Background: Polylysine-molossin is a 31 amino acid synthetic peptide that has previously been demonstrated to function as a DNA vector in vitro for cell lines and for the cornea. It incorporates the 15 amino acid integrin-binding domain of the venom of the American pit viper, Crotalus molossus molossus as the targeting moiety and a chain of 16 lysines as the DNA-binding moiety. The objective of this study was to evaluate several parameters of importance for in vivo applications.
Methods: Binding and tissue distribution of the vector/DNA complexes were followed by a monoclonal antibody to the vector, or by the use of fluorescein-labeled DNA. Standard in vitro transfections were used to monitor effective gene transfer.
Results: (1) Optimal DNA/vector concentration. Saturation of vector/DNA binding sites on the ECV304 cell line occurred at 6 microg/ml of DNA. The concentration of vector/DNA complexes required for optimal gene transfection was found to be 2-8 microg/ml of DNA, corresponding to the concentration needed for saturation binding. (2) Optimal target cell exposure time. Vector/ DNA complexes saturated target cell binding sites within 5 min of incubation. However, lengthy exposure times (>2-3 hr) to the transfection medium were essential for substantial gene transfer. This was a consequence of two complementary factors. First, it was important that target cells be exposed to vector/DNA complexes for approximately 1 hr at 37 degrees C. Saturation of target sites at 4 degrees C and then removal of the transfection medium was much less effective. Second, exposure to chloroquine for 8-10 hr after uptake of vector/DNA complexes was essential for optimal gene transfer. (3) Inhibitory effects of serum. Exposure of complexes to even 1% serum before transfection, markedly inhibited gene transfer. However, target cells previously saturated with vector/DNA complexes and then exposed to 10% serum showed substantial gene transfer. (4) Extravasation and binding stability in vivo. Cold ex vivo perfusion of rat hearts with vector/DNA complexes demonstrated that little, if any, complex moved out of the vascular system. After transplantation of the heart, most of the complex bound to the vasculature was lost within 30 min of reestablishing the blood circulation.
Conclusions: Careful attention to several parameters of little importance in vitro need to be paid for optimal in vivo application of DNA vector systems.