Enhancement of Lentiviral vector efficiency by modification of cis- and transacting factors affecting genomic RNA encapsidation.

通过修饰影响基因组 RNA 衣壳化的顺式和反式因子来增强慢病毒载体效率。

• 批准号: 1637872
• 金额: --
• 依托单位: University of Cambridge
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2015
• 资助国家: 英国
• 起止时间: 2015 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-1637872
• 关键词: Enhancement; Lentiviral; vector; efficiency; modification

Lentiviral vectors have been established as highly useful laboratory tools and for therapeutic use in humans for many years. Through continuous improvements, HIV-1 derived vectors have evolved into safer and more effective gene delivery vehicles. This vector system offers great advantages including sustained gene expression through stable vector integration, the capability of infecting both dividing and non-diving cells, broad tissue tropism, and a potentially safer integration site profile. More than two decades have passed since lentiviral vectors started being explored for gene therapy use. Despite further modifications, the lentiviral vector production system has not drastically changed since the introduction of the 3rd generation four plasmid system. Typically they comprise a transgene flanked by cis-acting sequences including the CMV promoter for transcription, the 5' leader of HIV containing the packaging signal sequences and a second sequence the Rev Response Element (RRE). Improvement of our understanding of the HIV-1 life cycle is a prerequisite for optimization of the current vector design. Our knowledge about the RNA packaging process has increased significantly since these vectors were first developed. We have a clearer knowledge of the 2D and 3D structure of the viral RNA in the packaging signal region and we understand more about how the RNA is selected and transported to the site of encapsidation into virions. There are still many unanswered questions that restrict us from improving viral titres and gene transfer efficiency. The following questions, including: Which are the important sequences that influence HIV-1 vectors to manage to traffic RNA to the budding site to become its genome? Which are the critical host cell proteins contributing to this process? Which are the finer details of RNA structure elsewhere in the virus that may influence packaging. Using recent findings about packaging the first aim is to make an informed mutational analysis of the packaging signal region to establish the minimal sequences required for efficient packaging. Mutational analysis will also be used to stabilize the RNA structure so that it favourites the formation of the U5-AUG interaction that promotes transcription and dimerization, but supresses translation. The infectious HIV-1 strain has to acquire flexible structures that serve various functions, which the HIV-1 derived vectors don't have to perform. The cellular chaperone proteins which accompany the viral RNA genome through the cell to the budding site will also be sought by RNA pull down and cross linking techniques and Mass Spectroscopy of proteins found linked to the genomic RNA. Their importance will be investigated using interfering RNA knockdown and/or cDNA overexpression experiments. Lastly, another aspect of this project will be the modification of the current cell lines used to produce lentiviral vectors. This will be achieved by either introducing vital replication factors for the virus or by identifying restriction factors whose genes will be knocked down. The overall aim of the project is to increase our understanding of the RNA encapsidation process from many angles and use the knowledge to optimise design of vectors to generate highly efficient versions with maximised transgene carrying and delivery capacity for treatment of human disease

慢病毒载体已被确立为高度有用的实验室工具，并用于人类的治疗用途多年。通过不断的改进，HIV-1衍生载体已经发展成为更安全、更有效的基因传递载体。该载体系统提供了巨大的优势，包括通过稳定的载体整合持续的基因表达，感染分裂和非分裂细胞的能力，广泛的组织嗜性，以及潜在的更安全的整合位点分布。自从慢病毒载体开始被探索用于基因治疗以来，已经过去了二十多年。尽管有进一步的修改，慢病毒载体生产系统自引入第三代四质粒系统以来没有发生重大变化。通常，它们包含侧接有顺式作用序列的转基因，所述顺式作用序列包括用于转录的CMV启动子、含有包装信号序列的HIV的5'前导序列和第二序列Rev应答元件（RRE）。 提高我们对HIV-1生命周期的理解是优化当前载体设计的先决条件。自从这些载体首次开发以来，我们对RNA包装过程的了解显著增加。我们对包装信号区中病毒RNA的2D和3D结构有了更清楚的了解，并且我们对RNA如何被选择并转运到病毒体中的位点有了更多的了解。目前仍有许多未解决的问题限制了我们提高病毒滴度和基因转移效率。以下问题，包括：哪些是影响HIV-1载体设法将RNA运输到萌芽位点成为其基因组的重要序列？哪些是促成这一过程的关键宿主细胞蛋白？它们是病毒中其他地方RNA结构的细节，可能会影响包装。 使用最近的研究结果包装的第一个目标是使一个明智的突变分析的包装信号区，以建立有效包装所需的最小序列。突变分析也将用于稳定RNA结构，以便其有利于促进转录和二聚化但抑制翻译的U 5-AUG相互作用的形成。感染性HIV-1病毒株必须获得灵活的结构，以提供各种功能，而HIV-1衍生载体不必执行这些功能。伴随病毒RNA基因组通过细胞到达出芽位点的细胞伴侣蛋白也将通过RNA下拉和交联技术以及发现与基因组RNA连接的蛋白质的质谱来寻找。将使用干扰RNA敲低和/或cDNA过表达实验来研究它们的重要性。最后，该项目的另一个方面将是用于生产慢病毒载体的现有细胞系的修饰。这将通过引入病毒的重要复制因子或通过鉴定其基因将被敲除的限制因子来实现。 该项目的总体目标是从多个角度增加我们对RNA聚合过程的理解，并利用这些知识优化载体的设计，以产生具有最大化转基因携带和递送能力的高效版本，用于治疗人类疾病。