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

Lee Barrett, Martin Berry, WB Ying, Matthew Hodgkin, LW Seymour, AM Gonzalez, Martin Read, Andrew Baird, Ann Logan

Research output: Contribution to journalArticle

15 Citations (Scopus)


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.
Original languageEnglish
Pages (from-to)429-438
Number of pages10
JournalJournal of Gene Medicine
Publication statusPublished - 1 Jan 2004


  • non-viral
  • gene transfer
  • PC12 cells
  • central nervous system
  • cholera toxin


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