Development of small-scale adeno-associated virus vector (AAV) production using Cell-Free Protein Synthesis (CFPS) technologies

Project ID: 2531bd1616

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Research Theme: Manufacturing The Future

Research Area(s): Synthetic Biology
Manufacturing technologies

UCL Lead department: Biochemical Engineering

Department Website

Lead Supervisor: Stefanie Frank

Project Summary:

Recombinant Adeno-Associated Virus (rAAV)-based vectors are widely used in gene therapy development, offering transformative potential for patients with severe, often rare, genetic diseases. However, rAAV production is costly, time-consuming, and difficult to scale. It requires mammalian expression systems, robust downstream purification, and removal of non-functional capsids. Thus, there is a growing need for more sustainable and scalable manufacturing approaches. Cell-free protein synthesis (CFPS) is a powerful, cost-effective platform for producing complex proteins at scales ranging from microscale to manufacturing. Its open nature allows easy manipulation, including post-translational modifications (PTMs). We have recently applied and optimised an in-house E. coli-based CFPS [1] to produce virus-like particles of the AAV5 serotype. The protein is expressed in soluble form and spontaneously assembles into capsid particles, which we demonstrated can be taken up by cells [2]. Building on this work, the aim of this project is to develop strategies for controlled capsid assembly and nucleotide packaging using cell-free protein synthesis technologies for simple and cost-effective production of rAAV at a small scale. This offers a practical route for early-stage development and will support rapid, high-quality, and cost-effective rAAV bioprocesses.

Key objectives include:

• Cell-free protein synthesis (CFPS) of rAAV capsid proteins and development of controlled assembly strategies.
• Development of particle loading strategies.
• Evaluating targeted delivery and uptake of CFPS-produced rAAVs.
• Assessing AAV CFPS scalability.

The candidate will gain expertise in synthetic biology, microbiology, CFPS, biochemical/biophysical methods, analytics, and bioprocessing. Results will be shared in supervisory meetings, conferences, and peer-reviewed publications.

References: [1] https://doi.org/10.3390/vaccines9030193; [2] https://doi.org/10.1021/acssynbio.4c00403