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

急性肝功能衰竭是一种严重的医疗状况，与相当高的发病率和死亡率相关，因为治疗选择很少。同种异体肝移植是这些患者最常见和最广泛使用的治疗方法，但由于缺乏合适的供体器官和受者的临床状况不佳，成功往往受到限制。体外肝脏异种灌流是治疗急性肝功能衰竭患者的一种很有前途的方法，特别是由于合适器官的无限可用性的优势。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相似（例如，体液排斥反应、凝血功能障碍、血细胞隔离），但有重要的区别：患者的肝脏保留在原位，有机会进行器官恢复，而猪肝可以提供肝功能，而无需进行高要求的手术干预。使用转基因肝脏应有助于克服或显著降低（免疫）障碍，以在异种体外肝脏灌注期间允许足够的器官功能，最终使患者的肝脏恢复（桥至恢复）或作为桥至移植。本项目旨在优化体外肝脏异种灌流治疗急性肝衰竭。建立猪肝离体灌注回路，敲除猪异种反应性关键基因，引入人类基因，结合抗炎吸附剂干预，建立临床可行的灌注方案。为此，将分别用猪全血和人全血灌注进行自体对照实验和异种对照实验。将在规定的时间点从灌注回路中获得血液样本，以确定肝脏的解毒和合成电位、凝血状态以及免疫激活状态。此外，组织病理学和免疫组织化学分析以及实时定量PCR用于评估免疫球蛋白沉积，补体成分和内皮活化标志物，将使用组织样本进行。在灌注过程中记录肝脏的一般功能参数（即动脉和门静脉灌注、血管阻力、胆汁生成和器官存活）。为了确定最佳的基因修饰，将在不同的实验环境下进行灌注实验。最后，敲除的最佳整体组合将与多个转基因组合。

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.