小柯机器人

新方法改进人类长期造血干细胞的基因编辑效率
2020-06-30 19:43

意大利IRCCS圣拉斐尔科学研究所Luigi Naldini小组在研究中取得进展。他们通过克隆追踪验证了人类长期造血干细胞(HSCs)的高效基因编辑。该项研究成果发表在2020年6月29日出版的《自然-生物技术》上。

研究人员将条形码策略应用于编辑细胞的克隆追踪(BAR-Seq)技术。尽管嫁接编辑克隆保留了多系和自我更新的能力,但研究人员发现编辑激活了p53,这大大缩小了血细胞嵌合小鼠的HSC克隆库。瞬时p53抑制恢复了多克隆移植物的组成。

研究人员通过瞬时表达腺病毒5 E4orf6/7蛋白来增加细胞周期进程并上调表达同源导向修复(HDR)复合物的组分,从而提高了HDR效率。E4orf6/7蛋白招募细胞周期控制器E2F到其靶基因。在长期人类移植物中,E4orf6/7表达和p53抑制作用相结合可使HDR的编辑效率高达50%,但不会干扰已编辑HSC的增殖和自我更新。该优化的编辑方法扩大HSC基因编辑的适用性,并为临床应用铺平道路。

据介绍,对HSCs进行基因修饰是治疗多种疾病的潜在方法。但是,HDR效率低以及该程序对克隆成分和移植动力学的未知影响阻碍了其在临床上的应用。

附:英文原文

Title: Efficient gene editing of human long-term hematopoietic stem cells validated by clonal tracking

Author: Samuele Ferrari, Aurelien Jacob, Stefano Beretta, Giulia Unali, Luisa Albano, Valentina Vavassori, Davide Cittaro, Dejan Lazarevic, Chiara Brombin, Federica Cugnata, Anna Kajaste-Rudnitski, Ivan Merelli, Pietro Genovese, Luigi Naldini

Issue&Volume: 2020-06-29

Abstract: Targeted gene editing in hematopoietic stem cells (HSCs) is a promising treatment for several diseases. However, the limited efficiency of homology-directed repair (HDR) in HSCs and the unknown impact of the procedure on clonal composition and dynamics of transplantation have hampered clinical translation. Here, we apply a barcoding strategy to clonal tracking of edited cells (BAR-Seq) and show that editing activates p53, which substantially shrinks the HSC clonal repertoire in hematochimeric mice, although engrafted edited clones preserve multilineage and self-renewing capacity. Transient p53 inhibition restored polyclonal graft composition. We increased HDR efficiency by forcing cell-cycle progression and upregulating components of the HDR machinery through transient expression of the adenovirus 5 E4orf6/7 protein, which recruits the cell-cycle controller E2F on its target genes. Combined E4orf6/7 expression and p53 inhibition resulted in HDR editing efficiencies of up to 50% in the long-term human graft, without perturbing repopulation and self-renewal of edited HSCs. This enhanced protocol should broaden applicability of HSC gene editing and pave its way to clinical translation. Transient p53 inhibition and induced cell-cycle progression increase clonal engraftment and homology-directed repair in hematopoietic stem cells.

DOI: 10.1038/s41587-020-0551-y

Source: https://www.nature.com/articles/s41587-020-0551-y

Nature Biotechnology:《自然—生物技术》,创刊于1996年。隶属于施普林格·自然出版集团,最新IF:31.864
官方网址:https://www.nature.com/nbt/
投稿链接:https://mts-nbt.nature.com/cgi-bin/main.plex


本期文章:《自然—生物技术》:Online/在线发表

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