AAV1.NT-3 gene therapy for X-linked Charcot-Marie-Tooth neuropathy type 1.

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.

Unraveling the genetic landscape of autosomal recessive Charcot-Marie-Tooth neuropathies using a homozygosity mapping approach.

Autosomal recessive forms of Charcot-Marie-Tooth disease (ARCMT) are rare but severe disorders of the peripheral nervous system. Their molecular basis is poorly understood due to the extensive genetic and clinical heterogeneity, posing considerable challenges for patients, physicians, and researchers. We report on the genetic findings from a systematic study of a large collection of 174 independent ARCMT families. Initial sequencing of the three most common ARCMT genes (ganglioside-induced differentiation protein 1­GDAP1, SH3 domain and tetratricopeptide repeats-containing protein 2­SH3TC2, histidine-triad nucleotide binding protein 1­HINT1) identified pathogenic mutations in 41 patients. Subsequently, 87 selected nuclear families underwent single nucleotide polymorphism (SNP) genotyping and homozygosity mapping, followed by targeted screening of known ARCMT genes. This strategy provided molecular diagnosis to 22% of the families. Altogether, our unbiased genetic approach identified pathogenic mutations in ten ARCMT genes in a total of 41.3% patients. Apart from a newly described founder mutation in GDAP1, the majority of variants constitute private molecular defects. Since the gene testing was independent of the clinical phenotype of the patients, we identified mutations in patients with unusual or additional clinical features, extending the phenotypic spectrum of the SH3TC2 gene. Our study provides an overview of the ARCMT genetic landscape and proposes guidelines for tackling the genetic heterogeneity of this group of hereditary neuropathies.

AAV1.NT-3 gene therapy in the SOD1KO mouse model of accelerated sarcopenia.

BACKGROUND: Sarcopenia, an age-related loss of muscle mass, is a critical factor that affects the health of the older adults. The SOD1KO mouse is deficient of Cu/Zn superoxide dismutase, used as an accelerated aging model. We previously showed that NT-3 improves muscle fibre size by activating the mTOR pathway, suggesting a potential for attenuating age-related muscle loss. This study assessed the therapeutic efficacy of AAV1.NT-3 in this accelerated aging model. METHODS: Twelve 6 months old SOD1KO mice were injected intramuscularly with a 1 × 1011 vg dose of AAV1.tMCK.NT-3, and 13 age-matched SOD1KO mice were used as controls. The treatment effect was evaluated using treadmill, rotarod and gait analyses as well as histological studies assessing changes in muscle fibre, and fibre type switch, in tibialis anterior, gastrocnemius, and triceps muscles, and myelin thickness by calculating G ratio in sciatic and tibial nerves. Molecular studies involved qPCR experiments to analyse the expression levels of mitochondrial and glycolysis markers and western blot experiments to assess the activity of mTORC1 pathway. RESULTS: Treatment resulted in a 36% (154.9 vs. 114.1; P < 0.0001) and 76% increase (154.3 vs. 87.6; P < 0.0001) in meters ran, with treadmill test at 3 and 6 months post gene delivery. In addition, the treated cohort stayed on rotarod 30% (52.7 s vs. 40.4 s; P = 0.0095) and 54% (50.4 s vs. 32.7 s; P = 0.0007) longer, compared with untreated counterparts at 3 and 6 months post injection. Gait analysis, performed at endpoint, showed that stride width was normalized to wild type levels (29.3 mm) by an 11% decrease, compared with untreated cohort (28.6 mm vs. 32.1 mm; P = 0.0014). Compared with wild-type, SOD1KO mice showed 9.4% and 11.4% fibre size decrease in tibialis anterior and gastrocnemius muscles, respectively, which were normalized to wild type levels with treatment. Fibre diameter increase was observed prominently in FTG fibre type. G ratio analysis revealed hypomyelination in the tibial (0.721) and sciatic (0.676) nerves of SOD1KO model, which was reversed in the NT-3 cohort (0.646 and 0.634, respectively). Fibre size increase correlated with the increase in the p-S6 and p-4E-BP1 levels, and in the glycolysis markers in tibialis anterior. Alterations observed in the mitochondrial markers were not rescued with treatment. Overall, response to NT-3 was subdued in gastrocnemius muscle. CONCLUSIONS: This study shows that AAV1.NT-3 gene therapy protected SOD1KO mouse from accelerated aging effects functionally and histologically. We further confirmed that NT-3 has potential to activate the mTOR and glycolytic pathways in muscle.

AAV1.NT-3 gene therapy prevents age-related sarcopenia.

Sarcopenia is progressive loss of muscle mass and strength, occurring during normal aging with significant consequences on the quality of life for elderly. Neurotrophin 3 (NT-3) is an important autocrine factor supporting Schwann cell survival and differentiation and stimulating axon regeneration and myelination. NT-3 is involved in the maintenance of neuromuscular junction (NMJ) integrity, restoration of impaired radial growth of muscle fibers through activation of the Akt/mTOR pathway. We tested the efficacy of NT-3 gene transfer therapy in wild type (WT)-aged C57BL/6 mice, a model for natural aging and sarcopenia, via intramuscular injection 1 × 1011 vg AAV1.tMCK.NT-3, at 18 months of age. The treatment efficacy was assessed at 6 months post-injection using run to exhaustion and rotarod tests, in vivo muscle contractility assay, and histopathological studies of the peripheral nervous system, including NMJ connectivity and muscle. AAV1.NT-3 gene therapy in WT-aged C57BL/6 mice resulted in functional and in vivo muscle physiology improvements, supported by quantitative histology from muscle, peripheral nerves and NMJ. Hindlimb and forelimb muscles in the untreated cohort showed the presence of a muscle- and sex-dependent remodeling and fiber size decrease with aging, which was normalized toward values obtained from 10 months old WT mice with treatment. The molecular studies assessing the NT-3 effect on the oxidative state of distal hindlimb muscles, accompanied by western blot analyses for mTORC1 activation were in accordance with the histological findings. Considering the cost and quality of life to the individual, we believe our study has important implications for management of age-related sarcopenia.

AAV1.NT-3 gene therapy in a CMT2D model: phenotypic improvements in GarsP278KY/+ mice.

Glycyl-tRNA synthetase mutations are associated to the Charcot-Marie-Tooth disease type-2D. The GarsP278KY/+ model for Charcot-Marie-Tooth disease type-2D is known best for its early onset severe neuropathic phenotype with findings including reduced axon size, slow conduction velocities and abnormal neuromuscular junction. Muscle involvement remains largely unexamined. We tested the efficacy of neurotrophin 3 gene transfer therapy in two Gars mutants with severe (GarsP278KY/+ ) and milder (GarsΔETAQ/+ ) phenotypes via intramuscular injection of adeno-associated virus setoype-1, triple tandem muscle creatine kinase promoter, neurotrophin 3 (AAV1.tMCK.NT-3) at 1 × 1011 vg dose. In the GarsP278KY/+ mice, the treatment efficacy was assessed at 12 weeks post-injection using rotarod test, electrophysiology and detailed quantitative histopathological studies of the peripheral nervous system including neuromuscular junction and muscle. Neurotrophin 3 gene transfer therapy in GarsP278KY/+ mice resulted in significant functional and electrophysiological improvements, supported with increases in myelin thickness and improvements in the denervated status of neuromuscular junctions as well as increases in muscle fibre size along with attenuation of myopathic changes. Improvements in the milder phenotype GarsΔETAQ/+ was less pronounced. Furthermore, oxidative enzyme histochemistry in muscles from Gars mutants revealed alterations in the content and distribution of oxidative enzymes with increased expression levels of Pgc1a. Cox1, Cox3 and Atp5d transcripts were significantly decreased suggesting that the muscle phenotype might be related to mitochondrial dysfunction. Neurotrophin 3 gene therapy attenuated these abnormalities in the muscle. This study shows that neurotrophin 3 gene transfer therapy has disease modifying effect in a mouse model for Charcot-Marie-Tooth disease type-2D, leading to meaningful improvements in peripheral nerve myelination and neuromuscular junction integrity as well as in a unique myopathic process, associated with mitochondria dysfunction, all in combination contributing to functional outcome. Based on the multiple biological effects of this versatile molecule, we predict neurotrophin 3 has the potential to be beneficial in other aminoacyl-tRNA synthetase-linked Charcot-Marie-Tooth disease subtypes.

Systemic delivery of AAVrh74.tMCK.hCAPN3 rescues the phenotype in a mouse model for LGMD2A/R1.

Limb girdle muscular dystrophy (LGMD) 2A/R1, caused by mutations in the CAPN3 gene and CAPN3 loss of function, is known to play a role in disease pathogenicity. In this study, AAVrh74.tMCK.CAPN3 was delivered systemically to two different age groups of CAPN3 knockout (KO) mice; each group included two treatment cohorts receiving low (1.17 × 1014 vg/kg) and high (2.35 × 1014 vg/kg) doses of the vector and untreated controls. Treatment efficacy was tested 20 weeks after gene delivery using functional (treadmill), physiological (in vivo muscle contractility assay), and histopathological outcomes. AAV.CAPN3 gene therapy resulted in significant, robust improvements in functional outcomes and muscle physiology at low and high doses in both age groups. Histological analyses of skeletal muscle showed remodeling of muscle, a switch to fatigue-resistant oxidative fibers in females, and fiber size increases in both sexes. Safety studies revealed no organ tissue abnormalities; specifically, there was no histopathological evidence of cardiotoxicity. These results show that CAPN3 gene replacement therapy improved the phenotype in the CAPN3 KO mouse model at both doses independent of age at the time of vector administration. The improvements were supported by an absence of cardiotoxicity, showing the efficacy and safety of the AAV.CAPN3 vector as a potential gene therapy for LGMDR1.

Gene therapy to promote regeneration in Charcot-Marie-Tooth disease.

The molecular pathogenesis underlying Charcot-Marie-Tooth (CMT) neuropathy subtypes is becoming increasingly variable and identification of common approaches for treatment, independently of the disease causing gene defect, is therefore much desirable. Gene therapy approach from the clinical translational view point is particularly challenging for the most common "demyelinating" CMT1 subtypes, caused by primary Schwann cell genetic defects. Studies have shown that impaired regenerative capacity of distal axons is major contributing factor to distal axonal loss in primary Schwann cell genetic defects and neurotrophin 3 (NT-3) improves impaired regeneration in CMT1 mouse models. This review surveys the evidence supporting the rationale for AAV1.NT-3 surrogate gene therapy to improve nerve regeneration in CMT1A. The translational process, from proof of principal studies to the design of the phase I/IIa trial evaluating scAAV1.tMCK.NTF3 gene therapy for treatment of CMT1A is summarized.

SIK2 attenuates proliferation and survival of breast cancer cells with simultaneous perturbation of MAPK and PI3K/Akt pathways.

Salt Inducible Kinase2 (SIK2) has been shown to contribute to tumorigenesis in multiple tumor types in a dichotomous manner. However, little is known about its contribution to breast malignancies. Here, we report SIK2 as a potential tumor suppressor in breast cancer whose expression was reduced in tumor tissues and breast cancer cell lines compared to normal counterparts. In vitro loss- and gain-of-function experiments combined with xenograft studies demonstrated that SIK2-mediated attenuation of proliferation and survival of breast cancer cells with parallel inhibition of both Ras/Erk and PI3K/Akt pathways. Our findings elucidated that SIK2 has also an inhibitory role in migration/invasion ability of breast cancer cells through regulation of epithelial mesenchymal transition. Immunostaining of patient tumors revealed that SIK2 protein level is frequently downregulated in invasive mammary carcinomas and negatively correlated with the mitotic activity of the cells in triple negative breast cancers and hormone positive tumors. Strikingly, patient survival analysis indicated that higher levels of SIK2 are significantly associated with better survival, especially in basal breast cancer cases. Overall, our findings suggest SIK2 as a potential tumor suppressor in the control of breast tumorigenesis, at least in part, via inhibiting PI3K/Akt and Ras/ERK signaling cascades simultaneously and a novel prognostic marker, especially in basal subtypes of breast cancer.

Ultrasonographic findings in hereditary neuropathy with liability to pressure palsies.

OBJECTIVES: The aims of this study were to evaluate the sonographic findings of patients with hereditary neuropathy with liability to pressure palsies (HNPP) and to examine the correlation between sonographic and electrophysiological findings. METHODS: Nine patients whose electrophysiological findings indicated HNPP and whose diagnosis was confirmed by genetic analysis were enrolled in the study. The median, ulnar, peroneal, and tibial nerves were evaluated by ultrasonography. RESULTS: We ultrasonographically evaluated 18 median, ulnar, peroneal, and tibial nerves. Nerve enlargement was identified in the median, ulnar, and peroneal nerves at the typical sites of compression. None of the patients had nerve enlargement at a site of noncompression. None of the tibial nerves had increased cross-sectional area (CSA) values. There were no significant differences in median, ulnar, and peroneal nerve distal motor latencies (DMLs) between the patients with an increased CSA and those with a normal CSA. In most cases, there was no correlation between electrophysiological abnormalities and clinical or sonographic findings. DISCUSSION: Although multiple nerve enlargements at typical entrapment sites on sonographic evaluation can suggest HNPP, ultrasonography cannot be used as a diagnostic tool for HNPP. Ultrasonography may contribute to the differential diagnosis of HNPP and other demyelinating polyneuropathies or compression neuropathies; however, further studies are required.

Exome Sequence Analysis Suggests that Genetic Burden Contributes to Phenotypic Variability and Complex Neuropathy.

Charcot-Marie-Tooth (CMT) disease is a clinically and genetically heterogeneous distal symmetric polyneuropathy. Whole-exome sequencing (WES) of 40 individuals from 37 unrelated families with CMT-like peripheral neuropathy refractory to molecular diagnosis identified apparent causal mutations in ∼ 45% (17/37) of families. Three candidate disease genes are proposed, supported by a combination of genetic and in vivo studies. Aggregate analysis of mutation data revealed a significantly increased number of rare variants across 58 neuropathy-associated genes in subjects versus controls, confirmed in a second ethnically discrete neuropathy cohort, suggesting that mutation burden potentially contributes to phenotypic variability. Neuropathy genes shown to have highly penetrant Mendelizing variants (HPMVs) and implicated by burden in families were shown to interact genetically in a zebrafish assay exacerbating the phenotype established by the suppression of single genes. Our findings suggest that the combinatorial effect of rare variants contributes to disease burden and variable expressivity.

Motor protein mutations cause a new form of hereditary spastic paraplegia.

OBJECTIVE: To identify a novel disease gene in 2 families with autosomal recessive hereditary spastic paraplegia (HSP). METHODS: We used whole-exome sequencing to identify the underlying genetic disease cause in 2 families with apparently autosomal recessive spastic paraplegia. Endogenous expression as well as subcellular localization of wild-type and mutant protein were studied to support the pathogenicity of the identified mutations. RESULTS: In 2 families, we identified compound heterozygous or homozygous mutations in the kinesin gene KIF1C to cause hereditary spastic paraplegia type 58 (SPG58). SPG58 can be complicated by cervical dystonia and cerebellar ataxia. The same mutations in a heterozygous state result in a mild or subclinical phenotype. KIF1C mutations in SPG58 affect the domains involved in adenosine triphosphate hydrolysis and microtubule binding, key functions for this microtubule-based motor protein. CONCLUSIONS: KIF1C is the third kinesin gene involved in the pathogenesis of HSPs and is characterized by a mild dominant and a more severe recessive disease phenotype. The identification of KIF1C as an HSP disease gene further supports the key role of intracellular trafficking processes in the pathogenesis of hereditary axonopathies.