Abstract
X-linked Charcot-Marie-Tooth neuropathy (CMTX) is caused by mutations in the gene encoding Gap Junction Protein Beta-1 (GJB1)/Connexin32 (Cx32) in Schwann cells. Neurotrophin-3 (NT-3) is an important autocrine factor supporting Schwann cell survival and differentiation and stimulating axon regeneration and myelination. Improvements in these parameters have been shown previously in a CMT1 model, TremblerJ mouse, with NT-3 gene transfer therapy. For this study, scAAV1.tMCK.NT-3 was delivered to the gastrocnemius muscle of 3-month-old Cx32 knockout (KO) mice. Measurable levels of NT-3 were found in the serum at 6-month post gene delivery. The outcome measures included functional, electrophysiological and histological assessments. At 9-months of age, NT-3 treated mice showed no functional decline with normalized compound muscle action potential amplitudes. Myelin thickness and nerve conduction velocity significantly improved compared with untreated cohort. A normalization toward age-matched wildtype histopathological parameters included increased number of Schmidt-Lanterman incisures, and muscle fiber diameter. Collectively, these findings suggest a translational application to CMTX1.
Highlights
We show that NT-3 gene therapy prevented functional decline and improved electrophysiological and histopathological phenotype of connexin 32 (Cx32) KO mouse model by intramuscular delivery of scAAV1. tMCK.NT-3
Results rAAV.NT-3 vector production and potency scAAV1.tMCK.NT-3 design (Supplementary Fig. S2a) and production followed previously described methods at Nationwide Children’s Hospital, Columbus [11]. scAAV1.tMCK.NT-3, at 1 × 1011 vg dose was delivered to the gastrocnemius muscle of Cx32 knockout (Cx32 KO) mice at 3 month of age; blood samples from anesthetized treated and untreated mice were collected by cardiac puncture at 6 months post gene injection and serum was assayed for NT-3 levels using a capture ELISA as previously reported [11]
Rotarod performance, tested at baseline and 6 months posttreatment showed that gene transfer in Cx32 KO mice preserved function (Median: 46 s. at baseline vs. 38 s. at endpoint; n = 11, p = 0.11) without significant decline (Fig. 1a), similar to the observations in the age-matched wild type (WT) mice (Fig. 1c)
Summary
Manifesting female carriers usually present with a milder phenotype due to random X-inactivation. Male patients often show intermediate slowing of motor nerve conduction velocity (NCV; 25–35 m/s) and females present mildly slowed motor NCVs (>35 m/s) [3]. The protein is localized to the non-compacted myelin of the paranodes and Schmidt- Lanterman incisures (SLIs) of SCs where it is thought to provide reflexive gap junction channels allowing for the exchange ions and small signaling molecules and metabolites [6,7,8,9]
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