Efficient episomal gene transfer to human hepatic cells using the pFAR4-S/MAR vector

Liver-directed gene therapy, using mainly viral vectors for the genetic cell modification, is a promising therapeutic approach for many genetic and metabolic liver diseases. The recent successful preclinical trials with AAV vectors expose the benefits as well as the limitations of the system. We focused on the development of an alternative non-viral episomal gene transfer system, by inserting the DNA element Scaffold/Matrix Attachment Region (S/MAR) into the free of antibiotic resistance gene miniplasmid vector (pFAR4). We produced pFAR4 derivative experimental vectors, carrying the eGFP gene driven by the composite HCRHPi liver-specific promoter and either lacking (pFAR4-noS/MAR) or containing the S/MAR element in an upstream (pFAR-S/MAR-IN) or downstream (pFAR4-S/MAR-OUT) configuration in relation to the poly-A signal of the eGFP expression cassette. Upon transfer into Huh7 cells by lipofection, vector pFAR4-S/MAR IN showed significantly higher transfection efficiency and eGFP expression than the control vector or the pFAR4-S/MAR-OUT (p < 0.005), estimated by fluorescent microscopy and flow cytometry. Stable transfections were produced only with cultures containing vector pFAR4-S/MAR IN, through the expansion of single colonies, which displayed sustained GFP expression and plasmid copy number per cell of 2.3 ± 0.4, at 3 months of culture. No vector integration events were detected in these cultures by FISH analysis, while the presence of free, circular plasmids was documented by plasmid rescue assay. The presence of S/MAR renders pFAR4 miniplasmid substantially more efficient regarding episomal gene transfer and is suitable for liver-directed studies towards gene therapy applications.