An enhanced EBNA1 variant with reduced IR3 domain for long-term episomal maintenance and transgene expression of oriP-based plasmids in human cells.

The latent replication of oriP-based plasmids in human cells depends on the viral oriP-binding transactivator EBNA1. In this report, the effect of the internal repeat 3 (IR3 or GlyAla repeat) domain of EBNA1 on long-term maintenance and transgene expression of OriP-based plasmids was examined in dividing human cells. To assess the potential contribution of different isoforms of EBNA1 specifically, the long-term stability of oriP-based plasmids was determined after stable transfection of various CMV-driven EBNA1 genes in EBV-negative human B cells. Episome copy number was quantified using a novel sensitive assay based on human mitochondrial DNA as an internal extrachromosomal control. Using this assay, the standard B95.8-derived EBNA1 was compared with its truncated IR3-deleted, form, as well as a new EBNA1 isoform cloned from Raji. The results of a 6-month study indicate that the isoforms of EBNA1 differ with respect to their efficiency of plasmid maintenance. While the EBNA-1 Raji encoding plasmid was the most stable, the oriP-based vector expressing the truncated EBNA1 (IR3del) gene was lost at a much higher rate than those transducing full size EBNA1s. In parallel, long-term reporter gene expression in various human B cell lines was shown to persist at the highest level with the oriP-based Raji EBNA-1 construct. These results show that the GlyAla domain can positively influence long-term plasmid stability and episomal transgene expression.