Controllable base editing therapy for DMD

项目摘要/摘要 Duchenne肌营养不良症(DMD)是一种致命的肌肉退行性疾病，90%以上的心肌病 心力衰竭是导致死亡的主要原因。DMD患者通常存在框外 导致肌营养不良蛋白完全丧失的突变。虽然CRISPR-基因编辑已经被使用 删除突变外显子以恢复dystrophin阅读框，这一策略提高了潜在的安全性 令人担忧的是，它依赖于修复CRISPR/Cas9造成的双链DNA断裂，这可能会导致 不想要的大片段缺失、DNA重排和病毒载体整合。开发新的方法来 准确和安全地纠正横纹肌的致病突变是对抗DMD的迫切需要。 碱基编辑的最新进展使我们能够探索精确纠正基因突变的可行性。 不产生双链DNA断裂的DMD动物模型。我们最近的研究表明， 高效纠正DMD小鼠模型横纹肌中的致病点突变 全身性递送腺相关病毒9(AAV9)。这为体内碱基的临床翻译铺平了道路 正在为DMD编辑。然而，迫切需要新的战略来解决几个与安全有关的问题(例如 在AAV投放后，随着碱基编辑剂的持续表达，非目标活动的积累； 使用了高剂量的AAV；宿主免疫反应)。在这里，我们建议开发一种新型的可控基 编辑系统(以减少非目标编辑事件的累积和基础编辑人员对主持人的暴露 免疫系统)在最近工程的嗜肌性AAV衣壳中传递(以增加肌肉转导 与AAV9相比，AAV的效率降低了10倍。治疗的有效性和安全性将得到广泛的推广 使用新建立的DMD兔模型进行研究，该模型真实地概括了DMD的临床症状 人类DMD中的肌营养不良和心肌病。我们将进一步改造免疫友好型载体和 开发一种新的免疫调节策略来减轻体内AAV递送的宿主免疫应答 基础编辑疗法。拟议研究的完成将大大推动我们的翻译工作，以 开发更安全的DMD精准药物。

PROJECT SUMMARY/ABSTRACT Duchenne muscular dystrophy (DMD) is a lethal muscle degenerative disease with cardiomyopathy in over 90% of patients, and heart failure is a leading cause of mortality. DMD patients commonly harbor out-of-frame mutations which result in complete loss of dystrophin protein. While CRISPR-gene editing has been employed to delete the mutant exon for restoration of the dystrophin reading frame, this strategy raises potential safety concerns as it relies on repair of the double strand DNA breaks created by CRISPR/Cas9, which may cause unwanted large deletion, DNA rearrangement and viral vector integration. Developing novel approaches to precisely and safely correct the disease-causing mutations in striated muscles is in urgent need to combat DMD. Recent advances in base editors allow us to explore the feasibility of precise correction of genetic mutations in animal models of DMD without creating double strand DNA breaks. Our recent studies demonstrate it is highly efficient to correct a disease-causing point mutation in the striated muscles of a mouse model of DMD using systemic delivery of adeno-associated virus 9 (AAV9). This paves the way for clinical translation of in vivo base editing for DMD. However, novel strategies are in urgent need to solve several safety-related issues (e.g. the accumulation of off-target activities with persistent expression of base editing agents following AAV delivery; high dose of AAV utilized; host immune responses). Here we propose to develop a novel controllable base editing system (to reduce accumulation of off-target editing events and exposure of base editor to the host immune system) delivered in recently engineered myotropic AAV capsids (to increase muscle transduction efficiency with 10-fold less of AAV as compared to AAV9). The therapeutic efficacy and safety will be extensively investigated using a newly established DMD rabbit model, which faithfully recapitulates the clinical signs of muscular dystrophy and cardiomyopathy in human DMD. We will further engineer immune-friendly vectors and develop a novel immune-modulating strategy to mitigate the host immune responses to the in vivo AAV-delivered base editing therapy. Completion of the proposed studies will significantly advance our translational efforts to develop safer precision medicine for DMD.