Impact of dual-baculovirus infection on the Sf9 insect cell transcriptome during rAAV 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. More recently, IC-BEVS has also been used as an alternative to produce recombinant adeno-associated virus (rAAV). 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 rAAV of serotype 2 (rAAV2) using a low multiplicity of infection, dual-baculovirus system was performed via single-cell RNA-sequencing (scRNA-seq). Before infection, the principal source of variability in Sf9 insect cells was associated with the cell cycle. Over the course of infection, an increase in transcriptional heterogeneity was detected, which was linked to the expression of baculovirus genes as well as to differences in rAAV transgenes (rep, cap and gfp) expression. Noteworthy, at 24 h post-infection, only 29.4% of cells enclosed all three necessary rAAV transgenes to produce packed rAAV2 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 into the rational cell and process engineering toward improved rAAV2 production in IC-BEVS.

Dissecting insect cell heterogeneity during influenza VLP production using single-cell transcriptomics.

The insect cell-baculovirus expression vector system (IC-BEVS) has been widely used to produce recombinant protein at high titers, including complex virus-like particles (VPLs). However, cell-to-cell variability upon infection is yet one of the least understood phenomena in virology, and little is known about its impact on production of therapeutic proteins. This study aimed at dissecting insect cell population heterogeneity during production of influenza VLPs in IC-BEVS using single-cell RNA-seq (scRNA-seq). High Five cell population was shown to be heterogeneous even before infection, with cell cycle being one of the factors contributing for this variation. In addition, infected insect cells were clustered according to the timing and level of baculovirus genes expression, with each cluster reporting similar influenza VLPs transgenes (i.e., hemagglutinin and M1) transcript counts. Trajectory analysis enabled to track infection progression throughout pseudotime. Specific pathways such as translation machinery, protein folding, sorting and degradation, endocytosis and energy metabolism were identified as being those which vary the most during insect cell infection and production of Influenza VLPs. Overall, this study lays the ground for the application of scRNA-seq in IC-BEVS processes to isolate relevant biological mechanisms during recombinant protein expression towards its further optimization.

Transcriptome analysis of Sf9 insect cells during production of recombinant Adeno-associated virus.

The insect cell-baculovirus expression vector system (IC-BEVS) has emerged as an alternative time- and cost-efficient production platform for recombinant Adeno-associated virus (AAV) for gene therapy. However, a better understanding of the underlying biological mechanisms of IC-BEVS is fundamental to further optimize this expression system toward increased product titer and quality. Here, gene expression of Sf9 insect cells producing recombinant AAV through a dual baculovirus expression system, with low multiplicity of infection (MOI), was profiled by RNA-seq. An 8-fold increase in reads mapping to either baculovirus or AAV transgene sequences was observed between 24 and 48 h post-infection (hpi), confirming a take-over of the host cell transcriptome by the baculovirus. A total of 336 and 4784 genes were identified as differentially expressed at 24 hpi (vs non-infected cells) and at 48 hpi (vs. infected cells at 24 hpi), respectively, including dronc, birc5/iap5, and prp1. Functional annotation found biological processes such as cell cycle, cell growth, protein folding, and cellular amino acid metabolic processes enriched along infection. This work uncovers transcriptional changes in Sf9 in response to baculovirus infection, which provide new insights into cell and/or metabolic engineering targets that can be leveraged for rational bioprocess engineering of IC-BEVS for AAV production.

Gene Expression Analysis of Adapted Insect Cells during Influenza VLP Production Using RNA-Sequencing.

Adaptive laboratory evolution has been used to improve production of influenza hemagglutinin (HA)-displaying virus-like particles (VLPs) in insect cells. However, little is known about the underlying biological mechanisms promoting higher HA-VLP expression in such adapted cell lines. In this article, we present a study of gene expression patterns associated with high-producer insect High Five cells adapted to neutral pH, in comparison to non-adapted cells, during expression of influenza HA-VLPs. RNA-seq shows a decrease in the amount of reads mapping to host cell genomes along infection, and an increase in those mapping to baculovirus and transgenes. A total of 1742 host cell genes were found differentially expressed between adapted and non-adapted cells throughout infection, 474 of those being either up- or down-regulated at both time points evaluated (12 and 24 h post-infection). Interestingly, while host cell genes were found up- and down-regulated in an approximately 1:1 ratio, all differentially expressed baculovirus genes were found to be down-regulated in infected adapted cells. Pathway analysis of differentially expressed genes revealed enrichment of ribosome biosynthesis and carbohydrate, amino acid, and lipid metabolism. In addition, oxidative phosphorylation and protein folding, sorting and degradation pathways were also found to be overrepresented. These findings contribute to our knowledge of biological mechanisms of insect cells during baculovirus-mediated transient expression and will assist the identification of potential engineering targets to increase recombinant protein production in the future.