Characterizing and Increasing Nuclear Entry of Electrotransfected Plasmid DNA

Abstract

Nonviral DNA transfection has important applications in gene therapy, both as an alternative to viral and mRNA-based gene therapies, and as an integral process in the production of viral gene therapies. Currently, nonviral DNA transfection is limited by relatively low efficiency compared to mRNA transfection and viral transduction. A major factor limiting DNA transfection efficiency is that transgenic DNA must enter the nucleus of cells for gene expression to occur, and this process is known to be inefficient. In my dissertation, I investigated the mechanisms of nuclear entry in nonviral DNA transfection as well as strategies to increase nuclear delivery of transfected plasmid DNA (pDNA). To this end, I studied nonviral DNA delivery using a multifaceted approach: measuring cellular uptake and nuclear entry of transfected DNA as well as subsequent gene expression at the mRNA and protein levels. Using this approach, I directly compared pDNA transfection in dividing and non-dividing cells and showed that pDNA nuclear entry occurred in most cells in both groups, but to a greater extent in dividing cells. During this study I observed intranuclear pDNA in both punctate and diffuse patterns and found some evidence to suggest that intranuclear punctate pDNA may be hubs of increased transcriptional activity. In separate studies, I showed that decreasing the size of non-virally transfected DNA is a viable strategy to increase nuclear entry and transfection efficiency and that conjugating nuclear localization signal (NLS) peptides to transfected DNA is not. Additionally, I developed a novel device and protocol to improve nonviral DNA delivery by squeezing cells after they have been transfected and showed that this technique can substantially increase cellular uptake and nuclear accumulation of transfected DNA. However, this approach is currently limited by side effects of cell squeezing, including increased chromatin condensation, that reduce transgene expression in squeezed cells. Together, my work contributed to the body of research aimed at understanding and improving nonviral DNA transfection, providing clues of a new mechanism for pDNA transcription and insight into effective strategies for increasing nuclear delivery of transfected DNA.