Investigation of the role of target cell factors in retrovirus transduction
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Gene therapy is the intracellular delivery of genetic material for a therapeutic effect and is currently being used in clinical trials for the treatment of cancer, AIDS and vascular diseases. Recombinant retroviral vectors are one of the most commonly used gene delivery vectors in clinical trials because they can permanently integrate the therapeutic gene into the genome of the target cell resulting in persistent gene expression. However, recombinant retroviral vectors suffer from several limitations. The gene transfer efficiency is not high enough to produce a desired therapeutic effect and the vectors lack the ability to genetically modify target tissue without producing unpredictable side- effects on healthy bystander tissue. The focus of this thesis is to determine target cell factors that affect efficiency and specificity of gene transfer of recombinant retroviruses. Successful gene transfer by recombinant retroviruses is a multi-step process and we have focused our efforts on those target cell factors that affect virus entry into the target cell. We have developed an experimental system to study the effect of pathway of virus entry and the intracellular trafficking itinerary of the targeted receptor, on the efficiency of gene transfer of targeted retroviruses. Our results indicate that interaction with a targeted receptor affects the efficiency of gene transfer of a targeted retrovirus by altering the residence time of the virus on the cell surface, by changing the region of the cell surface that the virus is exposed to, with respect to its natural receptor or by changing the pH that the virus is exposed to during intracellular transport. We have examined if recombinant retroviruses are capable of inducing signaling events in target cells to overcome barriers to efficient gene transfer. We have found that retroviruses are capable of activating actin-regulating-GTPase Rac1 while entering target cells. We have found that retroviruses use non-envelope and non-receptor molecules to induce Rac1 activation. Rac1 activity is important for efficient fusion and intracellular trafficking of the virus and blocking mediators of Rac1 activity on target cells affects the efficiency of gene transfer of recombinant retroviruses. The implications of our findings on efficient retrovirus-cell interactions are discussed.