Pre Clinical Assessment of AAVrh74.MCK.GNE Viral Vector Therapeutic Potential: Robust Activity Despite Lack of Consistent Animal Model for GNE Myopathy

Background: GNE myopathy is a unique adult onset rare neuromuscular disease caused by recessive mutations in the GNE gene. The pathophysiological mechanism of this disorder is not well understood and to date, there is no available therapy for this debilitating disease. We have previously established proof of concept that AAV based gene therapy can effectively deliver the wild type human GNE into cultured muscle cells from human patients and in mice, using a CMV promoter driven human wild type GNE plasmid delivered through an adeno associated virus (AAV8) based platform. Objective: In the present study we have generated a muscle specific GNE construct, driven by the MCK promoter and packaged with the AAVrh74 serotype for efficacy evaluation in an animal model of GNE Myopathy. Methods: The viral vector was systemically delivered at 2 doses to two age groups of a Gne–/– hGNED207V Tg mouse described as a preclinical model of GNE Myopathy, and treatment was monitored for long term efficacy. Results: In spite of the fact that the full described characteristics of the preclinical model could not be reproduced, the systemic injection of the rAAVrh74.MCK.GNE viral vector resulted in a long term presence and expression of human wt GNE in the murine muscles and in some improvements of their mild phenotype. The Gne–/– hGNED207V Tg mice are smaller from birth, but cannot be differentiated from littermates by muscle function (grip strength and Rotarod) and their muscle histology is normal, even at advanced age. Conclusions: The rAAVrh74.MCK.GNE vector is a robust tool for the development of GNE Myopathy therapies that supply the intact GNE. However, there is still no reliable animal model to fully assess its efficacy since the previously developed Gne–/– hGNED207V Tg mice do not present disease characteristics.

Long-term Evaluation Parameters in GNE Myopathy: A Five-year Observational Follow-up Natural History Study

BackgroundA number of clinical trials targeting GNE myopathy patients have been conducted. However, useful clinical parameters for post-marketing surveillance and long-term clinical observation have not yet been established. ObjectiveWe conducted a 5-year observational follow-up natural history study to identify evaluation parameters which may be useful for the long-term observation of GNE myopathy patients. MethodsThirty-three genetically-confirmed GNE myopathy patients were recruited and evaluated at study entry (baseline) and yearly in a 5-year follow-up. Hand-held dynamometer measurements of knee extension strength, grip power, and pinch power, summed Manual Muscle Testing (MMT) score of 17 muscles, Gross Motor Function Measure (GMFM), 6-minute walk test, percent vital capacity and percent force vital capacity (%FVC), lean body mass (whole body, arms, and legs), creatine kinase (CK), Barthel Index, modified Rankin Scale, and SF-36 national standard scores were examined. ResultsOf the 33 patients, 22 (66%) completed evaluations for the entire 5-year follow-up period. These patients had a significant reduction in summed MMT score (p=0.001), GMFM (p=0.001), grip power (p=0.013), pinch power (p<0.001), CK (p=0.030), %FVC (p<0.001), leg lean body mass (p=0.040), and the Physical Functioning subscale score of the SF-36 (p=0.015) at the 5th year evaluation relative to baseline. Among these parameters, summed MMT score, GMFM, pinch power, and %FVC showed significant changes even in non-ambulant patients.ConclusionsMMT, GMFM, pinch power, CK, %FVC, lean body mass, and Physical Functioning subscale score of the SF-36 are useful parameters for the long-term evaluation of GNE myopathy patients.

Visualizing Muscle Sialic Acid Expression in the GNED207VTgGne-/- Cmah-/- Model of GNE Myopathy: A Comparison of Dietary and Gene Therapy Approaches

Background: GNE myopathy (GNEM) is a rare, adult-onset, inclusion body myopathy that results from partial loss of function mutations in the GNE gene. GNE encodes UDP-GlcNAc epimerase/Mannose-6 kinase, a protein with two enzymatic activities that comprise the committed step in biosynthesis of sialic acid (SA), an essential glycan that appears on the terminal positions of many extracellular oligosaccharide chains. These GNE mutations can cause a reduction of SA in many tissues, although pathology is restricted to skeletal muscles through a poorly understood mechanism. Objective: Despite recent advances in the field, it remains unclear which therapeutic avenue is most promising for the restoration of SA level in skeletal muscle affected by GNEM. Our objective was to assess dietary and gene therapy strategies for GNEM in Cmah-deficient GNED207VTgGne-/- mice, a model that allows for the visualization of orally delivered N-glycolylneuraminic acid (Neu5Gc), one of the two predominant SA forms in muscle. Methods: Methods included in situ physiology studies of the tibialis anterior muscle, studies of ambulation and limb grip strength, and muscle staining using MAA, SNA, and anti-Neu5Gc antibody, along with qPCR, qRT-PCR, western blot, and HPLC studies to assess virally introduced DNA, GNE gene expression, GNE protein expression, and SA expression. Results: We found that a diet enriched in Neu5Gc-containing glycoproteins had no impact on Neu5Gc immunostaining in muscles of GNEM model mice. Delivery of a single high dose oral Neu5Gc therapy, however, did increase Neu5Gc immunostaining, though to levels below those found in wild type mice. Delivery of a single dose of GNE gene therapy using a recombinant Adeno Associated Virus (rAAV) vector with a liver-specific or a muscle-specific promoter both caused increased muscle Neu5Gc immunostaining that exceeded that seen with single dose monosaccharide therapy. Conclusions: Our findings indicate that dietary loading of Neu5Gc-containing glycoproteins is not effective in increasing muscle Neu5Gc expression, while single dose oral Neu5Gc monosaccharide or GNE gene therapy are. Neu5Gc immunostaining, however, showed greater changes than did lectin staining or HPLC analysis. Taken together, these results suggest that Neu5Gc immunostaining may be more sensitive technique to follow SA expression than other more commonly used methods and that liver expression of GNE may contribute overall muscle SA content.

GNE myopathy with thrombocytopenia: a case report and review of the literature

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Safety and efficacy of N-acetylmannosamine (ManNAc) in patients with GNE myopathy: an open-label phase 2 study

Purpose To evaluate the safety and efficacy of N-acetylmannosamine (ManNAc) in GNE myopathy, a genetic muscle disease caused by deficiency of the rate-limiting enzyme in N-acetylneuraminic acid (Neu5Ac) biosynthesis. Methods We conducted an open-label, phase 2, single-center (NIH, USA) study to evaluate oral ManNAc in 12 patients with GNE myopathy (ClinicalTrials.gov NCT02346461). Primary endpoints were safety and biochemical efficacy as determined by change in plasma Neu5Ac and sarcolemmal sialylation. Clinical efficacy was evaluated using secondary outcome measures as part of study extensions, and a disease progression model (GNE-DPM) was tested as an efficacy analysis method. Results Most drug-related adverse events were gastrointestinal, and there were no serious adverse events. Increased plasma Neu5Ac (+2,159 nmol/L, p < 0.0001) and sarcolemmal sialylation (p = 0.0090) were observed at day 90 compared to baseline. A slower rate of decline was observed for upper extremity strength (p = 0.0139), lower extremity strength (p = 0.0006), and the Adult Myopathy Assessment Tool (p = 0.0453), compared to natural history. Decreased disease progression was estimated at 12 (γ = 0.61 [95% CI: 0.09, 1.27]) and 18 months (γ = 0.55 [95% CI: 0.12, 1.02]) using the GNE-DPM. Conclusion ManNAc showed long-term safety, biochemical efficacy consistent with the intended mechanism of action, and preliminary evidence clinical efficacy in patients with GNE myopathy.