CRISPR editing therapy for Duchenne muscular dystrophy

项目描述 杜氏肌营养不良症（DMD）是由肌营养不良蛋白基因的无效突变引起的。CRISPR/Cas9 编辑有望从遗传学的根源上治疗DMD。由于DMD影响身体的所有肌肉，有效 DMD的治疗需要全身肌肉递送。腺相关病毒（AAV）载体是唯一的 输送系统，可以有效地达到所有的身体肌肉。出于这个原因，AAV一直是选择的载体 用于DMD的CRISPR介导的基因修复疗法。AAV载体导致持续的转基因表达。 连续的Cas9表达产生了两个问题。首先，它增加了脱靶编辑的可能性。第二、 对细菌衍生的Cas9蛋白的细胞毒性T淋巴细胞（CTL）应答可以消除经处理的细胞 废除治疗已经开发了许多方法来监控脱靶编辑并改进 基因编辑保真度然而，对Cas9特异性CTL应答进行建模一直是难以捉摸的。小鼠研究 显示了有限的细胞应答，其未能消除Cas9转导的细胞。事实上，我们和其他人 观察到mdx中几乎终生的Cas9表达、肌肉病理改善和功能改善 小鼠，最常用的小鼠DMD模型。与小鼠模型相反，考虑营养不良犬 为人体试验提供更好的模型。为了确定Cas9免疫是否是AAV介导的免疫系统的障碍， 在DMD CRISPR治疗中，我们在四个独立的犬模型（正常 犬和三种不同的犬DMD模型），使用Cas9和非Cas9 AAV载体，通过局部和非局部途径， 全身性给药。我们发现了令人信服的证据，表明Cas9，而不是非Cas9，AAV载体， 诱导了强大的CTL应答并消除了基因编辑的肌营养不良蛋白阳性肌纤维。我们的研究 建立了可靠的模型系统来研究Cas9免疫。重要的是，它为开发和 验证可以减轻临床相关大型动物中Cas9特异性CTL应答的策略 模型在这项提案中，我们将利用我们的发现来探索可能支持持久性的新策略。 在犬DMD模型中不诱导CTL应答的治疗性编辑。我们的研究将为 未来将AAV CRISPR疗法转化为DMD患者的方法。我们的研究结果也将告知 将AAV CRISPR疗法翻译用于其他人类疾病。

Project Description Duchenne muscular dystrophy (DMD) is caused by null mutations in the dystrophin gene. CRISPR/Cas9 editing holds promise to treat DMD at its genetic root. Since DMD affects all muscles in the body, effective therapy for DMD would require bodywide muscle delivery. Adeno-associated virus (AAV) vector is the only delivery system that can efficiently reach all body muscles. For this reason, AAV has been the vector of choice for CRISPR-mediated gene repair therapy for DMD. The AAV vector leads to persistent transgene expression. Continuous Cas9 expression creates two problems. First, it increases the odds of off-target editing. Second, the cytotoxic T lymphocyte (CTL) response to the bacterial-derived Cas9 protein can eliminate the treated cells and abolish the therapy. Many approaches have been developed to monitor off-target editing and improve gene editing fidelity. However, it has been elusive to model the Cas9-specific CTL response. Mouse studies revealed a limited cellular response that failed to eliminate Cas9 transduced cells. In fact, we and others have observed nearly lifelong Cas9 expression, muscle pathology amelioration, and function improvement in mdx mice, the most used mouse DMD model. In contrast to the mouse model, dystrophic canines are considered better models for informing human trials. To determine whether Cas9 immunity is a hurdle for AAV-mediated DMD CRISPR therapy, we performed a comprehensive study in four independent canine models (normal canines and three different canine DMD models) using both Cas9 and non-Cas9 AAV vectors via local and systemic delivery. We found compelling evidence suggesting that the Cas9, but not non-Cas9, AAV vector induced a robust CTL response and eliminated gene-edited dystrophin-positive myofibers. Our studies established a reliable model system to study Cas9 immunity. Importantly, it opens the door to developing and validating strategies that may mitigate the Cas9-specific CTL response in a clinically relevant large animal model. In this proposal, we will leverage our findings to explore novel strategies that may support persistent therapeutic editing without inducing the CTL response in the canine DMD model. Our studies will pave the way for translating AAV CRISPR therapy to DMD patients in the future. Our findings will also inform the translation of AAV CRISPR therapy for other human diseases.