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Contributed by Gail Mandel, September 22, 2017 (sent for review August 30, 2017; reviewed by Gordon G. Carmichael and Stuart Cobb)
Significance
Rett syndrome (RTT) is a neurological disease caused by mutations in the gene encoding the global transcriptional regulator, Methyl CpG Binding Protein 2 (MECP2). We exploit a strategy to repair mutant Mecp2 mRNA that if successful should reverse symptoms. The strategy utilizes the catalytic activity of a naturally occurring enzyme, Adenosine Deaminase Acting on RNA (ADAR2), which in brain alters the mRNA sequence and function of proteins. In cultured RTT neurons co-expressing a modified ADAR2 protein and an appropriate RNA guide, a human mutation in Mecp2 mRNA is repaired efficiently. RNA repair restores MeCP2 function, consistent with reversal of the pathological consequences of the RTT mutation. Our strategy holds promise for new therapeutic approaches to RTT and other neurological diseases.
Abstract
Rett syndrome (RTT) is a debilitating neurological disorder caused by mutations in the gene encoding the transcription factor Methyl CpG Binding Protein 2 (MECP2). A distinct disorder results from MECP2 gene duplication, suggesting that therapeutic approaches must restore close to normal levels of MECP2. Here, we apply the approach of site-directed RNA editing to repair, at the mRNA level, a disease-causing guanosine to adenosine (G > A) mutation in the mouse MeCP2 DNA binding domain. To mediate repair, we exploit the catalytic domain of Adenosine Deaminase Acting on RNA (ADAR2) that deaminates A to inosine (I) residues that are subsequently translated as G. We fuse the ADAR2 domain, tagged with a nuclear localization signal, to an RNA binding peptide from bacteriophage lambda. In cultured neurons from mice that harbor an RTT patient G > A mutation and express engineered ADAR2, along with an appropriate RNA guide to target the enzyme, 72% of Mecp2 mRNA is repaired. Levels of MeCP2 protein are also increased significantly. Importantly, as in wild-type neurons, the repaired MeCP2 protein is enriched in heterochromatic foci, reflecting restoration of normal MeCP2 binding to methylated DNA. This successful use of site-directed RNA editing to repair an endogenous mRNA and restore protein function opens the door to future in vivo applications to treat RTT and other diseases.
Footnotes
↵1J.R.S. and S.Y.K. contributed equally to this work.
- ↵2To whom correspondence should be addressed. Email: mandelg{at}ohsu.edu.
Author contributions: J.R.S., S.Y.K., G.M.C., H.N., J.P.A., and G.M. designed research; J.R.S., S.Y.K., G.M.C., and Z.S. performed research; H.N. contributed new reagents/analytic tools; J.R.S., S.Y.K., G.M.C., J.P.A., and G.M. analyzed data; and J.R.S., J.P.A., and G.M. wrote the paper.
Reviewers: G.G.C., University of Connecticut Health Center; and S.C., University of Glasgow.
The authors declare no conflict of interest.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1715320114/-/DCSupplemental.
Published under the PNAS license.
Site-directed RNA repair of endogenous Mecp2 RNA
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