A translatable RNAi-driven gene therapy silences PMP22/Pmp22 genes and improves neuropathy in CMT1A mice
Marina Stavrou, … , Scott Q. Harper, Kleopas A. Kleopa
Marina Stavrou, … , Scott Q. Harper, Kleopas A. Kleopa
Published May 17, 2022
Citation Information: J Clin Invest. 2022;132(13):e159814. https://doi.org/10.1172/JCI159814.
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Research Article Neuroscience

A translatable RNAi-driven gene therapy silences PMP22/Pmp22 genes and improves neuropathy in CMT1A mice

Abstract

Charcot-Marie-Tooth disease type 1A (CMT1A), the most common inherited demyelinating peripheral neuropathy, is caused by PMP22 gene duplication. Overexpression of WT PMP22 in Schwann cells destabilizes the myelin sheath, leading to demyelination and ultimately to secondary axonal loss and disability. No treatments currently exist that modify the disease course. The most direct route to CMT1A therapy will involve reducing PMP22 to normal levels. To accomplish this, we developed a gene therapy strategy to reduce PMP22 using artificial miRNAs targeting human PMP22 and mouse Pmp22 mRNAs. Our lead therapeutic miRNA, miR871, was packaged into an adeno-associated virus 9 (AAV9) vector and delivered by lumbar intrathecal injection into C61-het mice, a model of CMT1A. AAV9-miR871 efficiently transduced Schwann cells in C61-het peripheral nerves and reduced human and mouse PMP22 mRNA and protein levels. Treatment at early and late stages of the disease significantly improved multiple functional outcome measures and nerve conduction velocities. Furthermore, myelin pathology in lumbar roots and femoral motor nerves was ameliorated. The treated mice also showed reductions in circulating biomarkers of CMT1A. Taken together, our data demonstrate that AAV9-miR871–driven silencing of PMP22 rescues a CMT1A model and provides proof of principle for treating CMT1A using a translatable gene therapy approach.

Authors

Marina Stavrou, Alexia Kagiava, Sarah G. Choudury, Matthew J. Jennings, Lindsay M. Wallace, Allison M. Fowler, Amanda Heslegrave, Jan Richter, Christina Tryfonos, Christina Christodoulou, Henrik Zetterberg, Rita Horvath, Scott Q. Harper, Kleopas A. Kleopa

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Figure 6

Efficient PMP22/Pmp22 silencing and improvement of motor function and sciatic MNCVs but not CMAPs or blood biomarker phenotypes following late treatment of CMT1A mice.

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Efficient PMP22/Pmp22 silencing and improvement of motor function and sc...
(A) Design of the late (L.) and extended early (E.E.) treatment trial. RT-qPCR analysis of (B) PMP22 and Pmp22 and (C) Mpz, Cnp, Gldn, and Gjb1 gene expression in lumbar roots and sciatic and femoral nerves of late-treated CMT1A mice (n = 4/group). (D–I) Western blots and analysis of human PMP22, murine PMP22, murine tubulin, EGFP, and murine MPZ protein levels. (J–M) Behavioral analysis comparing noninjected WT and CMT1A mice (n = 10/group) and CMT1A-AAV9-miR871 and CMT1A-AAV9-miRLacZ mice (n = 16/group). (N) Hind limb opening angle estimation for 10-month-old noninjected WT and CMT1A mice (n = 6/group) and L.CMT1A-AAV9-miR871, E.E.CMT1A-AAV9-miR871, and CMT1A-AAV9-miRLacZ mice (n = 6/group). (O) MNCV and (P) CMAP analysis of 10-month-old WT and noninjected CMT1A mice (n = 6/group) and L.CMT1A-AAV9-miR871, E.E.CMT1A-AAV9-miR871, and CMT1A-AAV9-miRLacZ mice (n = 8/group). (Q) NF-L (n = 6/group) and (R) Gdf15 (n = 10/group) circulating biomarker analysis in 10-month-old L.CMT1A-AAV9-miR871 and L.CMT1A-AAV9-miRLacZ mice. Values represent the mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001, by unpaired, 1-tailed Student’s t test (B, C, Q, and R) and 1-way ANOVA with Tukey’s multiple-comparison test (G–P). For J–M, statistical significance is shown in Supplemental Figures 21 and 22.