AAV9-mediated SH3TC2 gene replacement therapy targeted to Schwann cells for the treatment of CMT4C.

Type 4C Charcot-Marie-Tooth (CMT4C) demyelinating neuropathy is caused by autosomal recessive SH3TC2 gene mutations. SH3TC2 is highly expressed in myelinating Schwann cells. CMT4C is a childhood-onset progressive disease without effective treatment. Here, we generated a gene therapy for CMT4C mediated by an adeno-associated viral 9 vector (AAV9) to deliver the human SH3TC2 gene in the Sh3tc2-/- mouse model of CMT4C. We used a minimal fragment of the myelin protein zero (Mpz) promoter (miniMpz), which was cloned and validated to achieve Schwann cell-targeted expression of SH3TC2. Following the demonstration of AAV9-miniMpz.SH3TC2myc vector efficacy to re-establish SH3TC2 expression in the peripheral nervous system, we performed an early as well as a delayed treatment trial in Sh3tc2-/- mice. We demonstrate both after early as well as following late treatment improvements in multiple motor performance tests and nerve conduction velocities. Moreover, treatment led to normalization of the organization of the nodes of Ranvier, which is typically deficient in CMT4C patients and Sh3tc2-/- mice, along with reduced ratios of demyelinated fibers, increased myelin thickness and reduced g-ratios at both time points of intervention. Taken together, our results provide a proof of concept for an effective and potentially translatable gene replacement therapy for CMT4C treatment.

Mitofusin 1 overexpression rescues the abnormal mitochondrial dynamics caused by the Mitofusin 2 K357T mutation in vitro.

BACKGROUND AND AIMS: Mitofusin 1 (MFN1) and MFN2 are outer mitochondrial membrane fusogenic proteins regulating mitochondrial network morphology. MFN2 mutations cause Charcot-Marie-Tooth type 2A (CMT2A), an axonal neuropathy characterized by mitochondrial fusion defects, which in the case of a GTPase domain mutant, were rescued following wild-type MFN1/2 (MFN1/2WT ) overexpression. In this study, we compared the therapeutic efficiency between MFN1WT and MFN2WT overexpression in correcting mitochondrial defects induced by the novel MFN2K357T mutation located in the highly conserved R3 region. METHODS: Constructs expressing either MFN2K357T , MFN2WT , or MFN1WT under the ubiquitous chicken ß-actin hybrid (CBh) promoter were generated. Flag or myc tag was used for their detection. Differentiated SH-SY5Y cells were single transfected with MFN1WT , MFN2WT , or MFN2K357T , as well as double transfected with MFN2K357T /MFN2WT or MFN2K357T /MFN1WT . RESULTS: SH-SY5Y cells transfected with MFN2K357T exhibited severe perinuclear mitochondrial clustering with axon-like processes devoid of mitochondria. Single transfection with MFN1WT resulted in a more interconnected mitochondrial network than transfection with MFN2WT , accompanied by mitochondrial clusters. Double transfection of MFN2K357T with either MFN1WT or MFN2WT resolved the mutant-induced mitochondrial clusters and led to detectable mitochondria throughout the axon-like processes. MFN1WT showed higher efficacy than MFN2WT in rescuing these defects. INTERPRETATION: These results further demonstrate the higher potential of MFN1WT over MFN2WT overexpression to rescue CMT2A-induced mitochondrial network abnormalities due to mutations outside the GTPase domain. This higher phenotypic rescue conferred by MFN1WT , possibly due to its higher mitochondrial fusogenic ability, may be applied to different CMT2A cases regardless of the MFN2 mutation type.

Gene delivery targeted to oligodendrocytes using a lentiviral vector.

BACKGROUND: Most leukodystrophies result from mutations in genes expressed in oligodendrocytes that may cause autonomous loss of function of cell structural proteins. Therefore, effective gene delivery to oligodendrocytes is necessary to develop future treatments. MATERIALS: To achieve this, we cloned a lentiviral vector in which the enhanced green fluorescent protein (EGFP) expression was driven by the oligodendrocyte specific 2,3-cyclic nucleotide 3-phosphodiesterase promoter. The vector was inserted into C57BL/6 neonatal mouse brain by combined intraventricular and parenchymal injections. RESULTS: Assessment of EGFP expression revealed a widespread distribution, specifically in cells of the oligodendrocyte linage, starting from postnatal day 6 (P6) in the subventricular zone and spreading through migrating oligodendrocyte precursors. By P30, it was detectable throughout the brain and persisted for at least 3 months, showing an increase both in the number of expressing cells and in intensity over time. EGFP expression was restricted to oligodendrocyte linage cells. On average, 20.3 ± 2.56% of all oligodendrocytes in different central nervous system areas were EGFP-positive, with regional variations. CONCLUSIONS: Lentiviral gene delivery using an oligodendrocyte-specific promoter may achieve widespread and long-lasting expression selectively in oligodendrocytes, offering a possibility for gene therapy in certain leukodystrophies, although the relatively low rates of oligodendrocyte transduction are a limitation that remains to be overcome.

Morvan syndrome: clinical and serological observations in 29 cases.

OBJECTIVE: A study was undertaken to describe the clinical spectrum, voltage-gated potassium channel (VGKC) complex antibody specificities, and central nervous system localization of antibody binding in 29 patients diagnosed with Morvan syndrome (MoS). METHODS: Clinical data were collected using questionnaires. Radioimmunoassay, cell-based assays, and mouse brain immunohistochemistry were used to characterize the serum antibodies. RESULTS: Neuromyotonia (100%), neuropsychiatric features (insomnia 89.7%, confusion 65.5%, amnesia 55.6%, hallucinations 51.9%), dysautonomia (hyperhidrosis 86.2%, cardiovascular 48.3%), and neuropathic pain (62.1%) were the most common manifestations. A total of 93.1% of MoS patients were male. VGKC-complex antibodies were present in 23 of 29 (79%) MoS patients at referral; 24 of 27 available sera had CASPR2, LGI1, or both CASPR2 and LGI1 antibodies (3 also with contactin-2 antibodies). CASPR2 antibodies were generally higher titer than LGI1 antibodies. Tumors (41.4%), mainly thymomas, were associated with CASPR2 antibodies and a poor prognosis, whereas LGI1 antibodies were associated with serum hyponatremia. In brain tissue regions including the hypothalamus, raphe, and locus coeruleus, commercial antibodies to LGI1 bound to neuronal cell bodies including the antidiuretic hormone-secreting and orexin-secreting hypothalamic neurons, whereas CASPR2 commercial antibodies bound more often to the neuropil. MoS antibodies bound similarly, but there was evidence of additional antibodies in some sera that were not adsorbed by LGI1- or CASPR2-expressing cells and bound to mouse Caspr2(-/-) tissue. INTERPRETATION: MoS is clinically distinct from other VGKC-complex antibody-associated conditions, and usually is associated with high-titer CASPR2 antibodies, often accompanied by lower-titer LGI1 antibodies. CASPR2 and LGI1 antibodies bind to multiple brain regions, which helps to explain the multifocal clinical features of this disease, but other antibodies are likely to play a role in some patients and need to be characterized in future studies.