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(NSCs)进行基因修饰的新策略的开发，将对NSCs介导的神经疾病细胞疗法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包装系统，用于基于单纯疱疹病毒(HSV)的扩增子载体，没有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100kb的基因组DNA插入可以包装成HSV病毒粒子，完整地传递，并在体外培养的细胞中表达。通过添加基于EB病毒(EBV)的复制子元件，带有基因组DNA插入的HSV扩增片段(HSV/EBV/基因组载体)被证明可以作为额外的染色体元件在感染细胞中复制和维持，并在较长时间内表达功能基因产物。该载体系统的独特性质使我们推测HSV/EBV/基因组载体可以作为一种平台技术来实现NSCs介导的细胞治疗的一些长期目标，即：1)补充疾病NSCs在体内外的遗传缺陷以治疗遗传性神经疾病，2)稳定和细胞类型特异性的转基因在从转导的NSCs分化而来的神经元和神经胶质细胞中的表达，以及3)NSCs的基因工程用于移植后在体内可控的增殖、迁移和分化。为了评估这一方法的可行性，本项目建议：1)验证HSV/EBV/基因组载体的上体复制和维护，并在培养的NSCs分离时评估载体上的转基因表达；2)在体外和体内验证基因组转基因在终末分化神经元和神经胶质细胞中的稳定和细胞类型特异性表达。我们将研究五个人类基因，次黄嘌呤磷酸核糖转移酶、巢蛋白、髓鞘碱性蛋白、神经胶质纤维酸性蛋白和微管相关蛋白tau，分别作为管家基因、神经干细胞特异性基因、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/基因组载体的了解，并确认基因组转基因和EBV元件的优势，从而为利用HSV/EBV/基因组载体进行神经干细胞的基因工程奠定坚实的基础

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