Improvement of protein production by engineering a novel antiapoptotic baculovirus vector to suppress the expression of<i>Sf‐caspase‐1</i>and<i>Tn‐caspase‐1</i>

Zhang, Xiaoyue; Zhao, Kaixia; Lan, Lan; Shi, Na; Nan, Hao; Shi, Yanan; Xu, Xiaodong; Chen, Hongying

doi:10.1002/bit.27807

Cited by 9 publications

(16 citation statements)

References 44 publications

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“…The inhibition of pre-apoptotic (e.g., dronc) [62] and apoptotic (e.g., caspase-1) gene expression has already been shown, indicating slight improvements. [63][64][65][66][67] However, a recent study repressing Caspase-1 activity does not correlate to these findings, indicating no significant increase in productivity and prolonged cell survival, [68] as Caspase-1 activity may already be inhibited by viral anti-apoptotic genes such as the viral P35 protein. [69] Nevertheless, other differential expressed apoptotic-related genes observed in this study may proof functionality in delaying apoptosis and/or improving productivity in IC-BEVS.…”

Section: Discussionmentioning

confidence: 99%

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

Virgolini

Hagan

Correia

et al. 2022

Biotechnology Journal

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The insect cell-baculovirus expression vector system (IC-BEVS) has emerged as an alternative time-and cost-efficient production platform for recombinant Adenoassociated 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.

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Section: Discussionmentioning

confidence: 99%

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

Virgolini

Hagan

Correia

et al. 2022

Biotechnology Journal

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“…During the course of this study, we were unable replicate previously reported results that the RNAi‐mediated suppression of Caspase‐1 in lepidopteran cells can improve BEVS yields. [ 15–17,41 ] There are several possible explanations to account for this result. Firstly, previous studies exploring RNAi knockdown of Caspase‐1 in lepidopteran cells almost exclusively utilized static culture and media containing fetal bovine serum to undertake BEVS infection experiments.…”

Section: Discussionmentioning

confidence: 99%

“…Previous reports have documented that RNAi-based silencing of Sf-Caspase1 in Sf9 cells could improve the recombinant protein production of the BEVS. [15,[39][40][41] It is possible that reducing Sf-Caspase-1 expression, rather than eliminating it completely, could account for the discrepancy between the effects of RNAi knockdowns and CRISPR-Cas9 knockouts on recombinant protein production. To explore this possibility, Sf9 cell lines stably expressing RNAi constructs targeting Sf-Caspase-1 were generated via transfection with plasmids designed to reproduce two previously published constructs.…”

Section: Establishment Of Rnai-expressing Sf9 Cell Lines Stably Suppr...mentioning

confidence: 99%

Knockout of Sf‐Caspase‐1 generates apoptosis‐resistant Sf9 cell lines: Implications for baculovirus expression

Malmanche

Marcellin

Reid

2022

Biotechnology Journal

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The Sf9 cell line, originally isolated from the insect Spodoptera frugiperda, is commonly used alongside the baculovirus expression vector system (BEVS) to produce recombinant proteins and other biologics. As more BEVS-derived vaccines and therapeutics are approved by regulators and manufactured at scale, there is increasing interest in improving the Sf9 cell line to improve bioprocess robustness and increase product yields. CRISPR-Cas9 is a powerful genome-editing tool with great potential to improve cell line characteristics. Nevertheless, reports of genome-editing in Sf9 cells are scarce, and targets for engineering are elusive. To evaluate the effectiveness of CRISPR-Cas9 to improve BEVS yields, we generated Sf9 cell lines with functional knockouts in the Sf-Caspase-1 gene, which encodes an effector caspase involved in the execution of apoptosis. Deletion of Sf-Caspase-1 abolished the hallmarks of apoptotic cell death including plasma membrane blebbing and effector caspase activity. Following infection of Sf-Caspase-1 knockout Sf9 cultures with a recombinant baculovirus expressing βgalactosidase, we did not observe any differences in cell death kinetics or increases in productivity. Similar results were obtained when Sf-Caspase-1 expression was suppressed via RNA interference. We anticipate that the CRISPR-Cas9 workflow reported here will spur future efforts to rationally engineer Sf9 cells for improved baculovirus expression.

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“…(B) Schematic diagram of Bac563-5 T used in this study. Bac563-5 T was modified from BAC10:KO1629 by knocking out ChiA/v-cath (Ac126-127) and p10 (Ac137) and inserting an shRNA expression cassette at the Ac137 locus to inhibit the virus infection-induced apoptosis (Je et al, 2001a;Zhang et al, 2021Zhang et al, ). 10.3389/fmicb.2023 Frontiers in Microbiology 05 frontiersin.org genome for better expression of foreign genes and insertion of larger exogenous DNA fragments, we generated 14 fragment-knockout (KO) bacmids based on BAC10:KO1629 (Table 2).…”

Section: Construction Of Acmnpv Fragment-knockout Bacmidsmentioning

confidence: 99%

Improvement of protein production in baculovirus expression vector system by removing a total of 10 kb of nonessential fragments from Autographa californica multiple nucleopolyhedrovirus genome

Zhang

Zong

et al. 2023

Front. Microbiol.

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Baculovirus expression vector system (BEVS) is a powerful and versatile platform for recombinant protein production in insect cells. As the most frequently used baculovirus, Autographa californica multiple nucleopolyhedrovirus (AcMNPV) encodes 155 open reading frames (ORFs), including a considerable number of non-essential genes for the virus replication in cell culture. Studies have shown that protein production in BEVS can be improved by removing some viral dispensable genes, and these AcMNPV vectors also offer the possibility of accommodating larger exogenous gene fragments. In this study, we, respectively, deleted 14 DNA fragments from AcMNPV genome, each of them containing at least two contiguous genes that were known nonessential for viral replication in cell culture or functionally unknown. The effects of these fragment-deletions on virus replication and exogenous protein production were examined. The results showed that 11 of the 14 fragments, containing 43 genes, were dispensable for the virus replication in cultured cells. By detecting the expression of intracellularly expressed and secreted reporter proteins, we demonstrated that nine of the fragment-deletions benefited protein production in Sf9 cells and/or in High Five cells. After combining the deletion of some dispensable fragments, we obtained two AcMNPV vectors shortened by more than 10 kb but displayed an improved capacity for recombinant protein production. The deletion strategies used in this study has the potential to further improve the BEVS.

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Improvement of protein production by engineering a novel antiapoptotic baculovirus vector to suppress the expression ofSf‐caspase‐1andTn‐caspase‐1

Cited by 9 publications

References 44 publications

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

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

Knockout of Sf‐Caspase‐1 generates apoptosis‐resistant Sf9 cell lines: Implications for baculovirus expression

Improvement of protein production in baculovirus expression vector system by removing a total of 10 kb of nonessential fragments from Autographa californica multiple nucleopolyhedrovirus genome

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