靶向DUX4多聚腺苷酸化信号的基因编辑：一种治疗面肩肱型肌营养不良症的方法？
Gene Editing Targeting the DUX4 Polyadenylation Signal: A Therapy for FSHD?
作者信息
Joubert Romains, Mariot Virginie, Charpentier Marine, Concordet Jean Paul, Dumonceaux Julie
机构信息
NIHR Biomedical Research Centre, University College London, Great Ormond Street Institute of Child Health and Great Ormond Street Hospital NHS Trust, London WC1N 1EH, UK.
Genome Structure and Instability laboratory, CNRS UMR 7196, Inserm U1154, National Museum of Natural History, 75005 Paris, France.
出版信息
J Pers Med. 2020 Dec 23;11(1):7. doi: 10.3390/jpm11010007.
Facioscapulohumeral dystrophy (FSHD, OMIM: 158900, 158901) is the most common dystrophy in adults and so far, there is no treatment. Different loci of the disease have been characterized and they all lead to the aberrant expression of the DUX4 protein, which impairs the function of the muscle, ultimately leading to cell death. Here, we used gene editing to try to permanently shut down DUX4 expression by targeting its poly(A) sequence. We used transcription activator-like effector nucleases (TALEN) and CRISPR-Cas9 nucleases in vitro on FSHD myoblasts. More than 150 TOPO clones were sequenced and only indels were observed in 4%. Importantly, in 2 of them, the DUX4 poly(A) signal was eliminated at the genomic level but DUX4 mRNA was still produced thanks to the use of a non-canonical upstream poly(A) signal sequence. These experiments show that targeting DUX4 PAS at the genomic level might not be an appropriate gene editing strategy for FSHD therapy.
面肩肱型肌营养不良症（FSHD，OMIM：158900，158901）是成人中最常见的肌营养不良症，迄今为止尚无治疗方法。该疾病的不同基因座已被鉴定，它们均导致DUX4蛋白的异常表达，这会损害肌肉功能，最终导致细胞死亡。在此，我们使用基因编辑技术，试图通过靶向DUX4的多聚腺苷酸（poly(A)）序列来永久关闭其表达。我们在体外对FSHD成肌细胞使用了转录激活样效应核酸酶（TALEN）和CRISPR-Cas9核酸酶。对150多个TOPO克隆进行了测序，仅4%的克隆中观察到插入缺失。重要的是，其中2个克隆中，虽然在基因组水平上消除了DUX4的poly(A)信号，但由于使用了非经典的上游poly(A)信号序列，仍产生了DUX4 mRNA。这些实验表明，在基因组水平上靶向DUX4的多聚腺苷酸化信号（PAS）可能不是FSHD治疗的合适基因编辑策略。
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