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.
Highlights
One of the major barriers to human gene therapy is safe, efficient delivery of genetic material and/or editing complexes to specific tissues or cell types
This review presents the mechanisms of several riboswitches with therapeutic potential, their performance in mammalian cells and animal models, and recent progress in improving their regulatory properties and developing methods for riboswitch screens and selections
Riboswitches, aptazyme and RNA interference (RNAi) switches, represent an attractive method for control of associated virus (AAV)-delivered therapeutic transgene expression due to their small sizes, non-immunogenicity, modular structures, and ability to function without protein switching elements
Summary
One of the major barriers to human gene therapy is safe, efficient delivery of genetic material and/or editing complexes to specific tissues or cell types. Pharmaceuticals 2021, 14, 554 repeats (ITRs) necessary for packaging and nuclear localization, rendering the virus completely replication deficient and severely limiting integration into the host genome [5] Regardless of these advantages, the small size of the AAV genome can present a challenge: AAV vectors can only package and deliver transgenes up to 4.7 kb in size, while this is reduced to 2.4 kb in scAAV [11]. There are several systems which enable exogenous control of gene expression: these include the Tet-On and Tet-Off systems which enable strong induction or inhibition of transgene expression in response to the small-molecule drug doxycycline, optogenetics approaches which allow highly specific spatial and temporal control of transgene expression using light, and even systems which control transgene expression using sound [16,17,18,19,20] Several of these systems have been used to regulate therapeutic transgenes in animal models, but they rely upon expression of non-mammalian proteins to function; in addition to being immunogenic, inclusion of the genes coding for regulatory proteins occupies precious space in the AAV genome.
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