Investigating Recombinant AAV (rAAV) Gene Therapy Viral Vector Production

Rationale: Recombinant adeno-associated virus (rAAV) vectors packaged with a target genome are the leading platform for in vivo gene therapy delivery for the treatment of a range of diseases.
However, gene therapy products are extremely expensive (e.g. Luxturna for treatment of inherited retinal dystrophy is US$425,000; Zolgensma for spinal muscular atrophy is the most expensive gene therapy or drug ever at US$2.3M). The high price is largely due to the cost of manufacturing viral vector based gene therapies which remains a challenge, with the majority of rAAV particles generated empty (anywhere from 20-98%) using cultured HEK293 cells and not containing the transgene of interest. Thus, without improvements in the manufacturing of rAAV and the efficiency of transgene packaging, their production will be insufficient to support widespread use in the clinic and potentially life changing/saving rAAV therapies may never reach the market.
Aims, Objectives, Approaches: This project will (a) characterise and map the fundamental HEK293 cellular responses to the demands of plasmid-based transient production of rAAV vectors via RNAseq and relate this to the cells ability to produce rAAV at high yield and quality; (b) analyse rAAV vector component expression and localisation during vector production by microscopy and protein/transcript analysis; (c) generate engineered HEK293 host systems from targets identified by RNAseq; (d) engineering of X-gene expression (a recently identified protein that is implicated in AAV replication); (e) undertake industrial validation of new HEK293 cell hosts ability to produce rAAV at the industrial partner site.
Impact: The project has the potential to deliver a step-change in our understanding of the fundamental biology underpinning the responses of HEK293 cells to an imposed rAAV load and how these define the efficiency of vector packaging, alongside application of this knowledge to generate new HEK293 hosts for improved manufacturing of rAAV vectors at vastly reduced cost.