Novel non-integrating DNA Nano-S/MAR vectors restore gene function in isogenic patient-derived pancreatic tumor models

Abstract We describe here non-integrating minimally sized Nano-S/MAR DNA vectors which can be used to genetically modify dividing cells in place of integrating vectors. They represent a unique genetic tool, which avoids vector-mediated damage. Previous work has shown that DNA vectors comprising a mammalian S/MAR element can provide persistent mitotic stability over hundreds of cell divisions, resisting epigenetic silencing and thereby allowing sustained transgene expression. The composition of the original S/MAR vectors does present some inherent limitations which can provoke cellular toxicity. Here, we present a new system, the Nano-S/MAR, which drives higher transgene expression and has improved efficiency of establishment, due to their minimal impact on cellular processes and perturbation of the endogenous transcriptome. We show that these features enable the hitherto challenging genetic modification of patient-derived cells to stably restore the tumour suppressor gene SMAD4 to a patient-derived SMAD4 knockout pancreatic cancer line. Nano-S/MAR modification does not alter the molecular or phenotypic integrity of the patient-derived cells in cell culture and xenograft mouse models. In conclusion, we show that these DNA vectors can be used to persistently modify a range of cells, providing sustained transgene expression while avoiding the risks of insertional mutagenesis and other vector mediated toxicity.