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 2

In vivo assessment of AAV9-miR transduction in PNS tissues and validation of AAV9-miR871 silencing efficiency in a CMT1A mouse model 6 weeks after injection.

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In vivo assessment of AAV9-miR transduction in PNS tissues and validatio...
(A) Lumbar spinal roots and sciatic nerve sections as well as teased femoral nerve fibers showing EGFP autofluorescence in SCs and axons. Arrowheads indicate examples of EGFP+ nuclei. Scale bars: 60 μm (lumbar root and sciatic nerve) and 20 μm (femoral nerve). (B) Quantification of EGFP-expressing PNS cells (n = 4/group). (C) VGCNs (n = 4/group) confirmed peripheral nerve transduction. RT-qPCR analysis of (D) Human (hu) PMP22 and murine (mu) Pmp22 and of murine (E) Mpz, Cnp, Gldn, and Gjb1 gene expression (n = 3/group). Fold changes in relative mRNA expression of CMT1A-AAV9-miR871 were calculated in comparison with expression levels in CMT1A-AAV9-miRLacZ mice. All samples were normalized to endogenous Gapdh. Quantification of (F) human PMP22 and (G) murine MPZ Western blot protein ODs, normalized to tubulin (Tub), in CMT1A-AAV9-miR871 and CMT1A-AAV9-miRLacZ mice in lumbar roots, sciatic nerves, and femoral nerves. Western blots showing human PMP22, murine tubulin, EGFP, and murine MPZ protein levels in (H) roots, (I) sciatic nerves, and (J) femoral nerves. Values represent the mean ± SD. *P < 0.05 and **P < 0.01, by 1-way ANOVA with Tukey’s multiple-comparison test. R, lumbar roots; S, sciatic nerves; F, femoral nerves; i.th., intrathecal; m.o., months old; Non Inj, noninjected.