In vitro investigation of factors important for the delivery of an integrin-targeted nonviral DNA vector in organ transplantation

Background. Polylysine-molossin is a 31 amino acid synthetic peptide that h as previously been demonstrated to function as a DNA vector in vitro for ce ll lines and for the cornea. It incorporates the 15 amino acid integrin-bin ding domain of the venom of the American pit viper, Crotalus molossus molos sus as the targeting moiety and a chain of 16 lysines as the DNA-binding mo iety. The objective of this study was to evaluate several parameters of imp ortance for in vivo applications. Methods. Binding and tissue distribution of the vector/DNA complexes were f ollowed by a monoclonal antibody to the vector, or by the use of fluorescei n-labeled DNA, Standard in vitro transfections were used to monitor effecti ve gene transfer. Results. (1) Optimal DNA/vector concentration. Saturation of vector/DNA bin ding sites on the ECV304 cell line occurred at 6 mu g/ml of DNA. The concen tration of vector/DNA complexes required for optimal gene transfection was found to be 2-8 mu g/ml of DNA, corresponding to the concentration needed f or saturation binding. (2) Optimal target cell exposure time. Vector/ DNA c omplexes saturated target cell binding sites within 5 min of incubation. Ho wever, lengthy exposure times (>2-3 hr) to the transfection medium were ess ential for substantial gene transfer. This was a consequence of two complem entary factors. First, it was important that target cells be exposed to vec tor/DNA complexes for similar to 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) Inhibito ry effects of serum. Exposure of complexes to even 1% serum before transfec tion, markedly inhibited gene transfer. However, target cells previously sa turated with vector/DNA complexes and then exposed to 10% serum showed subs tantial gene transfer. (4) Extravasation and binding stability in vivo. Col d ex vivo perfusion of rat hearts with vector/DNA complexes demonstrated th at little, if any, complex moved out of the vascular system. After transpla ntation 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 i n vitro need to be paid for optimal in vivo application of DNA vector syste ms.