Development of conditionally replicating integrase defective lentiviral vectors for Epstein-Barr virus gene therapy

Integrase defective lentiviral vectors show promise for achieving gene expression without integration, preserving some benefits of LVs, whereas reducing the potentially detrimental risk of insertional mutagenesis. Numerous reports supported the ability of these vectors to confer long-term gene expression in slowly dividing cell types for potentially corrective gene expression. These reports also highlighted additional applications of these vectors as delivery platforms for alternate integrative pathways, suicide gene therapy and vaccines. Nevertheless, in cell culture systems the transgene expression duration of IDLVs is highly transient, because of the rapid cell turnover, limiting their use to non-dividing or slowly dividing cells. The instability of the IDLVs in proliferating cells is mainly due to the lack of an origin of replication allowing vector’s DNA replication. In a work by Vargas et al. (2008), it has been demonstrated that if the transgene cassette carries a viral ORI, IDLV’s transgene expression persists if the necessary trans protein is supplied. In the work described in this thesis we have engineered IDLVs to selectively transduce EBNA1-expressing cells by incorporating the EBV oriP into the IDLV genome. Exploitation of EBNA1 to maintain DNA that contains the oriP has obvious use for gene therapy. Epstein–Barr virus (EBV) is widely associated with disease in both immunocompromised and immunocompetent hosts and has been implicated in approximately 1% of tumors worldwide. Current therapies are poorly effective and often toxic, therefore, the development of an effective, tumor-specific treatment is of high priority. The goal of IDLV-mediated suicide gene therapy of EBV-driven diseases would be to eliminate B cells that are latently infected with EBV using an IDLV that contains the EBV elements required for EBNA1-induced episome retention and transcriptional up-regulation.