Charcot-Marie-Tooth disease type 2CC due to NEFH variants causes a progressive, non-length-dependent, motor-predominant phenotype.

OBJECTIVE: Neurofilaments are the major scaffolding proteins for the neuronal cytoskeleton, and variants in NEFH have recently been described to cause axonal Charcot-Marie-Tooth disease type 2CC (CMT2CC). METHODS: In this large observational study, we present phenotype-genotype correlations on 30 affected and 3 asymptomatic mutation carriers from eight families. RESULTS: The majority of patients presented in adulthood with motor-predominant and lower limb-predominant symptoms and the average age of onset was 31.0±15.1 years. A prominent feature was the development of proximal weakness early in the course of the disease. The disease progressed rapidly, unlike other Charcot-Marie-Tooth disease (CMT) subtypes, and half of the patients (53%) needed to use a wheelchair on average 24.1 years after symptom onset. Furthermore, 40% of patients had evidence of early ankle plantarflexion weakness, a feature which is observed in only a handful of CMT subtypes. Neurophysiological studies and MRI of the lower limbs confirmed the presence of a non-length-dependent neuropathy in the majority of patients.All families harboured heterozygous frameshift variants in the last exon of NEFH, resulting in a reading frameshift to an alternate open reading frame and the translation of approximately 42 additional amino acids from the 3' untranslated region (3'-UTR). CONCLUSIONS: This phenotype-genotype study highlights the unusual phenotype of CMT2CC, which is more akin to spinal muscular atrophy rather than classic CMT. Furthermore, the study will enable more informative discussions on the natural history of the disease and will aid in NEFH variant interpretation in the context of the disease's unique molecular genetics.

Comparison of toxicity and transfection efficiency of amphiphilic block copolymers and polycationic polymers in striated muscles.

BACKGROUND: Gene delivery using synthetic vectors is frequently based on cationic compounds such as polyethyleneimine (PEI). However, few data have been published on the ability of PEI to mediate transgene expression in muscle tissue. Besides cationic vectors, there is increasing interest focusing on amphiphilic copolymers as gene carriers into striated muscles, although their mechanism of action is unknown. METHODS: Plasmid DNA was associated with three different polymers: the cationic polyethyleneimine and two amphiphilic copolymers displaying few (tetronic 304) or no charges (pluronic L64). The resulting formulations were investigated by dynamic light scattering, laser doppler velocimetry, gel retardation assay and transmission electron microscopy. The toxicity and efficiency of the carriers were assessed in both skeletal and cardiac muscles. RESULTS: PEI efficiently condenses plasmids into small complexes displaying a positive electrophoretic mobility. However, these PEI/DNA complexes lead to severe side-effects in vivo. The association between amphiphilic copolymers and DNA leads to no or only partial condensation of plasmids. Moreover, amphiphilic polymers do not interact strongly with DNA and tetronic-based systems are destabilized with a decreasing pH. Those vectors also display a negative electrophoretic mobility. Thus, characteristics of amphiphilic polymer/DNA systems might be considered unfavourable for transfection. However, safe and rather efficient gene expression was obtained in skeletal muscles, even at low DNA doses, but not in the myocardium. CONCLUSIONS: The present study highlights the interest in amphiphilic carriers for promoting DNA transfection in vivo. Gaining new insights into the properties of these vectors should allow their optimization.

Hydrosoluble polymers for muscular gene delivery.

Striated muscle tissue is an attractive target for gene delivery as it can be easily reached and can express exogenous proteins. However, administration of naked DNA results in low transfection levels, and the design and development of safe and efficient gene delivery systems are thus required. This review is focusing on the characteristics of the striated muscle tissue with regards to features possibly affecting gene transfer, as well as the different soluble polymers that have been evaluated as gene carriers. The described formulations are ranging from polymers displaying a high density of positive charges to non-ionic molecules. Nevertheless, polymers exhibiting few or no positive charges appear to our opinion as the most promising approach to achieve both safe and efficient transfection of the striated muscles.

Parameters affecting organization and transfection efficiency of amphiphilic copolymers/DNA carriers.

Amphiphilic block copolymers are attracting increasing interest in the field of gene therapy, especially for transfection of striated muscles. However, little is known about the parameters affecting their transfection efficiency in vivo. These copolymers can self-assemble as micelles in certain conditions. Since micellization strongly depends on the temperature and ionic content of the preparation medium, the present paper aimed at investigating the influence of these parameters in the context of gene delivery. We first assessed the micellization of pluronic L64 and tetronic 304 at various temperatures in water, saline or Tyrode's salts solution. Pluronic L64 can form micelles at temperatures above 37 degrees C in water or at 37 degrees C in the Tyrode's salts solution, in the range of concentration investigated. For tetronic 304, CMC was found to be far below the concentrations used to transfer DNA. Pluronic L64 interacted with DNA only in the presence of micelles. Moreover, in vivo evaluation demonstrated that significantly improved transfection efficiency was obtained at 37 degrees C in Tyrode's salts solution for pluronic L64 based formulations, compared to 4 degrees C and 20 degrees C. Such differences were not recorded with tetronic 304. Finally, optimized formulations of both tetronic 304 and pluronic L64 were able to mediate efficient transfection in dystrophic muscles.