**4.1 OriP/EBNA-1 episomal vectors**

The best characterized system for episomal maintenance is based on sequences derived from the EBV genome. EBV is a member of the herpesvirus family with a 172-kb genome that is latently maintained as an independently replicating episome in a small percentage of infected lymphocytes (Masucci & Ernberg, 1994). During the latent phase of its cycle, DNA replication occurs from the origin of replication *oriP* and only about 10 proteins are produced of which the only protein that is required for replication at *oriP* is the Epstein Barr Nuclear Antigen-1 (EBNA-1) (Yates et al., 1985). The interaction of *oriP* with EBNA-1 also enables the segregation of the viral genome between the daughter cells through the association of EBNA-1 with host metaphase chromosomes (Harris et al., 1985).

These features of the EBV have been exploited to develop a system for episomal maintenance of foreign DNA delivered into cells. It has been shown that plasmids carrying *oriP* and expressing EBNA-1 can replicate autonomously once per cell cycle when delivered into human cells and can segregate by attaching to the host chromosomes (Haase & Calos, 1991). The *oriP*/EBNA-1 system has also been shown to support long-term episomal maintenance without selection and expression of very large human genes, such as the *CFTR* (Huertas et al., 2000), the human hypoxanthine phosphoribosyltransferase (*HPRT*) (Wade-Martins et al., 2000) and the β*-globin* gene (Black & Vos, 2002). Following these promising results, a convenient system for adding the *oriP*/EBNA-1 sequences onto any BAC already containing a therapeutic gene has been developed (Magin-Lachmann et al., 2003) and will be analyzed later on.

The *oriP*/EBNA-1 retention system is easy to use and can provide extra-chromosomal maintenance to foreign DNA of hundred kilobases delivered into cells but has some major disadvantages. It provides random rather than equal segregation of the episomal vector to daughter cells which results in loss of the episomes at a rate of 2-8% per cell division (Sclimenti & Calos, 1998). This, along with the fact that it involves viral sequences particularly from the EBV which has been associated to several types of human malignancies (Cohen, 2000) limits the use of EBV vectors for safe gene therapy.
