In Vitro FVIII-Encoding Transgenic Mesenchymal Stem Cells Maintain Successful Coagulation in FVIII-Deficient Plasma Mimicking Hemophilia A

• Published in: Turkish journal of haematology 2023-05, Vol.40 (2), p.118-124
• Main Authors: Hemşinlioğlu, Cansu, Aslan, Elif Sibel, Taştan, Cihan, Çakırsoy, Didem, Turan, Raife Dilek, Seyis, Utku, Elek, Muhammer, Sır Karakuş, Gözde, Günaydın, Ömur Selin, Abanuz, Selen, Kançağı, Derya Dilek, Yurtsever, Bulut, Yalçın, Koray, Kasap, Murat, Ovalı, Ercüment
• Format: Article
• Language: English
• Subjects: Animals; Blood Coagulation; Factor VIII - genetics; Genetic Therapy - methods; Hemophilia A - genetics; Hemophilia A - therapy; Humans; Mesenchymal Stem Cells - metabolism
• ISSN: 1300-7777; 1308-5263
• DOI: 10.4274/tjh.galenos.2023.2022-0318

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