Evolution of biohybrid lung – Genetically modified anti-thrombogenic and immunological invisible endothelial cells used for endothelialization of gas exchange membranes for first in-vivo application

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In our previous studies we focused on the development of an implantable biohybrid lung (BH) as an alternative to lung transplantation. To support long-term application of the BH, we established the endothelialization of gas exchange membranes (GEM) to create a hemocompatible surface. Doubtless, primary autologous endothelial cells (ECs) would be the ideal cell source, but their generation for GEM endothelialization is impossible. Our generated low immunogenic MHC class I and class II silenced ECs are the optimal alternative. MHC-silenced ECs showed to be protected from MHC-dependent allogeneic cellular and humoral allogeneic immune responses. Although, silencing MHC-expression demonstrated to be an effective strategy to facilitate the use of allogeneic ECs to endothelialize the GEM and promote its use in clinical application, the risk for thrombogenic events remains a major concern. Hence, this project aims at the further refinement of the BH by genetically engineering the ECs towards deleting their thrombogenicity and protecting them against MHC and non-MHC related antibodies and allogeneic cellular immune responses. This approach will contribute to generate complete immunologically invisible ECs to MHC-related or non-related allogeneic immune responses and simultaneously prevent the initiation of thrombogenic events. For this purpose, immunologically invisible ECs will be generated by transduction with lentiviral vectors encoding for the shRNAs targeting β2-microglobulin and the class II transactivator (CIITA) and the negative complement regulatory proteins CD55 and CD59. Furthermore, they will be further transduced with vectors encoding for thrombomodulin or the serine protease S1 to enable the complete anti-thrombogenic and immunologically invisible endothelialization of the GEM. Despite the generation of these genetically modified “super-cells”, a variety of additional requirements need to be fulfilled by the EC monolayer on the GEM to ensure the needed long-term lung support, or even replace the patient’s lung function. By the successful transfer of our analyzing methods from the 2D-gas exchange foils to the clinically relevant 3D-HFM, establishment of suitable in-house coatings and 3D-HFM multi-layer applications, as well as the establishment of a small and large animal model, to test the BH application in the in-vivo setting, we will answer clinically relevant questions. By the successful implementation of this project, we will identify the best GEM coating and endothelialization procedure, which fulfills the crucial requirements for the secure and long-term applied biohybrid lung. The results generated within this proposal will pave the way for a future application of the biohybrid lung as alternative to lung transplantation as realistic final destination therapy.