title: Riboswitch-mediated Attenuation of Transgene Cytotoxicity Increases Adeno-associated Virus Vector Yields in HEK-293 Cells.
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.
authors.affiliation: Target Discovery Research, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach an der Riss, Germany.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Strobel
authors.affiliation: Department of Chemistry, University of Konstanz, Konstanz, Germany.
authors.forename: Benedikt
authors.initials: B
authors.lastname: Klauser
authors.affiliation: Department of Chemistry, University of Konstanz, Konstanz, Germany.
authors.forename: Jörg S
authors.initials: JS
authors.lastname: Hartig
authors.affiliation: Target Discovery Research, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach an der Riss, Germany.
authors.forename: Thorsten
authors.initials: T
authors.lastname: Lamla
authors.affiliation: Translational Medicine and Clinical Pharmacology, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach an der Riss, Germany.
authors.forename: Florian
authors.initials: F
authors.lastname: Gantner
authors.affiliation: Respiratory Diseases Research, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach an der Riss, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Kreuz
mesh_terms: D000818:Animals; D000229:Dependovirus / Q000235:genetics*; D015870:Gene Expression; D005786:Gene Expression Regulation / Q000187:drug effects; D023061:Gene Order; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics*; D006147:Guanine / Q000378:metabolism / Q000494:pharmacology; D057809:HEK293 Cells; D006801:Humans; D008024:Ligands; D051379:Mice; D058928:Riboswitch; D014161:Transduction, Genetic; D019076:Transgenes; D014779:Virus Replication / Q000187:drug effects
chemical_list: D008024:Ligands; D058928:Riboswitch; D006147:Guanine