Efficient gene editing of human long-term hematopoietic stem cells validated by clonal tracking.
Efficient gene editing of human long-term hematopoietic stem cells validated by clonal tracking.
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DOI：
10.1038/s41587-020-0551-y
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发表时间：
2020-11
影响因子：
46.9
通讯作者：
Naldini L
中科院分区：
文献类型：
--
作者：
Ferrari S;Jacob A;Beretta S;Unali G;Albano L;Vavassori V;Cittaro D;Lazarevic D;Brombin C;Cugnata F;Kajaste-Rudnitski A;Merelli I;Genovese P;Naldini L
定向基因编辑在造血干细胞(HSCs)是一种很有前途的治疗几种疾病的方法。然而，同源定向修复(HDR)在HSCs中的有限效率，以及该过程对移植后克隆组成和动力学的未知影响，阻碍了临床翻译。在这里，我们将条形码策略应用于编辑细胞的克隆跟踪(BAR-Seq)，并表明编辑激活了P53，这显著缩小了血液病小鼠的HSC克隆库，尽管移植的编辑克隆保留了多系和自我更新能力。短暂的P53抑制恢复了多克隆移植物成分。我们通过迫使细胞周期进展和通过瞬时表达腺病毒5E4orf6/7蛋白上调HDR机制的组件来提高HDR效率，该蛋白在其靶基因上招募细胞周期控制器E2F。联合应用E4orf6/7的表达和P53的抑制，在长期的人类移植中，HDR的编辑效率高达50%，而不会干扰编辑后的HSC的再繁殖和自我更新。这一增强的方案将扩大HSC基因编辑的适用性，并为临床翻译铺平道路。
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 upon 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 significantly shrinks the HSC clonal repertoire in hematochimeric mice, although engrafted edited clones preserved 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.
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影响因子：
8.8
作者：
Kuo CY;Long JD;Campo-Fernandez B;de Oliveira S;Cooper AR;Romero Z;Hoban MD;Joglekar AV;Lill GR;Kaufman ML;Fitz-Gibbon S;Wang X;Hollis RP;Kohn DB
通讯作者：
Kohn DB
影响因子：
64.8
作者：
Genovese, Pietro;Schiroli, Giulia;Escobar, Giulia;Di Tomaso, Tiziano;Firrito, Claudia;Calabria, Andrea;Moi, Davide;Mazzieri, Roberta;Bonini, Chiara;Holmes, Michael C.;Gregory, Philip D.;van der Burg, Mirjam;Gentner, Bernhard;Montini, Eugenio;Lombardo, Angelo;Naldini, Luigi
通讯作者：
Naldini, Luigi
影响因子：
20.3
作者：
Fares, Iman;Chagraoui, Jalila;Sauvageau, Guy
通讯作者：
Sauvageau, Guy
影响因子：
11.4
作者：
Komori, H;Enomoto, M;Ohtani, K
通讯作者：
Ohtani, K
影响因子：
23.9
作者：
Laurenti, Elisa;Frelin, Catherine;Xie, Stephanie;Ferrari, Robin;Dunant, Cyrille F.;Zandi, Sasan;Neumann, Andrea;Plumb, Ian;Doulatov, Sergei;Chen, Jing;April, Craig;Fan, Jian-Bing;Iscove, Norman;Dick, John E.
通讯作者：
Dick, John E.