Laboratory of Applied Human Genetics, National Cancer Centre, Singapore, Singapore.
SingHealth Experimental Medicine Centre, Singapore, Singapore.
J Cell Mol Med. 2018 Jul;22(7):3698-3702. doi: 10.1111/jcmm.13648. Epub 2018 Apr 23.
Genetically modified FVIII-expressing autologous bone marrow-derived mesenchymal stromal cells (BMSCs) could cure haemophilia A. However, culture-expanded BMSCs engraft poorly in extramedullary sites. Here, we compared the intramedullary cavity, skeletal muscle, subcutaneous tissue and systemic circulation as tissue microenvironments that could support durable engraftment of FVIII-secreting BMSC in vivo. A zinc finger nuclease integrated human FVIII transgene into PPP1R12C (intron 1) of culture-expanded primary canine BMSCs. FVIII-secretory capacity of implanted BMSCs in each dog was expressed as an individualized therapy index (number of viable BMSCs implanted × FVIII activity secreted/million BMSCs/24 hours). Plasma samples before and after implantation were assayed for transgenic FVIII protein using an anti-human FVIII antibody having negligible cross-reactivity with canine FVIII. Plasma transgenic FVIII persisted for at least 48 weeks after implantation in the intramedullary cavity. Transgenic FVIII protein levels were low after intramuscular implantation and undetectable after both intravenous infusion and subcutaneous implantation. All plasma samples were negative for anti-human FVIII antibodies. Plasma concentrations and durability of transgenic FVIII secretion showed no correlation with the therapy index. Thus, the implantation site microenvironment is crucial. The intramedullary microenvironment, but not extramedullary tissues, supported durable engraftment of genetically modified autologous FVIII-secreting BMSCs.
基因修饰的 FVIII 表达自体骨髓间充质基质细胞（BMSCs）可治愈血友病 A。然而，体外扩增的 BMSCs 在骨髓外部位的植入效果较差。在这里，我们比较了骨髓腔、骨骼肌、皮下组织和全身循环作为支持体内 FVIII 分泌 BMSC 持久植入的组织微环境。锌指核酸酶将人 FVIII 转基因整合到培养扩增的原代犬 BMSCs 的 PPP1R12C（内含子 1）中。每个犬的植入 BMSC 的 FVIII 分泌能力用个性化治疗指数（植入的有活力的 BMSC 数量×每百万 BMSC/24 小时分泌的 FVIII 活性）表示。植入前后的血浆样本使用抗人 FVIII 抗体进行转基因 FVIII 蛋白的检测，该抗体与犬 FVIII 具有可忽略的交叉反应性。在骨髓腔内植入后，转基因 FVIII 至少持续 48 周存在于血浆中。肌肉内植入后，转基因 FVIII 蛋白水平较低，静脉内输注和皮下植入后均无法检测到。所有血浆样本均未检测到抗人 FVIII 抗体。转基因 FVIII 分泌的血浆浓度和持久性与治疗指数无相关性。因此，植入部位微环境至关重要。骨髓内微环境而非骨髓外组织支持基因修饰的自体 FVIII 分泌 BMSC 的持久植入。
