Controllable base editing therapy for DMD

项目总结/摘要 Duchenne型肌营养不良症（DMD）是一种致死性肌肉退行性疾病，90%以上的患者伴有心肌病 心力衰竭是导致患者死亡的主要原因。DMD患者通常会出现帧外 导致肌营养不良蛋白质完全丧失的突变。虽然CRISPR基因编辑已经被应用于 删除突变外显子以恢复肌营养不良蛋白阅读框架，这种策略提高了潜在的安全性 因为它依赖于CRISPR/Cas9产生的双链DNA断裂的修复，这可能导致 不需要的大缺失、DNA重排和病毒载体整合。开发新的方法， 精确而安全地纠正横纹肌中的致病突变是对抗DMD的迫切需要。 碱基编辑器的最新进展使我们能够探索精确校正基因突变的可行性， DMD的动物模型，而不产生双链DNA断裂。我们最近的研究表明， 有效纠正DMD小鼠模型横纹肌中的致病点突变， 腺相关病毒9（AAV 9）的全身递送。这为体内基质的临床转化铺平了道路 编辑DMD然而，迫切需要新的策略来解决几个安全相关的问题（例如， 在AAV递送后，随着碱基编辑剂的持续表达，脱靶活性的积累; 使用高剂量AAV;宿主免疫应答）。在这里，我们建议开发一种新型的可控基础 编辑系统（以减少偏离目标的编辑事件的积累和基本编辑器对主机的暴露 免疫系统）在最近工程化的亲肌性AAV衣壳中递送（以增加肌肉转导 与AAV 9相比，AAV的效率低10倍）。其治疗效果和安全性将得到广泛关注。 使用新建立的DMD兔模型进行研究，该模型忠实地再现了DMD的临床体征。 肌营养不良和心肌病。我们将进一步设计免疫友好型载体， 开发一种新的免疫调节策略以减轻宿主对体内AAV递送的免疫应答， 基础编辑疗法完成拟议的研究将大大推进我们的翻译工作， 为DMD开发更安全的精准药物。

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.