SUMMARY Programmable RNA editing is gaining momentum as an approach to repair mutations, but its efficiency in repairing endogenous mutant RNA in complex tissue is unknown. Here we apply this approach to the brain and successfully repair a guanosine-to-adenosine mutation in methyl CpG binding protein 2 RNA that causes the neurodevelopmental disease Rett syndrome. Repair is mediated by hippocampal injections of juvenile Mecp2 317G>A mice with an adeno-associated virus expressing the hyperactive catalytic domain of adenosine deaminase acting on RNA 2 and Mecp2 guide. After 1 month, 50% of Mecp2 RNA is recoded in three different hippocampal neuronal populations. MeCP2 protein localization to heterochromatin is restored in neurons to 50% of wild-type levels. Whole-transcriptome RNA analysis of one neuronal population indicates that the majority of off-target editing sites exhibit rates of 30% or less. This study demonstrates that programmable RNA editing can be utilized to repair mutations in mouse models of neurological disease.

Rett syndrome is a neurological disease due to loss-of-function mutations in the transcription factor, Methyl CpG binding protein 2 (MECP2). Because overexpression of endogenous MECP2 also causes disease, we have exploited a targeted RNA-editing approach to repair patient mutations where levels of MECP2 protein will never exceed endogenous levels. Here, we have constructed adeno-associated viruses coexpressing a bioengineered wild-type ADAR2 catalytic domain (Editase wt ) and either Mecp2 -targeting or nontargeting gfp RNA guides. The viruses are introduced systemically into male mice containing a guanosine to adenosine mutation that eliminates MeCP2 protein and causes classic Rett syndrome in humans. We find that in the mutant mice injected with the Mecp2 -targeting virus, the brainstem exhibits the highest RNA-editing frequency compared to other brain regions. The efficiency is sufficient to rescue MeCP2 expression and function in the brainstem of mice expressing the Mecp2 -targeting virus. Correspondingly, we find that abnormal Rett-like respiratory patterns are alleviated, and survival is prolonged, compared to mice injected with the control gfp guide virus. The levels of RNA editing among most brain regions corresponds to the distribution of guide RNA rather than Editase wt . Our results provide evidence that a targeted RNA-editing approach can alleviate a hallmark symptom in a mouse model of human disease.

RNA base editing is gaining momentum as an approach to repair mutations, but its application to neurological disease has not been established. We have succeeded in directed transcript editing of a pathological mutation in a mouse model of the neurodevelopmental disease, Rett syndrome. Specifically, we directed editing of a guanosine to adenosine mutation in RNA encoding Methyl CpG Binding Protein 2 (MECP2). Repair was mediated by injecting the hippocampus of juvenile Rett mice with an adeno-associated virus expressing both an engineered enzyme containing the catalytic domain of Adenosine Deaminase Acting on RNA 2 and a Mecp2 targeting guide. After one month, 50% of Mecp2 RNA was recoded in three different hippocampal neuronal subtypes, and the ability of MeCP2 protein to associate with heterochromatin was similarly restored to 50% of wild-type levels. This study represents the first in vivo programmable RNA editing applied to a model of neurological disease.