Eggert Tanner W, Dhingani Dhruv Y, Kleiner Ralph E
Frick Chemistry Laboratory, Department of Chemistry, Princeton University, Princeton, NJ 08544, USA.
bioRxiv. 2025 Jul 12:2025.07.11.664364. doi: 10.1101/2025.07.11.664364.
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.
可编程RNA编辑是一种很有前景的治疗策略,用于纠正mRNA上的致病突变,但目前的方法主要依赖于内源性RNA编辑酶(即ADARs),其底物范围和效率有限。在这里,我们展示了利用进化的TadA衍生脱氨酶和2'-甲氧基乙基(MOE)修饰的反义寡核苷酸(ASO)进行可编程RNA编辑,以指导位点定向的A到I编辑。与ADAR酶不同,TadA蛋白修饰单链RNA(ssRNA)。我们对内源性和疾病相关的mRNA上基于ASO引导的TadA编辑器进行了分析,并开发了一种“凸起形成”的ASO结构,以将RNA编辑限制在靶位点。此外,我们证明,在试管中形成并通过脂质转染递送的共价腺苷脱氨酶-ASO“RNP”缀合物,与异位表达的RNA编辑酶相比,能实现靶向且高效的RNA编辑,同时转录组范围内的脱靶编辑显著降低。总之,我们的工作扩展了可编程RNA编辑方法的范围,对RNA行为的治疗调控具有广泛的意义。