外显子
吗啉
RNA剪接
分子生物学
外显子跳跃
突变
选择性拼接
成纤维细胞
剪接位点突变
信使核糖核酸
剪接
生物
癌症研究
基因
核糖核酸
化学
遗传学
细胞培养
斑马鱼
作者
Kristin A. Ham,R. Johnsen,Michel Tchan,Steve Wilton,May T. Aung-Htut
出处
期刊:Methods in molecular biology
日期:2022-11-19
卷期号:: 239-251
标识
DOI:10.1007/978-1-0716-2772-3_14
摘要
The mutation c.-32-13T>G in the GAA gene impacts normal exon 2 splicing and is found in two-thirds of late-onset Pompe disease cases. We have explored a therapeutic strategy using splice modulating phosphorodiamidate morpholino oligomers to enhance GAA exon 2 inclusion in the mature mRNA of patients carrying this common mutation. We performed in silico analysis of the GAA gene transcript for potential splicing silencers and designed oligomers targeting motifs predicted to enhance exon 2 retention in the mature mRNA. Two patient-derived fibroblasts were obtained from Coriell Institute for Medical Research, and seven fibroblast strains from unrelated patients were supplied by Westmead Hospital in Sydney, Australia. Both fibroblasts and forced-myogenic cells were treated with optimized phosphorodiamidate morpholino oligomers supplied by Sarepta Therapeutics. Total RNA and protein were extracted from the cells after incubation with phosphorodiamidate morpholino oligomers, and RT-PCR and RT-qPCR were performed to confirm exon 2 inclusion is enhanced. Acid α-glucosidase activity and expression levels were also assessed to confirm therapeutic potential.
科研通智能强力驱动
Strongly Powered by AbleSci AI