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

Objective: 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 FVIIImimicking 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). Materials and Methods: A lentiviral vector encoding a B domaindeleted 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. Results: 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, FVIIIdeficient 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). Conclusion: 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.

Anahtar Kelime:

İn Vitro FVIII İçeren Transgenik Mezenkimal Kök Hücreler Hemofili A’yı Taklit Eden FVIII İçermeyen Plazmada Başarılı Koagülasyon Sağlamaktadır

Öz:

Amaç: Hemofili A, pıhtılaşma faktörü VIII’in (FVIII) eksikliğine bağlı gelişen, hemofili hastalarının yaklaşık %80-85’ini oluşturan, X’e bağlı resesif geçiş gösteren bir kanama bozukluğudur. Kanama semptomlarını önlemek ve tedavi etmek için plazma kaynaklı tedaviler ya da rekombinant FVIII konsantreleri ile FVIII’i taklit eden monoklonal antikorlar kullanılmaktadır. Son zamanlarda EMA, hemofili A’nın ilk gen tedavisi için koşullu pazarlama onayı vermiştir. Bu çalışmada, hemofili A hastalığından sorumlu olan FVIII eksikliğini düzeltebilmek amacıyla FVIII salgılayan transgenik mezenkimal kök hücreler (MKH) ile koagülasyon etkinliğinin değerlendirilmesi amaçlanmaktadır. Gereç ve Yöntemler: CD45R0 truncated (CD45R0t) yüzey belirteci ile B domaini silinmiş FVIII cDNA dizisini kodlayan bir lentiviral vektör, MKH’leri transdükte ederek transgenik FVIII ekspresyonu sağlayan birincil bir hücre hattı geliştirilmiştir. MKH’lerden salgılanan FVIII proteininin etkinliği ve fonksiyonalitesi anti-FVIII ELISA, CD45R0t akım sitometrisi, FVIII western blot ve karışım testi (mixing test) analizleri ile in vitro olarak değerlendirilmiştir. Bulgular: Çalışmamızda transgenik MKH’lerin kalıcı FVIII sekresyonu sağladığı gösterilmiştir. Zamana bağlı yapılan analizlerde FVIII sekresyonunda anlamlı bir değişim olmadığı, MKH’lerde stabil FVIII ekspresyonu sağladığı gösterilmiştir. Koagülasyon analizlerinde, MKH süpernatanına sekrete olan FVIII proteininin fonksiyonalitesi, karışım testi kullanılarak saptanmıştır. Karışım testi analizlerinde, FVIII içermeyen insan plazma ürünleri salinle (kontrol grubu) ve FVIII-salgılayan MKH süpernatantı (çalışma grubu) ile karıştırılmıştır. Kontrol grubunun ortalama FVIII düzeylerinin 0,41±0,03 IU/dL olduğu, MKH süpernatant ile karıştırılan çalışma grubunda ise ortalama FVIII düzeylerinin 25,41±33,38 IU/ dL (p<0,01) olduğu gösterilmiştir. Kontrol grubunun ortalama aktive parsiyel tromboplastin zamanı (aPTT) 92,69±11,38 saniye iken, FVIIIsalgılayan MKH süpernatantı ile karıştırılan grubun ise ortalama aPTT seviyelerinin 38,60±13,38 saniyeye düştüğü gösterilmiştir (p<0,001). Sonuç: Bu in vitro çalışma, hemofili A’yı tedavi etmek için uygulanabilir umut verici yeni bir yöntem ortaya koymaktadır. Bu veriler ışığında FVIII knock-out hayvan modelinde FVIII salgılayan transgenik MKH çalışması başlatılacaktır.

Anahtar Kelime:

Belge Türü: Makale Makale Türü: Araştırma Makalesi Erişim Türü: Erişime Açık

1. Carcao MD. The diagnosis and management of congenital hemophilia. Semin Thromb Hemost 2012;38:727-734.
2. Stonebraker JS, Bolton-Maggs PH, Soucie JM, Walker I, Brooker M. A study of variations in the reported haemophilia A prevalence around the world. Haemophilia 2010;16:20-32.
3. Srivastava A, Santagostino E, Dougall A, Kitchen S, Sutherland M, Pipe SW, Carcao M, Mahlangu J, Ragni MV, Windyga J, Llinás A, Goddard NJ, Mohan R, Poonnoose PM, Feldman BM, Lewis SZ, van den Berg HM, Pierce GF; WFH Guidelines for the Management of Hemophilia panelists and coauthors. WFH Guidelines for the Management of Hemophilia, 3rd edition. Haemophilia 2020;26 (Suppl 6):1-158.
4. White GC 2nd, Rosendaal F, Aledort LM, Lusher JM, Rothschild C, Ingerslev J; Factor VIII and Factor IX Subcommittee. Definitions in hemophilia. Recommendation of the scientific subcommittee on factor VIII and factor IX of the scientific and standardization committee of the International Society on Thrombosis and Haemostasis. Thromb Haemost 2001;85:560. 5. Nathwani AC. Gene therapy for hemophilia. Hematology Am Soc Hematol Educ Program 2022;2022:569-578.
6. Perrin GQ, Herzog RW, Markusic DM. Update on clinical gene therapy for hemophilia. Blood 2019;133:407-414.
7. Rangarajan S, Walsh L, Lester W, Perry D, Madan B, Laffan M, Yu H, Vettermann C, Pierce GF, Wong WY, Pasi KJ. AAV5-factor VIII gene transfer in severe hemophilia A. N Engl J Med 2017;377:2519-2530.
8. High KA, George LA, Eyster ME, Sullivan SK, Ragni MV, Croteau SE, Samelson-Jones BJ, Evans M, Joseney-Antoine M, Macdougall A. A phase 1/2 trial of investigational SPK-8011 in hemophilia a demonstrates durable expression and prevention of bleeds. Blood 2018;132:487.
9. Konkle BA, Stine K, Visweshwar N, Harrington TJ, Leavitt AD, Giermasz A, Arkin S, Di Russo G, Snyder A, Woolfson A, Rouy D. Updated follow-up of the Alta study, a phase 1/2, open label, adaptive, dose-ranging study to assess the safety and tolerability of SB-525 gene therapy in adult patients with severe hemophilia A. Blood 2019;134(Suppl 1):2060.
10. Calcedo R, Vandenberghe LH, Gao G, Lin J, Wilson JM. Worldwide epidemiology of neutralizing antibodies to adeno-associated viruses. J Infect Dis 2009;199:381-390. 11. Ronzitti G, Gross DA, Mingozzi F. Human immune responses to adeno-associated virus (AAV) vectors. Front Immunol 2020;11:670.
12. Batty P, Sihn CR, Ishida J, Mo AM, Yates B, Brown C, Harpell L, Pender A, Russell C, Sardo Infirri S, Torres R, Winterborn A, Fong S, Lillicrap D. Long-term vector genome outcomes and immunogenicity of AAV FVIII gene transfer in the hemophilia A dog model. Res Pr Thromb Haemost 2020;4(Suppl 1)PB1087.
13. Schnepp BC, Jensen RL, Chen CL, Johnson PR, Clark KR. Characterization of adenoassociated virus genomes isolated from human tissues. J Virol 2005;79:14793- 14803.
14. Talmadge JE, Cowan KH. Gene therapy in oncology. In: Niederhuber JE, Armitage JO, Doroshow JH, Kastan MB, Tepper JE (eds). Abeloff’s Clinical Oncology, 6th Edition. Philadelphia, Elsevier, 2020.
15. Vannucci L, Lai M, Chiuppesi F, Ceccherini-Nelli L, Pistello M. Viral vectors: a look back and ahead on gene transfer technology. New Microbiol 2013;36:1-22.
16. Chuah MK, Evens H, VandenDriessche T. Gene therapy for hemophilia. J Thromb Haemost 2013;11(Suppl 1):99-110.
17. Pittman DD, Marquette KA, Kaufman RJ. Role of the B domain for factor VIII and factor V expression and function. Blood 1994;84:4214-4225.
18. Taştan C, Kançağı DD, Turan RD, Yurtsever B, Çakırsoy D, Abanuz S, Yılancı M, Seyis U, Özer S, Mert S, Kayhan CK, Tokat F, Açıkel Elmas M, Birdoğan S, Arbak S, Yalçın K, Sezgin A, Kızılkılıç E, Hemşinlioğlu C, İnce Ü, Ratip S, Ovalı E. Preclinical assessment of efficacy and safety analysis of CAR-T cells (ISIKOK-19) targeting CD19-expressing B-cells for the first Turkish academic clinical trial with relapsed/ refractory ALL and NHL patients. Turk J Hematol 2020;37:234-247.
19. Abcam. Human FVIII ELISA Kit. Cambridge, Abcam, 2020.
20. Franchini M, Mannucci PM. Hemophilia A in the third millennium. Blood Rev 2013;27:179-184.
21. Balkaransingh P, Young G. Novel therapies and current clinical progress in hemophilia A. Ther Adv Hematol 2018;9:49-61.
22. George LA, Monahan PE, Eyster ME, Sullivan SK, Ragni MV, Croteau SE, Rasko JEJ, Recht M, Samelson-Jones BJ, MacDougall A, Jaworski K, Noble R, Curran M, Kuranda K, Mingozzi F, Chang T, Reape KZ, Anguela XM, High KA. Multiyear factor VIII expression after AAV gene transfer for hemophilia A. N Engl J Med 2021;385:1961-1973.
23. Lin P, Lin Y, Lennon DP, Correa D, Schluchter M, Caplan AI. Efficient lentiviral transduction of human mesenchymal stem cells that preserves proliferation and differentiation capabilities. Stem Cells Transl Med 2012;1:886-897.
24. Picanço V, Heinz S, Bott D, Behrmann M, Covas DT, Seifried E, Tonn T. Recombinant expression of coagulation factor VIII in hepatic and non-hepatic cell lines stably transduced with third generation lentiviral vectors comprising the minimal factor VIII promoter. Cytotherapy 2007;9:785-794.
25. Doering CB, Denning G, Shields JE, Fine EJ, Parker ET, Srivastava A, Lollar P, Spencer HT. Preclinical Development of a Hematopoietic Stem and Progenitor Cell Bioengineered Factor VIII Lentiviral Vector Gene Therapy for Hemophilia A. Hum Gene Ther. 2018;29:1183-1201.

APA	Hemsinlioglu C, ASLAN E, Tastan C, Çakırsoy D, TURAN R, Seyis U, Elek M, Karakuş G, GÜNAYDIN Ö, Abanuz S, DİLEK KANÇAĞI D, yurtsever b, Yalçın K, KASAP M, Ovalı E (2023). In Vitro FVIII-Encoding Transgenic Mesenchymal Stem Cells Maintain Successful Coagulation in FVIII-Deficient Plasma Mimicking Hemophilia A. , 118 - 124. 10.4274/tjh.galenos.2023.2022-0318
Chicago	Hemsinlioglu Cansu,ASLAN ELIF SIBEL,Tastan Cihan,Çakırsoy Didem,TURAN Raife Dilek,Seyis Utku,Elek Muhammer,Karakuş Gözde Sır,GÜNAYDIN Ömür Selin,Abanuz Selen,DİLEK KANÇAĞI Derya,yurtsever bulut,Yalçın Koray,KASAP Murat,Ovalı Ercüment In Vitro FVIII-Encoding Transgenic Mesenchymal Stem Cells Maintain Successful Coagulation in FVIII-Deficient Plasma Mimicking Hemophilia A. (2023): 118 - 124. 10.4274/tjh.galenos.2023.2022-0318
MLA	Hemsinlioglu Cansu,ASLAN ELIF SIBEL,Tastan Cihan,Çakırsoy Didem,TURAN Raife Dilek,Seyis Utku,Elek Muhammer,Karakuş Gözde Sır,GÜNAYDIN Ömür Selin,Abanuz Selen,DİLEK KANÇAĞI Derya,yurtsever bulut,Yalçın Koray,KASAP Murat,Ovalı Ercüment In Vitro FVIII-Encoding Transgenic Mesenchymal Stem Cells Maintain Successful Coagulation in FVIII-Deficient Plasma Mimicking Hemophilia A. , 2023, ss.118 - 124. 10.4274/tjh.galenos.2023.2022-0318
AMA	Hemsinlioglu C,ASLAN E,Tastan C,Çakırsoy D,TURAN R,Seyis U,Elek M,Karakuş G,GÜNAYDIN Ö,Abanuz S,DİLEK KANÇAĞI D,yurtsever b,Yalçın K,KASAP M,Ovalı E In Vitro FVIII-Encoding Transgenic Mesenchymal Stem Cells Maintain Successful Coagulation in FVIII-Deficient Plasma Mimicking Hemophilia A. . 2023; 118 - 124. 10.4274/tjh.galenos.2023.2022-0318
Vancouver	Hemsinlioglu C,ASLAN E,Tastan C,Çakırsoy D,TURAN R,Seyis U,Elek M,Karakuş G,GÜNAYDIN Ö,Abanuz S,DİLEK KANÇAĞI D,yurtsever b,Yalçın K,KASAP M,Ovalı E In Vitro FVIII-Encoding Transgenic Mesenchymal Stem Cells Maintain Successful Coagulation in FVIII-Deficient Plasma Mimicking Hemophilia A. . 2023; 118 - 124. 10.4274/tjh.galenos.2023.2022-0318
IEEE	Hemsinlioglu C,ASLAN E,Tastan C,Çakırsoy D,TURAN R,Seyis U,Elek M,Karakuş G,GÜNAYDIN Ö,Abanuz S,DİLEK KANÇAĞI D,yurtsever b,Yalçın K,KASAP M,Ovalı E "In Vitro FVIII-Encoding Transgenic Mesenchymal Stem Cells Maintain Successful Coagulation in FVIII-Deficient Plasma Mimicking Hemophilia A." , ss.118 - 124, 2023. 10.4274/tjh.galenos.2023.2022-0318
ISNAD	Hemsinlioglu, Cansu vd. "In Vitro FVIII-Encoding Transgenic Mesenchymal Stem Cells Maintain Successful Coagulation in FVIII-Deficient Plasma Mimicking Hemophilia A". (2023), 118-124. https://doi.org/10.4274/tjh.galenos.2023.2022-0318

APA	Hemsinlioglu C, ASLAN E, Tastan C, Çakırsoy D, TURAN R, Seyis U, Elek M, Karakuş G, GÜNAYDIN Ö, Abanuz S, DİLEK KANÇAĞI D, yurtsever b, Yalçın K, KASAP M, Ovalı E (2023). In Vitro FVIII-Encoding Transgenic Mesenchymal Stem Cells Maintain Successful Coagulation in FVIII-Deficient Plasma Mimicking Hemophilia A. Turkish Journal of Hematology, 40(2), 118 - 124. 10.4274/tjh.galenos.2023.2022-0318
Chicago	Hemsinlioglu Cansu,ASLAN ELIF SIBEL,Tastan Cihan,Çakırsoy Didem,TURAN Raife Dilek,Seyis Utku,Elek Muhammer,Karakuş Gözde Sır,GÜNAYDIN Ömür Selin,Abanuz Selen,DİLEK KANÇAĞI Derya,yurtsever bulut,Yalçın Koray,KASAP Murat,Ovalı Ercüment In Vitro FVIII-Encoding Transgenic Mesenchymal Stem Cells Maintain Successful Coagulation in FVIII-Deficient Plasma Mimicking Hemophilia A. Turkish Journal of Hematology 40, no.2 (2023): 118 - 124. 10.4274/tjh.galenos.2023.2022-0318
MLA	Hemsinlioglu Cansu,ASLAN ELIF SIBEL,Tastan Cihan,Çakırsoy Didem,TURAN Raife Dilek,Seyis Utku,Elek Muhammer,Karakuş Gözde Sır,GÜNAYDIN Ömür Selin,Abanuz Selen,DİLEK KANÇAĞI Derya,yurtsever bulut,Yalçın Koray,KASAP Murat,Ovalı Ercüment In Vitro FVIII-Encoding Transgenic Mesenchymal Stem Cells Maintain Successful Coagulation in FVIII-Deficient Plasma Mimicking Hemophilia A. Turkish Journal of Hematology, vol.40, no.2, 2023, ss.118 - 124. 10.4274/tjh.galenos.2023.2022-0318
AMA	Hemsinlioglu C,ASLAN E,Tastan C,Çakırsoy D,TURAN R,Seyis U,Elek M,Karakuş G,GÜNAYDIN Ö,Abanuz S,DİLEK KANÇAĞI D,yurtsever b,Yalçın K,KASAP M,Ovalı E In Vitro FVIII-Encoding Transgenic Mesenchymal Stem Cells Maintain Successful Coagulation in FVIII-Deficient Plasma Mimicking Hemophilia A. Turkish Journal of Hematology. 2023; 40(2): 118 - 124. 10.4274/tjh.galenos.2023.2022-0318
Vancouver	Hemsinlioglu C,ASLAN E,Tastan C,Çakırsoy D,TURAN R,Seyis U,Elek M,Karakuş G,GÜNAYDIN Ö,Abanuz S,DİLEK KANÇAĞI D,yurtsever b,Yalçın K,KASAP M,Ovalı E In Vitro FVIII-Encoding Transgenic Mesenchymal Stem Cells Maintain Successful Coagulation in FVIII-Deficient Plasma Mimicking Hemophilia A. Turkish Journal of Hematology. 2023; 40(2): 118 - 124. 10.4274/tjh.galenos.2023.2022-0318
IEEE	Hemsinlioglu C,ASLAN E,Tastan C,Çakırsoy D,TURAN R,Seyis U,Elek M,Karakuş G,GÜNAYDIN Ö,Abanuz S,DİLEK KANÇAĞI D,yurtsever b,Yalçın K,KASAP M,Ovalı E "In Vitro FVIII-Encoding Transgenic Mesenchymal Stem Cells Maintain Successful Coagulation in FVIII-Deficient Plasma Mimicking Hemophilia A." Turkish Journal of Hematology, 40, ss.118 - 124, 2023. 10.4274/tjh.galenos.2023.2022-0318
ISNAD	Hemsinlioglu, Cansu vd. "In Vitro FVIII-Encoding Transgenic Mesenchymal Stem Cells Maintain Successful Coagulation in FVIII-Deficient Plasma Mimicking Hemophilia A". Turkish Journal of Hematology 40/2 (2023), 118-124. https://doi.org/10.4274/tjh.galenos.2023.2022-0318