Establishment of high-titre, stable LV producer cells with high-level genome RNA

将治疗性DNA输送到患者体内的最有效的基因转移方法之一是使用工程病毒，如慢病毒载体或LV。为了生产安全、不可复制的慢病毒载体，你需要在生产细胞中表达来自不同DNA的多种成分，包括病毒核心结构的Gag-Poll以及酶和包膜。另一个基本组成部分是载体基因组本身，它编码将被输送到患者细胞的治疗基因。细胞系发展的创新导致了生产型细胞系的建立，在这种细胞系中，包括载体基因组在内的所有病毒载体成分都稳定地整合到宿主细胞中。这些细胞系的开发是牛津生物医学和伦敦大学学院的关键活动。稳定的生产者细胞系是理想的，因为它们代表了更具可扩展性和健壮的制造过程。虽然所有组件都需要在LV生产者细胞中高水平表达，但本项目重点关注载体基因组RNA在生产者细胞中的水平。这里的一个关键假设是，高滴度的LV生产需要LV生产者中高水平的载体基因组RNA。然而，要稳定表达足够高水平的RNA基因组一直是一个挑战，在生产者培养上清液中增加全包装的、有功能的LV比例，而不是“空的”LV颗粒。UCL和OXB一直在探索提高基因组RNA水平的方法；具体地说，该项目将探索使用许多LVGenome副本作为实现高水平载体基因组的一种方式。这将支持高滴度LV的生产，并有助于为建立基因定义(更安全)、高滴度、稳定的LV生产商创建一个可复制的平台。将探索实现高载体基因组表达的替代策略。学生将研究这些策略背后的生物/分子机制(S)。除了这些细胞工程，学生还将研究向量分析和缩小制造，以展示在这个项目中工程细胞的质量和可扩展性。除了常见的LV分析(功能和物理滴定、污染物检测/定量等)外，还将探索采用即将推出的分析技术进行的单颗粒分析。

One of the most efficient gene transfer methodologies to deliver therapeutic DNA into patients is the use of engineered viruses, such as lentiviral vectors or LVs. To produce safe, non-replicating lentiviral vectors, you need to express multiple components from separate DNA in the producer cells, including gag-pol for viral core structure and enzymes and envelopes. The other essential component is the vector genome itself that encodes therapeutic genes to be delivered to patients' cells. Innovation in cell line development has led to the establishment of producer cell lines where all of the viral vector components, including the vector genome, are stably integrated into the host cell. The development of these cell lines are key activities at Oxford Biomedica and at UCL. Stable producer cell lines are desirable as they represent the more scalable and robust manufacturing process.While all components need to be expressed at high levels in the LV producer cell, this project focuses on the level of vector genome RNA in the producer cell. A key hypothesis here is that high-titre LV production requires high level of vector genome RNA in LV producers. However, it has been challenging to stably express high enough level of RNA genome, increasing proportion of fully-packaged, functional LVs against 'empty' LV particles in the producer culture supernatant. Both UCL and OXB have been exploring methods to boost genome RNA level; specifically this project will explore the use of many LVGenome copies as a way to achieve high levels of vector genome. This will support high-titre LV production and help create a reproducible platform for establishing genetically defined (safer), high-titre, stable LV producers. Alternative strategies towards high vector genome expression will be explored. The student will investigate the biological/molecular mechanism(s) behind these strategies.Besides these cell engineering, the student will also investigate vector analytics and scale-down manufacture to demonstrate the quality and scalability of cells engineered in this project. In addition to common LV analyses (functional and physical titres, contaminant detection/quantification etc), single particle analyses with upcoming analytic technologies will be explored.