Correction: Tickner, Z.J.; Farzan, M. Riboswitches for Controlled Expression of Therapeutic Transgenes Delivered by Adeno-Associated Viral Vectors. Pharmaceuticals 2021, 14, 554.

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

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

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.

Selection of High-Affinity RNA Aptamers That Distinguish between Doxycycline and Tetracycline.

Oligonucleotide aptamers 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 a 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 dissociation constants of approximately 7 nM, and tetracycline, which they bind undetectably. They thus function as orthogonal complements to the original tetracycline aptamer. Unexpectedly, doxycycline aptamers adopt a conformation distinct from that of the tetracycline aptamer and depend on constant regions originally installed as primer binding sites. We show that the fluorescence emission intensity of doxycycline 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.

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 components1-5. In this study, 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 enabled in vivo regulation of adeno-associated virus (AAV)-delivered transgenes, allowing dose-dependent and up to 223-fold regulation of protein expression over at least 43 weeks. To test the potential of these reversible on-switches in gene therapy for anemia of chronic kidney disease6, we demonstrated 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 of gene therapies and broaden their use.