Phenotypic suppression of DRPLA model mice using CRISPR / Cas9 system
Taisuke Kato1, Sachiko Hirokawa1, Kenta Kobayashi2, Shoji Tsuji3 and Osamu Onodera1,4 1Department of Molecular Neuroscience, Brain Research Institute, Niigata University, Niigata, Japan; 2Section of Viral Vector Development, National Institute Physiological Sciences, Aichi, Japan; 3Department of Neurology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan; 4Department of Neurology, Brain Research Institute, Niigata University, Niigata, Japan
Polyglutamine diseases are caused by the expansion of CAG repeats in the causative genes. The expanded polyglutamine stretch confers toxic functions leading to neurodegeneration. Dentatorubural pallidoluysian atrophy (DRPLA) is caused by the expansion of CAG repeat in Atrophin-1 (ATN1) gene. The reduction of the expression of ATN1 is a therapeutic strategy for DRPLA. However, an approach utilizing gene-silencing latently contains concerns about a loss of functions of target genes as that suppresses the gene expression not only from mutant allele but also from wild-type allele. Mice with null Atn1 show normal survival rate without deleterious phenotype, suggesting that DRPLA is a disease for which a non-allele-specific approach is applicable. CRISPR/Cas9 system is considered to be an excellent system for a gene-silencing approach. The ultimate goal of this study is gene therapy employing adeno-associated virus vector (AAV) for DRPLA. In that case, genome-editing is expected to be introduced in a mosaic pattern in the brain. As a first step, we investigated whether the therapeutic strategy of silencing of toxic gene expression in a mosaic fashion is effective in the treatment of DRPLA by creating transgenic mosaic mice. We have used the transgenic mice harboring a single copy human ATN1 gene with expanded CAG triplet repeat. We introduced guide-RNA for human ATN1 and mRNA of Cas9 using electroporation to fertilized eggs of the transgenic mouse and acquired mosaic mice with 75 % mosaicism of the ATN1 gene with editing. We found decreasing expression of human mutant ATN1. The mosaic mice showed lack of the phenotype of DRPLA (myoclonus, clasping, and seizure), and displayed a natural survival. These results indicate feasibility of the strategy with genome-editing for therapy of DRPLA.
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