Advancing AAV production with high-throughput screening & transcriptomics
Posted: 23 July 2024Christopher A Reid, Markus Hörer, Mohammad A Mandegar
Recombinant adeno-associated viral (rAAV) vector manufacturing processes must be improved if successful commercialization of many pipeline products is to be realized. The traditional design-of-experiment (DoE) approach to process development has led to some improvements, but more is needed. Ascend has used an integrated strategy that complements an iterative data-driven rational approach with high-throughput screening and transcriptomics to better understand the cellular processes involved in rAAV replication and assembly. This includes timely expression of AAV proteins, helper virus, and cellular gene products at appropriate levels; precise assembly of capsid proteins; as well as replication of vector genomes and their packaging into pre-formed empty capsids.
The data obtained from a high-throughput AAV screen showed similarities with the transcriptomics data collected during AAV production. Differentially expressed transcripts during AAV9 production were identified using RNA-sequencing and intersected with results obtained from a primary small-molecule screen of potential enhancers of AAV9 production. The top three shared pathways consisted of transcription, signal transduction, and cell-cycle regulation. The three most promising small-molecule enhancers were investigated by performing runs in shake flasks. Final validation of the enhancers thereby leveraging Ascend analytics platform is performed in Ambr®15 and Ambr®250 bioreactors from Sartorius, as data collected in these scale-down bioreactors is predictive for large-scale manufacturing platform.
These discoveries are being combined with others made at Ascend to offer an unmatched level of gene therapy development and support from gene to GMP. A modular rAAV manufacturing platform features proprietary and outsourced analytical techniques to balance the yield and quality of each process. All our work is focused on ensuring process scalability and regulatory compliance as well as product safety, efficacy, and cost-effectiveness across scales and serotypes.
To reference: Cell & Gene Therapy Insights 2024; 10(6), 821–840. DOI: 10.18609/CGTI.2024.095