Adeno-associated virus mediated gene transfer into primary rat brain neuronal and glial cultures: enhancement with the pH-sensitive surfactant dodecyl 2-(1′-imidazolyl) propionate

Abstract

This study evaluated the effects of a novel, pH-sensitive surfactant, dodecyl 2-(1′-imidazolyl) propionate (DIP), on cationic lipid mediated transfection in primary rat brain neuronal and glial cultures. The cationic lipid complex DOTAP/DOPE (1,2-dioleoyl-3-trimethylammonium propionate and dioleoyl phosphatidylethanolamine, respectively) was added over a range of concentrations (0–120 μg/ml) with DNA concentration kept constant (1.6 μg/ml). The neuron-specific enolase (NSE) and cytomegalovirus (CMV) promoters were found to drive green fluorescent protein (GFP) expression in neuron-enriched and glial cultures, respectively, using adeno-associated virus (AAV) derived constructs. NSE-driven GFP expression was not observed in glial cultures. Addition of DOTAP/DOPE increased transfection efficiency over a wide range of lipid concentrations (5–50 μg/ml) keeping DNA concentration constant (1.6 μg/ml). Addition of DIP to the lipid/DNA complex increased maximum transfection efficiencies in glial and neuronal cultures 2–3-fold. Transfection efficiencies were at their maximum with a similar total lipid concentration (50 μg/ml) in both cell-types in the presence of DIP. Neuronal cultures were more sensitive than glia to the toxic actions of DOTAP/DOPE, with or without DIP. These results indicate that AAV-mediated gene-transfer to neurons and glia can be facilitated by addition of a pH-sensitive surfactant to cationic liposome/DNA complexes and that endosomal escape could be a limiting factor in transgene expression.