1079. Effect of WPRE on Transgene Expression Using Different Promoters in the Context of Hydrodynamically Delivered Plasmid Vectors

The Woodchuck hepatitis virus (WHV) posttranscriptional regulatory element (WPRE) facilitates nucleocytoplasmic transport of RNA mediated by several alternative pathways that may be cooperative. This element has been included in many different genetherapy vectors including lentivirus, AAV, adenovirus etc., to stimulate heterologous cDNAs expression. In this work we have analyzed the vitro and in vivo effect of WPRE element over transgene expression using two different promoters in the context of a non-viral vector, naked DNA: an ubiquitous promoter AFR with moderate transcriptional activity, and a strong liver specific promoter AAT-EnhALb. Two different versions of the WPRE element were included in the study: the conventional one, which include 60 aminoacids of theHBV X protein aminoterminal region (named as WPRE long), and a shorter version in which only 30 aminoacids remained (named as WPRE short). For the in vitro studies, HEPG2 cells were transfected with 7.5 × 10e11copies of plasmid expressing luciferase, with or without WPRE elements, and a renilla-luciferase expressing plasmid, using lipofectamine. Enzyme activities were measured using the Dual-Luciferase Reporter Assay System (Promega). In vivo, 2.5 × 10e12 copies of each plasmid were administered using hydrodynamic injection via tail vein, thus, luciferase expression was mainly detected in the liver. Luciferase expression was analyzed using a cooled CCD camera at days 1, 7 and 21. 21 days after in vivo analysis, the mice were sacrificed and luciferase expression was measured in liver extracts. DNA and RNA were extracted from the livers, and plasmid copies and luciferase mRNA was quantified using quantitative PCR and RT-PCR. Striking differences on WPRE activity were observed depending on the promoter, both in vitro and in vivo, 1 day after injection. WPRE elements significantly increased luciferase expression when gene expression was under control of AFR promoter while the expression was not significantly altered when the promoter driving luciferase expression was the liver specific AAT promoter. However, when luciferase expression was analyzed 7 or 21 days after hydrodynamic injection, WPRE element increased luciferase expression independently on which promoter was used. Moreover, significant differences were observed depending on the WPRE version. Short and long WPREs similarly increased the transgene expression from AFR promoter, while the increase was significantly higher when AAT promoter was combined with the WPRE long, than in with WPRE short. In vitro analysis of luciferase activity on liver extract corroborated the in vivo data. In conclusion, the effect of modified versions of WPRE over transgene expression depends on the promoter and has to be analysed both in vivo and in vitro in order to get a correct picture of the interaction between the promoter and expression regulatory elements.