165. Theophylline-Dependent Aptazyme as a Novel Tool for Transgene Expression Regulation in Mammalian Cells

Abstract

Successful and safe gene therapy approaches rely on regulatory tools ensuring both specific and controlled expression of the therapeutic gene. To ensure safety, timing or dosing in patients, external control of therapeutic gene expression is desirable or even required. In this study, we explored the potential of artificial aptazymes, ligand-dependent self-cleaving ribozymes, as an innovative tool, for regulation of therapeutic gene expression. For cloning of dual-luciferase reporter plasmid pTA-luc, psiCheck-2 (Promega) was used as template vector. The theophylline-dependent aptazyme was inserted into the 3’-UTR of the Gaussia luciferase gene. Quantification of reporter activity after plasmid transfection of HEK293 cells showed the basal level of less than 10% of psiCheck-2, and dose-dependent turn-on regulation of protein expression by theophylline. The maximum magnitude of the induction versus basal level was ~4-fold. Furthermore, the induction level was responsive to a wide range of theophylline concentrations in the medium, from 0 to 1 mM, facilitating the fine-tuning of transgene expression. The similar results came after changing the reporter gene to Kallistatin, but the regulatory fold can be quite different. Actually, in our study, not every aptazyme can be improved in regulatory ability by mutating the upstream start codon from AUG to AAG to avoid nonspecific recognition by the small ribosomal subunit, which may lead to drastic reduction in regulation. However, by inserting aptazyme in different site or using the tandem one, we made it done. The regulatory range of the single insertion system appeared almost 3 times exactly wider than the original system, let alone the ability of the tandem one. And we have evidence to believe that the regulatory ability will be various according to the insertion site and the vector. The aptazyme-based control system was further extended to HeLa and A549 cells, and demonstrated to function in a rapid, reversible and dose-dependent manner, suggesting potential for therapeutic applications in humans. Unfortunately, due to the toxicity of theophlline, the regulatory ability can only be defined in theory, and the decrease of ligand concentration would cause the reduction of regulatory ability. More safer ligands are urgent required in the futrue. View Large Image | Download PowerPoint Slide