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...