In vivo validation of a reversible small molecule-based switch for synthetic self-amplifying mRNA regulation.

Abstract Synthetic mRNA therapeutics have the potential to revolutionize healthcare as they enable patients to produce therapeutic proteins inside their own bodies. However, convenient methods that allow external control over the timing and magnitude of protein production after in vivo delivery of synthetic mRNA are lacking. In this study, we validate the in vivo utility of a synthetic self-amplifying mRNA (RNA replicon) whose expression can be turned off using a genetic switch that responds to oral administration of trimethoprim (TMP), an FDA-approved small-molecule drug. After intramuscular electroporation, the engineered RNA replicon exhibited dose-dependent and reversible expression of its encoded protein upon TMP administration. The TMP serum level needed for maximal downregulation of protein translation was approximately 45-fold below that used in humans for therapeutic purposes. To demonstrate the therapeutic potential of the technology, we injected mice with a TMP-responsive RNA replicon encoding erythropoietin (EPO) and successfully controlled the timing and magnitude of EPO production as well as changes in hematocrit. This work demonstrates the feasibility of controlling mRNA kinetics in vivo, thereby broadly expanding the clinical versatility of mRNA therapeutics.