Increased recombinant adeno-associated virus production by HEK293 cells using small molecule chemical additives

Recombinant adeno-associated virus (rAAV) has established itself as a highly efficacious gene delivery vector with a well characterised safety profile allowing broad clinical application. Recent successes in rAAV-mediated gene therapy clinical trials will continue to drive demand for improved rAAV production processes to reduce costs. Here, we demonstrate that small molecule bioactive chemical additives can significantly increase recombinant AAV vector production by human embryonic kidney (HEK) cells up to three-fold. Nocodazole (an anti-mitotic agent) and M344 (a selective histone deacetylase inhibitor) were identified as positive regulators of rAAV8 genome titre in a microplate screening assay. Addition of nocodazole to triple-transfected HEK293 suspension cells producing rAAV arrested cells in G2/M phase, increased average cell volume and reduced viable cell density relative to untreated rAAV producing cells at harvest. Final crude genome vector titre from nocodazole treated cultures was >2-fold higher compared to non-treated cultures. Further investigation showed nocodazole addition to cultures to be time critical. Genome titre improvement was found to be scalable and serotype independent across two distinct rAAV serotypes, rAAV8 and rAAV9. Furthermore, a combination of M344 and nocodazole produced a positive additive effect on rAAV8 genome titre, resulting in a three-fold increase in genome titre compared to untreated cells.

 

Comments:

It sounds like you're describing a research study or a scientific paper related to the use of small molecule bioactive chemical additives to enhance recombinant adeno-associated virus (rAAV) vector production. This is a very exciting area of research, as improving production processes can significantly impact the availability and affordability of gene therapy, which has enormous potential for treating various genetic and acquired diseases.

From your description, it appears that the study demonstrates the effectiveness of two specific chemical additives, nocodazole (an anti-mitotic agent) and M344 (a selective histone deacetylase inhibitor), in enhancing rAAV vector production in human embryonic kidney (HEK) cells. These additives were found to increase rAAV8 genome titre up to three-fold in a microplate screening assay.

Nocodazole, by arresting cells in G2/M phase, increasing cell volume, and reducing viable cell density, led to a significant improvement in the final crude genome vector titre. The study also noted that the timing of nocodazole addition was critical for its effectiveness. Additionally, the effects of nocodazole were scalable and independent of rAAV serotypes, as it worked for both rAAV8 and rAAV9.

Furthermore, when nocodazole was combined with M344, there was a positive additive effect, resulting in a three-fold increase in rAAV8 genome titre compared to untreated cells. This combination of additives seems to be promising for further optimizing rAAV production processes, potentially making gene therapy more accessible by reducing production costs.

It's worth noting that the development of efficient and cost-effective production methods is a crucial step in the advancement of gene therapy. These findings could pave the way for future research and applications in the field of gene therapy and molecular medicine.