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Allele-specific RNA interference prevents neuropathy in Charcot-Marie-Tooth disease type 2D mouse models
Kathryn H. Morelli, … , Scott Q. Harper, Robert W. Burgess
Kathryn H. Morelli, … , Scott Q. Harper, Robert W. Burgess
Published September 26, 2019
Citation Information: J Clin Invest. 2019;129(12):5568-5583. https://doi.org/10.1172/JCI130600.
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Research Article Genetics Neuroscience

Allele-specific RNA interference prevents neuropathy in Charcot-Marie-Tooth disease type 2D mouse models

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Abstract

Gene therapy approaches are being deployed to treat recessive genetic disorders by restoring the expression of mutated genes. However, the feasibility of these approaches for dominantly inherited diseases — where treatment may require reduction in the expression of a toxic mutant protein resulting from a gain-of-function allele — is unclear. Here we show the efficacy of allele-specific RNAi as a potential therapy for Charcot-Marie-Tooth disease type 2D (CMT2D), caused by dominant mutations in glycyl-tRNA synthetase (GARS). A de novo mutation in GARS was identified in a patient with a severe peripheral neuropathy, and a mouse model precisely recreating the mutation was produced. These mice developed a neuropathy by 3–4 weeks of age, validating the pathogenicity of the mutation. RNAi sequences targeting mutant GARS mRNA, but not wild-type, were optimized and then packaged into AAV9 for in vivo delivery. This almost completely prevented the neuropathy in mice treated at birth. Delaying treatment until after disease onset showed modest benefit, though this effect decreased the longer treatment was delayed. These outcomes were reproduced in a second mouse model of CMT2D using a vector specifically targeting that allele. The effects were dose dependent, and persisted for at least 1 year. Our findings demonstrate the feasibility of AAV9-mediated allele-specific knockdown and provide proof of concept for gene therapy approaches for dominant neuromuscular diseases.

Authors

Kathryn H. Morelli, Laurie B. Griffin, Nettie K. Pyne, Lindsay M. Wallace, Allison M. Fowler, Stephanie N. Oprescu, Ryuichi Takase, Na Wei, Rebecca Meyer-Schuman, Dattatreya Mellacheruvu, Jacob O. Kitzman, Samuel G. Kocen, Timothy J. Hines, Emily L. Spaulding, James R. Lupski, Alexey Nesvizhskii, Pedro Mancias, Ian J. Butler, Xiang-Lei Yang, Ya-Ming Hou, Anthony Antonellis, Scott Q. Harper, Robert W. Burgess

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Figure 1

In vitro characterization of ΔETAQ mutation.

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In vitro characterization of ΔETAQ mutation.
(A) The position and evolut...
(A) The position and evolutionary conservation of the ΔETAQ (red) and P234KY (green) GARS mutations. (B) Initial aminoacylation rates (pmol/min) of WT (black), P234KY (green), and ΔETAQ (blue) GARS were plotted against tRNA concentrations and fit to the Michaelis-Menten equation. (C) Representative cultures of yeast strains lacking GRS1 to test for growth in the presence of each mutation (ΔETAQ or P234KY) modeled in the human GARS open reading frame. (D) WT, P234KY, or ΔETAQ GARS was expressed (with a V5 tag) and tested by immunoprecipitation with an anti-NRP1 antibody to detect aberrant interactions. Western blots were performed with anti-NRP1 and anti-V5 antibodies. Immunoprecipitation (IP), negative control (IgG), and input experiments are indicated.

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ISSN: 0021-9738 (print), 1558-8238 (online)

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