Targeted RNA editing by direct delivery of an adenosine deaminase-antisense oligo conjugate

Programmable RNA editing is a promising therapeutic strategy for correcting disease-causing mutations on mRNA, but current approaches rely primarily upon endogenous RNA editing enzymes (i.e. ADARs) that have restricted substrate scope and efficiency. Here, we demonstrate programmable RNA editing with evolved TadA-derived deaminases and 2-methoxyethyl (MOE)-modified antisense oligonucleotides (ASO) to guide site-directed A-to-I editing. In contrast to ADAR enzymes, TadA proteins modify single-stranded RNA (ssRNA). We profile ASO-guided TadA-based editors on endogenous and disease-relevant mRNAs and develop a "bulge-forming" ASO architecture to constrain RNA editing to the target site. Further, we demonstrate that a covalent adenosine deaminase-ASO "RNP" conjugate formed in the test tube and delivered by lipofection achieves targeted and efficient RNA editing with dramatically lower transcriptome-wide off-target editing as compared to ectopically expressed RNA editing enzymes. Taken together, our work expands the scope of programmable RNA editing methods with broad implications for therapeutic modulation of RNA behavior.