title: CTb targeted non-viral cDNA delivery enhances transgene expression in neurons.
abstract: 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-K(100)) was compared by fluorescence-activated cell sorting (FACS) analysis of green fluorescent protein (GFP) expression and luminometric measurement of beta-galactosidase (beta-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-K(100) 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-DNA(GFP), compared with untargeted K(100) polyplexes. 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.
authors.affiliation: Molecular Neuroscience Group, Department of Medicine, University of Birmingham, Birmingham B15 2TH, UK. L.B.Barrett@bham.ac.uk
authors.forename: L B
authors.initials: LB
authors.lastname: Barrett
authors.forename: M
authors.initials: M
authors.lastname: Berry
authors.forename: W-B
authors.initials: WB
authors.lastname: Ying
authors.forename: M N
authors.initials: MN
authors.lastname: Hodgkin
authors.forename: L W
authors.initials: LW
authors.lastname: Seymour
authors.forename: A-M
authors.initials: AM
authors.lastname: Gonzalez
authors.forename: M L
authors.initials: ML
authors.lastname: Read
authors.forename: A
authors.initials: A
authors.lastname: Baird
authors.forename: A
authors.initials: A
authors.lastname: Logan
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics; D002627:Chemistry, Physical / Q000379:methods; D002772:Cholera Toxin / Q000235:genetics* / Q000378:metabolism; D018076:DNA, Complementary; D005677:G(M1) Ganglioside / Q000378:metabolism; D015870:Gene Expression; D018014:Gene Transfer Techniques; D020932:Nerve Growth Factor / Q000494:pharmacology; D009474:Neurons / Q000502:physiology*; D016716:PC12 Cells; D011107:Polylysine / Q000235:genetics*; D051381:Rats; D014162:Transfection / Q000379:methods
chemical_list: D018076:DNA, Complementary; D011107:Polylysine; D005677:G(M1) Ganglioside; D002772:Cholera Toxin; D020932:Nerve Growth Factor