Atrophin-1 Antisense Oligonucleotide Provides Robust Protection from Pathology in a Fully Humanized DRPLA Model

Dentatorubral-pallidoluysian atrophy (DRPLA) is a fatal neurodegenerative disease arising from a CAG repeat expansion in the atrophin-1 (ATN1) gene. Because DRPLA, like many repeat expansion disorders (REDs), arises predominantly from toxic gain-of-function mechanisms, we hypothesized that ATN1 knockdown would have therapeutic potential. To test this, we established the first fully-humanized mouse model of a RED, in which one allele of mouse Atn1 is completely replaced by human ATN1, including 112 pure CAG repeats. This novel approach to exploring RED biology provides significant advantages, notably the ability to test sequence-specific therapeutics targeting human sequences, even in introns and untranslated regions of pre-mRNA. We found that our model—the Atn1Q112/+ mouse—recapitulates key features of human DRPLA, including behavioral alterations, reduced brain size and aggregate accumulation. We treated Atn1Q112/+ mice with antisense oligonucleotides (ASOs) targeting mouse Atn1 (to probe for loss of function concerns), human ATN1, or a combination. Treatment with human, but not mouse, ATN1-targeting ASOs provides remarkable protection from a range of disease-related behavioral phenotypes, including aggregation of mutant ATN1 (mATN1), and marked rescue of transcriptional dysregulation in the cerebellum. These results have helped motivate an ongoing human clinical study of ASOs targeting ATN1 for DRPLA. Overall design: Mice were treated with ASO from 2 days to 9 weeks of age. We sequenced n=5 per genotype (WT or ATN1.Q112/+) treated with Saline, Mo_ASO, or Hu_ASO. Total N=30

齿状核红核苍白球路易体萎缩症（Dentatorubral-pallidoluysian atrophy, DRPLA）是一种致命的神经退行性疾病，由萎缩蛋白-1（atrophin-1, ATN1）基因的CAG重复扩增引发。与多数重复扩增疾病（repeat expansion disorders, REDs）类似，DRPLA主要通过毒性功能获得性机制致病，因此我们推测敲低ATN1具备治疗潜力。
为验证这一假说，我们构建了首个完全人源化的RED小鼠模型：将小鼠Atn1的一个等位基因完全替换为携带112个纯合CAG重复的人源ATN1基因。这种用于探究RED生物学的全新方法具有显著优势，尤其可实现靶向人源序列（包括内含子及前体信使RNA（pre-mRNA）的非翻译区）的序列特异性治疗药物筛选。
我们发现该模型——Atn1Q112/+小鼠——可重现人类DRPLA的关键特征，包括行为异常、脑体积缩小以及致病蛋白聚集沉积。我们分别使用靶向小鼠Atn1（用于探究功能丧失相关风险）、人源ATN1的反义寡核苷酸（antisense oligonucleotides, ASOs），以及二者联合处理Atn1Q112/+小鼠。
实验结果显示，靶向人源ATN1而非小鼠Atn1的ASO治疗，可显著改善多种疾病相关行为表型，包括突变型ATN1（mATN1）的聚集情况，并显著挽救小脑的转录失调。上述结果推动了针对DRPLA的靶向ATN1的ASO人体临床试验的开展。
整体实验设计：小鼠于出生后2天至9周龄期间接受ASO处理。我们对每种基因型（野生型（wild type, WT）或ATN1.Q112/+）分别设置生理盐水、Mo_ASO、Hu_ASO处理组，每组样本量n=5，总样本量N=30