Therapeutic genome editing for amyotrophic lateral sclerosis

 描述（由申请人提供）：肌萎缩侧索硬化症（ALS）是一种破坏性和进行性神经退行性疾病，其特征在于脑干、脊髓和大脑皮层中的上下运动神经元的丧失。ALS导致肌肉萎缩、瘫痪，并最终在症状发作的3-5年内死于呼吸衰竭。ALS无法治愈，目前的治疗方法只能减缓疾病的进展。赋予Cu/Zn超氧化物歧化酶1（SOD 1）基因毒性功能的突变导致近25%的遗传性ALS病例，并且是这种疾病最常见的遗传原因。这些突变被认为会对运动神经元产生神经毒性。事实上，小鼠中致病性SOD 1蛋白的产生导致与ALS的特征非常相似的迟发性、进行性神经变性疾病。此外，许多研究表明，突变体SOD 1以非细胞自主方式驱动神经变性，致病性星形胶质细胞、少突胶质细胞和小胶质细胞对运动神经元有毒。ALS的破坏性影响强调了开发有效治疗这种疾病的根本原因的新疗法的迫切需要。基因治疗对许多人类疾病的治疗有很大的希望，并且是对抗神经退行性疾病的一种潜在的强有力的方法。迄今为止，原理验证研究表明，病毒载体介导的运动神经元和星形胶质细胞中突变体SOD 1基因的沉默可以增加ALS小鼠模型的寿命。然而，这些方法受到治疗不完整性的限制。位点特异性DNA核酸内切酶用于治疗目的代表了从基因治疗的角度解决ALS的潜在范式转变机会。与仅解决疾病症状的常规方法不同，工程化核酸酶能够纠正疾病的根本原因，从而通过基因组修饰永久消除症状。该研究的目的是开发一种基于核酸酶介导的SOD 1基因体内敲除的ALS基因治疗方法。通过基因组编辑靶向破坏突变体SOD 1，结合递送替代SOD 1基因，将用于延迟瘫痪的发作，改善运动功能，并延长ALS小鼠模型的生存期。腺相关病毒9（AAV 9）通过全身注射穿过新生小鼠的血脑屏障，将用于将基因组编辑货物递送到运动神经元和星形胶质细胞。为了确保有效递送至ALS影响的细胞，将进行定向进化以产生具有增强的靶向能力的新AAV载体。这些研究将证明治疗性基因组编辑治疗ALS的可行性，并为未来的临床转化奠定基础。

 DESCRIPTION (provided by applicant): Amyotrophic lateral sclerosis (ALS) is a devastating and progressive neurodegenerative disease characterized by loss of upper and lower motor neurons in the brainstem, spinal cord and cerebral cortex. ALS leads to muscle wasting, paralysis and, ultimately, death from respiratory failure within 3-5 years of symptom onset. There is no cure for ALS and current treatments only slow the progression of disease. Mutations that confer toxic function(s) to the Cu/Zn superoxide dismutase 1 (SOD1) gene are responsible for nearly 25% of inherited ALS cases and are the most common genetic cause for this disorder. These mutations are thought to impart neurotoxicity to motor neurons. Indeed, production of pathogenic SOD1 protein in mice results in a late- onset, progressive neurodegenerative disease that closely mimics the hallmarks of ALS. Moreover, numerous studies have shown that mutant SOD1 drives neurodegeneration in a non-cell autonomous manner by which pathogenic astrocytes, oligodendrocytes and microglia are toxic to motor neurons. The destructive impact of ALS underscores the urgent need for the development of new therapies that effectively treat the underlying cause of this disorder. Gene therapy holds great promise for the treatment of many human diseases and is a potentially powerful approach for combating neurodegeneration. To date, proof-of-principle studies have indicated that viral vector-mediated silencing of the mutant SOD1 gene in motor neurons and astrocytes can increase lifespan in mouse models of ALS. However, these approaches have been limited by the incomplete nature of the treatment. The use of site-specific DNA endonucleases for therapeutic purposes represents a potentially paradigm shifting opportunity to address ALS from the perspective of gene therapy. Unlike conventional methods, which only address disease symptoms, engineered nucleases are capable of correcting the underlying cause of the disorder, thereby permanently eliminating the symptoms via genome modification. The goal of the proposed research is to develop a gene therapy for ALS based on nuclease-mediated knockout of the SOD1 gene in vivo. Targeted disruption of mutant SOD1 via genome editing, in conjugation with delivery of a replacement SOD1 gene, will be used to delay the onset of paralysis, improve motor function, and extend survival in mouse models of ALS. Adeno-associated virus 9 (AAV9), which crosses the blood-brain barrier in neonatal mice via systemic injection, will be used to deliver the genome editing cargo to motor neurons and astrocytes. To ensure efficient delivery to ALS-affected cells, directed evolution will be performed to generate new AAV vectors with enhanced targeting capabilities. These studies will demonstrate the feasibility of therapeutic genome editing for treatment of ALS and lay the groundwork for future clinical translation.