Ex-vivo liver xenoperfusion for the treatment of acute liver failure

急性肝功能衰竭是一种与相当大的发病率和死亡率相关的危重疾病，因为治疗方案很少。同种异体肝移植是这些患者最常用和最广泛的治疗方法，但由于缺乏合适的供体器官和受体的临床条件较差，其成功往往受到限制。体外肝脏异种灌流为急性肝功能衰竭患者提供了一种很有前途的治疗方法--特别是由于适当器官的无限可获得性。尽管与这种治疗策略相关的临床问题几乎与异种肝移植后已知的问题(如体液排斥、凝血障碍、血细胞隔离)相似，但仍有重要的区别：患者的S肝脏仍然保留在原位，有机会进行器官恢复，而猪肝可以提供肝功能，而不需要苛刻的手术干预。转基因肝脏的使用应该有助于克服或显著降低(免疫)屏障，以在异种体外肝脏灌流期间允许足够的器官功能，最终使患者的S肝脏能够恢复(桥到恢复)或作为桥到移植。该项目旨在优化体外肝脏异种灌流治疗急性肝功能衰竭。通过利用猪肝建立体外灌流电路，通过敲除与异种反应性有关的猪关键基因并引入人类基因，结合基于抗炎吸附的干预，将建立临床上可行的灌流方案。为此，将分别对自体和异种对照实验进行猪和人的全血灌流。在规定的时间点从灌流回路中获取血液样本，以确定S肝脏的解毒和合成潜力、凝血状态以及免疫激活状态。此外，还将使用组织样本进行组织病理学和免疫组织化学分析，以及用于评估免疫球蛋白沉积、补体成分和内皮激活标记的实时定量聚合酶链式反应。在灌流过程中将记录肝脏的一般功能参数(即动脉和门静脉血流灌注、血管阻力、胆汁生成和器官存活)。为了确定最佳的遗传修饰，将在不同的实验环境中进行灌流实验。最后，最好的整体敲除组合将与多个转基因相结合。

Acute liver failure is a critical medical condition associated with considerable morbidity and mortality since therapeutic options are scarce. Allogeneic liver transplantation is the most commonly and widely used treatment for these patients, but success is often restricted due to the lack of suitable donor organs and the poor clinical condition of the recipients. Ex-vivo liver xenoperfusion represents a promising therapeutic approach in patients suffering from acute liver failure – especially due to the advantage of unlimited availability of appropriate organs. Although the clinical problems associated with this treatment strategy are almost similar to those known after xenogeneic liver transplantation (e.g. humoral rejection, coagulopathy, blood cell sequestration), there are important differences: The patient´s liver remains in place with the opportunity for organ recovery and the porcine liver can provide hepatic function without the risk of a demanding surgical intervention. The use of genetically modified livers should help to overcome or significantly lower the (immunological) barriers to allow sufficient organ function during xenogeneic extracorporeal liver perfusion, eventually enabling recovery of the patient´s liver (bridge-to-recovery) or as bridge-to-transplantation.This project aims to optimize ex-vivo liver xenoperfusion for the treatment of acute liver failure. A clinically feasible perfusion protocol will be established by setting up an ex-vivo perfusion circuit using porcine livers with targeted intervention by knock-out of porcine key genes regarding xenoreactivity and introduction of human genes in combination with an anti-inflammatory adsorber-based intervention. For this, perfusions with whole blood of porcine and human origin will be performed for autologous and xenogeneic control experiments, respectively. Blood samples will be obtained from the perfusion circuit at defined time points to determine the liver´s detoxification and synthesis potential, the coagulatory state as well as the state of immune activation. Additionally, histopathological and immunohistochemical analyses as well as real-time quantitative PCR for assessment of immunoglobulin deposits, complement components and markers of endothelial activation, will be performed using tissue samples. General functional parameters of the liver will be recorded during perfusion (i.e. arterial and portovenous perfusion, vascular resistance, bile production and organ survival). To identify the optimal genetic modification, perfusion experiments will be carried out in different experimental settings. Finally, the best overall combination of knock-outs will be combined with multiple transgenes.