基于CRISPR/Cas9定点编辑蛋白系统对Myo6突变遗传性耳聋的基因治疗研究

Genetic mutations are responsible for about 60% of all congenital hearing loss cases. The current standards of care for genetic hearing loss are hearing aids or cochlear implants, which provide limited compensation for the lost function in a relatively small patient population. Viral-based gene therapy has been developed as the main approach for potential treatment. However the constraints in viral system, including the insert size limitation, the maintenance of long-term expression and the risk associated with long-term presence of viral genome, severely limit the potential. Genome editing, especially CRISPR/Cas9-mediated system, has recently been shown to correct genetic mutations in animals and human cell lines, thus offers a new gene therapy paradigm to treat genetic hearing loss. ..We have recently demonstrated that Cas9 protein and single guide RNA (gRNA) can be complexed by cationic lipid transfection reagents and injected into mouse inner ear in vivo for high-efficiency genome editing, making it extremely attractive to be developed into a new type of gene therapy for genetic hearing loss. We also showed a peptide Aurein 1.2 improved protein delivery in vivo. In this application we propose to use protein/RNA delivery for genome editing that targets a mutation in the Myosin VI gene, p.C442Y, which causes dominant deafness in human. A knockin mouse model harbors the Myo6 mutation will be used to study genome editing by indels (insertion and deletions) that abolish the mutation and hearing recovery as the consequence. We will select the most potent gRNA for complexation with Cas9 or Aurein-Cas9 by lipofectamine2000 and for in vivo injection into mouse inner ear. Hearing will be studied by ABR (acoustic brain-stem response) and DPOAE (distorted product of acoustic emission) over a period of 1-17 months bi-monthly. Inner ears will be analyzed for cell survival, hair cell stereocilia morphology and connections between hair cells and neurons. The study has the potential to be the first to combine direct protein:RNA delivery into mammalian inner ear in vivo for genome editing that leads to hearing recovery in a genetic hearing loss model. The study will have tremendous impact for a new type of treatment for genetic hearing loss based on genome editing.

单基因突变导致的遗传性耳聋占语前聋的一半以上，原位基因治疗是最精准的治疗方法。CRISPR/Cas9是新发现强大的基因编辑工具, 为遗传性疾病有前景的治疗方法。申请人前期研究证实以Cas9蛋白质结合脂质体可对在体小鼠内耳毛细胞进行特异性基因编辑。以多肽Aurein修饰的蛋白效率更高。我们前期构建了首例模拟人类MYO6显性突变的小鼠模型，耳聋特点符合CRISPR/Cas9蛋白系统基因治疗的要求。本项目将以此模型为研究对象，以CRISPR/Cas9蛋白以及Aurein修饰的CRISPR/Cas9蛋白在体进行基因组定点编辑，敲除突变等位基因，干预后行听觉检测及内耳形态学研究，观察CRISPR/Cas9基因编辑蛋白对此耳聋模型的听觉修复效果及形态学作用，探索CRISPR/Cas9技术在显性突变的遗传性耳聋模型中永久敲除具有显性负效应的突变等位基因，以期为人类遗传性耳聋的原位精准基因治疗提供科学依据

据世界卫生组织官网报道，超过全球人口的5%，即4.66亿人患有致残性听力损失，其中3400万是儿童。先天性耳聋的儿童，60%是遗传因素所致，目前尚无可以治疗的药物。随着基因编辑和基因治疗技术的迅速发展，有望精准治愈遗传性耳聋。肌球蛋白VI是一种非常规肌球蛋白，对听觉和前庭功能至关重要。人类MYO6基因的致病变体导致常染色体显性或隐性形式的听力损失。对引起听力损失的Myo6基因突变的有效治疗是有限的。我们研究了腺相关病毒（AAV）-PHP.eB载体介导的金黄色葡萄球菌Cas9（SaCas9-KKH）-sgRNA复合物的体内编辑是否能改善Myo6 WT/C442Y小鼠模型的听力损失，该模型模拟了人类患者的表型。AAV-SaCas9-KKH-Myo6-g2系统对C442Y等位基因的体内编辑效率平均为4.05%，比对WT等位基因的编辑效率高约17倍。在Myo6 WT/C442Y小鼠中注射AAV-SaCas9-KKH-Myo6-g2后，观察到听觉功能可恢复至5个月。同时，与未经处理的小鼠相比，在AAV-SaCas9-KKH-Myo6-g2处理的小鼠中还观察到ABR I波的潜伏期变短，DPOAE阈值更低，毛细胞存活率提高，毛束形态更规则，钙电流恢复。本研究结果将为体内基因组编辑作为各种半显性形式的听力损失和其它半显性疾病的治疗方法提供了进一步证据。

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