Temporal Insights into Molecular and Cellular Responses during rAAV Production in HEK293T Cells

Abstract

The gene therapy field is actively pursuing cost-effective, large-scale production of recombinant adeno-associated virus (rAAV) vectors for therapeutic applications, which demand high dosages. Enhanced yield is essential but presents technical and cost challenges. Strategies like suspension cell culture, transfection optimization, and cultivation conditions have shown moderate success but fall short for large-scale applications. In this study, we explore the host cell proteome of HEK293T cell lines used for AAV2 production under different transfection conditions (standard, sub-optimal, optimal) using SWATH-MS. To understand molecular and cellular mechanisms, we created a tailored spectral library for HEK293T-AAV interactions. Our gene ontology and pathway analysis revealed significant protein expression variations, particularly in processes related to cellular homeostasis, metabolic regulation, vesicular transport, ribosomal biogenesis, and cellular proliferation under optimal transfection conditions. These conditions increased rAAV titre by 50% compared to standard protocols. Furthermore, we identified alterations in host cell proteins crucial for AAV mRNA stability and gene translation, particularly regarding AAV capsid transcripts in optimal transfection conditions. This study provides insights into cellular mechanisms during rAAV production in HEK293T cells and offers potential advancements in scalability and cost-efficiency for gene therapy vector production.SignificanceGenerating AAV is challenging and the amount produced is limited, despite the success of gene therapy. Thus, it is necessary to enhance the productivity of host cells through various engineering techniques. The replication and transduction of viruses cause numerous modifications to the host cell’s proteome. Viruses exploit the processes of the host cell, influencing the availability, post-translational adjustments, associations, or placement of its proteins during the replication/growth cycle. Comprehending the fluctuations in biological processes during AAV replication is essential to grasping how virus-host relations shape the result of infection and viral protein expression.