Restoration of FVIII Function and Phenotypic Rescue in Hemophilia A Mice by Transplantation of MSCs Derived From F8-Modified iPSCs.
Restoration of FVIII Function and Phenotypic Rescue in Hemophilia A Mice by Transplantation of MSCs Derived From F8-Modified iPSCs.
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通过移植源自 F8 修饰的 iPSC 的 MSC 来恢复 A 型血友病小鼠的 FVIII 功能和表型拯救
DOI：
10.3389/fcell.2021.630353
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发表时间：
2021
影响因子：
5.5
通讯作者：
Wu L
中科院分区：
文献类型：
--
作者：
Qiu L;Xie M;Zhou M;Liu X;Hu Z;Wu L
A 型血友病 (HA) 是一种 X 连锁隐性先天性出血性疾病，影响 80%–85% 的血友病患者。近一半的严重血友病病例是由破坏 F8 的 0.6 Mb 基因组倒位 (Inv22) 引起的。尽管基于病毒的基因疗法已显示出对B型血友病（HB）的治疗效果，但这种有前景的方法现阶段不适用于HA；这一限制主要是由于F8 cDNA的体积较大，远远超过了腺相关病毒（AAV）的包装能力。我们之前报道过使用 TALEN 对 HA 患者特异性诱导多能干细胞 (HA-iPSC) 中的 Inv22 进行原位遗传校正。我们还研究了一种靶向基因添加的替代策略，其中使用 TALENickase 将 B 结构域删除的 F8 (BDDF8) 的 cDNA 靶向 HA-iPSC 的 rDNA 基因座，以恢复 FVIII 功能。间充质干细胞（MSCs）免疫原性低，在生理条件下可分泌FVIII；在本研究中，MSC 与 F8 校正 iPSC、BDDF8-iPSC 和 HA-iPSC 相区分。对分化的 MSC 进行了表征，并在体外验证了 MSC 中的 FVIII 表达功效。通过静脉注射将三种类型的MSC引入HA小鼠体内。在移植 F8 校正 iMSC 和 BDDF8-iMSC 的 HA 小鼠中观察到长期植入并恢复 FVIII 功能和表型拯救。我们的研究结果表明，使用源自 F8 修饰的 iPSC 的 iMSC 进行离体基因治疗对于 HA 和其他遗传性出生缺陷（尤其是涉及大序列变异的缺陷）的治疗性基因编辑的临床转化来说是可行、有效且有前景的。
Hemophilia A (HA), an X-linked recessive congenital bleeding disorder, affects 80%–85% of patients with hemophilia. Nearly half of severe cases of hemophilia are caused by a 0.6-Mb genomic inversion (Inv22) that disrupts F8. Although viral-based gene therapy has shown therapeutic effects for hemophilia B (HB), this promising approach is not applicable for HA at the present stage; this limitation is mainly due to the large size of F8 cDNA, which far exceeds the adeno-associated virus (AAV) packaging capacity. We previously reported an in situ genetic correction of Inv22 in HA patient-specific induced pluripotent stem cells (HA-iPSCs) by using TALENs. We also investigated an alternative strategy for targeted gene addition, in which cDNA of the B-domain deleted F8 (BDDF8) was targeted at the rDNA locus of HA-iPSCs using TALENickases to restore FVIII function. Mesenchymal stem cells (MSCs) have low immunogenicity and can secrete FVIII under physiological conditions; in this study, MSCs were differentiated from F8-corrected iPSCs, BDDF8-iPSCs, and HA-iPSCs. Differentiated MSCs were characterized, and FVIII expression efficacy in MSCs was verified in vitro. The three types of MSCs were introduced into HA mice via intravenous injection. Long-term engraftment with restoration of FVIII function and phenotypic rescue was observed in HA mice transplanted with F8-corrected iMSCs and BDDF8-iMSCs. Our findings suggest that ex vivo gene therapy using iMSCs derived from F8-modified iPSCs can be feasible, effective, and promising for the clinical translation of therapeutic gene editing of HA and other genetic birth defects, particularly those that involve large sequence variants.
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