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

在我们以前的研究中，我们专注于开发可植入的生物混合肺（BH）作为肺移植的替代方案。为了支持BH的长期应用，我们建立了气体交换膜（GEM）的内皮化，以创建血液相容性表面。毫无疑问，原代自体内皮细胞（EC）将是理想的细胞来源，但它们的生成GEM内皮化是不可能的。我们产生的低免疫原性MHC I类和II类沉默EC是最佳的替代方案。MHC沉默的内皮细胞显示出对MHC依赖的同种异体细胞和体液同种异体免疫应答的保护。 尽管沉默MHC表达被证明是促进同种异体EC内皮化GEM并促进其在临床应用中的使用的有效策略，但血栓形成事件的风险仍然是一个主要问题。因此，该项目旨在通过基因工程改造EC以消除其血栓形成性并保护其免受MHC和非MHC相关抗体和同种异体细胞免疫应答的影响来进一步完善BH。这种方法将有助于产生完整的免疫学上不可见的EC，以MHC相关或非相关的同种异体免疫应答，同时防止血栓形成事件的发生。为此，将通过用编码靶向β2-微球蛋白和II类反式激活因子（CIITA）的shRNA以及阴性补体调节蛋白CD 55和CD 59的慢病毒载体转导来产生免疫学上不可见的EC。此外，它们将进一步用编码血栓调节蛋白或丝氨酸蛋白酶S1的载体转导，以实现GEM的完全抗血栓形成和免疫学上不可见的内皮化。尽管产生了这些转基因“超级细胞”，但GEM上的EC单层还需要满足各种额外要求，以确保所需的长期肺部支持，甚至取代患者的肺功能。通过将我们的分析方法从2D气体交换箔成功转移到临床相关的3D-HFM，建立合适的内部涂层和3D-HFM多层应用，以及建立小型和大型动物模型，以测试BH在体内环境中的应用，我们将回答临床相关问题。通过该项目的成功实施，我们将确定最佳的GEM涂层和内皮化程序，这满足了安全和长期应用的生物混合肺的关键要求。该提案中产生的结果将为生物混合肺作为肺移植的替代品作为现实的最终目的地治疗的未来应用铺平道路。

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.