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Intronization enhances expression of S-protein and other transgenes challenged by cryptic splicing

View ORCID ProfileKärt Tomberg, View ORCID ProfileLiliana Antunes, View ORCID ProfileYangYang Pan, View ORCID ProfileJacob Hepkema, View ORCID ProfileDimitrios A. Garyfallos, View ORCID ProfileAhmed Mahfouz, View ORCID ProfileAllan Bradley
doi: https://doi.org/10.1101/2021.09.15.460454
Kärt Tomberg
1CITIID, Department of Medicine, University of Cambridge, Cambridge, UK
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  • For correspondence: kt474@cam.ac.uk ab2592@cam.ac.uk
Liliana Antunes
1CITIID, Department of Medicine, University of Cambridge, Cambridge, UK
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YangYang Pan
1CITIID, Department of Medicine, University of Cambridge, Cambridge, UK
2College of Veterinary Medicine, Shanxi Agricultural University, Taigu, China
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Jacob Hepkema
3Wellcome Sanger Institute, Hinxton, UK
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Dimitrios A. Garyfallos
1CITIID, Department of Medicine, University of Cambridge, Cambridge, UK
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Ahmed Mahfouz
4Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands
5Delft Bioinformatics Lab, Delft University of Technology, Delft, The Netherlands
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Allan Bradley
1CITIID, Department of Medicine, University of Cambridge, Cambridge, UK
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  • For correspondence: kt474@cam.ac.uk ab2592@cam.ac.uk
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Abstract

The natural habitat of SARS-CoV-2 is the cytoplasm of a mammalian cell where it replicates its genome and expresses its proteins. While SARS-CoV-2 genes and hence its codons are presumably well optimized for mammalian protein translation, they have not been sequence optimized for nuclear expression. The cDNA of the Spike protein harbors over a hundred predicted splice sites and produces mostly aberrant mRNA transcripts when expressed in the nucleus. While different codon optimization strategies increase the proportion of full-length mRNA, they do not directly address the underlying splicing issue with commonly detected cryptic splicing events hindering the full expression potential. Similar splicing characteristics were also observed in other transgenes. By inserting multiple short introns throughout different transgenes, significant improvement in expression was achieved, including >7-fold increase for Spike transgene. Provision of a more natural genomic landscape offers a novel way to achieve multi-fold improvement in transgene expression.

Competing Interest Statement

KT and AB are named as inventors on a patent application covering the use of intronization for enhanced protein expression. The other authors declare no competing interests.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
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Posted September 15, 2021.
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Intronization enhances expression of S-protein and other transgenes challenged by cryptic splicing
Kärt Tomberg, Liliana Antunes, YangYang Pan, Jacob Hepkema, Dimitrios A. Garyfallos, Ahmed Mahfouz, Allan Bradley
bioRxiv 2021.09.15.460454; doi: https://doi.org/10.1101/2021.09.15.460454
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Intronization enhances expression of S-protein and other transgenes challenged by cryptic splicing
Kärt Tomberg, Liliana Antunes, YangYang Pan, Jacob Hepkema, Dimitrios A. Garyfallos, Ahmed Mahfouz, Allan Bradley
bioRxiv 2021.09.15.460454; doi: https://doi.org/10.1101/2021.09.15.460454

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