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

Abstract

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.

Human stem cell-based retina on chip as new translational model for validation of AAV retinal gene therapy vectors

“…We used AAV2, AAV8, AAV9, AAV2.7m8 (Dalkara et al, 2013), ShH10 (Klimczak et al, 2009), and AAV2.NN, AAV2.GL (Pavlou et al, 2021) with the ss expression cassettes ss-CMV-eGFP-SV40poly(A), ss-CMV-anti-FITC, or sc-CMV-eGFP-SV40poly(A) (Strobel et al, 2020), flanked by AAV2-derived inverted terminal repeats. AAV vectors were prepared as in Strobel et al (2019): HEK 293-H cells were cultured in DMEM + GlutaMAX-I + 10% FCS (Thermo Fisher Scientific) and transfected as previously described (Strobel et al, 2015).…”

Riboswitches for Controlled Expression of Therapeutic Transgenes Delivered by Adeno-Associated Viral Vectors

“…One tetracycline aptazyme, Tc40, enabled over 20-fold suppression in human cells and also achieved 7-fold suppression of an AAVdelivered transgene through oral administration of tetracycline in a mouse model. Strobel et al also recently demonstrated 15-fold induction of an AAV-delivered transgene in mice using a tetracycline-regulated aptazyme on-switch developed through a similar rational design and testing approach [154]. This result also represents a rare case in which switch performance was higher in an animal model than in previous results in cell culture [136].…”

Human stem cell-based retina-on-chip as new translational model for validation of AAV retinal gene therapy vectors

“…AAV2, AAV8, AAV9, AAV2.7m8 (Dalkara et al, 2013), ShH10 (Klimczak et al, 2009), and AAV2.NN, AAV2.GL (Pavlou et al, 2021) with the single stranded (ss) expression cassettes; ss-CMV-eGFP-SV40polyA, ss-CMV-antiFITC or self complementary (sc) sc-CMV-eGFP-SV40polyA, (Strobel et al, 2020) flanked by AAV2-derived inverted terminal repeats were applied. AAV vectors were prepared as previously described (Strobel et al, 2019).…”