(410c) Improved Nonviral Gene Delivery Systems for Stem Cell Therapy and DNA Vaccination Applications

Gene delivery is the delivery of exogenous genetic material to cells with the goal of altering molecular physiology to produce a cellular or systemic phenotype change. Unlike many conventional drugs, free nucleic acids are not readily internalized by eukaryotic cells due to size and charge, but many methods of their delivery to cells are the subject of intense research, including viral and non-viral methods. Non-viral gene delivery methods are much less efficient than viral methods, but flexibility in genetic cargo, ease of transfection protocols, and lack of safety issues make them advantageous alternatives. Strategies to engineer more effective non-viral gene delivery materials and methods are highly dependent on variable parameters such as cell type and application, and have focused on engineering increasingly more complex lipid and polymer vectors, but the rational design of new technologies is limited by our current knowledge of several key cellular barriers. To expand our knowledge of the “biology of transfection” our group has made efforts to understand the process of gene delivery, using diverse tools include modeling, gene expression analysis, high throughput screening, cell priming, and the development of new material systems. For mathematical modeling, we have described the process of gene delivery as nodes within a computer network, exchanging packets of information (i.e. plasmids) with different queuing rates to predict the physical distribution of pDNA within cells. Using these models to identify and quantify pDNA losses gives us perspective into complex cellular mechanisms that are largely unknown. We have improved these models to integrate mitosis, toxicity, and transgene expression, which must be balanced with improved nuclear pDNA import for optimized gene delivery outcomes. In addition to our modeling studies, have characterized the mechanisms underlying gene delivery using microarray analysis techniques, where RNA transcripts from cells that were sorted into successfully- and unsuccessfully-transfected populations after treatment with lipid- and polymer DNA complexes were measured to identify differentially expressed endogenous genes important to transfection. The identified genes were later targeted by pharmacological activation or inhibition to demonstrate ‘priming’ with these compounds can up- or down-regulate transgene expression by modulating the cellular response to gene delivery. Reversing this approach, we have also identified several clinically-approved drug groups that significantly up- or down-regulate gene delivery success in a high-throughput screen. The known effects of the drugs identified overlap with many of the functions of genes identified in our previous microarray studies. Furthermore, a specific family of drugs, glucocorticoids, were identified as potential priming agents, and we have now applied that family of drugs to a priming strategy to enhance gene delivery to human mesenchymal stem cells (hMSCs). hMSCs are under intense study for applications of cell and gene therapeutics because of their unique immunomodulatory and regenerative properties. Safe and efficient genetic modification of hMSCs could increase their clinical potential by allowing expression of therapeutic transgenes and control over behavior and differentiation. We have demonstrated that priming hMSCs with dexamethasone (DEX), a glucocorticoid drug, significantly increases transfection success across many different human donors. Furthermore, we have shown that DEX priming of transfection is mediated by binding of the cytosolic/nuclear glucocorticoid receptor (GR) and the DEX effect acts independent of classical nuclear import pathways. In addition, we have now expanded DEX priming to hMSCs derived from several different tissues and human donors, and demonstrate significant variability of transfection and responsiveness to DEX priming between tissue and donor source, reiterating the concern of variability in clinically relevant cell types. Finally, we have used our knowledge of the biology of transfection to design novel, hybrid particles, consisting of zein and chitosan, to orally deliver DNA for vaccine applications, and demonstrate induction of IgA antibodies indicative of an early mucosal immunity response. Together our studies demonstrate increased understanding of the nonviral gene delivery process, which can lead to improved delivery strategies in clinical applications.