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. 2021 Apr;18(2):1414-1425.
doi: 10.1007/s13311-021-01040-7. Epub 2021 Apr 8.

Targeting Gys1 with AAV-SaCas9 Decreases Pathogenic Polyglucosan Bodies and Neuroinflammation in Adult Polyglucosan Body and Lafora Disease Mouse Models

Emrah Gumusgoz  1 , Dikran R Guisso  1 , Sahba Kasiri  1 , Jun Wu  1 , Matthew Dear  1 , Brandy Verhalen  1   2 , Silvia Nitschke  1 , Sharmistha Mitra  1 , Felix Nitschke  1 , Berge A Minassian  3
Affiliations

Targeting Gys1 with AAV-SaCas9 Decreases Pathogenic Polyglucosan Bodies and Neuroinflammation in Adult Polyglucosan Body and Lafora Disease Mouse Models

Emrah Gumusgoz et al. Neurotherapeutics. 2021 Apr.

Abstract

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.

Keywords: AAV9; APBD; CRISPR/Cas9; EPM2A; EPM2B; GBE1; GYS1.

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Figures

Fig. 1
Fig. 1
AAV-SaCas9 targeting murine Gys1. Schematic representation of AAV vector, guide RNA sequence, and Gys1-Exon1 target site. ITR inverted terminal repeats, Myc c-myc, NLS nuclear localization signal sequence, bGH-pA bovine growth hormone polyadenylation signal, PAM protospacer adjacent motif
Fig. 2
Fig. 2
AAV-SaCas9 virus distribution as detected by SaCas9 immunohistochemistry. Representative images show SaCas9 expressing cells in cortex a and hippocampus b. Scale bars, 1 mm
Fig. 3
Fig. 3
Gys1 targeting AAV-SaCas9 disrupts the Gys1 gene, reduces Gys1 mRNA and protein levels, and decreases insoluble glycogen and PB accumulation in brains of the Gbe1Y239S APBD mouse model. Neonatal mice (P2) were injected with PBS (N = 12 for each experiment) or AAV-SaCas9 (N = 12 for each experiment), and mice were sacrificed at 3 months for brain tissue analysis. Indel percentage a and Gys1 mRNA level b were measured by ddPCR. Representative brain GYS1 western blots with stain-free gel as loading control c. Quantification of GYS1 western blots normalized to stain-free gel shown in d. Polyglucosan body (PB) quantification in the hippocampus e and degradation resistant glycogen content f. Representative micrographs of PASD stained hippocampus of PBS g and i vs AAV-SaCas9 h and j treated mice. Scale bars, 300 µm. Data are presented as mean ± SEM. Significance levels are indicated as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001
Fig. 4
Fig. 4
Gys1 targeting AAV-SaCas9 disrupts the Gys1 gene, reduces Gys1 mRNA and protein levels, and decreases insoluble glycogen and PB accumulation in brains of the Epm2a−/− LD mouse model. Neonatal mice (P2) were injected with PBS (N = 13 for each experiment) or AAV-SaCas9 (N = 8 in WB quantification and N = 10 for other experiments), and mice were sacrificed at 3 months for brain tissue analysis. Indel percentage a and Gys1 mRNA level b were measured by ddPCR. Representative brain GYS1 western blots with stain-free gel as loading control c. Quantification of GYS1 western blots normalized to stain-free gel shown in d. Polyglucosan body (PB) quantification in the hippocampus e and degradation resistant glycogen content f. Representative micrographs of PASD stained hippocampus of PBS g and i vs AAV-SaCas9 h and j treated mice. Scale bars, 300 µm. Data are presented as mean ± SEM. Significance levels are indicated as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001
Fig. 5
Fig. 5
Gys1 targeting AAV-SaCas9 disrupts the Gys1 gene, reduces Gys1 mRNA and protein levels, and decreases insoluble glycogen and PB accumulation in brains of the Epm2b−/− LD mouse model. Neonatal mice (P2) were injected with PBS (N = 10 for each experiment) or AAV-SaCas9 (N = 14 for each experiment), and mice were sacrificed at 3 months for brain tissue analysis. Indel percentage a and Gys1 mRNA level b were measured by ddPCR. Representative brain GYS1 Western blots with stain-free gel as loading control c. Quantification of GYS1 Western blots normalized to stain-free gel shown in d. Polyglucosan body (PB) quantification in the hippocampus e and degradation resistant glycogen content f. Representative micrographs of PASD stained hippocampus of PBS g and i vs AAV-SaCas9 h and j treated mice. Scale bars, 300 µm. Data presented as mean ± SEM. Significance levels are indicated as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001
Fig. 6
Fig. 6
Effect of AAV-SaCas9 treatment on polyglucosan body associated immune system activation. Neonatal mice (P2) were injected with PBS or AAV-SaCas9 and mice were sacrificed at 3 months for brain tissue analysis. WT indicates the wild type control group. For each panel, from left to right, Cxcl10, Ccl5, Lcn2, and C3 were used as neuroinflammation markers, and relative mRNA expression levels were analyzed by qRT-PCR for Gbe1Y239S a, Epm2a−/− b, and Epm2b−/− c mice. In panel a, WT (N = 13), PBS (N = 12), and AAV-SaCas9 (N = 12). In panel b, WT (N = 8), PBS (N = 13), and AAV-SaCas9 (N = 10). In panel c, WT (N = 11), PBS (N = 10), and AAV-SaCas9 (N = 14). Data are presented as mean ± SEM. Significance levels are indicated as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001
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