Gene engineering of nerve stem using HSV amplicon vector

描述（由申请人提供）：在体外和体内对神经干细胞/祖细胞（NSCs）进行基因修饰的新策略的发展将对神经系统疾病的NSCs介导的细胞治疗的发展以及基础发育神经生物学的进步产生重大影响。我们最近开发了一种单纯疱疹病毒（HSV）扩增子载体的包装系统，使其免于辅助病毒污染或病毒基因表达。利用这项技术，我们已经证明了超过100千碱基的基因组DNA插入物可以包装成HSV病毒粒子，完整地传递，并在体外培养细胞中表达。通过添加eb病毒（Epstein-Barr virus， EBV）复制子元件，发现带有基因组DNA插入的HSV扩增子（HSV/EBV/基因组载体）在感染细胞中作为染色体外元件复制并维持，并长时间表达功能基因产物。该载体系统的独特特性使我们假设HSV/EBV/基因组载体可以作为平台技术，实现nscs介导细胞治疗发展的一些长期目标，即：1)在体外和体内补充病变NSCs的遗传缺陷，治疗遗传性神经系统疾病；2)在转导的NSCs分化的神经元和胶质细胞中实现稳定的、细胞类型特异性的转基因表达；3)对NSCs进行基因工程，控制移植后在体内的增殖、迁移和分化。为了评估该方法的可行性，本项目拟：1)验证HSV/EBV/基因组载体的体外复制和维持，并评估其在培养分化的NSCs中的功能性转基因表达；2)验证基因组转基因在终末分化的神经元和神经胶质细胞中的稳定表达和细胞类型特异性表达。我们将分别研究五种人类基因：次黄嘌呤-磷酸核糖基转移酶、巢蛋白、髓鞘碱性蛋白、胶质纤维酸性蛋白和微管相关蛋白tau，它们分别作为一种管家基因、nsc特异性基因、少突胶质细胞特异性基因、星形胶质细胞特异性基因和神经元特异性基因。这些研究将进一步加深对HSV/EBV/基因组载体的认识，确认基因组转基因和EBV元件的优势，为利用HSV/EBV/基因组载体进行NSCs基因工程提供坚实的基础

DESCRIPTION (provided by applicant): The development of a novel strategy to genetically modify neural stem/progenitor cells (NSCs) both in vitro and in vivo will have a significant impact on the development of NSCs-mediated cell therapy for neurological disorders as well as on the advance of basic developmental neurobiology. We recently have developed a packaging system for herpes simplex virus (HSV)-based amplicon vectors free from helper virus contamination or viral gene expression. Using this technology, we have demonstrated that genomic DNA inserts over 100 kilobases can be packaged into HSV virions, delivered intact, and expressed in cultured cells in vitro. By adding Epstein-Barr virus (EBV)-based replicon elements, HSV amplicons with genomic DNA inserts (HSV/EBV/genomic vectors) were shown to replicate and be maintained as extra chromosomal elements in infected cells, and express functional gene products for a long period of time. The unique properties of this vector system lead us to hypothesize that HSV/EBV/genomic vectors can be a platform technology to achieve some of the long-term goals toward the development of NSCs-mediated cell therapy, namely: 1) Complementing genetic defects of diseased NSCs in vitro and in vivo to treat genetic neurological disorders, 2) stable and cell type-specific transgene expression in neurons and glial cells differentiated from the transduced NSCs, and 3) genetic engineering of NSCs for controlled proliferation, migration, and differentiation in vivo after transplantation. To evaluate the feasibility of this approach, this project proposes to: 1) Verify episomal replication and maintenance of HSV/EBV/genomic vectors and evaluate furictional transgene expression from the vectors in dividing NSCs in culture, and 2) verify stable and cell-type-specific expression of genomic transgenes in terminally differentiated neurons and glial cells both in vitro and in vivo. We will examine five human genes, hypoxanthine-phosphoribosyltransferase, nestin, myelin basic protein, glial fibrillary acidic protein, and microtubule-associated protein tau as a housekeeping, NSC-specific, oligodendrocyte-specific, astrocyte-specific, and neuron-specific gene, respectively. These studies will provide further understanding of HSV/EBV/genomic vectors and confirm the advantages of genomic transgenes and EBV elements, which then should provide a strong basis for genetic engineering of NSCs using HSV/EBV/genomic vectors