Slow accumulation of plasmid in muscle cells: Supporting evidence for a mechanism of DNA uptake by receptor-mediated endocytosis

Intramuscular plasmid DNA injection results in long-term but low and variab le expression of the injected genes. Optimization is difficult because the mechanism of naked DNA uptake by the cells in vivo is not yet determined. H ere we used injections of plasmid DNA encoding luciferase to further charac terize this mechanism. We analyzed the kinetics of naked DNA uptake by mean s of DNase I or heparin injections, using the level of luciferase expressio n as the indicator of DNA uptake. We demonstrated that in vivo heparin inhi bits DNA uptake without affecting the expression of DNA internalized by mea ns of electric pulses. Inhibition by heparin is dose dependent and compatib le with the competition for the binding to a receptor. As shown also with D Nase 1, DNA uptake by muscle cells is slow: a progressive accumulation of t he DNA in the myofibers can be found for at least 4 hours after naked DNA i njection. Physical presence of DNA molecules during the uptake period, but not later, was confirmed by the facilitation of DNA uptake with appropriate electric pulses. Therefore, uptake proceeds for the entire time during whi ch intact DNA is present in the extracellular compartment. Our results supp ort evidence for a DNA uptake mechanism based on receptor- mediated endocyt osis.