Application of an artificial deaminase system for restoration of mutated mRNAs in the macular mouse caused by T>C mutation

Abstract

Abstract RNA editing is a significant mechanism underlying genetic variation and protein molecule alteration; C-to-U RNA editing, specifically, is important in regulation of mammalian genetic diversity. The ability to define and limit access of the enzymatic machinery, to avoid modification of unintended targets, is key to the success of RNA editing. Identification of the core component of the apoB RNA editing holoenzyme, APOBEC, and investigation of new candidate genes encoding other elements of the complex could reveal further details of APOBEC mediated mRNA editing. Menkes disease is a recessive X chromosome-linked hereditary syndrome in humans, caused by defective copper metabolism due to mutations in the ATP7A gene, which encodes a copper-transport protein. Here, we generated plasmids encoding the MS2 system and the APOBEC1 deaminase domain and used a guide RNA with flanking MS2 sites to restore mutated Atp7a in fibroblasts from the macular mouse model of Menkes disease having T > C mutation. Around 35% of the mutated C nucleotide was restored to U, demonstrating that our RNA editing system is reliable and has potential for therapeutic clinical application. RNA base editing via human RNA-guided cytidine deaminases is a potentially attractive approach for in vivo therapeutic application and provides opportunities for new developments in this field.