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Recombinant AAV for Gene Therapy

Adeno-Associated Viruses (AAVs) are small viruses made of a single stranded DNA genome (4.7 kb) encapsidated in a viral capsid made by assembly of three proteins (VP1, VP2 and VP3). Wild-type AAVs require co-infection with a helper virus, such as adenovirus (Ad), herpes simplex virus, or vaccinia virus, to allow productive infection and replication. Upon active infection, the AAV genome persists in the host cell nucleus in an episomal form with sustained RNA expression and can also integrate the host DNA into chromosome 19.

Recombinant adeno-associated viruses (rAAV) are used as a DNA delivery vector for human gene therapy, relying on the ability of AAVs to infect human cells while being non-pathogenic. rAAVs are produced and administered without any helper virus, which lowers the risk of genome integration and confers these vectors with an interesting safety profile. The various AAV serotypes enable specificity to deliver the therapeutic Gene of Interest (GOI) into the targeted organs.

The demand for rAAV is growing rapidly as clinical pipelines expand and new gene therapy approvals are granted. However, the manufacturing process for AAV remains complex and challenging when it comes to scaling-up and achieving high yields. There is a strong need to find the optimal conditions contributing to large-scale AAV production process intensification.

Currently, the most common approach to manufacture rAAVs at large scale is the use of triple transfection in suspension cultured HEK293 cells followed by a purification process.

Transient Transfection for rAAV Production

Production of rAAVs commonly involves chemical-based transfection of plasmid DNA delivering genetic material into HEK293 cells. Cell machinery is rerouted to enable the production of viral proteins required to build novel rAAV particles that carry the therapeutic GOI.