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Thesis (Ph. D.)--Virginia Commonwealth University, 2006. / Prepared for: Dept. of Biomedical Engineering. Bibliography: leaves 202-220. Also available online.

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.

The study of drug delivery for the treatment of illnesses and injuries is an important area of pharmaceutical technology. A relatively new area of drug delivery being explored is gene therapy, which utilizes the idea that genes can be used as an alternative treatment. The exploration of gene delivery brought major advancements in the treatment of cancers and tumors as well as many challenges. In this study, the challenges of maintaining a stable vehicle for delivery, delivering genes into the cells, and the efficacy of the gene delivery vehicle were explored. Seven co-polymers of 12% (w/v) poly (D, L-lactic glycolide) (PDLG) were used to find a biodegradable polymer composition as an implant that temporarily controls the delivery of the genes. Of the formulations tested, 65/35 DL 3A and 50/50 DLG 4A were observed to show degradation time frames that best fit our purposes. Also, nanoparticles have been used to aid in the targeting of drugs to desired cells in delivery. One drawback of using nanoparticles is the potential toxic side effects they might cause. Zinc oxide nanoparticles coated with poly (vinyl pyrrolidone) (PVP) used as drug carriers were observed to have an effect on cell viability in previous studies. The cytotoxic effects of ZnO nanoparticles and PVP have on NIH 3T3 mouse fibroblast cells were investigated to see if there is a direct correlation between the level of PVP and zinc nanoparticles to the amount of cell death. It was found that an increase in concentration of ZnO nanoparticles correlates to a decrease in viability of the cells. It was also noted that the method of cell death is likely to be apoptosis. To confirm the efficacy of gene therapy through transfection, the transfection of the serum response factor (SRF) gene plasmid DNA and short interfering RNA (siRNA) were investigated. The efficiency of the transfection method were tested for both twodimensional and three-dimensional transfection of the SRF plasmid and siRNA. Experiments with two-dimensional transfection of the SRF plasmid and siRNA were successful, and transfer of the gene in the three-dimensional environment was observed with promising results with the siRNA.

Thesis (Ph. D.)--University of Texas at Austin, 2001. / Vita. Includes bibliographical references. Available also from UMI/Dissertation Abstracts International.

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

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