Abstract
Charcot-Marie-Tooth disease type 4C is the most common recessively inherited demyelinating neuropathy that results from loss of function mutations in the SH3TC2 gene. Sh3tc2-/- mice represent a well characterized disease model developing early onset progressive peripheral neuropathy with hypo- and demyelination, slowing of nerve conduction velocities and disturbed nodal architecture. The aim of this project was to develop a gene replacement therapy for treating Charcot-Marie-Tooth disease type 4C to rescue the phenotype of the Sh3tc2-/- mouse model. We generated a lentiviral vector LV-Mpz.SH3TC2.myc to drive expression of the human SH3TC2 cDNA under the control of the Mpz promoter specifically in myelinating Schwann cells. The vector was delivered into 3-week-old Sh3tc2-/- mice by lumbar intrathecal injection and gene expression was assessed 4-8 weeks after injection. Immunofluorescence analysis showed presence of myc-tagged human SH3TC2 in sciatic nerves and lumbar roots in the perinuclear cytoplasm of a subset of Schwann cells, in a dotted pattern co-localizing with physiologically interacting protein Rab11. Quantitative PCR analysis confirmed SH3TC2 mRNA expression in different peripheral nervous system tissues. A treatment trial was initiated in 3 weeks old randomized Sh3tc2-/- littermate mice which received either the full or mock (LV-Mpz.Egfp) vector. Behavioural analysis 8 weeks after injection showed improved motor performance in rotarod and foot grip tests in treated Sh3tc2-/- mice compared to mock vector-treated animals. Moreover, motor nerve conduction velocities were increased in treated Sh3tc2-/- mice. On a structural level, morphological analysis revealed significant improvement in g-ratios, myelin thickness, and ratios of demyelinated fibres in lumbar roots and sciatic nerves of treated Sh3tc2-/- mice. Finally, treated mice also showed improved nodal molecular architecture and reduction of blood neurofilament light levels, a clinically relevant biomarker for axonal injury/degeneration. This study provides a proof of principle for viral gene replacement therapy targeted to Schwann cells to treat Charcot-Marie-Tooth disease type 4C and potentially other similar demyelinating inherited neuropathies.
Highlights
Charcot-Marie-Tooth disease type 4 (CMT4) includes several recessively inherited demyelinating neuropathy forms caused by mutations in different genes, which lead to loss of function of the encoded proteins
We examined the expression of myc-tagged SH3TC2 in sciatic nerve teased fibres from intrathecally-injected mice
Since nodal abnormalities consisting of nodal widening have been detected as early as 1 month of age in Sh3tc2À/À mice and in biopsied nerves from CMT4C patients (Arnaud et al, 2009), we examined nodal pathology to clarify whether gene replacement could reverse some of these early onset pathological features of CMT4C
Summary
Charcot-Marie-Tooth disease type 4 (CMT4) includes several recessively inherited demyelinating neuropathy forms caused by mutations in different genes, which lead to loss of function of the encoded proteins. Almost all patients develop foot deformities and scoliosis, often requiring surgery (Kessali et al, 1997; Gabreels-Festen et al, 1999; Azzedine et al, 2006). Electrophysiological studies in CMT4C patients confirm the demyelinating process with mean median motor nerve conduction velocity (MNCV) of 22.6 m/s. Nerve biopsies were characterized by an increase of basement membranes around myelinated, demyelinated, and unmyelinated axons, relatively few onion bulbs, and, most typically, large cytoplasmic extensions of Schwann cells (Kessali et al, 1997; Gabreels-Festen et al, 1999; Senderek et al, 2003)
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