874. Long-Term Transgene Expression In Vivo from a Helper Dependent Adenovirus—Epstein-Barr Virus Hybrid Vector

Abstract

Helper Dependent Adenovirus (HDA) vectors deleted in all viral genes persist poorly in vivo due to viral genome loss during mitosis. We have addressed this limitation by using elements from Epstein-Barr Virus (EBV), the genome of which persists as an extra-chromosomal episome in replicating B cells. Maintenance is mediated by the virally-expressed Epstein-Barr Nuclear Antigen 1 (EBNA-1) protein that binds the episome and tethers it to metaphase chromosomes for segregation into daughter cells during mitosis. In our hybrid binary system, an HDA vector (HDA-EBV) is used to deliver a linearized, EBV-based episome to target cells. Co-infection with a second HDA vector expressing Cre recombinase (HDA.Cre) leads to the excision and circularization of the loxP-flanked episome sequence, and places a promoter upstream of the transgene. The episome also contains a human origin and the EBNA-1 binding site for replication and segregation in mitotic cells. We have shown that co-infection with HDA.Cre and an HDA-EBV vector carrying either a Cyan Fluorescence Protein (CFP) reporter gene or Puromycin AcetylTransferase (PAC) drug resistance gene produces circular episomes in vitro and that the EBV elements significantly prolong transgene expression1.