A reversible RNA on-switch that controls gene expression of AAV-delivered therapeutics in vivo

Widespread use of gene therapy technologies is limited in part by the lack of small genetic switches with wide dynamic ranges that control transgene expression without the requirement of additional protein components(1–5). Here, we engineered a class of type III hammerhead ribozymes to develop RNA switches that are highly efficient at cis-cleaving mammalian mRNAs and showed that they can be tightly regulated by a steric-blocking antisense oligonucleotide. Our variant ribozymes enable in vivo regulation of adeno-associated virus (AAV)-delivered transgenes, allowing dose-dependent control of protein expression up to 223-fold over at least 43 weeks. To test the potential of these reversible on-switches in gene therapy for anemia of chronic kidney disease(6), we demonstrate regulated expression of physiological levels of erythropoietin with a well-tolerated dose of the inducer oligonucleotide. These small, modular, and efficient RNA switches may improve the safety and efficacy, and broaden the use of gene therapies.