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

翻译后摘要：CFTR无义突变导致严重的囊性纤维化（CF）表型，对电流无反应， 小分子疗法通过在气道上皮细胞中安装一个CFTR基因， 使用腺嘌呤碱基编辑器（ABE）的单核苷酸A>G改变应恢复CFTR介导的Cl- 分泌到健康水平。我们已经优化了两亲性穿梭肽，用于递送CRISPR相关的CRISPR。 核酸酶核糖核蛋白（RNP）的小鼠气道和ABE RNP的恒河猴，实现治疗 编辑水平≥ 5%。我们建议将ABE RNP递送至携带人CFTR R553 X的小鼠的气道， 无义突变在该模型中，小鼠CFTR外显子12被人外显子12序列完全替换。 虽然对野生型人序列的取代对小鼠没有负面影响，但引入 在取代的外显子12中的人R553 X突变引起CF表型（断奶时肠梗阻， 气道上皮和肠类器官中CFTR功能的丧失）。这是唯一一个 具有疾病表型的CFTR无义突变。我们以前证明了体外ABE RNP 使用穿梭肽递送的R553 X可以有效地逆转人气道细胞中的R553 X突变。我们 首要目标是1）优化ABE 8 e RNP编辑，2）修复气道中的R553 X突变， 以足够的效率恢复CFTR功能，和3）研究安全性，毒性， 以及编辑方法的脱靶效应，同时用这种方法进行临床试验。

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.