CTb targeted non-viral cDNA delivery enhances transgene expression in neurons

Background Efficient neuronal gene therapy is a goal for the long‐term repair and regeneration of the injured central nervous system (CNS). We investigated whether targeting cDNA to neurons with cholera toxin b chain conjugated non‐viral polyplexes led to increased efficiency of non‐viral gene transfer in the CNS. Here, we illustrate the potential for this strategy by demonstrating enhanced transfection of a differentiated neuronal cell type, PC12. Methods In vitro transfection efficiency of a cholera toxin b chain–poly(D‐lysine) molecular conjugate (CTb‐K100) was compared by fluorescence‐activated cell sorting (FACS) analysis of green fluorescent protein (GFP) expression and luminometric measurement of β‐galactosidase (β‐gal) expression, to untargeted poly(D‐lysine) (K100) in undifferentiated and NGF‐differentiated PC12 cells. Results Transfection of undifferentiated PC12 cells with CTb‐K100 polyplexes resulted in a 36‐fold increase in levels of pCMV‐DNALacZ expression and a 20‐fold increase in the frequency of transduction with pCMV‐DNAGFP, compared with untargeted K100 polyplexes. Treatment of PC12 cells with 50 ng/ml/day of NGF for 14 days led to differentiation to a neuronal phenotype. Transfection of NGF‐differentiated cells with CTb‐K100 polyplexes resulted in a 133‐fold increase in levels of pCMV‐DNALacZ expression and a 11‐fold increase in the percentage of cells transduced with pCMV‐DNAGFP, compared with untargeted K100 polyplexes. Transfection was dependent on CTb, with CTb‐K100‐mediated transfections competitively inhibited with free CTb in both PC12 phenotypes. Conclusions Non‐viral systems for gene transfer in damaged CNS show superior toxicological profiles to most viruses but are limited by inefficient and non‐selective gene expression in target tissue. Cholera toxin is known to interact preferentially with neuronal cells of the central and peripheral nervous systems, mediating binding through the b subunit, CTb, and the pentasaccharide moiety of the gangliosaccharide, GM1, which is present at high levels on the neuronal cell surface. Here, we show that a molecular conjugate of the CTb subunit, covalently linked to poly(D‐lysine), is able to successfully target and significantly enhance transfection of a neuronal cell type, NGF‐differentiated rat PC12 pheochromocytoma cells. This observation encourages the further development of non‐viral strategies for the delivery of therapeutic genes to neurons. Copyright © 2004 John Wiley & Sons, Ltd.