490. Histone-Targeted Polyplexes Avoid Endosomal Escape and Enter the Nucleus During Post-Mitotic Redistribution of ER Membranes

Gene therapy has garnered significant interest over the past two decades as a method to advance the treatment of a wide variety of diseases including many types of cancer, cardiovascular diseases, blood diseases, and vision disorders. In recent years, non-viral methods of delivery have received particular attention because of their low toxicity, biocompatibility, and improved controllability compared to viral vectors. However, inefficient trafficking to the nucleus is a common cause of limited DNA delivery. Improving the efficacy of non-viral vehicles necessitates improved strategies to understand and engage native pathways for endocytic uptake and subcellular trafficking to the nucleus. To address these issues, we sought to develop gene targeting approaches able to capitalize on nature's own mechanisms for gene packaging and transfer, and to unravel the detailed trafficking behavior that allows such carriers to reach the nucleus. Building on histone biology, we developed histone-targeted polyplexes that could efficiently exploit native, histone-based processing pathways, and we showed that histone targeting promoted nuclear accumulation, DNA release, transcription, and enhanced transfection by polyplexes. In this paper, we describe a series of studies leading to the identification of key mechanisms controlling the improved nuclear delivery and retention by these structures.Most nonviral strategies rely upon endosomal escape designs; however, endosomal escape is often uncorrelated with improved gene transfer and membranolytic structures are often cytotoxic. In contrast, histone 3 (H3)-targeted polyplexes trafficked to the nucleus through an alternative route involving caveolae and the Golgi and ER, using pathways similar to several pathogens. We hypothesized that the efficacy of these polyplexes was due to an increased utilization of native vesicular trafficking as well as regulation by histone effectors. Using confocal microscopy and cellular fractionation, we determined that a key effect of histone-targeting was to route polyplexes away from clathrin-mediated recycling pathways by harnessing endomembrane transfer routes regulated by histone methyltransferases. Specifically, super resolution microscopy and fluorescence correlation spectroscopy indicated that the polyplexes remained associated with ER membranes until mitosis, when they were redistributed into the nucleus. These unprecedented findings suggest that histone targeting allows polyplexes to partition into the nucleus by engaging ER-mediated nuclear envelope reassembly.To further understand and exploit the mechanisms enabling nuclear uptake and retention, we have investigated whether the H3 importin, importin4 is involved in mediating polyplex nuclear accumulation. Up to 42% of the polyplexes co-localized with importin4, both inside the nucleus and on the nuclear periphery, and siRNA-mediated silencing of importin4 reduced gene expression by 23%. These findings suggest that importin4 may regulate nuclear partitioning and/or nuclear retention by H3-targeted polyplexes. These novel findings highlight alternative mechanisms to subvert endolysosomal trafficking and harness histone effectors to enhance gene transfer.