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输送到患者中的最有效的基因转移方法之一是使用工程病毒，例如慢病毒载体或LVS。为了产生安全的，非复制的慢病毒载体，您需要从生产细胞中的单独DNA中表达多个成分，包括病毒核心结构以及酶和信封的GAG-POL。另一个必不可少的成分是载体基因组本身，该基因组本身编码要递送给患者细胞的治疗基因。细胞系发育中的创新导致建立生产者细胞系，其中所有病毒载体成分（包括矢量基因组）都稳定地整合到宿主细胞中。这些细胞系的发展是牛津生物群和UCL的关键活性。稳定的生产商细胞系是可取的，因为它们代表了更可扩展和健壮的制造过程。虽然所有组件都需要在LV生产商细胞中高水平表达，但该项目集中于生产商细胞中的矢量基因组RNA水平。这里的一个关键假设是，高贴左右的生产需要高水平的LV生产者中的载体基因组RNA。但是，稳定表达足够高的RNA基因组，增加了针对生产者培养物上清液中“空” LV颗粒的全部功能LV的比例，这是一项挑战。 UCL和OXB都一直在探索提高基因组RNA水平的方法。具体而言，该项目将探讨许多LVGENOME副本作为获得高水平矢量基因组的一种方式。这将支持高贴级别的LV生产，并有助于创建一个可再现的平台，以建立遗传定义（更安全），高端，稳定的LV生产商。将探讨针对高矢量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。底面这些细胞工程，学生还将研究矢量分析和缩小制造制造，以证明该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）外，还将探索使用即将进行的分析技术的单个粒子分析。