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Many adult and most childhood neurological diseases have a genetic basis. CRISPR/Cas9 biotechnology holds great promise in neurological therapy, pending the clearance of major delivery, efficiency, and specificity hurdles. We applied CRISPR/Cas9 genome editing in its simplest modality, namely inducing gene sequence disruption, to one adult and one pediatric disease. Adult polyglucosan body disease is a neurodegenerative disease resembling amyotrophic lateral sclerosis. Lafora disease is a severe late childhood onset progressive myoclonus epilepsy. The pathogenic insult in both is formation in the brain of glycogen with overlong branches, which precipitates and accumulates into polyglucosan bodies that drive neuroinflammation and neurodegeneration. We packaged Staphylococcus aureus Cas9 and a guide RNA targeting the glycogen synthase gene, Gys1, responsible for brain glycogen branch elongation in AAV9 virus, which we delivered by neonatal intracerebroventricular injection to one mouse model of adult polyglucosan body disease and two mouse models of Lafora disease. This resulted, in all three models, in editing of approximately 17% of Gys1 alleles and a similar extent of reduction of Gys1 mRNA across the brain. The latter led to approximately 50% reductions of GYS1 protein, abnormal glycogen accumulation, and polyglucosan bodies, as well as ameliorations of neuroinflammatory markers in all three models. Our work represents proof of principle for virally delivered CRISPR/Cas9 neurotherapeutics in an adult-onset (adult polyglucosan body) and a childhood-onset (Lafora) neurological diseases., (© 2021. The American Society for Experimental NeuroTherapeutics, Inc.)

Lafora disease (LD) and adult polyglucosan body disease (APBD) are glycogen storage diseases characterized by a pathogenic buildup of insoluble glycogen. Mechanisms causing glycogen insolubility are poorly understood. Here, in two mouse models of LD (Epm2a -/- and Epm2b -/- ) and one of APBD (Gbe1 ys/ys ), the separation of soluble and insoluble muscle glycogen is described, enabling separate analysis of each fraction. Total glycogen is increased in LD and APBD mice, which, together with abnormal chain length and molecule size distributions, is largely if not fully attributed to insoluble glycogen. Soluble glycogen consists of molecules with distinct chain length distributions and differential corresponding solubility, providing a mechanistic link between soluble and insoluble glycogen in vivo. Phosphorylation states differ across glycogen fractions and mouse models, demonstrating that hyperphosphorylation is not a basic feature of insoluble glycogen. Lastly, model-specific variances in protein and activity levels of key glycogen synthesis enzymes suggest uninvestigated regulatory mechanisms., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Lafora disease (LD, OMIM #254780) is a rare, recessively inherited neurodegenerative disease with adolescent onset, resulting in progressive myoclonus epilepsy which is fatal usually within ten years of symptom onset. The disease is caused by loss-of-function mutations in either of the two genes EPM2A (laforin) or EPM2B (malin). It characteristically involves the accumulation of insoluble glycogen-derived particles, named Lafora bodies (LBs), which are considered neurotoxic and causative of the disease. The pathogenesis of LD is therefore centred on the question of how insoluble LBs emerge from soluble glycogen. Recent data clearly show that an abnormal glycogen chain length distribution, but neither hyperphosphorylation nor impairment of general autophagy, strictly correlates with glycogen accumulation and the presence of LBs. This review summarizes results obtained with patients, mouse models, and cell lines and consolidates apparent paradoxes in the LD literature. Based on the growing body of evidence, it proposes that LD is predominantly caused by an impairment in chain-length regulation affecting only a small proportion of the cellular glycogen. A better grasp of LD pathogenesis will further develop our understanding of glycogen metabolism and structure. It will also facilitate the development of clinical interventions that appropriately target the underlying cause of LD., Competing Interests: The authors declare no conflict of interest.