CRISPR/Cas9-based cell engineering approaches to boost recombinant AVV productivity.

Recombinant adeno-associated viral vectors (rAAV) have gained substantial attention following the commercialisation of new gene therapy products. However, current batch production methods utilising human embryonic kidney (HEK) 293 cells are inefficient, hindering cost-effective manufacturing. While various approaches have marginally improved rAAV yields, poor understanding of host cell biology may prevent further yield gains.

In our pursuit of increased rAAV titres, we devised a scalable, transient transfection process [VD1] using a suspension-adapted HEK cell line. Operational parameters were optimized via design of experiments (DoE) on the Sartorius Ambr@15 system, establishing an optimized design space. Scale-up to the Sartorius Ambr@250 system validated the process, yielding comparable titres to small-scale screening studies.

Additionally, we developed GMP-ready monoclonal HEK293 cell lines from the suspension-adapted HEK line. The optimised process was validated across two monoclonal lines, facilitating productivity-based ranking. Subsequent omics studies unveiled differentially enriched pathways, including antiviral defence pathways as potential cause for inefficient AAV productivity. To test this hypothesis, we initiated a proof-of-concept study targeting multiple genes in the cGAS–STINhG pathway using CRISPR/Cas9 technology to assess the impact on rAAV productivity.

Successful development of the multi-gene knockout strategy enables the generation of a CRISPR-mediated target validation platform, which can be applied to other candidate pathways identified through our omics studies. Whilst enhancing our understanding of key biological mechanisms in rAAV production, this will lead to the generation of novel, GMP-ready, engineered HEK lines with a potentially higher productivity, which can increase the industry gene therapy manufacturing capacity.