White, Elaine Joanna; (2000) Evaluation of receptor-mediated gene transfer using an integrin -targeting vector as a potential form of therapy for lysosomal storage diseases. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Lysosomal storage diseases (LSD) result from deficiencies of enzymes or structural proteins involved in the catabolism of macromolecules inside the lysosome. In this study non-viral receptor-mediated gene transfer into fibroblasts from patients with the LSDs, fucosidosis and Fabry disease, which result from deficiencies of a- L- fucosidase and a- galactosidase A respectively, has been investigated as a possible form of therapy for LSDs. The biochemical and molecular basis of the deficiency of a- L- fucosidase in the fucosidosis patients used in the study was investigated. The residual a- L- fucosidase activity and the amount of material cross-reacting with anti- a- L- fucosidase antibodies were determined. Patient genomic DNA was haplotyped for the Q/R281 polymorphism and analysed by single strand conformation polymorphism analysis (SSCP) followed by sequencing to identify the mutations in the a- L- fucosidase gene. In this study a non-viral vector, which exploits receptor-mediated endocytosis to target and enter cells, was used for gene delivery. This vector (LID complex) consisted of plasmid DNA complexed with the poly-lysine domain of a peptide containing an integrin- targeting domain and Lipofectin to aid endosomal escape of the complex. Transfection of fucosidosis fibroblasts with the luciferase reporter gene was shown to be less efficient than transfection of normal fibroblasts. Transfection of LSD patient fibroblast cultures with the normal cDNA of the gene that is defective resulted in a large increase in the enzyme activity of the defective enzyme. This activity was mainly secreted into the culture medium suggesting that the introduced gene product is not being correctly targeted to the lysosome. However, secretion of enzyme would be of some advantage for gene therapy if the enzyme were taken up by other cells via the mannose-6-phosphate receptor in the plasma membrane. Transfection of fibroblasts with LID complexes caused a small decrease in the intracellular activity of endogenous lysosomal enzymes and a small increase in their extracellular activity. This finding highlights the need for further basic studies on non-viral gene delivery systems to examine their modes of delivery and their effects on cells.

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