Gene replacement therapy after neuropathy onset provides therapeutic benefit in a model of CMT1X.

X-linked Charcot-Marie-Tooth disease (CMT1X), one of the commonest forms of inherited demyelinating neuropathy, results from GJB1 gene mutations causing loss of function of the gap junction protein connexin32 (Cx32). The aim of this study was to examine whether delayed gene replacement therapy after the onset of peripheral neuropathy can provide a therapeutic benefit in the Gjb1-null/Cx32 knockout model of CMT1X. After delivery of the LV-Mpz.GJB1 lentiviral vector by a single lumbar intrathecal injection into 6-month-old Gjb1-null mice, we confirmed expression of Cx32 in lumbar roots and sciatic nerves correctly localized at the paranodal myelin areas. Gjb1-null mice treated with LV-Mpz.GJB1 compared with LV-Mpz.Egfp (mock) vector at the age of 6 months showed improved motor performance at 8 and 10 months. Furthermore, treated mice showed increased sciatic nerve conduction velocities, improvement of myelination and reduced inflammation in lumbar roots and peripheral nerves at 10 months of age, along with enhanced quadriceps muscle innervation. Plasma neurofilament light (NEFL) levels, a clinically relevant biomarker, were also ameliorated in fully treated mice. Intrathecal gene delivery after the onset of peripheral neuropathy offers a significant therapeutic benefit in this disease model, providing a proof of principle for treating patients with CMT1X at different ages.

Intrathecal gene therapy in mouse models expressing CMT1X mutations.

Gap junction beta-1 (GJB1) gene mutations affecting the gap junction protein connexin32 (Cx32) cause the X-linked Charcot-Marie-Tooth disease (CMT1X), a common inherited neuropathy. Targeted expression of virally delivered Cx32 in Schwann cells following intrathecal injection of lentiviral vectors in the Cx32 knockout (KO) mouse model of the disease has led to morphological and functional improvement. To examine whether this approach could be effective in CMT1X patients expressing different Cx32 mutants, we treated transgenic Cx32 KO mice expressing the T55I, R75W or N175D CMT1X mutations. All three mutants were localized in the perinuclear compartment of myelinating Schwann cells consistent with retention in the ER (T55I) or Golgi (R75W, N175D) and loss of physiological expression in the non-compact myelin. Following intrathecal delivery of the GJB1 gene we detected the virally delivered wild-type (WT) Cx32 in non-compact myelin of T55I KO mice, but only rarely in N175D KO or R75W KO mice, suggesting dominant-negative effects of the R75W and N175D mutants but not of the T55I mutant on co-expressed WT Cx32. GJB1 treated T55I KO mice showed improved motor performance, lower ratios of abnormally myelinated fibers and reduction of inflammatory cells in spinal roots and peripheral nerves compared with mock-treated littermates. Either partial (N175D KO) or no (R75W KO) improvement was observed in the other two mutant lines. Thus, certain CMT1X mutants may interfere with gene addition therapy for CMT1X. Whereas gene addition can be used for non-interfering CMT1X mutations, further studies will be needed to develop treatments for patients harboring interfering mutations.

Efficacy of AAV serotypes to target Schwann cells after intrathecal and intravenous delivery.

To optimize gene delivery to myelinating Schwann cells we compared clinically relevant AAV serotypes and injection routes. AAV9 and AAVrh10 vectors expressing either EGFP or the neuropathy-associated gene GJB1/Connexin32 (Cx32) under a myelin specific promoter were injected intrathecally or intravenously in wild type and Gjb1-null mice, respectively. Vector biodistribution in lumbar roots and sciatic nerves was higher in AAVrh10 injected mice while EGFP and Cx32 expression rates and levels were similar between the two serotypes. A gradient of biodistribution away from the injection site was seen with both intrathecal and intravenous delivery, while similar expression rates were achieved despite higher vector amounts injected intravenously. Quantified immune cells in relevant tissues were similar to non-injected littermates. Overall, AAV9 and AAVrh10 efficiently transduce Schwann cells throughout the peripheral nervous system with both clinically relevant routes of administration, although AAV9 and intrathecal injection may offer a more efficient approach for treating demyelinating neuropathies.

Assessing the effects of three dental impression materials on the isolated sciatic nerve of rat and frog.

The effects on nerve tissue of three dental impression pastes were compared in this study. Two of the impression pastes, Examix and Express 3M, contained vinyl polysiloxane while the other, Xanthopren, did not. An in vitro model based on the isolated sciatic nerve of the frog and rat was used. As an indication of the proper functioning of the fibres in the nerve, the amplitude of evoked compound action potential (CAP) was monitored continuously. The results clearly showed that the number of active nerve fibres in the isolated sciatic nerves of either rat or frog exposed directly to impression pastes containing vinyl polysiloxane, decreased much faster than those of the nerves in contact to impression material without vinyl polysiloxane. When the nerve of the frog was exposed to Xanthopren there was a decrease in the CAP to 50% of the control values within 56.87+/-2.42 h (n=6). This value was called inhibition time to 50%, IT(50) and for Examix it was found to be 9.97+/-1.53 h. When the nerve of the rat was exposed to Xanthopren, the IT(50) was 15.34+/-2.97 h (n=6) for the Xanthopren and only 2.86+/-1.20 h for Examix and 2.76+/-0.48 h for Express 3M (n=6). There was no significant difference between the action of the last two compounds (P=0.85). This fast nerve fibre inactivation could be caused either by the chemical used for the synthesis of the two impression pastes, Examix and Express 3M, or by the unusual constriction of the nerve when it is embedded in the materials with vinyl polysiloxane. There is strong evidence to support the first case, since the incubation of the nerve in the presence of Examix, Express 3M and Xantopren in a way so the nerve was not in contact with the impression pastes, shows a much faster decrease of the CAP in the presence of the first two pastes. The decrease is caused by the death of nerve fibres, since there is no recovery in the CAP after the removal of Examix from the incubating saline.