以重組腺相關病毒載體在肝臟表現自體抗原PDC-E2來治療原發性膽管硬化症

Abstract

中文摘要 原發性膽管硬化症是慢性且進程緩慢的肝臟自體免疫疾病，患者肝臟的門脈三角出現自體反應T淋巴球浸潤以及肝內膽管遭受破壞，抗粒線體抗體的產生為原發性膽管硬化症的指標，大約95%的患者體內可測得此抗體。由於目前治療方法的治療效果相當有限，因此發展有效的治療方式為當務之急。 先前研究已知，患者的自體反應T淋巴球和抗粒線體抗體以位於粒線體基質的2-oxo-acid dehydrogenase enzyme complex (2-OADC) 家族為攻擊目標，而E2 subunit of pyruvate dehydrogenase complex (PDC-E2) 為主要的自體抗原。從已知患者與小鼠模式的研究結果，推測對PDC-E2自體抗原失去免疫耐受性是原發性膽管硬化症產生的原因。 肝臟為一個傾向引發免疫耐受性的淋巴器官，是因為其具有對外來抗原產生免疫耐受性的特殊抗原呈現細胞，因此我們假設以具有肝臟特異性的腺相關病毒載體，攜帶有肝臟侷限性表現的PDC-E2，能夠回復Xenobiotic引發原發性膽管硬化症的小鼠的免疫耐受性， 我們構築腺相關病毒載體，其攜帶具有肝臟侷限性於粒腺體或細胞膜表現的PDC-E2或truncated PDC-E2，而transgene的表現以及在細胞的表現位置已經在細胞和小鼠體內被證實。在動物實驗中，我們發現PDC-E2和膜結合PDC-E2幾乎沒有抑制Xenobiotic引發anti-PDC-E2抗體產生的效果，相反地，膜結合truncated PDC-E2明顯抑制重組PDC-E2蛋白免疫引發anti-PDC-E2抗體產生的效果，此結果為將來探討小鼠模式中病理反應奠定基礎。 基於我們目前的發現，在未來的實驗中，將專注於探討膜結合truncated PDC-E2是否能減緩Xenobiotic引發的病理現象的研究。

Primary biliary cirrhosis (PBC) is a chronic, slow progressive liver autoimmune disease. It is characterized by the infiltration of auto-reactive T cells in portal tracts and the destruction of the intrahepatic bile ducts. Anti-mitochondrial antibodies (AMA), the hallmark of PBC, are detected in approximately 95% of patients. Since current therapy of PBC has been disappointing, it is of an urgent need to develop an more effective therapy for PBC. Considerable data shown, the auto-reactive T cells and AMAs of PBC patients target members of the 2-oxo-acid dehydrogenase enzyme complexes (2-OADC) which are located in the inner mitochondrial matrix. Among them, the major autoantigen is the E2 subunit of pyruvate dehydrogenase complex (PDC-E2) . Indeed, data in patients and in PBC animal models have shown that break of tolerance to PDC-E2 causes pathology of PBC. Liver is a lymphoid organ favoring induction of immune tolerance, which may be correlated with the tolerogenic properties of hepatic antigen presenting cells. We hypothesized that hepatotropic adeno-associated virus delivery and hepatocyte-restricted PDC-E2 expression could restore immune tolerance in Xenobiotic induced PBC model. We constructed AAV vectors expressing hepatocyte restricted- PDC-E2 or truncated PDC-E2 in mitochondria or on cell membrane. Expression and cellular localization of the transgene was confirmed in vitro and in vivo. In animal experiments, we found native and membrane-bound PDC-E2 had little effect on 2OA-BSA-induced anti-PDC-E2 antibodies. In contrast, the membrane-bound form of truncated PDC-E2 greatly improved its surface expression and significantly suppressed anti-PDC-E2 antibodies induced by recombinant PDC-E2 immunization. The finding lay the foundation for investgating the influence of PBC pathology by hepatic expression PDC-E2. Based on our current finding, we will focus on whether PBC pathology can be alleviated by hepatic expression of membrane-bound truncated PDC-E2.