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

Abstract

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.

Novel Design of RNA Aptamers as Cancer Inhibitors and Diagnosis Targeting the Tyrosine Kinase Domain of the NT-3 Growth Factor Receptor Using a Computational Sequence-Based Approach

“…Riboswitches are the regulatory segments of mRNA molecules (often <100 nt) that regulate the expression of the proteins, due to the binding of a ligand or a small molecule [ 28 ]. They comprise two fundamental domains: an aptamer domain that binds the ligand and an expression domain that controls the gene expression via various mechanisms [ 29 ].…”

SHAPE-enabled fragment-based ligand discovery for RNA

“…Our screening strategy ultimately identified binders of the riboswitch and revealed a ligand binding site not targeted by a natural ligand of the riboswitch. Z1 is not a synthetic mimetic of the natural riboswitch ligand, should not bind TPP-dependent human enzymes, and thus provides an entry to using this riboswitch as an RNA-specific genetic control element ( 46 , 47 ).…”

RNAi-mediated rheostat for dynamic control of AAV-delivered transgenes

“…RNA-based molecular switches are emerging as attractive candidates to regulate transgene expression from AAVs owing to their small size, sequence specificity, and reduced potential for immunogenicity. 19 Recent developments in riboswitches based on steric oligo blockade of self-cleaving ribozyme activity and small molecule regulation of alternative RNA splicing have shown promise in preclinical models but their applicability in clinical settings is unknown. 38, 39 We report a generalizable and clinically viable approach for dosage control of rAAV-delivered cargos involving dynamic, robust, and reversible control via RNAi with benefit of infrequent dosing.…”

“…[16][17][18] Prior designs for RNAi-based on-switches have leveraged ligand binding to control the processing of shRNAs delivered alongside the therapeutic transgene or to modulate accessibility of endogenous microRNAs to their cognate binding sites on virally-delivered mRNAs. [19][20][21][22][23][24] However, their applicability has been limited due either to low dynamic range for modulation, off-target risks, or a lack of validation in the clinical setting. 19 In contrast, supplying chemically stabilized siRNAs exogenously overcomes the reliance on endogenous miRNAs and offers precise and flexible control of dosage.…”

“…[19][20][21][22][23][24] However, their applicability has been limited due either to low dynamic range for modulation, off-target risks, or a lack of validation in the clinical setting. 19 In contrast, supplying chemically stabilized siRNAs exogenously overcomes the reliance on endogenous miRNAs and offers precise and flexible control of dosage. As an alternative to siRNAs that require repeated, though infrequent administration, RNAi via shRNAs that can be stably introduced into AAV vectors in a gene therapy setting allow continuous regulation of the expressed transgene in cis as a single treatment.…”