Conditional control of suicide gene expression in tumor cells with theophylline-responsive ribozyme

Abstract

Numerous synthetic RNA-based controls for integrating sensing switches with function devices have been demonstrated in a variety of organisms for gene regulation. Although potential advantages of RNA-based genetic control strategies have been shown in clinical applications, successfully extending these engineered systems into medical applications has seldom been reported. Here, a synthetic RNA-based ribozyme system and its application in advancing rationally designed cellular therapy were described. The theophylline-responsive, ribozyme-based device provided a powerful platform for suicide gene expression regulation in tumor cells. Moreover, we demonstrate the ability of our synthetic controller to modulate effectively the viability of the cells in response to drug input. Our RNA-based regulatory system could dose-dependently fine-tune transgene expression in mammalian cells and address urgent limitations in existing genetic control strategies for gene- and cell-based therapies in the future.

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

“…Nonetheless, some aptazymes selected in prokaryotes or yeast can regulate transgene expression in mammals. Zhang et al synthesized three theophylline aptazyme libraries with different architectures and randomized CMs, used FACS to select theophylline-responsive aptazymes in bacteria, and screened candidate switches in human and yeast cells [169]. The resulting switches could suppress reporter gene expression by 4.8-fold in HEK293T cells, and the authors demonstrated theophylline-regulated cell killing by ganciclovir in a model system similar to that used by Kim et al [75].…”

Detection and beyond: challenges and advances in aptamer-based biosensors

“…180 When small molecules like metabolites bind to the ribozymes, autocatalytic cleavage is induced to regulate downstream gene expression. 181 Moreover, DNAzymes are frequently combined with FRET technology and applied for quantitative detection of diverse metal ions. 182 For instance, anchoring of DNAzyme-based sensors to the cell membrane enabled real-time monitoring of metal ions in the cellular microenvironment; 183 this task is extremely difficult using other contemporary biotechniques.…”

“…The cells are programmed to recognize the molecular entities among the cellular environments and can report the presence of molecules in a variety of ways, such as fluorescence emission or cellular suicide. 181,189 If microorganism or cell sensors are created, their production would be inexpensive, and these living sensors have the potential to be applied for disease theranostics and environmental monitoring. These applications require precise control of the whole-cell biosensors, which can be realized by aptamers.…”

Intracellular Selection of Theophylline-Sensitive Hammerhead Aptazyme

“…In the work conducted by Hartig et al, 21 the performance of theo-HHAz, 19 P1F5, 21 and 5.3 21 were evaluated together, and P1F5 showed better performance. Similarly, theo-HHAz, 19 L2bulgeOff2, 11 and MW (not named in the original paper) 22 were evaluated together by Pei et al, 38 in which MW showed better switching ability. Thus, to conduct a fair comparison and evaluate the performance of TAP-1 under a unified strategy, the chosen HHAz variants, M5, 20 P1F5, MW, H4, 23 and Theo5 17 (shown in Figure 1C) have been inserted at the same position to downregulate the expression of mCherry, and HEK293T cell was chosen to harbor the constructed vectors.…”