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

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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).