Therapeutic potential of base editing strategies to convert CAG to CAA in Huntington's Disease

总结/摘要 碱基编辑策略将CAG转化为CAA在亨廷顿病中的治疗潜力 亨廷顿氏病（HD）尽管其遗传原因众所周知，但仍无法开发有效的治疗方法 以及模型系统中涉及的许多机制，反映了基于模型的药物的有限临床效用。 调查事务所相比之下，对HD患者的观察可能会揭示实际上对人类有效的治疗方法。 所有的HD病例都是由于亨廷顿基因中CAG重复序列的扩增。然而，临床表现的年龄 差异很大，突变大小在发病年龄上的不明原因的差异显示了遗传性，表明HD是 被基因改变了。因此，我们进行了全基因组关联研究，发现重复 不稳定性相关DNA修复基因修饰HD发作。重要的是，我们发现，重复和损失的 亨廷顿CAG重复中的CAG-CAA中断分别显著延迟和加速HD发病年龄。 总之，我们的数据表明，HD的发生率在很大程度上取决于不间断CAG重复序列的大小。 并通过重复不稳定性进行修饰，为疾病的驱动因素和治疗策略提供了见解。 利用这些临床相关的人类观察，我们构思了新的治疗基础编辑 (BE)旨在通过减少CAG的大小来延迟临床表现的CAG向CAA转化的策略 不间断的CAG重复和潜在地进一步抑制重复扩增。我们的新型治疗BE 这些策略与其它基因靶向方法相比具有许多优点。患者观察结果 表明CAG向CAA的转化产生非常强的治疗益处（即，延迟发作超过 10年）。此外，我们针对疾病根源的BE策略不会改变亨廷顿蛋白 因为CAG和CAA都编码谷氨酰胺。因此，相同的单一治疗策略可应用于所有 HD患者产生等位基因特异性益处。在这里，我们建议确定选定的治疗潜力， 使用HD的相关细胞和动物模型将CAG转化为CAA的BE策略。简单地说，我们将1）评估 转换效率和等位基因特异性的BE策略具有高效率，2）测试是否CAG到- CAA转化影响HTT表达水平、神经元分化和其他分子表型，3） 确定CAG至CAA转化对CAG重复不稳定性的影响，和4）评估脱靶效应，和 进一步优化，以揭示具有最高可行性和治疗潜力的BE策略。本研究 我将1）为不同的基础编辑器和转换的组合产生一个完整的评估图表 策略，2）产生关于等位基因特异性、脱靶和分子后果的知识，3） 为后续优化提供考虑因素，4）当BE策略 适用于HD患者。因此，我们的研究测试了HD的新的和创新的治疗途径， 显著地有助于开发用于HD和其它CAG扩增病症的有效治疗剂。

SUMMARY / ABSTRACT Therapeutic potential of base editing strategies to convert CAG to CAA in Huntington's disease Huntington's disease (HD) defies development of effective treatments despite its long-known genetic cause and numerous mechanisms implicated in model systems, reflecting limited clinical utility of model-based investigations. By contrast, observations in HD patients may reveal therapeutics that actually works in people. All cases of HD are due to an expanded CAG repeat in huntingtin gene. However, age at clinical manifestation varies widely, and unexplained variance in age at onset by the mutation size shows heritability, indicating HD is modified by genes. Therefore, we performed genome-wide association study, and discovered that repeat instability-related DNA repair genes modify HD onset. Importantly, we revealed that duplicated and loss of CAG-CAA interruption in the huntingtin CAG repeat robustly delay and hasten HD onset age, respectively. Together, our data indicate that the rate of HD is largely determined by the size of uninterrupted CAG repeat and modified by repeat instability, providing insights into driver of the disease and therapeutic strategies. Capitalizing on these clinically relevant observations in humans, we conceived novel therapeutic Base Editing (BE) strategies to convert CAG to CAA aiming at delaying clinical manifestation by decreasing the size of uninterrupted CAG repeat and potentially further suppressing repeat expansion. Our novel therapeutic BE strategies have a number of advantages over other gene targeting approaches. Observations in patients suggest that CAG-to-CAA conversion produces very strong therapeutic benefit (i.e., delaying onset more than 10 years). In addition, our BE strategies, targeting the root cause of the disease, do not alter huntingtin protein since both CAG and CAA encode glutamine. Therefore, same single treatment strategies can be applied to all HD patients to produce allele-specific benefits. Here, we propose to determine therapeutic potential of selected BE strategies to convert CAG to CAA using relevant cell and animal models of HD. Briefly, we will 1) evaluate conversion efficiencies and allele specificity of BE strategies with high efficiencies, 2) test whether CAG-to- CAA conversion affects HTT expression levels, neuronal differentiation, and other molecular phenotypes, 3) determine impacts of CAG-to-CAA conversion on CAG repeat instability, and 4) evaluate off-target effects, and further optimize to reveal the BE strategy with the highest feasibility and therapeutic potential. This research will 1) produce a complete evaluation chart for combinations of different base editors and conversion strategies, 2) generate knowledge regarding allele specificity, off-targeting, and molecular consequences, 3) provides considerations for subsequent optimization, and 4) produce expected outcomes when BE strategies are applied to HD patients. Our research testing novel and innovative therapeutic routes for HD therefore will significantly contribute to the development of effective therapeutics for HD and other CAG expansion disorders.