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 Family of Repeats (FR) region of the EBV episome, and tethers it to metaphase chromosomes for segregation during telophase. In our hybrid binary system, an HDA vector (HDA.EBV) is used to deliver a linear EBV-based episome to target cells. Co-infection with a second HDA expressing Cre recombinase (HDA.Cre) leads to the excision and circularization of the loxP-flanked episome sequence, and places a CMV promoter upstream of the transgene. The episome also contains a human origin and the FR 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 EBNA-1 and the FR significantly prolong transgene expression (in publication).
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