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Pharmacogenomics screening and pharmacokinetics modeling of the nonviral gene delivery system
Inefficient transfection from nonviral gene delivery systems is the critical barrier preventing their application in gene therapy, tissue engineering, and biomaterials. The design of efficient delivery systems is hampered by i) lack of understanding of the molecules that facilitate the gene transfer process; and ii) limited ability to predict how changes to gene delivery systems impact transfection. The goals of this dissertation were to help fill those gaps in knowledge. To identify molecules that facilitate transfection, pharmacogenomics screens using microarrays were used to temporally investigate polymer- and lipid-mediated transfections. After two hours of exposure of DNA complexes to HEK 293T cells, a cellular shutdown of transcription was observed from either transfection system, with no genes similarly expressed. However, similar mechanisms were downregulated in cells treated with either system, including filopodia, GTPase signaling, or membrane trafficking. Next, gene expression profiles from cells that were successfully transfected were compared to untransfected cells. Transfected cells upregulated RAP1A, ATF3, and HSPA6 in either transfection system, though polyplex-mediated DNA delivery elicited a much greater cytotoxic response. Perturbing those genes with pharmacologic agents enhanced transfection up to 5-fold and confirmed their role in gene transfer. Pharmacokinetics modeling approaches were used to predict bottlenecks in transfection and how theoretical changes to the gene delivery system can improve transfection. A novel telecommunication model was constructed in MATLAB to simulate lipoplex delivery of the GFP gene to HeLa cells. Mitosis and toxicity events were included in the model, and simulated outputs of nuclear internalization and transfection efficiency were compared to experimental data. A potential new barrier to transfection was found, in that, DNA can be degraded during its distribution to daughter cells during mitosis. A priori predictions based on model sensitivity analysis suggest that increasing endosomal escape and decreasing lysosomal degradation, protein degradation, and GFP-induced toxicity can improve transfection efficiency by three-fold. Efforts in this dissertation led to the identification of specific molecules that facilitate gene transfer and mechanistic targets for engineering improved gene delivery systems.
Martin, Timothy Michael, "Pharmacogenomics screening and pharmacokinetics modeling of the nonviral gene delivery system" (2014). ETD collection for University of Nebraska - Lincoln. AAI3618598.