139. Controllable Target-Specific Transgene Induction Using an Allosteric trans-Splicing Ribozyme

Abstract

Gene therapeutic approaches are needed that can achieve inducible, adjustable, and well-controlled expression of therapeutic genes while simultaneously suppressing expression of disease-causing genes for maximization of their efficacy. In this study, we proposed a new approach to address one of the main challenges that current gene therapeutic techniques meet, which is based on group I intron ribozyme. To this end, we established an exogeneously and genetically controllable system through design of an allosteric ribozyme that comprises a Tetrahymena group I-based trans-splicing ribozyme as an active domain for RNA replacement, a small molecule-specific RNAaptamer as a sensor domain, and a communication module as an active transfer domain. This allosteric trans-splicing ribozyme can be used as a system that is able to specifically target, and thus reduce the level of, certain disease-specific RNAs, and simultaneously provides the ability to artificially control transgene expression through RNA replacement in therapeutic targets by using exogenous small molecules to activate the function of the ribozyme. The effectiveness of this approach was assessed by constructing various ribozymes in combination with a theophylline-binding aptamer to identify an allosteric ribozyme whose activity is controlled by theophylline both in vitro and in cell culture assays. Moreover, to confirm allosteric regulation of transgene expression, we constructed adenoviral vectors encoding the ribozymes and validated theophylline-dependent RNA replacement in target RNA-expressing cells. Results demonstrate that an allosteric trans-splicing ribozyme is an applicable RNA-based framework for engineering external ligand-controlled gene expression regulatory systems that exhibit adjustable regulation, design modularity (therefore, broad applicability), and target specificity. Such ribozyme could therefore be useful to monitor and/or modulate fate of any cells (such as cancer cells) using specific endogenous or exogenous cues.