Abstract：
BACKGROUND: Cystic Fibrosis (CF) is an autosomal recessive genetic disease caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein for which there is no cure. One approach to cure CF is to correct the underlying mutations in the CFTR gene. We have used triplex-forming peptide nucleic acids (PNAs) loaded into biodegradable nanoparticles (NPs) in combination with donor DNAs as reagents for correcting mutations associated with genetic diseases including CF. Previously, we demonstrated that PNAs induce recombination between a donor DNA and the CFTR gene, correcting the F508del CFTR mutation in human cystic fibrosis bronchial epithelial cells (CFBE cells) and in a CF murine model leading to improved CFTR function with low off-target effects, however the level of correction was still below the threshold for therapeutic cure.
METHODS: Here, we report the use of next generation, chemically modified gamma PNAs (γPNAs) containing a diethylene glycol substitution at the gamma position for enhanced DNA binding. These modified γPNAs yield enhanced gene correction of F508del mutation in human bronchial epithelial cells (CFBE cells) and in primary nasal epithelial cells from CF mice (NECF cells).
RESULTS: Treatment of CFBE cells and NECF cells grown at air-liquid interface (ALI) by NPs containing γtcPNAs and donor DNA resulted in increased CFTR function measured by short circuit current and improved gene editing (up to 32 %) on analysis of genomic DNA.
CONCLUSIONS: These findings provide the basis for further development of PNA and NP technology for editing of the CFTR gene.
METHODS: Here, we report the use of next generation, chemically modified gamma PNAs (γPNAs) containing a diethylene glycol substitution at the gamma position for enhanced DNA binding. These modified γPNAs yield enhanced gene correction of F508del mutation in human bronchial epithelial cells (CFBE cells) and in primary nasal epithelial cells from CF mice (NECF cells).
RESULTS: Treatment of CFBE cells and NECF cells grown at air-liquid interface (ALI) by NPs containing γtcPNAs and donor DNA resulted in increased CFTR function measured by short circuit current and improved gene editing (up to 32 %) on analysis of genomic DNA.
CONCLUSIONS: These findings provide the basis for further development of PNA and NP technology for editing of the CFTR gene.
摘要：
背景：囊性纤维化（CF）是一种常染色体隐性遗传疾病，由编码囊性纤维化跨膜传导调节因子（CFTR）蛋白的基因突变引起，目前尚无治愈方法。治愈CF的一种方法是纠正CFTR基因中的潜在突变。我们已经使用加载到可生物降解的纳米颗粒（NP）中的三链体形成肽核酸（PNA）与供体DNA组合作为校正与包括CF在内的遗传疾病相关的突变的试剂。以前,我们证明了PNA诱导供体DNA和CFTR基因之间的重组，纠正人囊性纤维化支气管上皮细胞（CFBE细胞）和CF鼠模型中的F508delCFTR突变，从而改善CFTR功能，降低脱靶效应，然而,矫正水平仍低于治疗性治愈的阈值.
方法：这里，我们报告了下一代的使用情况，化学修饰的γPNA（γPNA）在γ位置包含二甘醇取代，以增强DNA结合。这些修饰的γPNA在人支气管上皮细胞（CFBE细胞）和CF小鼠的原代鼻上皮细胞（NECF细胞）中增强了F508del突变的基因校正。
结果：用含有γtcPNA和供体DNA的NP处理在气-液界面（ALI）生长的CFBE细胞和NECF细胞导致通过短路电流测量的CFTR功能增加，并改善了基因组DNA分析中的基因编辑（高达32％）。
结论：这些发现为进一步开发用于编辑CFTR基因的PNA和NP技术提供了基础。
方法：这里，我们报告了下一代的使用情况，化学修饰的γPNA（γPNA）在γ位置包含二甘醇取代，以增强DNA结合。这些修饰的γPNA在人支气管上皮细胞（CFBE细胞）和CF小鼠的原代鼻上皮细胞（NECF细胞）中增强了F508del突变的基因校正。
结果：用含有γtcPNA和供体DNA的NP处理在气-液界面（ALI）生长的CFBE细胞和NECF细胞导致通过短路电流测量的CFTR功能增加，并改善了基因组DNA分析中的基因编辑（高达32％）。
结论：这些发现为进一步开发用于编辑CFTR基因的PNA和NP技术提供了基础。
