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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 (Editasewt) 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 Editasewt. Our results provide evidence that a targeted RNA-editing approach can alleviate a hallmark symptom in a mouse model of human disease.

Recruiting the endogenous editing enzyme adenosine deaminase acting on RNA (ADAR) with tailored guide RNAs for adenosine-to-inosine (A-to-I) RNA base editing is promising for safely manipulating genetic information at the RNA level. However, the precision and efficiency of editing are often compromised by bystander off-target editing. Here, we find that in 5′-U A N triplets, which dominate bystander editing, G•U wobble base pairs effectively mitigate off-target events while maintaining high on-target efficiency. This strategy is universally applicable to existing A-to-I RNA base-editing systems and complements other suppression methods such as G•A mismatches and uridine (U) depletion. Combining wobble base pairing with a circularized format of the CLUSTER approach achieves highly precise and efficient editing (up to 87%) of a disease-relevant mutation in the Mecp2 transcript in cell culture. Virus-mediated delivery of the guide RNA alone realizes functional MeCP2 protein restoration in the central nervous system of a murine Rett syndrome model with editing yields of up to 19% and excellent bystander control in vivo. Using G•U wobble base pairs at specific loci increases RNA base-editing precision and efficiency.

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.

The Summer Internship for Indigenous Peoples in Genomics Canada (SING Canada) is an annual, weeklong training program organized by the Indigenous Science, Technology, and Society program in the Faculty of Native Studies at the University of Alberta. During the 2019 annual program, Indigenous students, nation members, elders, and early career Indigenous scientists were invited to participate in an intensive training program on chronic wasting disease (CWD) of cervids (deer, moose, elk, and caribou). At the closing of SING Canada 2019, participants collaborated in a talking circle to capture their impressions on the directions of CWD management, research, and engagement of Indigenous peoples in these processes. The results of this discussion indicated that research on CWD lacks Indigenous input, resulting in adverse outcomes for Indigenous people. These findings point to a greater need for Indigenous engagement and consultation on CWD and inclusion of the more holistic Indigenous perspectives that place value and emphasis on the interconnection between living and nonliving beings. By engaging more critical Indigenous perspectives, future directions of CWD research and management can begin to identify and meaningfully address Indigenous peoples' needs.