Gene Therapy Strategies for Treating Epstein–Barr Virus-Associated Lymphomas: Comparison of Two Different Epstein–Barr Virus-Based Vectors

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Abstract
B cell lymphomas in immunocompromised patients frequently contain the Epstein–Barr virus (EBV) genome (MacMahon et al., 1991), suggesting that gene therapy strategies that target EBV-positive cells for destruction might be useful for the therapy of such tumors. We have previously shown that stable expression of the cytosine deaminase (CD) gene in EBV-positive lymphoblastoid cell lines induces cell killing in the presence of the prodrug 5-fluorocytosine, with a substantial bystander killing effect (Rogers et al., 1996). To promote specific killing of EBV-positive tumor cells, we have constructed two different EBV-based vectors containing the cytosine deaminase gene. The first vector (OriP-CD), which contains the intact EBV oriP enhancer/replication element, replicates as an episome specifically in EBV-positive cells and likewise enhances transcription in an EBV-specific manner. The OriP-CD vector cannot be packaged or spread from cell to cell. The second vector (OriLyt-CD) contains the EBV lytic origin of replication (oriLyt), the EBV packaging sequences (located in the viral termini), the oriP enhancer element (but not the complete replication origin), and the EBV BZLF1 gene (which induces expression of the EBV proteins required for replication of oriLyt). The OriLyt-CD vector is replicated through the oriLyt origin specifically in EBV-positive cells and packaged as an EBV pseudovirion. The packaged oriLyt-CD virion can subsequently infect cells containing the EBV receptor, CD21, and initiate another round of replication in EBV-positive cells. Here we demonstrate that each of these two different EBV-based gene therapy strategies induces specific killing of EBV-positive B cells in vitro (in the presence of 5-FC). The advantages and disadvantages of each strategy are discussed. We have constructed two different EBV-based vectors for expressing toxic genes in EBV-positive tumors. One vector (OriP-CD) is designed to be specifically expressed and replicated in latently EBV-infected tumor cells. The other vector (OriLyt-CD) induces lytic EBV infection when introduced into EBV-positive cells, and is then replicated and packaged using lytic EBV proteins. The packaged lytic EBV vector can then be spread from cell to cell, in contrast to the latent vector. Here we show that both vectors express the cytosine deaminase gene specifically in EBV-positive tumor cells in vitro and promote cell killing in the presence of the prodrug, 5-FC. Thus, either strategy might be useful for treating EBV-positive tumors in vivo.