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

将治疗性DNA传递给患者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。为了生产安全的、不可复制的慢病毒载体，你需要在产生细胞中表达来自不同DNA的多种成分，包括用于病毒核心结构的gag-pol、酶和包膜。另一个重要组成部分是载体基因组本身，它编码治疗基因，将其传递到患者的细胞中。细胞系发展的创新导致了生产者细胞系的建立，其中所有病毒载体成分，包括载体基因组，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学院和伦敦大学学院的主要活动。稳定的生产者细胞系是可取的，因为它们代表了更具可扩展性和健壮的制造过程。虽然所有成分都需要在LV产生细胞中高水平表达，但本项目侧重于产生细胞中载体基因组RNA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，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完全包装的功能性LV的比例，以对抗“空”LV颗粒。UCL和OXB都在探索提高基因组RNA水平的方法；具体来说，这个项目将探索使用许多LVGenome拷贝来实现高水平的载体基因组。这将支持高滴度的LV生产，并帮助创建一个可复制的平台，以建立基因定义（更安全）、高滴度、稳定的LV生产商。将探讨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。除了这些细胞工程，学生还将研究矢量分析和按比例缩小的制造，以展示在这个项目中设计的细胞的质量和可扩展性。除了常见的LV分析（功能和物理滴度，污染物检测/定量等），单颗粒分析与即将到来的分析技术将被探索。

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.