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. RNA-based switches 7-15 have two key strengths over protein-dependent transcriptional switches for gene-therapy applications. First, these switches are generally small (<200 bp), and thus can be easily incorporated into gene-therapy vectors with limited packaging capacity, for example those based on adeno-associated virus (AAV) 16. Second, RNA switches do not require a potentially immunogenic non-self protein such as the rtTA protein for the Tet-On transcriptional activation system 5. However, most RNA-based switches suffer from a narrow regulatory range, which usually precludes their use in vivo 10-14. This narrow

Vectors developed from adeno-associated virus (AAV) are powerful tools for in vivo transgene delivery in both humans and animal models, and several AAV-delivered gene therapies are currently approved for clinical use. However, AAV-mediated gene therapy still faces several challenges, including limited vector packaging capacity and the need for a safe, effective method for controlling transgene expression during and after delivery. Riboswitches, RNA elements which control gene expression in response to ligand binding, are attractive candidates for regulating expression of AAV-delivered transgene therapeutics because of their small genomic footprints and non-immunogenicity compared to protein-based expression control systems. In addition, the ligand-sensing aptamer domains of many riboswitches can be exchanged in a modular fashion to allow regulation by a variety of small molecules, proteins, and oligonucleotides. Riboswitches have been used to regulate AAV-delivered transgene therapeutics in animal models, and recently developed screening and selection methods allow rapid isolation of riboswitches with novel ligands and improved performance in mammalian cells. This review discusses the advantages of riboswitches in the context of AAV-delivered gene therapy, the subsets of riboswitch mechanisms which have been shown to function in human cells and animal models, recent progress in riboswitch isolation and optimization, and several examples of AAV-delivered therapeutic systems which might be improved by riboswitch regulation.

Oligonucleotide adapters are found in prokaryotes and eukaryotes, and they can be selected from large synthetic libraries to bind protein or small-molecule ligands with high affinities and specificities. Aptamers can function as biosensors, as protein recognition elements, and as components of riboswitches allowing ligand-dependent control of gene expression. One of the best studied laboratory selected aptamers binds the antibiotic tetracycline, but it binds with much lower affinity to the closely related but more bioavailable antibiotic doxycycline. Here we report enrichment of doxycycline-binding aptamers from a selectively-randomized library of tetracycline-aptamer variants over four selection rounds. Selected aptamers distinguish between doxycycline, which they bind with approximately 7 nanomolar dissociation constants, and tetracycline, which they bind undetectably. They thus function as orthogonal complements to the original tetracycline aptamer. Unexpectedly, doxycycline aptamers adopt a distinct conformation from the tetracycline aptamer and depend on constant regions originally installed as primerbinding sites. We show that doxycycline fluorescence emission intensity increases upon aptamer binding, permitting their use as biosensors. This new class of aptamers can be used in multiple contexts where doxycycline detection, or doxycycline-mediated regulation, is necessary.

In the original article, Renzl, C [...]