A Small-Molecule-Responsive Riboswitch Enables Conditional Induction of Viral Vector-Mediated Gene Expression in Mice

• Published in: ACS synthetic biology 2020-06, Vol.9 (6), p.1292-1305
• Main Authors: Strobel, Benjamin, Düchs, Matthias J, Blazevic, Dragica, Rechtsteiner, Philipp, Braun, Clemens, Baum-Kroker, Katja S, Schmid, Bernhard, Ciossek, Thomas, Gottschling, Dirk, Hartig, Jörg S, Kreuz, Sebastian
• Format: Article
• Language: English
• Subjects: 3' Untranslated Regions; Animals; Anti-Bacterial Agents - pharmacology; Aptamers, Nucleotide - genetics; Aptamers, Nucleotide - metabolism; Cell Line; Dependovirus - genetics; Gene Expression - drug effects; Genetic Vectors - genetics; Genetic Vectors - metabolism; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; Humans; Liver - metabolism; Lung - metabolism; Mice; RNA, Catalytic - genetics; RNA, Catalytic - metabolism; Tetracycline - pharmacology
• ISSN: 2161-5063; 2161-5063
• DOI: 10.1021/acssynbio.9b00410

Adeno-associated viral (AAV) vector-mediated gene therapy holds great potential for future medical applications. However, to facilitate safer and broader applicability and to enable patient-centric care, therapeutic protein expression should be controllable, ideally by an orally administered drug. The use of protein-based systems is considered rather undesirable, due to potential immunogenicity and the limited coding space of AAV. Ligand-dependent riboswitches, in contrast, are small and characterized by an attractive mode-of-action based on mRNA-self-cleavage, independent of coexpressed foreign protein. While a promising approach, switches available to date have only shown moderate potency in animals. In particular, ON-switches that induce transgene expression upon ligand administration so far have achieved rather disappointing results. Here we present the utilization of the previously described tetracycline-dependent ribozyme K19 for controlling AAV-mediated transgene expression in mice. Using this tool switch, we provide first proof for the feasibility of clinically desired key features, including multiorgan functionality, potent regulation (up to 15-fold induction), reversibility, and the possibility to fine-tune and repeatedly induce expression. The systematic assessment of ligand and reporter protein plasma levels further enabled the characterization of pharmacokinetic-pharmacodynamic relationships. Thus, our results strongly support future efforts to develop engineered riboswitches for applications in clinical gene therapy.