. Further, our work also emphasizes the importance of the time scale of the DNA delivery process to the dynamics of observed antisense effects. Implications of these results for the rational design of novel vectors will be discussed.