In this revised proposal, we continue to develop an approach to repair base mutations at the level of RNA, for attenuating symptoms in mouse models of human neurological disease. The experiments are an outgrowth of a pilot NIH Director?s Transformative Research Award that supported both recently published and preliminary results attesting to feasibility of this approach. Our work is currently focused on Rett syndrome, a devastating neurological disease due to mutations in the gene encoding the transcription factor, MECP2. We focused initially on a human patient guanosine (G) to adenosine (A) mutation in MECP2, MECP2317G>A, which interferes severely with its ability to bind to chromatin and results in Rett syndrome. We showed, for the first time, that endogenous Mecp2317G>A RNA can be recoded to the wild type amino acid efficiently in non-dividing neurons cultured from a Mecp2317G>A mouse line that exhibits severe Rett-like symptoms. The recoding occurred through site- directed deamination by a hijacked catalytic domain of Adenosine Acting on RNA 2 (ADAR2) (Editase), fused to a bacteriophage RNA binding peptide, which we targeted to the Mecp2 mutation by an RNA guide. In this revised application, we present new data indicating that recoding also occurs in vivo, in 3 different hippocampal neuronal populations, after direct hippocampal injection of AAV encoding the hybrid ADAR2 protein and Mecp2 RNA guides. Moreover, recoding resulted in amount of MeCP2 localization to chromatin consistent with amount of editing at the RNA level. We have developed the tools and reagents that now place us in an ideal position to address critical unanswered questions for reversing neurological phenotypes of Rett syndrome, and for testing hypotheses related to site-directed repair.
In Aim 1, we test the hypotheses that brain-wide repair of Mecp2317G>A RNA, by site-directed editing, can be tuned to high efficiency and specificity in mice, and restores proper chromatin interaction. For this purpose, we perform whole transcriptomic RNA seq analysis across the brain after peripheral injections of an efficient brain AAV serotype virus encoding optimized editing or control components.
In Aim 2, we test new guides for the ability to recruit endogenous ADAR2 to mutant Mecp2 RNA in vivo, circumventing potential immune responses to the bacteriophage moiety in the hybrid ADAR2 protein and potentially minimizing off-target editing. Our initial model is Mecp2311G>A that has the ideal nonsense codon for this approach.
In Aim 3, we inject peripherally the virus encoding our current and optimized editing components, or controls, to test our hypothesis that site-directed RNA editing can stabilize/reverse Rett-like symptoms in both Mecp2G>A mouse lines. In addition to Rett syndrome, our approach has the potential to cure thousands of additional pathogenic G>A mutations.

Public Health Relevance

This proposal extends successful work funded by a NIH Director's Transformative Research Award to apply a site-directed RNA editing approach to repair mutations in neurological disorders. We focus on guanosine to adenosine mutations that underlie several neurological disorders, including Rett syndrome, our model for testing basic and translational hypotheses related to the technology because of symptom reversibility in Rett mice. Our recent results show, for the first time with any editing method, widespread in vivo repair of an endogenous protein (MeCP2) in CNS neurons.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Research Project (R01)
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Molecular Neurogenetics Study Section (MNG)
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Mamounas, Laura
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Oregon Health and Science University
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United States
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