Enhancing Stability of Destabilized Green Fluorescent Protein Using Chimeric mRNA Containing Human Beta-Globin 5' and 3' Untranslated Regions.

ABSTRACT

BACKGROUND: In spite of recent progress in mRNA technologies and their potential applications for treatment of human diseases, problems such as the transient nature of mRNA limit the stability of gene up-regulation and, thus, potentially reduce mRNA efficiency for gene therapy. Using human ß-globin 5' and 3' untranslated regions (UTRs), this study aimed to develop the different chimeric constructs of mRNAs to increase the stability of destabilized green fluorescent protein (EGFPd2) in HEK 293 cells. METHODS: Purified human ß-globin (HBG) 5'-3'UTRs, and the coding sequence of destabilized green fluorescent protein (EGFPd2) were amplified separately and ligated to each other using SOEing PCR method in a different format. As controls, the original construct of EGFPd2 under the control of T7 promoter was used. Following in vitro transcription, HEK 293 cells were then transfected with several constructs and incubated at 37°C in a CO2 incubator. They were monitored under a fluorescence microscope every four hours for the first 24 hr, then every 12 hr afterwards. The resulting fluorescence was measured as a surrogate for translation efficiency and duration. RESULTS: By monitoring the HEK cells over 48 hr, cells transfected with mRNA with various HBG UTRs showed significantly different fluorescence intensity and stability in comparison with the pEGFPd2 prototype (control transcript) overtime. Overall, the images show that replacement of the 3' UTR end of the prototype vector pGFPd2 with the 3' end of ß-globin mRNA increases the half-life of the chimeric mRNA for more than 32 hr. CONCLUSION: This result indicates that ß-globin 3' UTR would definitely increase the half-life of mRNA and may help to decrease the mRNA therapeutic dosage in the treatment of diseases associated with mRNA therapy.