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

摘要/摘要 将CAG转换为CAA的碱基编辑策略在亨廷顿病中的治疗潜力 尽管亨廷顿病(HD)的遗传原因早已为人所知，但仍无法开发出有效的治疗方法 以及模型系统中涉及的众多机制，反映了基于模型的临床实用有限 调查。相比之下，对HD患者的观察可能会揭示出在人类身上实际有效的疗法。 所有HD病例均由Huntingtin基因CAG重复序列扩增所致。然而，临床表现的年龄 差异很大，发病年龄因突变大小而无法解释的差异表明是遗传性的，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 不稳定相关的DNA修复基因改变了HD的发病。重要的是，我们透露了复制和丢失的 CAG-CAA阻断在CAG重复中显著延缓和加速HD的发病年龄。 总之，我们的数据表明，HD的速率在很大程度上取决于不间断CAG重复的大小 并通过重复不稳定进行修改，为疾病的驱动因素和治疗策略提供了见解。 利用这些与临床相关的人类观察，我们构思了新的治疗基础编辑 (BE)将CAG转换为CAA的策略，旨在通过减少CAG的大小来延迟临床表现 不间断的CAG重复，并潜在地进一步抑制重复扩展。我们的新型治疗性BE 与其他基因靶向方法相比，该策略具有许多优势。对患者的观察 提示CAG到CAA的转换产生了非常强的治疗益处(即，比起 10年)。此外，我们针对疾病根本原因的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以应用于所有 使HD患者产生等位基因特异性的益处。在这里，我们建议确定选定的治疗潜力 利用HD的相关细胞和动物模型将CAG转化为CAA的策略。简而言之，我们将1)评估 高效的BE策略的转换效率和等位基因专一性；2)检测CAG-to-BE CAA转换影响HTT的表达水平、神经元分化和其他分子表型，3) 确定CAG到CAA转换对CAG重复不稳定性的影响，以及4)评估偏离目标的影响，以及 进一步优化，揭示具有最高可行性和治疗潜力的BE策略。这项研究 将1)为不同基础编辑和转换的组合生成完整的评估表 策略，2)产生关于等位基因专一性、非靶向性和分子后果的知识，3) 提供后续优化的考虑因素，以及4)在BE策略时产生预期结果 适用于HD患者。因此，我们的研究测试HD的新的和创新的治疗路线 对HD和其他CAG扩张性疾病的有效治疗方法的开发做出了重大贡献。

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.