Riboswitch-mediated Attenuation of Transgene Cytotoxicity Increases Adeno-associated Virus Vector Yields in HEK-293 Cells

Strobel, Benjamin; Klauser, Benedikt; Hartig, Jörg S.; Lamla, Thorsten; Gantner, Florian; Kreuz, Sebastian

Riboswitch-mediated Attenuation of Transgene Cytotoxicity Increases Adeno-associated Virus Vector Yields in HEK-293 Cells

Files

Strobel_0-305407.pdf(368.54 KB)

Date

2015

Authors

Strobel, Benjamin

Klauser, Benedikt

Hartig, Jörg S.

Lamla, Thorsten

Gantner, Florian

Kreuz, Sebastian

Publication type

Journal article

Publication status

Published

Published in

Molecular Therapy ; 23 (2015), 10. - pp. 1582-1591. - ISSN 1525-0016. - eISSN 1525-0024

Abstract

Cytotoxicity of transgenes carried by adeno-associated virus (AAV) vectors might be desired, for instance, in oncolytic virotherapy or occur unexpectedly in exploratory research when studying sparsely characterized genes. To date, most AAV-based studies use constitutively active promoters (e.g., the CMV promoter) to drive transgene expression, which often hampers efficient AAV production due to cytotoxic, antiproliferative, or unknown transgene effects interfering with producer cell performance. Therefore, we explored artificial riboswitches as novel tools to control transgene expression during AAV production in mammalian cells. Our results demonstrate that the guanine-responsive GuaM8HDV aptazyme efficiently attenuates transgene expression and associated detrimental effects, thereby boosting AAV vector yields up to 23-fold after a single addition of guanine. Importantly, riboswitch-harboring vectors preserved their ability to express functional transgene at high levels in the absence of ligand, as demonstrated in a mouse model of AAV-TGFβ1-induced pulmonary fibrosis. Thus, our study provides the first application-ready biotechnological system-based on aptazymes, which should enable high viral vector yields largely independent of the transgene used. Moreover, the RNA-intrinsic, small-molecule regulatable mode of action of riboswitches provides key advantages over conventional transcription factor-based regulatory systems. Therefore, such riboswitch vectors might be ultimately applied to temporally control therapeutic transgene expression in vivo.

Subject (DDC)

540 Chemistry

Cite This

ISO 690

STROBEL, Benjamin, Benedikt KLAUSER, Jörg S. HARTIG, Thorsten LAMLA, Florian GANTNER, Sebastian KREUZ, 2015. Riboswitch-mediated Attenuation of Transgene Cytotoxicity Increases Adeno-associated Virus Vector Yields in HEK-293 Cells. In: Molecular Therapy. 23(10), pp. 1582-1591. ISSN 1525-0016. eISSN 1525-0024. Available under: doi: 10.1038/mt.2015.123

BibTex

@article{Strobel2015Ribos-32750,
  year={2015},
  doi={10.1038/mt.2015.123},
  title={Riboswitch-mediated Attenuation of Transgene Cytotoxicity Increases Adeno-associated Virus Vector Yields in HEK-293 Cells},
  number={10},
  volume={23},
  issn={1525-0016},
  journal={Molecular Therapy},
  pages={1582--1591},
  author={Strobel, Benjamin and Klauser, Benedikt and Hartig, Jörg S. and Lamla, Thorsten and Gantner, Florian and Kreuz, Sebastian}
}

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Bibliography of Konstanz

Yes