A Novel Cell-Sheet Technology That Achieves Durable Factor VIII Delivery in a Mouse Model of Hemophilia A

Kohei Tatsumi,David Lillicrap,Midori Shima,Mitsuhiko Sugimoto,Hideto Matsui,Kazuo Ohashi,Teruo Okano,Toshiyuki Miyata

doi:10.1371/journal.pone.0083280

Kohei Tatsumi, David Lillicrap + Show 6 more

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https://doi.org/10.1371/journal.pone.0083280

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Abstract

Gene- or cell-based therapies aimed at creating delivery systems for coagulation factor VIII (FVIII) protein have emerged as promising options for hemophilia A treatment. However, several issues remain to be addressed regarding the efficacies and adverse events of these new classes of therapies. To improve an existing cell-based therapy involving the subcutaneous transplantation of FVIII-transduced blood outgrowth endothelial cells (BOECs), we employed a novel cell-sheet technology that allows individual dispersed cells to form a thin and contiguous monolayer without traditional bioabsorbable scaffold matrices. Compared to the traditional methodology, our cell-sheet approach resulted in longer-term and 3–5-fold higher expression of FVIII (up to 11% of normal) in recipient hemophilia A mice that lacked a FVIII humoral immune response due to transient immunosuppression with cyclophosphamide. Histological studies revealed that the transplanted BOEC sheets were structured as flat clusters, supporting the long-term expression of therapeutic FVIII in plasma from an ectopic subcutaneous space. Our novel tissue-engineering approach using genetically modified BOEC sheets could aid in development of cell-based therapy that will allow safe and effective in vivo delivery of functional FVIII protein in patients with hemophilia A.

Highlights

Hemophilia A is an inherited bleeding disorder caused by a deficiency of coagulation factor VIII (FVIII)
We previously reported that therapeutic levels of plasma FVIII can be successfully achieved in hemophilia A mice by subcutaneous implantation of lentivirally engineered blood outgrowth endothelial cells (BOECs) mixed with Matrigel [9]
Recent preclinical and clinical studies using adeno-associated viral (AAV) vectors for hemophilia B demonstrated that the safety profile is partly determined by vector dose, and that immune responses to AAV-capsid proteins with subsequent hepatocyte toxicity require transient immunosuppression in order to achieve sustained transgene expression [17,18,19,20]

Summary

Introduction

Hemophilia A is an inherited bleeding disorder caused by a deficiency of coagulation factor VIII (FVIII). Patients with hemophilia A are treated with plasma-derived or recombinant FVIII concentrates [1] This form of protein-replacement therapy has improved management of bleeding in hemophilia A patients. This method is problematic because of the requirement for frequent venous access as well as the limited availability and high costs of FVIII concentrates. In that system we observed gradual loss of plasma FVIII, probably due to breakdown of the scaffold material or cell death To overcome these issues, we employed cell-sheet technology, an innovative tissue-engineering approach that allows individual dispersed cells to form a thin and contiguous monolayer; this method has recently shown great promise in regenerative medicine [10,11]. We report a unique and effective tissue-engineering approach using BOEC sheets as a new class of potential cellbased treatment for hemophilia A

Materials and Methods

Results and Discussion

Conclusion