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Looking Beyond Plasmids: What’s on the Horizon for Gene Therapy Manufacture?

Plasmid-based methods have, for good reason, been a trusted method for the manufacture of adeno-associated viral (AAV) vectors for many years. As 2022 closes and we look towards 2023, plasmids will likely continue to be useful for gene therapies already in the pipeline. The refinement of robust plasmid-based manufacturing platforms, in particular, will further support drug developers as they prepare for the later stages of their therapeutic journey to commercialisation. Improvements in viral vector yield, as well as reduced costs and timelines, are of particular importance and are possible to achieve using platform approaches.

Key priorities for 2023 and beyond will be to reduce the costs of gene therapies and expand their accessibility to patients who need them, once novel therapies obtain regulatory approval. The ability of plasmid-based processes to meet increasingly ambitious challenges is likely to be limited and novel, disruptive technologies are keenly needed. One such disruptive technology, TESSA™ technology, has the potential to substantially increase yields compared with plasmid-based systems; could it be the future of gene therapy manufacture?

Plasmids: An Old Standard but a Gold Standard

AAV vectors are commonly manufactured in HEK293 cells transiently transfected with a set of plasmids, containing all the genetic instructions needed to produce AAV. Advancements in plasmid engineering have reinforced the successes of plasmid-based manufacturing platforms. Reconfiguration of the RepCap genes, as well as the removal of additional hexon genes in the adenoviral helper plasmid, have been found to enhance viral vector yield and performance. In addition to plasmid optimisation, fine-tuning of other aspects of platform approaches, which hold benefits in terms of pre-validated methods, equipment, and in-stock raw materials, can bring down timelines and costs.

There are, however, inherent limitations of working with plasmids. Transient transfection of plasmids into HEK293 cell lines is variable, limiting the maximum yield of AAV and introducing batch-to-batch variability. Plasmids may be genetically unstable, limiting quality and consistency when scaled up to the substantial quantities needed for treatment. The plasmids supply chain is beset with bottlenecks, with relatively few facilities able to produce plasmids to GMP grade.

Introducing Plasmid-free AAV Manufacture

One option for a high-yielding, plasmid-free approach is to use wildtype adenovirus to manufacture AAV. However, this method introduces the real possibility of adenovirus contaminating the final product, with expensive decontamination steps required to remove this contamination. Although high yielding, the decontamination costs may be a barrier to success.