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

建立具有高水平基因组RNA的高滴度、稳定的慢病毒生产细胞

• 批准号: 2881202
• 金额: --
• 依托单位: University College London
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2881202
• 关键词: Establishment; titre; stable; LV; producer

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.

将治疗性DNA递送到患者体内的最有效的基因转移方法之一是使用工程化病毒，例如慢病毒载体或LV。为了生产安全、非复制的慢病毒载体，需要在生产细胞中表达来自不同DNA的多种组分，包括用于病毒核心结构和酶以及包膜的gag-pol。另一个重要的组成部分是载体基因组本身，它编码要递送到患者细胞的治疗基因。细胞系开发中的创新已经导致生产细胞系的建立，其中所有病毒载体组分（包括载体基因组）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UCL的关键活动。稳定的生产细胞系是理想的，因为它们代表了更大规模和更稳健的生产工艺。虽然所有组分都需要在LV生产细胞中以高水平表达，但本项目重点关注生产细胞中载体基因组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，增加生产者培养物上清液中完全包装的功能性LV相对于“空”LV颗粒的比例一直是具有挑战性的。UCL和OXB一直在探索提高基因组RNA水平的方法;具体来说，该项目将探索使用许多LV基因组拷贝作为实现高水平载体基因组的方法。这将支持高滴度LV生产，并有助于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。除了这些细胞工程，学生还将研究载体分析和规模缩小的制造，以证明在这个项目中工程化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）外，还将探索使用即将到来的分析技术进行单颗粒分析。