Long-Term Effects of In Vivo Genome Editing in the Mouse Retina Using Campylobacter jejuni Cas9 Expressed via Adeno-Associated Virus
Long-Term Effects of In Vivo Genome Editing in the Mouse Retina Using Campylobacter jejuni Cas9 Expressed via Adeno-Associated Virus
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DOI：
10.1016/j.ymthe.2018.10.009
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发表时间：
2019-01-02
影响因子：
12.4
通讯作者：
Kim, Jeong Hun
中科院分区：
文献类型：
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作者：
Jo, Dong Hyun;Koo, Taeyoung;Kim, Jeong Hun
使用CRISPR系统进行基因组编辑提供了前所未有的机会，通过灭活不可药物化的靶点（如转录因子）来调节病理条件下的细胞反应。之前，我们证明了迄今为止最小的Cas9直系同源物，来自空肠弯曲杆菌（CjCas 9），靶向Hif 1a并在腺相关病毒（AAV）载体中递送，有效抑制了小鼠视网膜中的病理性脉络膜新生血管形成。在实施CjCas 9作为体内治疗模式之前，必须研究通过AAV介导的CjCas 9体内递送的靶基因破坏的长期作用。在这项研究中，组织学和视网膜电图分析表明，靶向Hif 1a的CjCas 9在视网膜中没有诱导任何明确的毒性，尽管靶基因在视网膜或视网膜色素上皮细胞中以45%至79%的频率突变。重要的是，在注射后14个月，在使用Digenome-seq和CasOFPs鉴定的潜在脱靶位点没有检测到indel，表明CjCas 9的长期表达不会加重脱靶效应。总之，我们的结果表明，玻璃体内注射编码靶向Hif 1a的CjCas 9的AAV有效地诱导并维持视网膜组织中的突变超过1年，并且不影响视网膜组织学完整性或功能。
Genome editing with CRISPR systems provides an unprecedented opportunity to modulate cellular responses in pathological conditions by inactivating undruggable targets, such as transcription factors. Previously, we demonstrated that the smallest Cas9 ortholog characterized to date, from Campylobacter jejuni (CjCas9) targeted to Hif1a and delivered in an adeno-associated virus (AAV) vector, effectively suppressed pathological choroidal neovascularization in the mouse retina. Before implementation of CjCas9 as an in vivo therapeutic modality, it is essential to investigate the long-term effects of target gene disruption via AAV-mediated delivery of CjCas9 in vivo. In this study, histologic and electroretinographic analyses demonstrated that CjCas9 targeted to Hif1a did not induce any definite toxicity in the retina, although the target gene was mutated with a frequency ranging from 45% to 79% in retinal or retinal pigment epithelial cells. Importantly, at 14 months after injection, no indels were detected at potential off-target sites identified using Digenome-seq and CasOFFinder, suggesting that long-term expression of CjCas9 does not aggravate off-target effects. Taken together, our results show that intravitreal injection of AAV encoding CjCas9 targeted to Hif1a effectively induced and maintained mutations in retinal tissues for more than 1 year and did not affect retinal histologic integrity or functions.