344. En Route to Non-Viral Genetic Engineering: Kinetics of DNA Uptake and Transgene Expression Following Repeated Transfection with Multiple Episomal Plasmids in Human Primary Fibroblast

Non-viral approach to cellular reprogramming or genome editing of mammalian cells often involve co-delivery of multiple types of nucleic acid molecules. Whether the method involves co-transfection with multiple episomal plasmid DNAs, a mixture of mRNA/gRNA oligomers or a combination of both DNA and RNA molecules, the efficacy of these modular approaches hinges upon the efficiency at which all the molecular factors are co-delivered and co-expressed at their optimal stoichiometric ratios. A significant rate-limiting step thus lies in the lack of an efficient co-transfection, in which a subset of the transfected population may be devoid of one, two, or more of the factors required, but the proportion at which these event occur is not clear. Further, because non-viral transfection is a transient process, repeated transfection is often employed to sustain transgene expression, as is often the case in non-viral episomal based cellular reprogramming. However, the effectiveness of these subsequent rounds of transfection in maintaining transgene expression among the transfected cells is presently unclear. In this study, we examined the kinetics of DNA uptake and transgene expression following cationic reagent-mediated non-viral transfection of primary human neonatal foreskin fibroblast with multiple episomal plasmid DNAs. To measure the level of DNA uptake and transgene expression following co-transfection, we employed two fluorescent reporter gene plasmids (eGFP and mtagBFP2) and covalently labeled them with either FITC or Cy5. Cells were transfected using XtremeGENE HP with either one or both of the labeled plasmids. More than 90% of the cells transfected were positive for either FITC or Cy5 plasmid DNA. When the labeled plasmids were mixed at 1:1 ratio or diluted with unlabeled DNA, there was a proportional decrease in the level of fluorescence in the respective fluorescent channel consistent with the relative input ratios between the two labeled DNAs. We also saw a strong correlation in the co-expression of both reporter genes following co-transfection with the majority of the transfected cells dually expressing both GFP and BFP (64%), however, there were subsets of singly transfected cells that express only GFP (8%) or BFP (27%). We next looked at the effectiveness of repeated transfection in enhancing/sustaining transgene expression in transfected cells. In order to distinguish the population of repeatedly transfected ones from new transfection events in subsequent rounds of transfection, we employed the same two reporter gene set-up (eGFP/mTagBFP2) in which cells were transfected with GFP first, followed by a second transfection with BFP a few days later; cells that were repeatedly transfected would then express BFP in addition to GFP. Our result showed that, while the overall transfection efficiency was higher with repeated transfection, to our surprise, the majority of the transfected cells remained GFP+; only a subset of the 40% of the transfected cells were positive for both GFP and BFP (~9% total), with the remaining attributed to newly transfected cells expressing only BFP. Taken together, these data suggest that while cationic reagent can efficiently co-deliver and co-transfect multiple episomal factors, the effectiveness of this modular approach in non-viral genetic engineering may be limited in cases where sustained expression is required due to the majority of the transfected cells being refractory to subsequent rounds of transfection. Addressing these rate-limiting steps should help increase the utility and efficiency of the system.

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