Abstract
ABSTRACT The episomal vector cannot integrate into the host cell chromosome, which has no potential risk in gene therapy. However, the low level of transgene expression driven by episomal vectors needs to be solved. In this study, we investigated the effects of enhancers, promoters and promoter variants on transgene expression levels driven by episomal vectors in HEK293, Chang liver and primary cells. Results showed that all eight cis-acting elements used could increase transfection efficiency and transient eGFP expression in transfected HEK293 and Chang liver cells. In stably transfected mammalian cells, the elongation factor-1 alpha (EF-1α) promoter and mutant-404 showed high and stable transgene expression. The mechanisms might be related to the type and quantity of transcription factor regulatory elements. Moreover, quantitative reverse transcription polymerase chain reaction analysis showed that mRNA expression levels were not directly proportional to protein expression levels. Furthermore, the EF-1α promoter conferred high transgene expression levels in primary cells, and the plasmid was also present in the episomal state. Taken together, these results provided valuable information for improving transgene expression with episomal vectors in mammalian cells.
Highlights
Gene therapy involves the delivery of a therapeutic gene into a patient’s cells as a drug to treat diseases
We evaluated the effects of different cis-acting elements on transgene expression levels in HEK293, Chang liver, and primary cells (PEF cells)
We demonstrated that enhancer elements, mutant CMV and CAG and EF-1α promoters increased transfection efficiency and recombinant protein transient expression
Summary
Gene therapy involves the delivery of a therapeutic gene into a patient’s cells as a drug to treat diseases. Vector systems play the key role in driving the expression of transgenes in gene therapy [1,2]. Episomal vectors can replicate in synchrony with each cell cycle division within the host genome for sustained, nonviral and nonintegrating transgene expression in vitro and in vivo [3,4,5], which does not cause insertional mutagenesis in gene therapy [6]. The first nonviral and episomal plasmid vector pEPI based on the matrix attachment region (MAR), which can replicate autonomously with low copy numbers in all cells tested, was developed by Piechaczek et al [7]. The pEPI vector has several limitations, such as low copy number, unstable expression and low expression level [8,9]
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