(310g) Intracellular Signaling Pathways in Nonviral Gene Delivery: Microarray Analysis of Gene Expression Profiles in Transfected Cells

Nonviral gene delivery approaches provide a mechanism to directly alter gene expression within a cell population; however inefficient gene delivery is a critical factor limiting its use in therapeutic applications including gene therapy or tissue engineering. While some studies have investigated trafficking pathways of DNA complexes within the cell, there have been few efforts to understand or engineer the molecular signaling pathways that dictate the efficacy of gene transfer. The importance of cell signaling in achieving successful nonviral gene transfer has not been thoroughly examined, though it clearly plays a role in regulation of cellular responses that may affect the ability of cells to become transfected, through inducing cell states that make the cells more or less responsive to gene transfer. We have used microarray analysis to identify signaling pathways that are modulated during nonviral gene transfer. Nonviral DNA complexes (composed of cationic lipids or polymers complexed with plasmid DNA encoding for GFP) were delivered to HEK293T human embryonic kidney epithelial cells, a widely used cell line in transfection experiments. Flow cytometry was then used to sort GFP-positive cells after 24 hours, allowing isolation of a population of transfected cells. These cells were then lysed and their mRNA collected and purified using standard techniques. Control and experimental RNA samples were then hybridized to separate Affymetrix GeneChip expression arrays, in triplicate. Expression patterns were compared between transfected and nontransfected samples, which revealed approximately 100 genes that were statistically differentially expressed between the two samples, including 99 genes considered to be statistically upregulated (2-fold or greater change in expression) and four genes determined to be significantly downregulated (2-fold or greater change in gene expression). Of particular interest, three genes were substantially upregulated in the transfected cells, including Rap1a, a GTPase implicated in a variety of cell behaviors including integrin-mediated cell adhesion, HSP70B' a stress-inducible gene that may be important for maintaining cell viability, and ATF3, a transcription factor that may activate or repress transcription to allow cells to adapt to various responses. Furthermore, a gene involved in endosomal acidification (ATPase lysosomal subunit) was also shown to be upregulated in transfected cells. Gene upregulation was correlated with transfection studies performed in the presence and absence of activators and inhibitors of these genes. Taken together, these results suggest that a cell's ability to respond to stress may determine the efficacy of gene transfer, and implicates several key cell processes involved in the trafficking of DNA complexes, including cell adhesion and endosomal escape. With a greater understanding of key signaling pathways involved in gene delivery, we hope to understand the mechanisms that render cells responsive to DNA transfer to develop more efficient nonviral delivery schemes.