In-vitro generation of modified platelets with enhanced function from induced pluripotent stem cells

• Applicants: Professorin Dr. Ute Modlich, Ph.D.; Professor Dr. Thomas Moritz
• Subject Area: Hematology, Oncology ; Cardiology, Angiology
• Term: from 2016 to 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 316622279

Platelets (thrombocytes) exert many of their functions in hemostasis, inflammation and angiogenesis by the controlled secretion of proteins and small molecules stored in their granules, rendering platelets an interesting target for the controlled delivery of therapeutic proteins. The therapeutic proteins may be loaded into the platelet granules, where they are shielded from the immune system and will be delivered locally upon platelet activation. The functionality of such platelets has been established for the treatment of factor VIII deficiency and the transplantation of gene modified hematopoietic stem cells, but many obstacles to a more general application remain. Thus, to modify platelets genetically, the procedure has to take place in a nucleated precursor cell, ideally a self-renewing induced pluripotent stem cells (iPSC) to test the efficacy and safety of the intervention on a clonal level. However, the in vitro generation of platelets from iPSCs, although principally feasible, is inefficient and transgene expression during this process is problematic. We therefore here propose to establish a tool-box for the in vitro generation of designer platelets with enhanced functionality (EF-platelets). Specifically we aim (i) to improve the in vitro differentiation of platelets from human iPSCs. We will modify the process by the ectopic expression of megakaryocytic transcription factors and modulators of platelet release. (ii) For genetic modification of human iPSCs we will establish vectors allowing for the controlled and sustained expression of transgenes applying different strategies to overcome silencing. (iii) Finally, we will modify candidate therapeutic proteins with targeting domains of granule proteins for granule-specific targeting. In our initial proof-of-concept studies the angiopoietins and their receptors as soluble or membrane-bound, dominant-negative form will serve as therapeutic proteins. In vitro generated platelets will be extensively characterized for phenotype and function including the evaluation in vivo mouse models. Our studies have wide-spread implications for biotechnology as well as basic medical research and will provide valuable insights into the control of gene/transgene expression in pluripotent cells and their progeny as well as into the regulation of hematopoiesis and megakaryopoiesis. The modification of platelet function will allow to study the role of platelets in disease states such as intravascular thrombus or plaque formation, overshooting or deficient vascularization, tumor growth, or inflammation and will offer new tools to interfere with these processes.

• DFG Programme: Research Grants