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Background  The episomal replication of the prototype vector pEPI-1 depends on a transcription unit starting from the constitutively expressed  Cytomegalovirus  immediate early promoter (CMV-IEP) and directed into a 2000 bp long  matrix attachment region sequence  (MARS) derived from the human β-interferon gene. The original pEPI-1 vector contains two mammalian transcription units and a total of 305 CpG islands, which are located predominantly within the vector elements necessary for bacterial propagation and known to be counterproductive for persistent long-term transgene expression.    Results  Here, we report the development of a novel vector pEPito, which is derived from the pEPI-1 plasmid replicon but has considerably improved efficacy both  in vitro  and  in vivo . The pEPito vector is significantly reduced in size, contains only one transcription unit and 60% less CpG motives in comparison to pEPI-1. It exhibits major advantages compared to the original pEPI-1 plasmid, including higher transgene expression levels and increased colony-forming efficiencies  in vitro , as well as more persistent transgene expression profiles  in vivo . The performance of pEPito-based vectors was further improved by replacing the CMV-IEP with the  human CMV enhancer/human elongation factor 1 alpha promoter  (hCMV/EF1P) element that is known to be less affected by epigenetic silencing events.    Conclusions  The novel vector pEPito can be considered suitable as an improved vector for biotechnological applications  in vitro  and for non-viral gene delivery  in vivo .

pEPito: a significantly improved non-viral episomal expression vector for mammalian cells