Priming Human Repopulating Hematopoietic Stem and Progenitor Cells for Cas9/sgRNA Gene Targeting.
Priming Human Repopulating Hematopoietic Stem and Progenitor Cells for Cas9/sgRNA Gene Targeting.
复制标题
DOI：
10.1016/j.omtn.2018.04.017
复制
发表时间：
2018-09-07
期刊：
影响因子：
--
通讯作者：
Porteus MH
中科院分区：
文献类型：
--
作者：
Charlesworth CT;Camarena J;Cromer MK;Vaidyanathan S;Bak RO;Carte JM;Potter J;Dever DP;Porteus MH
通过造血干细胞和祖细胞（HSPC）中的同源重组（HR）的工程化核酸酶介导的基因靶向具有治疗多种遗传性血液学和免疫学病症的潜力。在这里，我们确定了关键参数，以在HSPC中的β-珠蛋白（HBB）基因座处可重复地实现高频率的RNA引导的（单引导RNA [sgRNA]; CRISPR）-Cas9核酸酶（Cas9/sgRNA）和rAAV 6介导的HR。我们确定，通过在电穿孔后用rAAV 6转导HSPC，存在大于2倍的rAAV 6内吞的电穿孔辅助转导（EAT），其中大约70%的细胞群体在2小时内经历了转导。当在HBB靶向之前以低密度（1 × 105个细胞/mL）培养HSPC时，HSPC扩增速率与体外HR频率以及体内免疫缺陷NSG小鼠中的细胞再增殖显著正相关。我们还表明，在存在小分子UM 171和SR 1的情况下，以低细胞密度培养荧光激活细胞分选（FACS）富集的HBB靶向HSPC，刺激了基因编辑的HSPC的扩增，如通过免疫缺陷小鼠中较高的植入水平所测量的。这项工作不仅作为在HBB位点编辑HSPC以治疗β-血红蛋白病的优化方案，而且还作为在其他位点编辑HSPC以进行基础和转化研究的基础。
Engineered nuclease-mediated gene targeting through homologous recombination (HR) in hematopoietic stem and progenitor cells (HSPCs) has the potential to treat a variety of genetic hematologic and immunologic disorders. Here, we identify critical parameters to reproducibly achieve high frequencies of RNA-guided (single-guide RNA [sgRNA]; CRISPR)-Cas9 nuclease (Cas9/sgRNA) and rAAV6-mediated HR at the β-globin (HBB) locus in HSPCs. We identified that by transducing HSPCs with rAAV6 post-electroporation, there was a greater than 2-fold electroporation-aided transduction (EAT) of rAAV6 endocytosis with roughly 70% of the cell population having undergone transduction within 2 hr. When HSPCs are cultured at low densities (1 × 105 cells/mL) prior to HBB targeting, HSPC expansion rates are significantly positively correlated with HR frequencies in vitro as well as in repopulating cells in immunodeficient NSG mice in vivo. We also show that culturing fluorescence-activated cell sorting (FACS)-enriched HBB-targeted HSPCs at low cell densities in the presence of the small molecules, UM171 and SR1, stimulates the expansion of gene-edited HSPCs as measured by higher engraftment levels in immunodeficient mice. This work serves not only as an optimized protocol for genome editing HSPCs at the HBB locus for the treatment of β-hemoglobinopathies but also as a foundation for editing HSPCs at other loci for both basic and translational research.
登录
查看更多内容
影响因子：
8.8
作者：
Gundry MC;Brunetti L;Lin A;Mayle AE;Kitano A;Wagner D;Hsu JI;Hoegenauer KA;Rooney CM;Goodell MA;Nakada D
通讯作者：
Nakada D
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：
14.8
作者：
Bak RO;Dever DP;Porteus MH
通讯作者：
Porteus MH
影响因子：
46.9
作者：
Hendel A;Bak RO;Clark JT;Kennedy AB;Ryan DE;Roy S;Steinfeld I;Lunstad BD;Kaiser RJ;Wilkens AB;Bacchetta R;Tsalenko A;Dellinger D;Bruhn L;Porteus MH
通讯作者：
Porteus MH
影响因子：
20.3
作者：
Fares, Iman;Chagraoui, Jalila;Sauvageau, Guy
通讯作者：
Sauvageau, Guy