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Intravenous AAV-delivered CRISPR / Cas9 Gene therapy for DRPLA mice.
Osamu Onodera1, 2，
1Department of Neurology, Brain Research Institute, Niigata University; 2Department of Molecular Neuroscience, Brain research Institute, Niigata University;
Dentatorubural-pallidoluysian atrophy (DRPLA) is a neurodegenerative disorder characterized by myoclonus, epilepsy, cerebellar ataxia, choreoathetosis, and dementia. The expansion of CAG repeats in the Atrophin-1 (ATN1) gene causes DRPLA. Intranuclear accumulation of the expanded polyglutamine stretches is considered to confer toxic functions leading to neurodegeneration. The reduction of the expression of ATN1 is expected to provide a promising therapeutic strategy for DRPLA. Mice with null Atn1 show normal survival rates without deleterious phenotypes, suggesting that a non-allele-specific approach for gene-silencing can be applicable for DRPLA.
To proof this concept, We have utilized DRPLA Tg mice harboring the human ATN1 gene with expanded CAG-repeats. To achieve permanent suppression of mutant ATN1 expression, we have applied CRISPR/Cas9 mediated gene silencing using AAV vector (serotype PHP.eB). We intravenously administered the AAV-vector through retro-orbital sinus at 2.0×1012 vg/mouse after disease onset. NHEJ (non-homologous end-joining) drop-off assay with ddPCR revealed that approximately 20 to 30% editing of the target human ATN1 gene was achieved with this method in the brain. Regarding motor function, the rotarod score of non-treated Tg mice was decreased with age, and eventually, they could not stand on the rod due to severe imbalance by about eight months of age. On the other hand, AAV-treated Tg mice have maintained a constant rotarod score over eleven months of observation, even though the score was lower than that of wild-type mice. Moreover, epileptic seizure was suppressed. These results indicate the feasibility of the strategy with genome editing-mediated gene-silencing in vivo for therapy of DRPLA.
Ref.1) Chan KY et al., Nat Neurosci. 2017 8:1172-1179.
Ref.2) Findlay SD et al., PLoS One. 2016 4:e0153901
