Center for Reproductive Medicine, International Peace Maternity and Child Health Hospital, Innovative Research Team of High-level Local Universities in Shanghai, School of Medicine, Shanghai Jiao Tong University, Shanghai 200030, China.
Institute of Neuroscience, State Key Laboratory of Neuroscience, Key Laboratory of Primate Neurobiology, CAS Center for Excellence in Brain Science and Intelligence Technology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
Protein Cell. 2023 Jun 7;14(6):416-432. doi: 10.1093/procel/pwac043.
Approximately 140 million people worldwide are homozygous carriers of APOE4 (ε4), a strong genetic risk factor for late onset familial and sporadic Alzheimer's disease (AD), 91% of whom will develop AD at earlier age than heterozygous carriers and noncarriers. Susceptibility to AD could be reduced by targeted editing of APOE4, but a technical basis for controlling the off-target effects of base editors is necessary to develop low-risk personalized gene therapies. Here, we first screened eight cytosine base editor variants at four injection stages (from 1- to 8-cell stage), and found that FNLS-YE1 variant in 8-cell embryos achieved the comparable base conversion rate (up to 100%) with the lowest bystander effects. In particular, 80% of AD-susceptible ε4 allele copies were converted to the AD-neutral ε3 allele in human ε4-carrying embryos. Stringent control measures combined with targeted deep sequencing, whole genome sequencing, and RNA sequencing showed no DNA or RNA off-target events in FNLS-YE1-treated human embryos or their derived stem cells. Furthermore, base editing with FNLS-YE1 showed no effects on embryo development to the blastocyst stage. Finally, we also demonstrated FNLS-YE1 could introduce known protective variants in human embryos to potentially reduce human susceptivity to systemic lupus erythematosus and familial hypercholesterolemia. Our study therefore suggests that base editing with FNLS-YE1 can efficiently and safely introduce known preventive variants in 8-cell human embryos, a potential approach for reducing human susceptibility to AD or other genetic diseases.
全球约有 1.4 亿人是 APOE4（ε4）的纯合子携带者，这是晚发性家族性和散发性阿尔茨海默病（AD）的一个强烈遗传风险因素，其中 91%的人会比杂合子携带者和非携带者更早发病。通过靶向编辑 APOE4，可以降低 AD 的易感性，但需要一种控制碱基编辑器脱靶效应的技术基础，才能开发出低风险的个性化基因疗法。在这里，我们首先在四个注射阶段（1-细胞期至 8-细胞期）筛选了 8 种胞嘧啶碱基编辑器变体，发现 8 细胞胚胎中的 FNLS-YE1 变体具有最低的旁观者效应，达到了相当高的碱基转换率（高达 100%）。特别是，在携带 APOE4 的人类胚胎中，80%的 AD 易感 ε4 等位基因拷贝被转化为 AD 中性的 ε3 等位基因。严格的控制措施结合靶向深度测序、全基因组测序和 RNA 测序表明，FNLS-YE1 处理的人类胚胎及其衍生的干细胞中没有 DNA 或 RNA 脱靶事件。此外，FNLS-YE1 的碱基编辑对胚胎发育到囊胚阶段没有影响。最后，我们还证明 FNLS-YE1 可以在人类胚胎中引入已知的保护性变异，从而潜在地降低人类对系统性红斑狼疮和家族性高胆固醇血症的易感性。因此，我们的研究表明，FNLS-YE1 可以有效地在 8 细胞人类胚胎中安全地引入已知的预防变异，这可能是降低人类对 AD 或其他遗传疾病易感性的一种方法。
