Collins, Louise; (1999) A non-viral vector system for efficient gene transfer via membrane integrins. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

An integrin-targeted non-viral vector has been developed for receptor-mediated gene delivery. It consists of the RGD-containing portion of the venom from the American pit viper, Crotalus molossus molossus, synthesised together with a sixteen lysine chain for electrostatic binding and condensation of DNA. The poly-K-molossin peptide is able to complex DNA rapidly and aids efficient binding and internalisation of cells expressing ay and as integrins. Binding to the cell surface appears to be a co-operative event, involving both the RGD-integrin interaction and electrostatic binding due to the high positive charge of the vector. However, it is only at the point of integrin attachment that internalisation occurs. The poly-K-molossin/DNA vector system has been optimised to deliver reporter genes in vitro to ECV304 endothelial cells. Approximately 50 % transfection efficiencies are achieved under optimal conditions. It has also been tested on cultured rabbit cornea and expressed the reporter gene in approximately 30 % of the endothelial cells. Cell transfection in both systems requires the presence of chloroquine as an endosomolytic agent. The precise requirements of the cells to the poly-K-molossin/DNA complexes and chloroquine were identified, as well as the influence of temperature and serum upon the delivery system. Using a monoclonal antibody, raised to the RGD-containing portion of the vector, it was found that the peptide bound to a diverse range of cell types, establishing suitable targets for in vivo investigations. Preliminary perfusion studies with the poly-K-molossin/DNA vector complexes were carried out on the rat heart model. In conclusion, a novel non-viral gene therapy vector system has been developed, which results in efficient gene expression in both cell lines and corneal endothelium. The diversity of binding within tissue sections has shown the enormous potential of this vector in vivo, for future clinical applications.

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