Polycations and cationic lipids enhance adenovirus transduction and transgene expression in tumor cells

Abstract

Replication-deficient adenovirus vectors are efficient vehicles for delivering therapeutic genes into mammalian cells. However, the high doses required to produce effective gene transfer in vivo can also cause unwanted cellular toxicity. To improve replication-deficient adenovirus transgene expression while minimizing adverse reactions, we have tested polycationic compounds for their ability to enhance adenovirus adsorption. We demonstrate increased transgene expression after mixing adenovirus preparations with polycations, cationic lipids, and CaCl₂ prior to transduction in vitro. An E1-deleted adenovirus vector was admixed with various polycations, and β-galactosidase (β-gal) activity was evaluated. The optimal polycation concentrations for augmenting adenovirus-mediated gene transfer were 5–10 μg/mL polybrene, 400 μg/mL protamine sulfate, 10 μg/mL N-(1-[2,3-dioleoyloxy]propyl)-N,N,N-trimethylammonium methylsulfate (DOTAP), 2.5 μg/mL Lipofectamine, and 62.5 mM CaCl₂. Polycations enhanced β-gal expression in three of six established cell lines. Similar results were obtained using primary tumor cell cultures, where β-gal expression was increased 1.5- to 10.7-fold (mean = 3.6) by polybrene, 1.8- to 7.5-fold (mean = 3.4) by DOTAP, and 2.3- to 10.4-fold (mean = 4.8) by protamine sulfate. Adenovirus transduction efficiency in two primary leukemia isolates was improved by 3- and 4.5-fold. We were unable to demonstrate any benefit when adenovirus was admixed with protamine sulfate prior to intratumoral injection in a xenogeneic severe combined immunodeficient mouse melanoma tumor model. Further studies will determine whether polycations can improve intratumoral gene transfer.