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

多年来，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。通过不断的改进，HIV-1衍生载体已经进化成更安全、更有效的基因输送工具。这种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，感染分裂细胞和非潜水细胞的能力，广泛的组织趋向性，以及潜在更安全的整合位点轮廓。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20多年。尽管进行了进一步的修改，但自引入第三代四质粒系统以来，慢病毒载体的生产系统并没有发生重大变化。它们通常包括一个转基因，两侧是顺式作用序列，包括用于转录的CMV启动子、含有包装信号序列的HIV的5‘前导序列和第二个序列REV Response Element(RRE)。提高我们对HIV-1生命周期的了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包装过程的了解已经大大增加。我们对病毒RNA在包装信号区的2D和3D结构有了更清楚的了解，我们对RNA是如何被选择并运输到包裹成病毒粒子的位置有了更多的了解。仍然有许多悬而未决的问题制约着我们提高病毒滴度和基因转移效率。以下问题，包括：影响HIV-1载体设法将RNA运输到萌芽位置成为其基因组的重要序列是什么？在这一过程中，哪些是关键的宿主细胞蛋白？这些是病毒中其他地方的RNA结构的更精细的细节，可能会影响包装。利用最近关于包装的发现，第一个目标是对包装信号区进行知情的突变分析，以建立有效包装所需的最小序列。突变分析也将被用于稳定RNA结构，以便有利于U5-Aug相互作用的形成，该相互作用促进转录和二聚化，但抑制翻译。传染性的HIV-1毒株必须获得灵活的结构，这些结构可以服务于各种功能，而HIV-1衍生的载体不需要执行这些功能。伴随病毒RNA基因组通过细胞到达萌芽位置的细胞伴侣蛋白也将通过RNA下拉和交联技术以及与基因组RNA连接的蛋白质的质谱学来寻找。它们的重要性将通过干扰RNA敲除和/或cDNA过表达实验来研究。最后，该项目的另一个方面将是对目前用于生产慢病毒载体的细胞系进行改造。这将通过引入对病毒至关重要的复制因子或通过识别其基因将被破坏的限制因子来实现。该项目的总体目标是从多个角度增加我们对RNA包裹过程的了解，并利用这些知识来优化载体的设计，以产生具有最大限度的转基因携带和输送能力的高效版本，用于治疗人类疾病。

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