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Impact of dual baculovirus infection on the Sf9 insect cell transcriptome during AAV production using single-cell RNA-seq
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  • Nikolaus Virgolini,
  • Marco Silvano,
  • ryan hagan,
  • Ricardo Correia,
  • Paula Alves,
  • Colin Clarke,
  • Antonio Roldao,
  • Inês Isidro
Nikolaus Virgolini
Instituto de Biologia Experimental e Tecnologica, ITQB NOVA, Instituto de Tecnologia Química e Biológica António Xavier
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Marco Silvano
Instituto de Biologia Experimental e Tecnologica, ITQB NOVA, Instituto de Tecnologia Química e Biológica António Xavier
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ryan hagan
National Institute for Bioprocessing Research and Training, School of Chemical and Bioprocess Engineering, University College Dublin
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Ricardo Correia
Instituto de Biologia Experimental e Tecnologica, ITQB NOVA, Instituto de Tecnologia Química e Biológica António Xavier
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Paula Alves
Instituto de Biologia Experimental e Tecnologica, ITQB NOVA, Instituto de Tecnologia Química e Biológica António Xavier
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Colin Clarke
National Institute for Bioprocessing Research and Training, School of Chemical and Bioprocess Engineering, University College Dublin
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Antonio Roldao
Instituto de Biologia Experimental e Tecnologica, ITQB NOVA, Instituto de Tecnologia Química e Biológica António Xavier
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Inês Isidro
Instituto de Biologia Experimental e Tecnologica, ITQB NOVA, Instituto de Tecnologia Química e Biológica António Xavier

Corresponding Author:[email protected]

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Abstract

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 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 on product titer and/or quality. In this study, transcriptomics analysis of Sf9 insect cells during the production of recombinant AAV 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 to 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 showed to enclose all three necessary AAV transgenes to produce packed AAV particles, indicating limitations of the dual baculovirus system. In addition, the trajectory analysis herein performed highlighted biological processes such as protein folding, metabolic processes, translation and stress response has 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 AAV production in IC-BEVS.
15 Dec 2022Submitted to Biotechnology and Bioengineering
16 Dec 2022Submission Checks Completed
16 Dec 2022Assigned to Editor
16 Dec 2022Review(s) Completed, Editorial Evaluation Pending
24 Dec 2022Reviewer(s) Assigned
01 Feb 2023Editorial Decision: Revise Minor
18 Feb 20231st Revision Received
18 Feb 2023Submission Checks Completed
18 Feb 2023Assigned to Editor
18 Feb 2023Review(s) Completed, Editorial Evaluation Pending
01 Mar 2023Reviewer(s) Assigned
10 Mar 2023Editorial Decision: Accept