EBV VECTORS FOR TARGETED GENE THERAPY OF B-LYMPHOMAS

本申请的目的是 有效和选择性地消除B细胞来源淋巴瘤和白血病 使用天然靶向病毒载体系统在癌症患者中的细胞中进行。到 为了实现这一目标，申请人提出开发一种新的组合基因， 应用于Burkett B淋巴瘤的治疗方法基于i）最小的 "无基因" B嗜淋巴细胞性EB病毒（miniEBV）载体，和ii）a 基因增强的"超自杀" HSV 1胸苷激酶（super-TK）。 具体而言，将进行以下研究：目的1）测试 亲B淋巴细胞的miniEBV载体递送和表达自杀病毒的功效 基因进入人类B细胞淋巴瘤后，嗜瘤性和溶瘤策略。 这种方法是基于病毒胸苷激酶（TK）基因的转移 转化为B细胞衍生的淋巴瘤，使其对前药敏感 更昔洛韦（GCV）。为此，将使用miniEBV/sTK感染 体外培养的B淋巴瘤细胞和植入体内的瞬时感染细胞 动物模型分析前药介导的淋巴瘤根除， 体内方案。目的2）建立体外培养的包装细胞系统 以产生无辅助病毒的感染性miniEBV。这将涉及克隆基因组 在基于BAC的载体中的EBV的同源序列，以通过同源序列缺失包装序列， 重组此外，将评价EBV阴性细胞系的 对于miniEBV复制和包装成感染性病毒体是有利的。目的 3)使用目标1 - 2中获得的结果测试miniEBV系统的 在SCID-人淋巴瘤/白血病动物模型中使用 体内方案。通过静脉注射在体内预先建立的人B淋巴瘤 （静脉注射）还将对使用该系统的喷射进行评价，以消除 来自动物各器官的播散性B淋巴瘤。

DESCRIPTION: (Applicant's Abstract): The goal of this application is to effectively and selectively eliminate B-cell derived lymphoma and leukemia cells in cancer patients using a naturally targeted viral vector system. To achieve this goal, the applicant proposes to develop a novel combinatorial gene therapy approach as applied to Burkett B lymphoma based on i) a minimal "gene-less" B lymphotropic Epstein-Barr virus (miniEBV) vector, and ii) a genetically enhanced "hyper-suicide" HSV1 thymidine kinase (super-TK). Specifically, the following studies will be undertaken: Aim 1) To test the efficacy of B lymphotropic miniEBV vectors to deliver and express a suicide gene into human B-cell lymphomas following oncotropic and oncolytic strategies. This approach is based on the transfer of the viral thymidine kinase (TK) gene into B-cell derived lymphomas rendering them sensitive to the prodrug ganciclovir (GCV). For this endeavor miniEBV/sTK will be used to infect B-lymphoma cells in vitro and the transiently infected cells implanted in an animal model to analyze prodrug mediated eradication of the lymphoma using ex vivo protocol. Aim 2) Development of an in vitro cultured packaging cell system to produce helper-free infectious miniEBV. This will involve cloning the genome of EBV in a BAC based vector to delete the packaging sequence by homologous recombination. In addition, EBV negative cell lines will be evaluated for their permissivity to miniEBV replication and packaging into infectious virions. Aim 3) Use the results obtained in Aims 1-2 to test the miniEBV system for its efficiency and safety in a SCID-Human lymphoma/leukemia animal model using an in vivo protocol. Pre-established human B-lymphoma in vivo by intravenous (i.v.) injection with this system will be also evaluated in order to eliminate the disseminated B-lymphoma from various organs of the animal.