Abstract
We describe herein 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 that can provoke cellular toxicity. Herein, we present a new system, the nano-S/MAR, which drives higher transgene expression and has improved efficiency of establishment, due to the 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 tumor 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.
Highlights
Rescuing the function of mutated genes in tumor cells can help to define their molecular role and provide an insight into their interactions and the processes that drive the transformation of a normal cell toward cancer without disturbing other cellular processes
scaffold/matrix attachment region (S/MAR) vectors are a unique class of DNA constructs that can provide long-term transgene expression and mitotic stability in mammalian cells without relying on toxic viral components or random integration that can potentially disrupt the molecular behavior of the targeted cells
This new generation of DNA vectors was compared to the respective traditional plasmids in HEK293T cells, and we could show that the nano-S/MAR vector (nS/MAR) vectors had a higher establishment of efficacy and that they sustained more robust transgene expression in the established clones over time for a minimum of 35 days
Summary
Rescuing the function of mutated genes in tumor cells can help to define their molecular role and provide an insight into their interactions and the processes that drive the transformation of a normal cell toward cancer without disturbing other cellular processes. Previous work has established that DNA vectors comprising a nuclear scaffold/matrix attachment region (S/MAR) element and mammalian promoters allow long-term transgene expression in cancer cell lines, both in vitro and in vivo.[2,3,4] S/MARs mediate the binding of episomal vectors to the chromosomal scaffold during mitosis, providing sustained expression and mitotic stability over hundreds of cell divisions.[5,6] In the context of minicircle vectors, these motifs lead to a higher and more sustained transgene expression when compared to conventional plasmids, presumably due to the lack of bacterial sequences often characterized by the presence of CpG dinucleotides, responsible for the initiation of the vector silencing.[7] the production of minicircles is a laborious process that implies intramolecular recombination[8] followed by purification steps designated to separate the producer vector from the minicircle To overcome these problems, an alternative antibiotic-free selection system was established by Luke et al.[9] for the production of minimally sized plasmids. We directly compare plasmid vectors to nanovectors and show that nS/SMAR DNA vectors
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