Optimization of nonviral gene transfer of vascular smooth muscle cells in vitro and in vivo

Gene therapy strategies for the prevention of restenosis postangioplasty ar e promising. Nonviral gene transfer to the arterial wall in vivo has so far been limited by poor efficiency. This study aimed to optimize transfection of primary vascular smooth muscle cells using cationic nonviral formulatio ns based on cholesterol derivates (DC-, DAC-, DCQ-, and Sp-Chol), double-ch ained amphiphils (LipofectAMINE, DOTMA, DOSGA, DOSPER, and DOCSPER), or het erogeneous reagents (Superfect, Effectene, and Tfx-50). Estimation of trans fection efficiencies was performed using galactosidase assays at different ratios of transfection reagent to plasmid DNA with reporter gene. Toxicity was monitored by analyzing cell metabolism. Transfer efficiency and safety were determined in a porcine restenosis model for local gene therapy using morphometry, histology, galactosidase assays, and reverse-transcriptase pol ymerase chain reaction. The highest in vitro transfection efficiency was ac hieved using the recently developed DOCSPER liposomes, with transfer rates of at least 20% in vascular smooth muscle cells. Transfer efficiency was fu rther enhanced up to 20% by complexing with poly-L-lysine. Transfection eff iciency in vivo in a porcine restenosis model was up to 15% of adventitial cells using DOCSPER versus 0.1% using LipofectAMINE. Toxicity in vivo and i n vitro was lowest using DOCSPER. Increased biological effects were demonst rated following optimization of transfer conditions.