Acıbadem Labcell Cellular Therapy Laboratory, İstanbul, Türkiye
Biruni University, Department of Molecular Biology and Medical Genetics, İstanbul, Türkiye
Turk J Haematol. 2023 May 29;40(2):118-124. doi: 10.4274/tjh.galenos.2023.2022-0318. Epub 2023 Apr 6.
Hemophilia A is an X-linked recessive bleeding disorder caused by a deficiency of plasma coagulation factor VIII (FVIII), and it accounts for about 80%-85% of all cases of hemophilia. Plasma-derived therapies or recombinant FVIII concentrates are used to prevent and treat the bleeding symptoms along with FVIII-mimicking antibodies. Recently, the European Medicines Agency granted conditional marketing approval for the first gene therapy for hemophilia A. The aim of this study was to determine the effectiveness of coagulation in correcting FVIII deficiency with FVIII-secreting transgenic mesenchymal stem cells (MSCs).
A lentiviral vector encoding a B domain-deleted FVIII cDNA sequence with CD45R0 truncated (CD45R0t) surface marker was designed to develop a transgenic FVIII-expressing primary cell line by transducing MSCs. The efficacy and functionality of the FVIII secreted from the MSCs was assessed with anti-FVIII ELISA, CD45R0t flow cytometry, FVIII western blot, and mixing test analysis in vitro.
The findings of this study showed that the transgenic MSCs maintained persistent FVIII secretion. There was no significant difference in FVIII secretion over time, suggesting stable FVIII expression from the MSCs. The functionality of the FVIII protein secreted in the MSC supernatant was demonstrated by applying a mixing test in coagulation analysis. In the mixing test analysis, FVIII-deficient human plasma products were mixed with either a saline control or FVIII-secreted MSC supernatant. The mean FVIII level of the saline control group was 0.41±0.03 IU/dL, whereas the mean level was 25.41±33.38 IU/dL in the FVIII-secreting MSC supernatant mixed group (p<0.01). The mean activated partial thromboplastin time (aPTT) of the saline control group was 92.69±11.38 s, while in the FVIII-secreting MSC supernatant mixed group, the mean aPTT level decreased to 38.60±13.38 s (p<0.001).
The findings of this in vitro study suggest that the new method presented here is promising as a possible treatment for hemophilia A. Accordingly, a study of FVIII-secreting transgenic MSCs will next be initiated in a FVIII-knockout animal model.
血友病 A 是一种 X 连锁隐性遗传性出血性疾病，由血浆凝血因子 VIII（FVIII）缺乏引起，约占所有血友病病例的 80%-85%。血浆来源的治疗方法或重组 FVIII 浓缩物用于预防和治疗出血症状以及 FVIII 模拟抗体。最近，欧洲药品管理局批准了首个血友病 A 的基因治疗有条件上市。本研究的目的是确定 FVIII 分泌转基因间充质干细胞（MSCs）纠正 FVIII 缺乏的凝血效果。
设计了一种慢病毒载体，编码带有 CD45R0 截断（CD45R0t）表面标记的 B 结构域缺失 FVIII cDNA 序列，通过转导 MSCs 来开发转基因 FVIII 表达原代细胞系。通过抗 FVIII ELISA、CD45R0t 流式细胞术、FVIII Western blot 和体外混合试验分析评估 MSC 分泌的 FVIII 的功效和功能。
本研究发现，转基因 MSCs 持续分泌 FVIII。随着时间的推移，FVIII 分泌没有显著差异，提示 MSCs 稳定表达 FVIII。通过凝血分析中的混合试验证明 MSC 上清液中分泌的 FVIII 蛋白的功能。在混合试验分析中，将 FVIII 缺乏的人血浆制品与生理盐水对照或 FVIII 分泌 MSC 上清液混合。生理盐水对照组的平均 FVIII 水平为 0.41±0.03 IU/dL，而 FVIII 分泌 MSC 上清液混合组的平均水平为 25.41±33.38 IU/dL（p<0.01）。生理盐水对照组的平均活化部分凝血活酶时间（aPTT）为 92.69±11.38 s，而在 FVIII 分泌 MSC 上清液混合组中，平均 aPTT 水平降低至 38.60±13.38 s（p<0.001）。
本体外研究的结果表明，这里提出的新方法有望成为治疗血友病 A 的一种潜在方法。因此，下一步将在 FVIII 敲除动物模型中开展 FVIII 分泌转基因 MSCs 的研究。
