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 (M-K,00) was compared by fluorescence-activated cell sorting (FACS) analysis of green fluorescent protein (GFP) expression and luminometric measurement of beta-galactosidase (p-gal) expression, to untargeted poly(D-lysine) (K-100) in undifferentiated and NGF-differentiated PC12 cells. Results Transfection of undifferentiated PC12 cells with CTb-K-100 polyplexes resulted in a 36-fold increase in levels of pCMV-DNA(LacZ) expression and a 20-fold increase in the frequency of transduction with pCmv-DNA(GFP), compared with untargeted K-100 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-DNA(LacZ) expression and a 11-fold increase in the percentage of cells transduced with pCmv-DNAGFP, compared with untargeted K100 polypleyes. Transfection was dependent on CTb, with CTb-K-100-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 (C) 2004 John Wiley Sons, Ltd.