SCGE Disease Models Studies Supplement: Repair of a CFTR Nonsense Mutation Using Adenine Base Editing

摘要：cftr无义突变导致严重囊性纤维化(CF)表型，对电流无反应。 小分子疗法。在5-15%的呼吸道上皮细胞中安装一种 使用腺嘌呤碱基编辑程序(ABE)的单核苷酸A和G改变应能恢复cftr介导的Cl− 分泌物恢复到健康水平。我们已经对CRISPR相关的两亲性穿梭多肽进行了优化 核酸酶核糖核蛋白(RNP)对小鼠呼吸道和ABE RNP对猕猴，实现治疗 编辑级别≥为5%。我们建议将ABE RNP运送到携带人CFTRR553X的小鼠的呼吸道 无稽之谈的突变。在该模型中，小鼠CFTR12外显子被完全替换为人外显子12序列。 虽然野生型人类序列的替代对小鼠没有负面影响，但引入 人类R553X外显子12突变导致CF型(断奶时肠梗阻， 呼吸道上皮和肠道器官中CFTR功能的丧失)。这是仅有的动物模型之一 带有疾病表型的cftr无义突变。我们之前在体外证明了ABE RNP 使用穿梭多肽可以有效地逆转人呼吸道细胞中的R553X突变。我们的 首要目标是1)优化ABE8e RNP编辑，2)修复呼吸道中的R553X突变 小鼠上皮细胞具有足够的效率来恢复CFTR功能；3)研究其安全性、毒性、 以及在使用该方法进行临床试验的同时编辑方法的非目标效果。

Abstract: CFTR nonsense mutations cause a severe cystic fibrosis (CF) phenotype, unresponsive to current small molecule therapies. Correcting CFTR nonsense mutations in 5–15% of airway epithelia by installing a single nucleotide A>G change using an adenine base editor (ABE) should restore CFTR mediated Cl− secretion to healthy levels. We have optimized amphiphilic shuttle peptides for delivery of CRISPR associated nuclease ribonucleoproteins (RNP) to mouse airways and ABE RNP to rhesus monkeys, achieving therapeutic editing levels ≥5%. We propose to deliver ABE RNP to the airways of mice bearing the human CFTR R553X nonsense mutation. In this model, mouse CFTR exon 12 was entirely replaced with human exon 12 sequence. While substitution for wildtype human sequence has no negative consequences on mice, introduction of human R553X mutation in the substituted exon 12 causes a CF phenotype (intestinal obstruction at weaning, loss of CFTR function in airway epithelia and intestinal organoids). This is one of the only animal models of a CFTR nonsense mutation with a disease phenotype. We previously demonstrated ex vivo that ABE RNP delivered using shuttle peptides can efficiently revert the R553X mutation in human airway cells. Our overarching goals are 1) to optimize ABE8e RNP editing, 2) to repair the R553X mutation in the airway epithelia of mice with sufficient efficiency to restore CFTR function, and 3) to investigate the safety, toxicity, and off-target effects of the editing approach while advancing towards clinical trials with this method.