HSV amplicon vectors for neuroscience research

描述(由申请人提供)：病毒载体介导的基因传递到神经系统已被证明是一种强大的方法学，具有基础和治疗应用。尤其是重组AAV载体和慢病毒载体，由于它们的简单性、良好的安全性和持久的长期基因表达，在这方面越来越受欢迎。然而，这些载体的转基因能力被限制在分别容纳4.5kb和8kb的外源DNA。在过去的十年里，我们一直在研究和开发基于HSV的扩增子系统，作为一种替代的基因输送系统。单纯疱疹病毒扩增子载体是以复制缺陷的质粒为基础的载体，携带不到整个单纯疱疹病毒基因组的1%，并且包括DNA复制起点和DNA裂解/包装信号。该载体系统的主要优点是其巨大的转基因能力，可容纳高达150kb的外源DNA。这一独特的功能使扩增载体能够整合和传递完整的基因组位点、多个表达盒、用于调控基因表达的可诱导系统和报告基因，从而能够对融合蛋白、启动子活性或转导细胞进行定量和可视化。这种多功能扩增载体的开发和使用无疑将对基础神经科学和翻译神经科学的研究产生影响。例如，可以设计表达用于实时监测各种细胞活动的一个或多个报告基因的指示性扩增载体，或者可以创建在细胞类型特定的或可诱导的启动子的控制下表达一个或多个转基因和/或短发夹(Sh)RNA的可调节扩增载体，以调节外源和/或内源基因的表达。值得注意的是，这种大小高达150kb的复杂遗传结构可以很容易地转移到培养中几乎任何可感染HSV的细胞中，甚至在活着的动物中也是如此。然而，构建如此复杂的载体质粒需要精心设计的克隆策略和相当高的技术技能。此外，将一种DNA元件交换为另一种元件或将新的DNA元件添加到这种复杂的载体质粒中往往需要重新设计整个结构和广泛的分子克隆程序。为了克服这些问题，我们提出了一种新的策略，即将DNA元件和表达盒分别克隆到单独的穿梭质粒中，然后通过顺序使用多个位点特异的DNA重组酶组装成单个扩增子质粒。在这个拟议的项目中，我们将开发这样一个高效和通用的工具包来构建复杂的HSV扩增载体载体，并展示它们在促进HSV扩增载体在神经科学应用中的更广泛使用和充分利用方面的有效性。公共卫生相关性：病毒载体将基因传递到神经系统已被证明是一种强大的方法，无论是基础应用还是治疗应用。我们建议开发和改进一个基于单纯疱疹病毒(HSV)的扩增载体系统，用于神经科学研究。这项工作可能为研究神经系统的正常发育和神经系统疾病的发病机制提供新的方法，所开发的技术可能被应用于改进神经疾病的新疗法。

DESCRIPTION (provided by applicant): Viral vector-mediated gene delivery to the nervous system has proven a powerful methodology with both basic and therapeutic applications. Recombinant AAV vectors and lentiviral vectors, in particular, are increasingly popular in this context because of their simplicity, excellent safety profiles, and durable long-term gene expression. However, the transgene capacity of these vectors is limited to accommodate up to 4.5 and 8 kb of exogenous DNA, respectively. We have been studying and developing the HSV-based amplicon system as an alternative gene delivery system over the past decade. HSV amplicon vectors represent plasmid-based, replication-deficient vectors that carry less than 1% of the entire HSV genome and that include an origin of DNA replication and a DNA cleavage/packaging signal. The major strength of the vector system is its large transgene capacity to accommodate up to 150 kb of exogenous DNA. This unique feature allows amplicon vectors to incorporate and deliver an entire genomic locus, multiple expression cassettes, inducible systems for regulated gene expression, and reporter genes to enable quantitation and visualization of fusion proteins, promoter activity, or transduced cells. Development and use of such multi-functional amplicon vectors will undoubtedly impact research in both basic and translational neuroscience. For example, an indicator amplicon vector could be engineered that expresses one or multiple reporter genes for real-time monitoring of various cellular activities or a regulatable amplicon vector could be created that expresses one or multiple transgenes and/or short hairpin (sh) RNAs under the control of a cell type-specific or inducible promoter for regulated expression of exogenous and/or endogenous genes of interest. Significantly, such complex genetic constructs with sizes up to 150 kb can be readily transferred to virtually any HSV-infectable cells in culture and even in living animals. However, construction of such complex vector plasmids requires well designed cloning strategies and considerable technical skills. Moreover, exchanging one DNA element for another or adding a new DNA element to such complex vector plasmids often requires redesign of the entire construct and extensive molecular cloning procedures. To overcome these problems, we have come up with a new strategy in which DNA elements and expression cassettes are individually cloned into separate shuttle plasmids and then assembled into a single amplicon plasmid by sequential use of multiple site-specific DNA recombinases. In this proposed project, we will develop such an efficient and versatile tool kit to build complex HSV amplicon vector plasmids and demonstrate their usefulness in facilitating wider use and full exploitation of HSV amplicon vectors in neuroscience applications. PUBLIC HEALTH RELEVANCE: Viral vector delivery of genes to the nervous system has proven a powerful approach with both basic and therapeutic applications. We propose to develop and improve a herpes simplex virus (HSV)-based amplicon vector system for neuroscience research. The work may provide new methodologies to study the normal development of the nervous system and the pathogenesis of neurological disorders, and the developed technologies may be applied to improving novel therapeutics for neurological disorders.