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

Abstract

BackgroundThe 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.ResultsHere, 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.ConclusionsThe novel vector pEPito can be considered suitable as an improved vector for biotechnological applications in vitro and for non-viral gene delivery in vivo.

A Seven-Year Storage Report of Good Manufacturing Practice–Grade Naked Plasmid DNA: Stability, Topology, andIn Vitro/In VivoFunctional Analysis

“…This is because of their intrinsic biosafety characteristics and many technological improvements, which made nonviral vectors a promising alternative over viral vectors in gene therapy. The attractiveness of nonviral vectors is based on their favorable characteristics, such as low toxicity and immunogenicity, in association with continuously improving expression efficiencies (Gill et al, 2009;Tolmachov, 2009;Haase et al, 2010). For the use of plasmid DNA in clinical applications, there are two main requirements: the high product purity and product stability for the final formulation.…”

Characteristic element of matrix attachment region mediates vector attachment and enhances nerve growth factor expression in Chinese hamster ovary cells

“…Additionally, the intravenous administration of NGF does not allow for optimum efficiency. In addition to its high cost, these factors limit the application of NGF to the treatment of disorders and defects in the central nervous system (Haase et al, 2010;Liu et al, 2013). Gene transfection is a novel tool that can be used in the application of multipleneurotrophic factors.…”

In Vitro Replication Assay with Mammalian Cell Extracts

“…The pEPito vector is signifi cantly smaller in size, contains only one transcription unit and has 60 % less CpG motives as compared to pEPI-1 plasmid. It shows enhanced performance due to the presence of the human CMV enhancer/human elongation factor 1 alpha promoter that is known to be minimally affected by epigenetic silencing events [ 30 ]. The pEPito contains MAR sequence hence is applicable to study in vitro DNA replication; however, it has not been tested for mammalian replication assays yet.…”

“…This chapter will describe in detail the protocols to be followed for conducting an in vitro replication assay using pEPI-1 plasmid. A novel improvised vector pEPito, derived from the pEPI-1 plasmid replicon, has been developed recently to study biotechnological assays in vitro ( see Note 1 ) [ 30 ]. The assay involves the preparation of cytosolic and nuclear extracts from mammalian cells, purifi cation of the pEPI-1 plasmid, and the replication assay itself.…”