PDF(Pubmed)
Abstract：
Heart transplantation is associated with major hurdles, including the limited number of available organs for transplantation, the risk of rejection due to genetic discrepancies, and the burden of immunosuppression. In this study, we demonstrated the feasibility of permanent genetic engineering of the heart during ex vivo perfusion. Lentiviral vectors encoding for short hairpin RNAs targeting beta2-microglobulin (shβ2m) and class II transactivator (shCIITA) were delivered to the graft during two hours of normothermic EVHP. Highly efficient genetic engineering was indicated by stable reporter gene expression in endothelial cells and cardiomyocytes. Remarkably, swine leucocyte antigen (SLA) class I and SLA class II expression levels were decreased by 66% and 76%, respectively, in the vascular endothelium. Evaluation of lactate, troponin T, and LDH levels in the perfusate and histological analysis showed no additional cell injury or tissue damage caused by lentiviral vectors. Moreover, cytokine secretion profiles (IL-6, IL-8, and TNF-α) of non-transduced and lentiviral vector-transduced hearts were comparable. This study demonstrated the ex vivo generation of genetically engineered hearts without compromising tissue integrity. Downregulation of SLA expression may contribute to reduce the immunogenicity of the heart and support graft survival after allogeneic or xenogeneic transplantation.
摘要：
心脏移植与主要障碍有关，包括可供移植的器官数量有限，由于遗传差异而导致排斥的风险，和免疫抑制的负担。在这项研究中,我们证明了在离体灌注期间对心脏进行永久性基因工程的可行性。在常温EVHP的两个小时内，将编码靶向β2-微球蛋白（shβ2m）和II类反式激活因子（shCIITA）的短发夹RNA的慢病毒载体输送到移植物。在内皮细胞和心肌细胞中稳定表达的报告基因表明了高效的基因工程。值得注意的是,猪白细胞抗原(SLA)Ⅰ类和SLAⅡ类的表达水平分别下降了66%和76%,分别,在血管内皮。乳酸的评价，肌钙蛋白T,灌注液中的LDH水平和组织学分析显示，没有由慢病毒载体引起的其他细胞损伤或组织损伤。此外，未转导和慢病毒载体转导的心脏的细胞因子分泌谱（IL-6，IL-8和TNF-α）相当。这项研究证明了在不损害组织完整性的情况下离体产生基因工程心脏。SLA表达的下调可能有助于降低心脏的免疫原性并支持同种异体或异种移植后的移植物存活。
