14. High Efficiency and Long-Term Persistence In Vivo from a Helper Dependent Adenovirus/Epstein-Barr Virus Hybrid Vector
Authors: Sean D. Gallaher, Jose S. Gil, Arnold J. Berk
Journal: Molecular Therapy (2006)

Current strategies for gene replacement therapy are constrained by a number of factors. Adenovirus-based vectors can confer high transduction efficiency, but may persist poorly in mitotic cells. Integrating vectors, e.g. retrovirus vectors, can cause insertional mutagenesis/oncogenesis. Epstien-Barr virus episomes can persist extra-chromosomally, but delivery is inefficient. Our goal is to create a novel gene delivery vehicle that leverages the transduction efficiency of adenovirus with the non-integrating, but long-term persistence of an EBV episome. In our hybrid vector system, an EBV-based episome bearing a therapeutic or reporter transgene is delivered to target cells via a Helper Dependent Adenovirus (HDA or "gutted") vector. The episome contains an expressions cassette for the EBV Nuclear Antigen-1 (EBNA-1) protein, and its target binding site. It has been shown that these elements can tether an episome to host cell chromosomes, thus conferring long-term extra-chromosomal maintenance. A human origin in the episome allows for replication during S-phase. These sequences are present in linear form flanked by Cre recombinase targets in the HDA backbone. Upon transduction of target cells, Cre expressed from a second HDA vector excises and circularizes the episome sequence. This recombination places a promoter upstream of the transgene and EBNA-1. Previously, we have shown that our HDA-EBV hybrid produces circular episomes in vitro and that the EBV elements significantly prolong transgene expression. In the present study, we examine the benefit of using the hybrid vector for gene expression in mouse hepatocytes in vivo. The Renilla luciferase (RL) reporter was chosen as a transgene because its activity can be assayed repeatedly and non-invasively in mouse. In this vector, the RL transgene is only expressed following Cre-mediated circularization of the episome. Thus, the magnitude, location, and duration of bioluminescence indicates the efficiency of episome delivery and its persistence. Currently, 28 mice have been injected i.v. with either 5×109 genomes of an HDA-EBV expressing RL or various controls. An HDA vector with the same RL expression cassette but lacking the EBV elements was included in the control group. Transduction by the HDA-EBV vector was found to be highly efficient when assayed by confocal fluorescence microscopy on harvested liver tissue. Bioluminescence studies on the HDA-EBV reporter indicate that RL activity is detectable in the mouse as early as three days post injection and is localized to the liver. Luminosity then increases up to 100,000 times by day 30 before dropping about ten-fold by day 60. Continuing analysis shows that expression remains at this high level for at least four months. These results demonstrate how efficiently HDA vectors can be used to deliver EBV episomes to mouse hepatocytes in vivo. We propose that this vector system is ideally suited for applications such as FIX gene replacement for hemophilia where long-term hepatic expression of a therapeutic transgene is desirable.