EBV VECTORS FOR TARGETED GENE THERAPY OF B-LYMPHOMAS

描述：(申请者摘要)：本申请的目标是 有效和选择性地消除B细胞性淋巴瘤和白血病 使用自然靶向病毒载体系统的癌症患者的细胞。至 为了实现这一目标，申请人提议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 “无基因”嗜B淋巴细胞EB病毒(微型EBV)载体，以及ii)a 基因增强型“超级自杀”HSV1胸苷激酶(Super-TK)。 具体来说，将进行以下研究：目的1)测试 B淋巴细胞微型EBV载体传递和表达自杀的效果 通过致癌和溶瘤策略将基因导入人类B细胞淋巴瘤。 这种方法是基于病毒胸苷激酶(TK)基因的转移。 转化为B细胞来源的淋巴瘤，使其对前药敏感 更昔洛韦(GCV)。为此，将使用微型EBV/StK来感染 B-淋巴瘤细胞的体外培养和瞬时感染细胞的体内移植 前药介导EX根除淋巴瘤的动物模型分析 VIVO协议。目的2)体外培养包装细胞系统的研制 生产无辅助者的传染性迷你EBV。这将涉及到克隆基因组 在基于BAC的载体中通过同源删除包装序列 重组。此外，还将对EBV阴性细胞系进行评估 允许小型EBV复制和包装成具有感染性的病毒粒子。目标 3)使用在AIMS 1-2中获得的结果测试微型EBV系统的ITS 一种SCID-人类淋巴瘤/白血病动物模型的有效性和安全性 体内试验方案。静脉注射体内预先建立的人B淋巴瘤 (静脉注射)还将对使用该系统的注射进行评估，以消除 动物不同器官的播散性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.