Jianfei Xie 2015.07.19

1. Jianfei Xie 2015.07.19 Hemophilia A

2. Introduction Hemophilia A is an X-linked recessive bleeding disorder (Morvarid Moayeri et al. 2004).

3. Introduction Hemophilia A is characterized by inability to clot blood because of FVIII gene mutations and deficiency of this coagulation factor (Follenzi et al. 2012). 3D structure of FVIII, PDB ID: 2R7E

4. FVIII deficiency results in a severe bleeding phenotype leading to death, significant illness (Yi Lin et al. 2002). Joint injury Severe bleeding

5. Hemophilia A affects one in 5000 males, or about 400,000 individuals worldwide (Mannucci et al. 2014).

6. Treatment protocols 1. FVIII injection 2. Gene therapy 3. Cell therapy

7. FVIII injection Currently, Hemophilia A is treated by administration of plasma derived or recombinant FVIII, but this strategy is complicated by the development of inhibitory antibodies (Diego Zanolini et al. 2015). These so-called inhibitors may jeopardize the patient’s life and make therapeutic management more complex, and costly(Thierry Calvez et al. 2014). Inhibitors

8. FVIII Modification [1] Recombinant canine B-domain deleted FVIII exhibits high specific activity and is safe in the canine hemophilia A model. Denise et al. Blood. 2009. [2] Minimal modification in the factor VIII B-domain sequence ameliorates the murine hemophilia A phenotype. Joshua et al. Blood. 2013. [3] Reduction of the inhibitory antibody response to human factor VIII in hemophilia A mice by mutagenesis of the A2 domain B-cell epitope. Ernest et al. Blood. 2004 [4] Noncovalent stabilization of the factor VIII A2 domain enhances efficacy in hemophilia A mouse vascular injury models.Lilley Leong et al. Blood. 2014.

9. Gene therapy Naked DNA transfer of Factor VIII induced transgene-specific, species-independent immune response in hemophilia A mice. Mol Ther. Ye et al. 2004. FVIII disappearance correlated with the generation of high-titer, inhibitory anti-FVIII antibodies. Inhibitors

10. Cell therapy The goal of cell therapy is to introduce long-term expression of therapeutic levels of FVIII in vivo resulting in a cure of the disease .

11. Where is factor VIII synthesized? Controversial ! Hepatic and Extrahepatic • Hepatic • Liver sinusoidal endothelial cells (LSEC): Main source • (Everett LA et al, 2014; Fahs SA et al, 2014; Diego Zanolini et al, 2015) • 2. Hepatocytes and Küpffer cells: Low level • (Diego Zanolini et al 2015)

12. Extrahepatic 1. Peripheral or cord blood derived cells, including: (1) Endothelial progenitor/endothelial cells. (2) Monocytes, macrophages and megakaryocytes from hematopoietic stem cells. 2. Bone marrow derived cells, including Monocytes, macrophages and endothelial cells. These extrahepatic cells could synthesize factor VIII in sufficient amount to ameliorate the bleeding phenotype in hemophilic mice (Diego Zanolini et al 2015)

13. Cell therapy protocols: Hepatic and Extrahepatic Therapy Model: 1. Mouse to mouse 2. Human to mouse 3. Human to human

14. Mouse to mouse [1] Transplanted endothelial cells repopulate the liver endothelium and correct the phenotype of hemophilia A mice. Antonia Follenzi et al, 2008. [2] Transplantation of endothelial cells corrects the phenotype in hemophilia A mice. V. Kumaran et al. 2005. [3] Factor VIII Can Be Synthesized in Hemophilia A Mice Liver by Bone Marrow Progenitor Cell-Derived Hepatocytes and Sinusoidal Endothelial Cells. NeelamYadav. 2012. [4] Role of bone marrow transplantation for correcting hemophilia A in mice. Antonia Follenzi. Thrombosis and hemostasis. 2012. [5] The therapeutic effect of bone marrow–derived liver cells in the phenotypic correction of murine hemophilia A. NeelamYadav. Thrombosis and hemostasis. 2009. [6] Phenotypic correction of murine hemophilia A using an IPS cell-based therapy. Dan Xu. 2009 [7] Sustained phenotypic correction of hemophilia a mice following oncoretroviral-mediated expression of a bioengineered human factor VIII gene in long-term hematopoietic repopulating cells. The Journal of Gene Medicine. Jiro Kikuchi. 2004.

15. Human to mouse Hepatic • [1] Production of Factor VIII by Human Liver Sinusoidal Endothelial Cells Transplanted in Immunodeficient uPA Mice. Marina E. Fomin. 2013. • [2] Human hepatocyte propagation system in the mouse livers functional maintenance of the production of coagulation and anticoagulation factors. Cell Transplantation. Kohei Tatsumi et al. 2012. • uPA/SCID mice model • This mice model have a feature to develop an active damage of their own hepatic parenchymal cell, provide a hepatic environment that is more conductive to the engraftment and proliferation of human cells (Tateno et al. 2004; Kohei Tatsumi et al. 2012).

16. Human to mouse Extrahepatic (4 papers) 1. Use of blood outgrowth endothelial cells for gene therapy for hemophilia A. Yi Lin et al. Blood. 2002 Human venous blood  Mononuclear  Endothelial cells Transduced with GFP-FVIII expression vector NOD/SCID mice model

17. Yi Lin et al. Blood. 2002

18. Yi Lin et al Blood. 2002

19. Human to mouse Extrahepatic 2. Sustained transgene expression by human cord blood derived CD34+ cells transduced with simian immunodeficiency virus agmTYO1-based vectors. Kikuchi et al. J Gene Med. 2004. NOD/SCID mice Human cord blood CD34+ cells Transduced with GFP-FVIII expression vector NOD/SCID mice model

20. Presence of human CD 45+ cells Kikuchi et al. J Gene Med. 2004

21. Plasma FVIII levels in transplanted NOD/SCID mice Kikuchi et al. J Gene Med. 2004

22. Expression of lineage markers in human CD45+ cells and detection of human CD41+ platelets . Kikuchi et al. J Gene Med. 2004.

23. Human to mouse • Extrahepatic • 3. Extrahepatic sources of factor VIII potentially contribute to the coagulation cascade correcting the bleeding phenotype of mice with hemophilia A. Diego Zanolini. Haematologica. 2015 • Human cord blood CD34+ cells • NOD/SCID-γNull Hemophilia A mice (NSG-HA) model • Superior for transplanting human cells (Ito M et al, 2002)

24. Diego Zanolini. Haematologica. 2015

25. Diego Zanolini. Haematologica. 2015

26. Expression of FVIII in human Liver sinusoidal endothelial cells Diego Zanolini. Haematologica. 2015

27. Quantitative PCR showing FVIII mRNA expression in human liver and isolated human LSEC, KC and hepatocytes Diego Zanolini. Haematologica. 2015

28. Diego Zanolini. Haematologica. 2015

29. Transplanted human cells were identified in mouse blood by cytofluorimetry for human CD45 marker Diego Zanolini. Haematologica. 2015.

30. Human chimerism in BM and spleen of transplanted mice Diego Zanolini. Haematologica. 2015.

31. FVIII activity in mice transplanted with human CD34+ cells Diego Zanolini. Haematologica. 2015.

32. Three months after transplantation mice were challenged with tail clipping and nine out of 12 (75%) survived Diego Zanolini. Haematologica. 2015.

33. Human to mouse Extrahepatic 4. Platelet gene therapy corrects the hemophilic phenotype in immunocompromised hemophilia A mice transplanted with genetically manipulated human cord blood stem cells. Qizhen Shi. Blood. 2014. Human cord blood CD34+ cells  Transduced with 2bF8LV vector FVIII expression is under the control of the platelet-specific glycoprotein IIb promoter (2bF8).  NOD/SCID-γNull Hemophilia A mice (NSG-HA) model

34. Human cell chimerism in mice Qizhen Shi. Blood. 2014.

35. Human cell chimerism in mice Qizhen Shi. Blood. 2014.

36. Analysis of platelet FVIII expression in mice Qizhen Shi. Blood. 2014.

37. Survival rate after tail chipping Qizhen Shi. Blood. 2014.

38. Human to human [1] Cord blood hematopoietic stem cell transplantation in an adolescent with haemophilia. Caselli D. Haemophilia. 2012;18(2): 48-49. The authors described that during follow-up the FVIII levels in the plasma of this patient were increased and that, on the basis of the international classification, the degree of his coagulation defect prior to transplantation qualified him as a severe case whereas after transplant he should be defined as having moderate hemophilia. [2] Allogeneic bone marrow transplantation in a child with severe aplastic anemia and hemophilia A. Ostronoff M. Bone Marrow Transplant. 2006;37(6):627-628. The FVIII level did not change after transplantation, suggesting that bone marrow does not contribute significantly to FVIII production

39. Our Research Preclinical Therapy of Expanded and Differentiated Endothelial Progenitor Cells from Human and Non-human Primates Related articles: [1] Sustained Expansion and Transgene Expression of Coagulation Factor VIII-Transduced Cord Blood-Derived Endothelial Progenitor Cells. Christian Herder et al. Arterioscler Thromb Vasc Biol. 2003. [2] Storage and regulated secretion of factor VIII in blood outgrowth endothelial cells Biggelaar et al. Haematologica 2009. (1) In these two research human cells were transduced with a lentivirus encoding FVIII-GFP. (2) These two papers did not have mouse model.

40. Next key content 1. Human FVIII detection in mice. 2. Survival rate after tail chipping. My question Can Endothelial Progenitor/Endothelial Cells transplant in liver without the Liver sinusoidal endothelial cells injury step?

41. Protocol 1. Obtain immunodeficient hemophilia A mice 2. Immunosuppression (immunosuppressive reagent or T cells) [1] Immunomodulation of transgene responses following naked DNA transfer of human factor VIII into hemophilia A mice. Carol H. Miao. Blood 2006. [2] Donor antigen-primed regulatory T cells permit liver regeneration and phenotype correction in hemophilia A mouse by allogeneic bone marrow stem cells. Kochat V. Stem Cell Res Ther. 2015 3. Encapsulation [1] Encapsulated human primary myoblasts deliver functional hFIX in hemophilic mice. J Gene Med. Jianping Wen. 2007. [2] Delivery of human factor IX in mice by encapsulated recombinant myoblasts a novel approach towards allogeneic gene therapy of hemophilia B. Hortelano et al. Blood. 1996.

42. Thank you very much!