Impact of dual baculovirus infection on the Sf9 insect cell transcriptome during AAV production using single-cell RNA-seq.

The insect cell-baculovirus expression vector system (IC-BEVS) has shown to be a powerful platform to produce complex biopharmaceutical products, such as recombinant proteins and virus-like particles (VLPs). More recently IC-BEVS has been also used as an alternative to produce adeno-associated virus (AAV). However, little is known about the variability of insect cell populations and the potential effect of heterogeneity (e.g., stochastic infection process and differences in infection kinetics), on product titer and/or quality. In this study, transcriptomics analysis of Sf9 insect cells during the production of recombinant AAV of serotype 2 (AAV2) using a low multiplicity of infection, dual-baculovirus system was performed via single-cell RNA-seq (scRNA-seq). Before infection, the principal source of variability in Sf9 insect cells was associated with cell cycle. Over the course of infection, an increase in transcriptional heterogeneity was detected, this being linked to the expression of baculovirus genes as well as to differences in AAV transgenes (rep, cap and gfp) expression. Noteworthy, at 24 hours post-infection (hpi) only 29 % of cells enclosed all three necessary AAV transgenes to produce packed AAV2 particles, indicating limitations of the dual baculovirus system. In addition, the trajectory analysis herein performed highlighted that biological processes such as protein folding, metabolic processes, translation and stress response have been significantly altered upon infection. Overall, this work reports the first application of scRNA-seq to the IC-BEVS and highlights significant variations in individual cells within the population, providing insight for rational cell and process engineering towards improved AAV2 production in IC-BEVS. This article is protected by copyright. All rights reserved.