Department of Pediatrics, University of Iowa, Iowa City, IA, USA.
Molecular Medicine Graduate Program, Pappajohn Biomedical Institute, University of Iowa, Iowa City, IA, USA.
Nucleic Acids Res. 2021 Oct 11;49(18):10558-10572. doi: 10.1093/nar/gkab788.
Mutations in the CFTR gene that lead to premature stop codons or splicing defects cause cystic fibrosis (CF) and are not amenable to treatment by small-molecule modulators. Here, we investigate the use of adenine base editor (ABE) ribonucleoproteins (RNPs) that convert A•T to G•C base pairs as a therapeutic strategy for three CF-causing mutations. Using ABE RNPs, we corrected in human airway epithelial cells premature stop codon mutations (R553X and W1282X) and a splice-site mutation (3849 + 10 kb C > T). Following ABE delivery, DNA sequencing revealed correction of these pathogenic mutations at efficiencies that reached 38-82% with minimal bystander edits or indels. This range of editing was sufficient to attain functional correction of CFTR-dependent anion channel activity in primary epithelial cells from CF patients and in a CF patient-derived cell line. These results demonstrate the utility of base editor RNPs to repair CFTR mutations that are not currently treatable with approved therapeutics.
导致提前终止密码子或剪接缺陷的 CFTR 基因突变会导致囊性纤维化（CF），并且不能用小分子调节剂进行治疗。在这里，我们研究了腺嘌呤碱基编辑器（ABE）核糖核蛋白（RNP）的用途，该核糖核蛋白可将 A•T 转换为 G•C 碱基对，作为治疗三种 CF 致病突变的策略。使用 ABE RNP，我们纠正了人呼吸道上皮细胞中的提前终止密码子突变（R553X 和 W1282X）和剪接位点突变（3849+10kbC>T）。在 ABE 递送至细胞后，DNA 测序显示这些致病突变的校正效率达到 38-82%，同时最小化了旁观者编辑或插入缺失。这种编辑范围足以实现 CF 患者原代上皮细胞和 CF 患者衍生细胞系中 CFTR 依赖性阴离子通道活性的功能校正。这些结果表明，碱基编辑器 RNP 可用于修复目前无法用批准的治疗药物治疗的 CFTR 突变。
