Engineering a DNA-Protein Complex for Improved Nuclear Uptake and Gene Expression

• Kaitlyn Tien, Chemical Engineering, University of Delaware

• Millicent Sullivan, Chemical and Biomolecular Engineering, University of Delaware

DNA-based therapies can provide prolonged gene expression compared to less stable mRNA therapies and restore expression of missing or defective proteins, making them promising for treating chronic diseases. Despite these advantages, efficient delivery of plasmid DNA into the cell nucleus remains a major challenge to successful gene therapy. Lipid nanoparticles (LNPs) have emerged as the leading non-viral gene delivery platform but remain underdeveloped with DNA delivery. This project aims to improve nuclear DNA delivery by engineering a DNA-protein complex through the incorporation of a repeating DNA binding sequence designed to interact with proteins containing nuclear localization sequences (NLSs). Previous work incorporating NLS tags did increase nuclear uptake and expression using a native system. Currently, we are trying to expand this using a non-native system, requiring further design and testing. MLE-12 cells were transfected with LNP-encapsulated plasmid DNA and lipofectamine-complexed mRNA encoding for the DNA-binding protein. Both simultaneous and delayed mRNA dosing strategies were evaluated. For delayed dosing, mRNA was delivered to the MLE cells at 6, 12, or 24 hours after DNA delivery. At each dosing, the LNPs were removed before the mRNA was added. Luciferase assays were done 24 hours after transfection to quantify gene expression. The delivery of the LacI protein and plasmid containing four repeating LacI binding sites did not enhance gene expression, suggesting that nuclear uptake was not improved. These results indicate that the DNA-protein may not have formed the intended DNA-protein complex within the cytoplasm of the cell. A possible explanation for this is the lack of opportunities for the DNA and protein to meet each other. Future studies will focus on producing the DNA and protein separately and assembling the DNA-protein complex in vitro to determine whether complex formation is limiting nuclear uptake.