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在CFTR中通过CRISPR-Cas9基因编辑进行深层内拼接突变的分子和功能纠正

Amy J Walker¹, Carina Graham¹, Miriam Greenwood¹

¹Genetics and Genomic Medicine Department, UCL Great Ormond Street Institute of Child Health, London, UK.

Molecular therapy. Methods & clinical development

|November 29, 2023

概括

基因编辑成功地纠正了气道细胞中常见的囊性纤维化突变. 这种方法恢复了CFTR基因功能,为治疗由深层内突变引起的遗传疾病提供了潜力.

科学领域:

分子生物学分子生物学
基因工程是一种基因工程.
细胞生物学细胞生物学

背景情况:

囊性纤维化 (CF) 是一种由CFTR基因突变引起的遗传疾病.
一种常见的CF突变 (3849+10kb C>T) 破坏了正确的CFTR mRNA拼接.
针对深层内基突变仍然是基因疗法的挑战.

研究的目的:

开发和测试一种基因编辑策略,以纠正3849+10kb的C>T CFTR突变.
在患者衍生细胞中恢复CFTR基因功能和蛋白质表达.
评估基因编辑方法的安全性和有效性.

主要方法:

使用CRISPR-Cas9核糖蛋白 (RNP) 复合体与双导向RNA (gRNA) 来切除突变.
使用非病毒性,受体向的纳米复合物向CF基底上皮细胞传递RNP.
在空气-液体界面培养中评估了CFTR mRNA拼接,蛋白质表达和电生理功能.

主要成果:

基因编辑方法成功切除了CF细胞中的3849+10kbC>T突变.
恢复了正规的CFTRmRNA拼接和CFTR蛋白质表达.
同时在气道上皮培养物中观察到电生理功能的恢复.
通过选择基因组部位的桑格测序,没有检测到非目标编辑.

结论:

通过CRISPR-Cas9调解的基因编辑可以纠正深度内源性CF突变.
非病毒纳米复合物输送促进了气道上皮细胞中的基因编辑.
这一策略对治疗CF和其他具有内基突变的遗传疾病具有前景.

关键词:

克里斯普尔-Cas9是什么意思囊性纤维化症是什么纳米颗粒是一种纳米粒子.拼接突变拼接突变有针对性的切除术.

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