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), a US Food and Drug Administration (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.
Subject(s)
Erythropoietin/metabolism , Folic Acid Antagonists/administration & dosage , Protein Biosynthesis , RNA, Messenger/metabolism , Replicon , Trimethoprim/administration & dosage , Animals , Electroporation , Erythropoietin/genetics , Female , Genetic Therapy , Injections, Intramuscular , Mice , Mice, Inbred BALB C , RNA, Messenger/geneticsABSTRACT
Synthetic mRNA is an attractive vehicle for gene therapies because of its transient nature and improved safety profile over DNA. However, unlike DNA, broadly applicable methods to control expression from mRNA are lacking. Here we describe a platform for small-molecule-based regulation of expression from modified RNA (modRNA) and self-replicating RNA (replicon) delivered to mammalian cells. Specifically, we engineer small-molecule-responsive RNA binding proteins to control expression of proteins from RNA-encoded genetic circuits. Coupled with specific modRNA dosages or engineered elements from a replicon, including a subgenomic promoter library, we demonstrate the capability to externally regulate the timing and level of protein expression. These control mechanisms facilitate the construction of ON, OFF, and two-output switches, with potential therapeutic applications such as inducible cancer immunotherapies. These circuits, along with other synthetic networks that can be developed using these tools, will expand the utility of synthetic mRNA as a therapeutic modality.
Subject(s)
Gene Regulatory Networks , Genetic Therapy/methods , Promoter Regions, Genetic , RNA, Messenger/chemistry , RNA-Binding Proteins/chemistry , RNA/chemistry , Animals , Cell Line , Cricetinae , DNA/chemistry , Gene Library , Genetic Engineering , HEK293 Cells , Humans , Immunotherapy , Mice , RNA, Small Interfering/metabolism , Synthetic BiologyABSTRACT
The design of biomaterials for increasingly complex tissue engineering applications often requires exogenous presentation of biomolecular signals. Integration of gene delivery vectors with a biomaterial scaffold offers the potential to bypass the use of expensive and relatively inefficient growth factor supplementation strategies to augment cell behavior. However, integration of cationic polymer based gene delivery vectors within three-dimensional biomaterials, particularly matrices which can carry significant surface charge, remains poorly explored. We examined the potential of polyethylenimine (PEI) as a gene delivery vector for three-dimensional collagen-glycosaminoglycan (CG) scaffolds under development for tendon repair. While acetylated versions of PEI have demonstrated improved transfection efficiency in 2D culture assays, we investigated translation of this effect to a 3D biomaterial that contains significant electrostatic charge. A reporter gene was used to examine the impact of polymer modification, polymer:DNA ratio, and the degree of sulfation of the biomaterial microenvironment on gene delivery in vitro. We observed highest transgene expression in acetylated and unmodified PEI at distinct polymer:DNA ratios; notably, the enhancement often seen in two-dimensional culture for acetylated PEI did not fully translate to three-dimensional scaffolds. We also found highly sulfated heparin-based CG scaffolds showed enhanced initial luciferase expression but not prolonged activity. While PEI constructs significantly reduced tenocyte metabolic health during the period of transfection, heparin-based CG scaffolds showed the greatest recovery in tenocyte metabolic health over the full 2 week culture. These results suggest that the electrostatic environment of three-dimensional biomaterials may be an important design criterion for cationic polymer-based gene delivery.
