Dissecting ELANE neutropenia pathogenicity by human HSC gene editing.
Dissecting ELANE neutropenia pathogenicity by human HSC gene editing.
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DOI：
10.1016/j.stem.2020.12.015
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发表时间：
2021-05-06
期刊：
影响因子：
23.9
通讯作者：
Bauer DE
中科院分区：
文献类型：
--
作者：
Rao S;Yao Y;Soares de Brito J;Yao Q;Shen AH;Watkinson RE;Kennedy AL;Coyne S;Ren C;Zeng J;Serbin AV;Studer S;Ballotti K;Harris CE;Luk K;Stevens CS;Armant M;Pinello L;Wolfe SA;Chiarle R;Shimamura A;Lee B;Newburger PE;Bauer DE
重度先天性中性粒细胞减少症（SCN）是一种危及生命的疾病，最常见的原因是干扰中性粒细胞成熟的ELANE显性突变。我们在人类造血干细胞和祖细胞（HSPC）中进行了合并CRISPR筛选，将ELANE突变与中性粒细胞成熟潜力关联起来。早期外显子的高效基因编辑引发了无义介导的衰变（NMD），克服了ELANE突变SCN患者HSPC中的中性粒细胞成熟停滞，并产生了正常的造血移植功能。相反，模拟SCN相关突变的末端外显子移码等位基因逃脱了NMD，重演了中性粒细胞成熟停滞，并建立了ELANE突变SCN的动物模型。令人惊讶的是，只有-1框插入缺失阻碍了中性粒细胞的成熟，而-2框晚期外显子插入缺失抑制翻译并支持中性粒细胞的成熟。原代HSPC的基因编辑允许忠实地鉴定变体致病性，以阐明疾病的分子机制，并鼓励对ELANE突变型中性粒细胞减少症的通用治疗方法，恢复正常的中性粒细胞产生并保留HSPC功能。Rao及其同事在人类原代细胞中进行了全面的基因编辑，将ELANE突变与中性粒细胞成熟潜力联系起来。触发无义介导的衰变（NMD）的插入缺失规避了严重的先天性中性粒细胞减少症（SCN）突变。逃避NMD的插入缺失通过异种移植模型SCN，尽管-2框插入缺失抑制翻译并使中性粒细胞成熟。
Severe congenital neutropenia (SCN) is a life-threatening disorder most often caused by dominant mutations of ELANE that interfere with neutrophil maturation. We conducted a pooled CRISPR screen in human hematopoietic stem and progenitor cells (HSPCs) that correlated ELANE mutations with neutrophil maturation potential. Highly efficient gene editing of early exons elicited nonsense-mediated decay (NMD), overcame neutrophil maturation arrest in HSPCs from ELANE mutant SCN patients, and produced normal hematopoietic engraftment function. Conversely, terminal exon frameshift alleles that mimic SCN-associated mutations escaped NMD, recapitulated neutrophil maturation arrest, and established an animal model of ELANE mutant SCN. Surprisingly, only −1 frame indels impeded neutrophil maturation, while −2 frame late exon indels repressed translation and supported neutrophil maturation. Gene editing of primary HSPCs allowed faithful identification of variant pathogenicity to clarify molecular mechanisms of disease and encourage a universal therapeutic approach to ELANE mutant neutropenia, returning normal neutrophil production and preserving HSPC function. Rao and colleagues perform comprehensive gene editing in human primary cells to associate ELANE mutations with neutrophil maturation potential. Indels that trigger nonsense mediated decay (NMD) circumvent severe congenital neutropenia (SCN) mutations. Indels that escape NMD model SCN by xenograft although −2 frame indels repress translation and enable neutrophil maturation.
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