Cell-Free Protein Synthesis (CFPS) for improved production strategies of adeno-associated virus vectors

研究背景：开发中最广泛使用的基因治疗产品之一是基于重组腺相关病毒（AAV）的载体，用于基因治疗适应症中的递送。重组AAV生产是复杂且极其耗时的，需要哺乳动物细胞系、稳健的下游纯化和去除空的非功能性衣壳。AAV载体的开发主要集中在优化基因递送，然而，越来越需要开发基于平台的制造工艺和新型载体的筛选工具。无细胞蛋白质合成（CFPS）是一个强大的具有成本效益的筛选技术平台，适用于从微型到生产规模的各种复杂蛋白质产品。CFPS的可行性尚待探索，以解决AAV载体生产的挑战，是本项目的重点。目的和目标：本项目的目的是开发新的解决方案，快速AAV生物过程中的应用合成生物学和无细胞蛋白质合成技术的快速筛选能力和控制翻译后修饰。AAV衣壳蛋白的无细胞蛋白质合成（CFPS）和组装成空AAV颗粒。衣壳蛋白文库的产生和筛选。2.用遗传物质装载空AAV颗粒，最初编码模型蛋白（即荧光蛋白），随后是感兴趣的基因。3.细胞培养物中AAV颗粒的摄取。4.翻译后修饰的范围要求和CFPS的可扩展性。应用和益处：该项目将是使用CFPS作为筛选和/或制造技术的试验台，重点是AAV载体生产。CFPS能够在受控条件下快速体外生产和分析衣壳组装体。与细胞培养系统相比，开放系统如CFPS的一个优点是快速筛选能力（即用于衣壳蛋白工程、改善的稳定性和不同的血清型）。CFPS还提供对翻译后修饰（糖基化、泛素化、磷酸化、SUMO化、乙酰化、肉豆蔻酰化）的控制。关于制造，可以改进分离、纯度和分析，因为颗粒将不需要从细胞培养物中纯化。合成AAV颗粒的应用包括用于基因治疗的遗传有效载荷、小分子药物的细胞递送和疫苗开发。研究方法：候选人将培养生物信息学，微生物学，分子生物学，CFPS，蛋白质纯化，生物化学/生物物理方法，分析和生物加工方面的技能。UCL的专业知识和培训将与Pall在AAV病毒载体生产和纯化以及无细胞衍生AAV颗粒性能测试能力方面的生物工艺专业知识高度互补。研究结果将在与监督小组的定期会议上、会议上公布，并发表在同行评审的期刊上。与EPSRC的战略和研究领域保持一致：该项目与EPSRC的主题“制造未来”和“医疗技术”保持一致。它位于生物化学工程系，生物过程研究的世界领导者，创造新的工程解决方案，以支持未来的生物制造过程。该部门通过生物化学和系统工程师，合成生物学家和疫苗学家团队的中心提供研究，这些团队共同工作。该项目与未来目标医疗保健制造中心和未来疫苗制造研究中心（Vax Hub）保持一致，其愿景是通过整合发现和生物工艺来制造下一代疫苗。该项目是UCL-Pall Biotech Centre of Excellence（CoE）的一部分。

Context of research: One of the most widely used gene therapy products in development are recombinant Adeno-Associated Virus (AAV)-based vectors, used for delivery in gene therapy indications. Recombinant AAV production is complex and extremely time consuming, requiring mammalian cell lines, robust downstream purification and removal of empty non-functional capsids. The AAV vector development has largely focused on optimising gene delivery, however there is an increasing need to develop platform-based manufacturing processes and screening tools of novel vectors. Cell-free protein synthesis (CFPS) is a powerful cost-effective screening technology platform for a variety of complex protein products from microscale to manufacturing scale. CFPS has yet to be explored for its feasibility to address AAV vector manufacturing challenges and is the focus of this project.Aims and Objectives: The aim of this project is to develop novel solutions for rapid AAV bioprocesses by application of synthetic biology and cell-free protein synthesis technologies for rapid screening capability and control over posttranslational modifications.To meet these goals the following objectives will be pursued: 1. Cell-free protein synthesis (CFPS) of AAV capsid proteins and assembly into empty AAV particles. Generating and screening of a library of capsid proteins. 2. Loading empty AAV particles with genetic material, initially coding for a model protein (i.e. fluorescent protein) followed by genes of interest. 3. Uptake of AAV particles in cell culture. 4. Scoping requirements for posttranslational modifications and scalability of the CFPS. Applications and Benefits:This project will be a test bed for the use of CFPS as screening and/or manufacturing technology with focus on AAV vector production. CFPS could enable quick in vitro production and analysis of capsid assemblies under controlled conditions. One advantage of an open system like CFPS over a cell-culture system is the rapid screening capability (i.e. for capsid protein engineering, improved stability and different serotypes). CFPS also offers control over post translational modifications (glycosylation, ubiquitination, phosphorylation, SUMOylation, acetylation, myristoylation). With regards to manufacturing, separation, purity and analytics may be improved as particles will not need to be purified from cell culture. Applications of synthetic AAV particles include genetic payloads for gene therapy, cellular delivery of small-molecule drugs and vaccine development. The research methodology: The candidate will develop skills in bioinformatics, microbiology, molecular biology, CFPS, protein purification, biochemical/biophysical methods, analytics and bioprocessing. UCL's expertise and training will be highly complementary with Pall's bioprocesses expertise in AAV viral vector production and purification as well as testing capabilities for the performance of cell-free derived AAV particles. Results will be presented at regular meetings with the supervisory team, at conferences and published in peer-reviewed journals. Alignment to EPSRC's strategies and research areas: This project is aligned with the EPSRC themes 'manufacturing the future' and 'healthcare technologies'. It is based in the Department of Biochemical Engineering, a world leader in bioprocess research creating novel engineering solutions to underpin future biomanufacturing processes. The Department delivers research through hubs which are teams of biochemical and system engineers, synthetic biologists and vaccinologists, that work together. The project is aligned with the Future Targeted Healthcare Manufacturing Hub and the The Future Vaccine Manufacturing Research Hub (Vax-Hub) whose vision it is to manufacture the next-generation vaccines by integrating discovery and bioprocess. The project is part of the UCL-Pall Biotech Centre of Excellence (CoE).