Department of Pediatrics, Stanford University, Stanford, CA 94305, USA.
Department of Pediatrics, Stanford University, Stanford, CA 94305, USA.
Mol Ther. 2022 Jan 5;30(1):223-237. doi: 10.1016/j.ymthe.2021.03.023. Epub 2021 Mar 29.
Cystic fibrosis (CF) is a monogenic disease caused by impaired production and/or function of the CF transmembrane conductance regulator (CFTR) protein. Although we have previously shown correction of the most common pathogenic mutation, there are many other pathogenic mutations throughout the CF gene. An autologous airway stem cell therapy in which the CFTR cDNA is precisely inserted into the CFTR locus may enable the development of a durable cure for almost all CF patients, irrespective of the causal mutation. Here, we use CRISPR-Cas9 and two adeno-associated viruses (AAVs) carrying the two halves of the CFTR cDNA to sequentially insert the full CFTR cDNA along with a truncated CD19 (tCD19) enrichment tag in upper airway basal stem cells (UABCs) and human bronchial epithelial cells (HBECs). The modified cells were enriched to obtain 60%-80% tCD19 UABCs and HBECs from 11 different CF donors with a variety of mutations. Differentiated epithelial monolayers cultured at air-liquid interface showed restored CFTR function that was >70% of the CFTR function in non-CF controls. Thus, our study enables the development of a therapy for almost all CF patients, including patients who cannot be treated using recently approved modulator therapies.
囊性纤维化（CF）是一种由 CF 跨膜电导调节蛋白（CFTR）的产生和/或功能受损引起的单基因疾病。尽管我们之前已经证明了对最常见的致病突变的纠正，但 CF 基因中还有许多其他的致病突变。一种自体气道干细胞治疗方法，其中 CFTR cDNA 被精确地插入 CFTR 基因座，可能为几乎所有 CF 患者开发出持久的治愈方法，而与致病突变无关。在这里，我们使用 CRISPR-Cas9 和携带 CFTR cDNA 两半的两种腺相关病毒（AAV），依次在上气道基底干细胞（UABCs）和人支气管上皮细胞（HBECs）中插入完整的 CFTR cDNA 以及截断的 CD19（tCD19）富集标签。对修饰后的细胞进行富集，从 11 位具有多种突变的不同 CF 供体中获得 60%-80%的 tCD19 UABCs 和 HBECs。在气液界面培养的分化上皮单层中，CFTR 功能得到恢复，其恢复程度超过非 CF 对照中 CFTR 功能的 70%。因此，我们的研究为几乎所有 CF 患者开发了一种治疗方法，包括那些无法使用最近批准的调节剂治疗的患者。
