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

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 post
translational 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).

The CDT has a proven track record of delivering impact from its research and training activities and this will continue in the new Centre. The main types of impact relate to: (i) provision of highly skilled EngD and sPhD graduates; (ii) generation of intellectual property (IP) in support of collaborating companies or for spin-out company creation; (iii) knowledge exchange to the wider bioprocess-using industries; (iv) benefits to patients in terms of new and more cost effective medicines, and (v) benefits to the wider society via involvement in public engagement activities and impacts on policy.

With regard to training, provision of future bioindustry leaders is the primary output of the CDT and some 96% of previous EngD graduates have progressed to relevant bioindustry careers. These highly skilled individuals help catalyse private sector innovation and biomanufacturing activity. This is of enormous importance to capitalise on emerging markets, such as Advanced Therapy Medicinal Products (ATMPs), and to create new jobs and a skilled labour force to underpin economic growth. The CDT will deliver new, flexible on-line training modules on complex biological products manufacture that will be made available to the wider bioprocessing community. It will also provide researchers with opportunities for international company placements and cross-cohort training between UCL and SSPC via a new annual Summer School and Conference.

In terms of IP generation, each industry-collaborative EngD project will have direct impact on the industry sponsor in terms of new technology generation and improvements to existing processes or procedures. Where substantial IP is generated in EngD or sPhD programmes, this has the potential to lead to spin-out company creation and job creation with wider economic benefit. CDT research has already led to creation of a number of successful spin-out companies and licensing agreements. Once arising IP is protected the existing UCL and NIBRT post-experience training programmes provide opportunities for wider industrial dissemination and impact of CDT research and training materials.

CDT projects will address production of new ATMPs or improvements to the manufacture of the next generation of complex biological products that will directly benefit healthcare providers and patients. Examples arising from previous EngD projects have included engineered enzymes for greener pharmaceutical synthesis, novel bioprocess operations to reduce biopharmaceutical manufacturing costs and the translation of early stem cell therapies into clinical trials. In each case the individual researchers have been important champions of knowledge exchange to their collaborating companies.

Finally, in terms of wider public engagement and society, the CDT has achieved substantial impact via involvement of staff and researchers in activities with schools (e.g. STEMnet), presentations at science fairs (Big Bang, Cheltenham), delivery of high profile public lectures (Wellcome Trust, Royal Institution) as well as TV and radio presentations. The next generation of CDT researchers will receive new training on the principles of Responsible Innovation (RI) that will be embedded in their research and help inform their public engagement activities and impact on policy.