(205c) Investigating the Molecular Factors That Facilitate Nonviral Gene Delivery through Targeted Cellular Pathway Priming Studies and Telecommunications Modeling of DNA Transfer

Gene delivery has emerged as a promising approach for use in gene therapeutics, diagnostics, functional genomics, medical devices, and tissue engineering. While nonviral gene delivery techniques are less efficient than viral systems, they are considered an attractive alternative because of low toxicity and immunogenicity, lack of pathogenicity, inexpensive synthesis, and easy modification. Because of these advantages many investigations have approached the problem of inefficient transfection by focusing on the delivery system itself. For modeling efforts, kinetics of the gene delivery system have been identified and incorporated into computer models in order to identify barriers which act as bottlenecks to transfection. Those efforts have provided targets for focusing engineering efforts on physiochemical modification of the gene delivery system to overcome cellular barriers. Despite these efforts limited successes in improved transfection have resulted. We have taken a different approach to attack the problem of low transfection by focusing on the biology of the cell in both in vitro transfection and in silicomodeling efforts.

We previously identified molecules and pathways which facilitate transfection using microarray and pathway analysis. During the internalization phase of the complexes we found that cells exhibit a shutdown of transcriptional activity in processes involved in filopodia production, GTPase signaling, and membrane trafficking. At later time points when nuclear accumulation of plasmids occurs, we identified molecules involved in nucleic acid binding and NFκB protein complex. We have also previously developed a mathematical model to investigate bottlenecks in transfection. Our model is based on telecommunications queuing theory, in which we are the first to include biological effects such as mitosis and toxicity and simulate outputs on the period of days where those biological effects affect transfection. We are able to account for subcellular trafficking of plasmids and complexes and found that the model agreed perfectly with experimental data for transfection efficiency and subcellular trafficking of plasmids in the nucleus and validated its use for testing hypotheses. In this work, we investigated those molecular factors that facilitate nonviral gene delivery through targeted cellular pathway priming studies and telecommunications modeling of DNA transfer.

For cell priming studies, we delivered pharmacologic agents to the cells to cause the cell to over- or under- express the target molecules. We then delivered complexes to the treated cells and, after 24 h, we evaluated transfection using FACS or luciferase protein assays. In studies targeting internalization processes of HEK293T cells, relative to control, transfection could be enhanced (up to 5.3 fold when SNX24 was activated using 4-hydroxytamoxifen) or decreased (up to 7.6 fold when ALMS1 was activated using phenethicillin) when using Lipofectamine 2000 (LF2000) or polyethylenimine (PEI) gene carriers. In studies targeting molecules involved in nuclear entry, transfection could be enhanced (up to 10 fold when IREB1 was activated using dexamethasone) in multiple cell types (hMSCs; HEK293T) and with multiple vectors (LF2000 and LTX, and PEI). We hypothesized increased transfection was due to enhanced nuclear accumulation of plasmids since dexamethasone has been shown to dilate the nuclear pores of oocytes. We tested that hypothesis in our model, which demonstrated that transfection would be enhanced if the number of nuclear plasmids increased, a result consistent with our in vitro DEX studies. In vitroexperiments confirmed a 10-fold enhancement in nuclear accumulation of plasmids in hMSCs over cells not primed by dexamethasone.

In this work we demonstrated that cell priming based on identified molecular targets using pharmacologic agents has successes in enhancing transfection. Hence, we extended the genes-to-drugs cell priming approach, to a drugs-to-genes approach, in which, we performed a high throughput drug screen to identify drugs which drastically alter transfection. Cefatrizine, a β-lactam cephalosporin with anti-bacterial properties which may affect DNA polymerase and mitochondrial DNA replication in eukaryotes, was identified to knockout transfection 130 fold, relative to positive control without affecting cell viability. The identification of the specific molecular and cellular pathway involved is the target of ongoing efforts. Increasing the understanding of molecular factors and signaling pathways that either serve to promote or inhibit transfection is critical to the design of delivery agents that can efficiently transfer DNA.