The goal of this New Innovator project is to advance gene delivery applications for stem cell therapies through innovative new delivery systems designed using information gained from priming of endogenous cellular targets and telecommunications (mathematical) modeling of nonviral DNA transfer. Gene delivery requires a carrier to transport exogenous genes as plasmid DNA (pDNA) to cells to produce an encoded protein (i.e. transfection). Nonviral delivery systems have been extensively investigated as a technique to accomplish transfection, however they suffer from low efficiency, thereby limiting their application in functional genomics, tissue engineering, medical devices, and gene therapy. In particular, gene delivery to adult-derived stem cells has proven especially difficult. Empirical investigations into enhancing transfection of nonviral systems have often focused on modifications to the pDNA carrier. However, those studies have had limited successes in enhancing transfection largely due to the lack of information of the molecular factors that facilitate the delivery process, as well as an incomplete understanding of the process itself. Recently, two different approaches have been investigated by the PI to improve our understanding of the gene delivery process: pharmacogenomics screens (i.e. microarray analysis) and pharmacokinetics models. Microarray studies in the PI's laboratory have been used to profile transfected and untransfected (but treated) cell populations, providing molecular targets to enhance nonviral transfection. This project proposes to use those targets for cell priming to both increase our understanding of the role of the targets and develop simple new protocols to enhance transfection. In addition, we will identify more candidate priming agents through a screen of clinically approved drugs, which can also be analyzed for their effect on specific gene pathways, to further our understanding of the process. In parallel with previous microarray analysis efforts, computational models have been developed, providing insight into barriers and kinetic parameters of the gene transfer process. However previous models are limited in their ability to output mechanistic information. Here a novel kinetic model of gene delivery using telecommunications queuing theory will overcome challenges encountered by previous mathematical models and allow for integration of cell priming modes of transfection. In this telecommunications model, delivery of DNA to the cell nucleus is considered in the same way as the delivery of a packet of information (DNA) to a destination computer (nucleus) within a packet- switched computer network (barriers to DNA transfer). This model will allow for a priori predictions about novel transfection systems. With information gained from this project, I propose to develop new delivery strategies that incorporate drug priming to chemically modulate key cellular barriers as a simple and clinically-translatable approach to improve transfection of adult human stem cells, for use in tissue engineering and regenerative medicine, the delivery and secretion of therapeutic proteins, organ transplantation, and cancer therapy.
PROJECT RELEVANCE Delivering a correct form of a gene to a cell could result in advanced therapies for a variety of diseases. However, delivery of DNA encoding that gene has proven to be very difficult, especially when considering techniques that do not make use of modified viruses. The objective of this project is to investigate if drug molecules can improve the delivery of DNA, and use mathematical algorithms to determine rules by which DNA nanoparticles, which do not contain viral elements, may be more effective in gene therapy applications.
