ssODN-Mediated In-Frame Deletion with CRISPR/Cas9 Restores FVIII Function in Hemophilia A-Patient-Derived iPSCs and ECs
ssODN-Mediated In-Frame Deletion with CRISPR/Cas9 Restores FVIII Function in Hemophilia A-Patient-Derived iPSCs and ECs
复制标题
ssODN 介导的 CRISPR/Cas9 框内删除可恢复 A 型血友病患者衍生的 iPSC 和 EC 中的 FVIII 功能
DOI：
10.1016/j.omtn.2019.05.019
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发表时间：
2019-09-06
影响因子：
8.8
通讯作者：
Liang, Desheng
中科院分区：
文献类型：
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作者：
Hu, Zhiqing;Zhou, Miaojin;Liang, Desheng
鉴于F8的cDNA太大而不能包装到腺相关病毒（AAV）衣壳中，已经尝试了用于血友病A（HA）治疗的某些形式的B结构域缺失的F8（BDD-F8）的基因转移，并取得了有希望的结果。在这里，我们描述了在HA患者来源的诱导多能干细胞（HA-iPSC）中通过单链寡脱氧核苷酸（ssODN）介导的在F8的B结构域内用CRISPR/Cas9进行的框内缺失的有效基因校正。FVIII的表达和活性在体外和体内在校正的HA-iPSC衍生的诱导内皮祖细胞（C-iEPCs）中恢复。在C-iEPC输注后，HA小鼠中的出血表型得到挽救。我们的结果证明了一种有效的原位基因校正方法，该方法通过使用ssODN和CRISPR/Cas9引入微小缺失来重构F8转录物并恢复HA-iPSC衍生的EPC中的FVIII功能，在HA基因治疗中具有潜在的临床影响。我们首次在体外和体内证明了由具有部分缺失的B结构域的内源性F8基因编码的FVIII功能。这项工作也为其他基因移码突变的遗传校正提出了一种适用的策略。
Given that the cDNA of F8 is too large to be packaged into adeno-associated virus (AAV) capsids, gene transfer of some versions of B-domain-deleted F8 (BDD-F8) for hemophilia A (HA) treatment has been attempted with promising results. Here, we describe an efficient gene correction via single-stranded-oligodeoxynucleotide (ssODN)-mediated in-frame deletion within the B domain of F8 with CRISPR/Cas9 in HA-patient-derived induced pluripotent stem cells (HA-iPSCs). The expression and activity of FVIII was restored in corrected HA-iPSC-derived induced endothelial progenitor cells (C-iEPCs) in vitro and in vivo. The bleeding phenotype was rescued in HA mice after C-iEPC infusion. Our results demonstrate an efficient approach for in situ gene correction via introduction of a tiny deletion using ssODN and CRISPR/Cas9 to reframe the F8 transcript and restore FVIII function in HA-iPSC-derived EPCs with potential clinical impact in HA gene therapy. For the first time, we demonstrated in vitro and in vivo the FVIII function that is encoded by the endogenous F8 gene with a partially deleted B domain. This work also suggests an applicable strategy for genetic correction of other gene frameshift mutations.