(326g) Award Submission: Effect of Uptake Pathway On Non-Viral Gene Delivery in Vitro and in Vivo

Efficient non-viral gene delivery often involves the conjugation of a cell-specific ligand to the vector, which directs the vector to its intended target. However, little is known in terms of how the process of endocytosis affects the performance of the delivery vehicle. Previously, we have demonstrated that caveolin-mediated endocytosis is important to in vitro polyethylenimine (PEI)-mediated gene delivery in HeLa cells through the use of small molecule drugs and small-interfering RNA. The goal of this project is to elucidate the effects of cellular uptake mechanism on non-viral gene delivery in vitro and in vivo utilizing small hairpin RNA (shRNA) Tet-on system. Specifically, we are investigating the efficacy of PEI-mediated gene delivery upon internalization via clathrin-dependent endocytosis or caveolin-dependent endocytosis in HeLa and MDA-MB-231 cell cultures and xenograft tumors. We attached transferrin and folate ligands to PEI to direct internalization by clathrin- and caveolin-mediated mechanisms, respectively, and transfected HeLa and MDA-MB-231 cells with targeted and untargeted PEI in the presence of small molecule drugs that inhibit clathrin- or caveolin-mediated endocytosis. We have demonstrated that caveolin-dependent endocytosis is important to successful PEI-mediated gene delivery in both HeLa and MDA-MB-231 cells. In order to further understand the impact of endocytic pathways on gene delivery efficiency in vitro, we have modified HeLa and MDA-MB-231 cells to express small hairpin RNA (shRNA) that target clathrin, caveolin, or lamin A/C (negative control) protein expression (HeLa-CLTC, HeLa-CAV, HeLa-LAM, 231-CLTC, 231-CAV, and 231-LAM)  in the presence of tetracycline. Using the shRNA Tet-on system, we are able to demonstrate ~90% clathrin knockdown and ~60% caveolin knockdown in both modified HeLa and MDA-MB-231 cells.  Using this shRNA knockdown mechanism, we have confirmed that caveolin-dependent endocytosis is more important than clathrin-dependent endocytosis in PEI-mediated gene delivery, which corroborates the results observed in unmodified cell lines. We will also report the effect of clathrin-dependent and caveolin-dependent endocytosis on in vivo PEI-mediated gene delivery in a xenograft murine tumor model.  Such studies of intracellular trafficking and its effect on PEI gene delivery efficacy in cancer cell lines and tumors enable researchers to design more efficient non-viral gene delivery vectors for cancer gene therapy, by synergistically combining the design of cell targeting ligands and the understanding of cellular uptake mechanism for polymeric carriers.