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Paper on E. coli-based cell-free protein synthesis (CFPS) of AAV published in ACS Synthetic Biology

11 September 2024

A paper titled “Adeno-Associated Virus 5 Protein Particles Produced by E. coli Cell-Free Protein Synthesis” by Danielle Deuker, Daniel Bracewell and Stefanie Frank at UCL Biochemical Engineering and Ernest Asilonu from Cytiva has been published in ACS Synthetic Biology

Danielle Deuker

Danielle is an EngD researcher at UCL Biochemical Engineering who works as part of the UCL-Cytiva Centre of Excellence (CoE). This industrial research initiative, valued at £2.7m, funds researchers and research activities between the two organisations.
Recombinant adeno-associated viruses (rAAVs), are non-infectious viruses that have proven effective for delivery of gene therapies. They are promising alternatives to other traditional viral vectors, such as Adenovirus and Lentivirus vectors. This is because rAAVs have low immunogenicity and can exhibit long-term gene expression, making them both safe and lasting. Additionally, unlike some other vectors, different serotypes of AAVs carry different tissue tropisms, allowing targeted therapeutics.

Current AAV manufacturing is high cost, time-consuming and resource-intensive, in part due to long processes and the removal of process impurities such as empty capsids. Currently approved Hemgenix, an AAV-based drug used to treat haemophilia B, costs ~$3.5 million per patient. The research team investigated the use of an E. coli cell-free protein synthesis system to produce empty AAV particles. The method has the potential to reduce the production time and the need for prolonged sterile culture techniques normally required for cell culture.

“This shows that AAVs have the potential to get therapies to market faster for a number of reasons: gene therapies can be easily screened for suitable targets, and the speed of manufacture is improved. It could help make personalised medicine more accessible by making it faster and cheaper and could also lower the carbon footprint of making medicines.” Said Danielle.

It should be noted that this is a very early stage of the research. Cell-free synthesis of AAVs has not been previously reported, and this study is very much a proof of concept that uses only the most abundant capsid protein to make empty particles. The team aims to improve yield in the next stage and produce a product that more closely resembles a traditional AAV.

Danielle told us “This would mean including the other capsid proteins and trying to load the capsids with cargo such as DNA. We would then need to test if capsids made this way can still be effective gene therapy treatments by integrating into the nucleus and delivering genetic information.”

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