先导编辑和腺嘌呤碱基编辑体外纠正耳聋基因SLC26A4致病变异  

Correction of the pathogenic mutation in the deafness gene SLC26A4 via prime editor and adenine base editor in vitro

作　　者：金疆 吕技能 陈磊 谢丽丽 Jin Jiang;Lyu Jineng;Chen Lei;Xie Lili(Department of Laboratory,Wenzhou People′s Hospital,Wenzhou Third Clinical College of Wenzhou Medical University,Wenzhou Maternal and Child Health Hospital,Wenzhou325027,China;State Key Laboratory of Ophthalmology,Optometry and Vision Science,Eye Hospital,Wenzhou Medical University,Wenzhou325027,China)

机构地区：[1]温州市人民医院、温州医科大学温州市第三临床学院、温州市妇幼保健院检验科,温州325027 [2]温州医科大学附属眼视光医院眼科学、视光学与视觉科学国家重点实验室,温州325027

出　　处：《中华耳鼻咽喉头颈外科杂志》2025年第1期34-41,共8页Chinese Journal of Otorhinolaryngology Head and Neck Surgery

基　　金：温州市科技计划项目(Y2023784)。

摘　　要：目的探讨体外先导编辑(prime editor)和腺嘌呤碱基编辑(adenine base editor)纠正人耳聋基因SLC26A4 c.1229C>T致病变异的可行性。方法2023年3月至2024年4月针对SLC26A4 c.1229C>T变异位点使用先导编辑和腺嘌呤碱基编辑,设计并构建编辑向导RNA(prime editing guide RNA,pegRNA)以及单链向导RNA(single guide RNA,sgRNA)表达载体并在人HEK293T变异模型进行纠正实验,通过深度测序进行纠正效率分析。结果成功构建了SLC26A4 c.1229C>T的变异细胞模型。通过先导编辑和腺嘌呤碱基编辑在SLC26A4 c.1229C>T变异细胞模型中实现了纠正,深度测序分析结果显示碱基纠正效率分别为(31.89±0.77)%和(41.07±2.28)%。结论本研究提出了一种新的基于人类耳聋基因SLC26A4的碱基纠正策略,为SLC26A4基因变异所致耳聋的基因治疗提供了可行性参考。Objective To investigate the feasibility of in vitro prime editor(PE)and adenine base editor(ABE)for correction the pathogenic variant of the human deafness gene SLC26A4 c.1229C>T.Methods From March 2023 to April 2024,prime editing guide RNA(pegRNA)expression vectors as well as single guide RNA(sgRNA)were designed and constructed for the SLC26A4 c.1229C>T variant,and the feasibility of correction was performed in the HEK293T mutation model,the correction efficiency was analyzed by deep sequencing.Results A mutant cell model of SLC26A4 c.1229C>T was successfully established.Correction was achieved in the SLC26A4 c.1229C>T mutant cell model using PE and ABE8e.Deep sequencing analysis revealed the correction efficiencies of(31.89±0.77)%and(41.07±2.28)%,respectively.Conclusion In this study,a new base correction strategy based on the human deafness gene SLC26A4 is proposed,which provides a viable reference for gene therapy of deafness caused by SLC26A4 gene mutation.

关 键 词：听觉丧失 感音神经性 突变 SLC26A4 先导编辑 腺嘌呤碱基编辑 体外研究 

分 类 号：R764.43[医药卫生—耳鼻咽喉科]