Kenney S, Ge J Q, Westphal E M, Olsen J
UNC Lineberger Comprehensive Cancer Center, Department of Medicine, University of North Carolina at Chapel Hill, 27599-7295, USA.
Hum Gene Ther. 1998 May 20;9(8):1131-41. doi: 10.1089/hum.1998.9.8-1131.
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.
免疫功能低下患者的B细胞淋巴瘤常常含有爱泼斯坦-巴尔病毒（EBV）基因组（MacMahon等人，1991年），这表明针对EBV阳性细胞进行破坏的基因治疗策略可能对这类肿瘤的治疗有用。我们先前已表明，在EBV阳性淋巴母细胞系中稳定表达胞嘧啶脱氨酶（CD）基因会在存在前药5-氟胞嘧啶的情况下诱导细胞杀伤，且具有显著的旁观者杀伤效应（Rogers等人，1996年）。为促进对EBV阳性肿瘤细胞的特异性杀伤，我们构建了两种不同的基于EBV的载体，它们都含有胞嘧啶脱氨酶基因。第一种载体（OriP-CD）含有完整的EBV oriP增强子/复制元件，作为附加体在EBV阳性细胞中特异性复制，同样以EBV特异性方式增强转录。OriP-CD载体不能被包装或在细胞间传播。第二种载体（OriLyt-CD）含有EBV裂解性复制起点（oriLyt）、EBV包装序列（位于病毒末端）、oriP增强子元件（但不是完整的复制起点）以及EBV BZLF1基因（它诱导oriLyt复制所需的EBV蛋白表达）。OriLyt-CD载体通过oriLyt起点在EBV阳性细胞中特异性复制，并包装成EBV假病毒粒子。包装好的oriLyt-CD病毒粒子随后可感染含有EBV受体CD21的细胞，并在EBV阳性细胞中启动新一轮复制。在此我们证明，这两种不同的基于EBV的基因治疗策略在体外（在5-FC存在的情况下）均能诱导对EBV阳性B细胞的特异性杀伤。并讨论了每种策略的优缺点。
