A Reversible RNA On-Switch That Controls Expression of Aav-Delivered Transgenes 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 component. Self-cleaving ribozyme-based RNA switches, with their small size and lack of dependence on exogenous proteins, make ideal regulators of therapeutic transgenes. In this study, by converting a previously optimized type I Schistosoma mansoni hammerhead ribozyme to a type III ribozyme, and sequentially optimizing the ribozyme’s stem III, stem I and loop I sequences, we engineered a class of hammerhead ribozymes that are highly efficient in cis-cleaving mammalian mRNAs. We then developed efficient RNA on-switches by using octaguanidine dendrimer-conjugated phosphorodiamidate morpholino oligonucleotide, a steric-blocking antisense oligonucleotide (ASO), to tightly regulate these ribozyme’s self-cleavage activity. This RNA switch system enables efficient and reversible regulation of adeno-associated virus (AAV)-delivered transgenes in vivo, allowing dose-dependent and up to 200-fold regulation of protein expression over at least 43 weeks in mice. To test the potential of these RNA switches in gene-therapy applications, we demonstrated regulated expression of physiological levels of erythropoietin (Epo) with a well-tolerated dose of the inducer oligonucleotide. These ASO-regulated, small, modular and efficient RNA switches have the potential to improve the safety and efficacy of gene therapies and broaden their use. A figure (Figure 1) is provided to serve as a graphical abstract for a better illustration of the study. A manuscript related to this study has been accepted for publication by Nature Biotechnology (Zhong G*, Wang H, He W, Li Y, Mou H, Tickner Z, Tran M, Ou T, Yin Y, Diao H, Farzan M*. A reversible RNA on-switch that controls gene expression of AAV-delivered therapeutics in vivo. Nature Biotechnology. 2019.).