(560c) Cellular and Molecular Factors Contribute to An in Vitro Refractory Period Between Sequential DNA Complex Deliveries

Repeated delivery of DNA complexes to cells, tissues, or organisms is required for multiple biological applications such as the maintenance of a therapeutic effect in gene therapy, the triggering of specific cellular responses to alter transfection, and the study of genetics in general. Previous studies report on a putative in vivo refractory period, where cells are unlikely to be transfected a second time after previously being transfected. To further understand this effect, in vitro studies were conducted to determine the  molecular factors attributed to the refractory event.

Microarray analysis, RT-PCR, and flow cytometry studies were conducted to identify the genes and cellular mechanisms that might contribute to the refractory period. For the microarray studies, DNA complexes were delivered to HEK 293T cells, and gene expression data were collected 2 to 24 hours post complex delivery. At these same time points, post complex delivery, total RNA was extracted from cells and the amount was quantified via RT-PCR. Finally, flow cytometry analysis was conducted on HEK 293T cells that were first treated with complexes containing a plasmid encoding for a fluorescent reporter gene, then, 2 to 24 hours afterwards, treated with complexes containing a secondary fluorescent reporter gene. Flow cytometry was conducted 24 hours after the second plasmid was delivered.

Microarray data revealed altered gene expression profiles in cells recently treated with complexes, as compared to cells treated at later time points. These data indicate different molecular mechanisms guiding initial cell responses to complex delivery as opposed to those mechanisms promoting transfection. In addition, results from RT-PCR demonstrates a decrease in total RNA at earlier time points, indicating a possible shut-down of mRNA production immediately following DNA complex delivery. Furthermore, flow cytometry analysis shows decreased transfection efficiency of a secondary plasmid, as compared to the efficiency of single plasmid delivery. Together, these data demonstrate that cellular mechanisms are initiated after DNA complex delivery that may be responsible for the refractory period during which cells are less likely to respond to additional DNA complex treatments. Elucidation of the cellular and molecular factors underlying the refractory period will allow for the exploitation of these molecules by cell priming or modification of DNA delivery systems to overcome the effects of the refractory event and facilitate more efficient transfection of repeat DNA deliveries.