Single AAV-mediated mutation replacement genome editing in limited number of photoreceptors restores vision in mice
Single AAV-mediated mutation replacement genome editing in limited number of photoreceptors restores vision in mice
复制标题
DOI：
10.1038/s41467-019-14181-3
复制
发表时间：
2020-01-24
影响因子：
16.6
通讯作者：
Nakazawa, Toru
中科院分区：
文献类型：
--
作者：
Nishiguchi, Koji M.;Fujita, Kosuke;Nakazawa, Toru
用腺相关病毒（AAV）补充功能缺陷基因的野生型拷贝是临床上正在探索的用于各种视网膜营养不良的策略。然而，这种载体的低负荷限制允许其仅用于一小部分在相对小的致病基因中具有突变的患者。为了克服这个问题，我们开发了一种单一的AAV平台，该平台允许基于组合的CRISPR-Cas9和微同源介导的末端连接（MMEJ）用其野生型对应物局部替换突变序列。在失明的小鼠中，突变替代挽救了大约10%的光感受器，导致光敏感性的改善和视觉敏锐度的提高。这些效果与基因补充介导的恢复相当，基因补充针对更多的光感受器。这种策略可以应用于治疗由较大基因突变引起的遗传性疾病，常规的基因补充疗法目前尚不可行。使用腺相关病毒（AAV）用野生型拷贝替换突变基因已被探索用于治疗遗传性视网膜病，但低负荷限制限制了其使用。在这里，作者描述了一个单一的AAV平台，该平台允许基于组合的CRISPR-Cas9和微同源介导的末端连接，用其野生型对应物局部替换突变序列。
Supplementing wildtype copies of functionally defective genes with adeno-associated virus (AAV) is a strategy being explored clinically for various retinal dystrophies. However, the low cargo limit of this vector allows its use in only a fraction of patients with mutations in relatively small pathogenic genes. To overcome this issue, we developed a single AAV platform that allows local replacement of a mutated sequence with its wildtype counterpart, based on combined CRISPR-Cas9 and micro-homology-mediated end-joining (MMEJ). In blind mice, the mutation replacement rescued approximately 10% of photoreceptors, resulting in an improvement in light sensitivity and an increase in visual acuity. These effects were comparable to restoration mediated by gene supplementation, which targets a greater number of photoreceptors. This strategy may be applied for the treatment of inherited disorders caused by mutations in larger genes, for which conventional gene supplementation therapy is not currently feasible. Replacing mutant genes with wildtype copies using adeno-associated virus (AAV) has been explored for the treatment of inherited retinopathies, but the low cargo limit restricts its use. Here the authors describe a single AAV platform that allows local replacement of a mutated sequence with its wildtype counterpart, based on combined CRISPR-Cas9 and micro-homology-mediated end joining.