Improved liver function and regeneration with A20

描述(由申请人提供)：肝细胞的坏死和凋亡是与肝损伤相关的重要病理特征。肝细胞凋亡是病毒性肝炎、缺血性肝损伤、脓毒症、胆汁淤积症的一个特征，也是暴露于乙醇、对乙酰氨基酚和细胞抑制药物等肝脏毒性物质的结果。大量肝细胞的凋亡和坏死导致暴发性肝功能衰竭。被诊断为FHF的患者中，只有14%的人通过药物治疗痊愈。原位肝移植(OLT)极大地改善了这些患者的命运(49%接受了OLT)，但37%的患者在等待OLT期间死亡。这幅黯淡的图景被肝脏独特的再生能力所平衡。肝细胞复制可在手术、病毒或化学性肝损伤后1-2周内完全恢复肝功能和肿块。我们认为，保护肝细胞免于凋亡和促进其增殖是两种可能有益于FHF的策略。我们的初步数据表明，A20促进肝细胞增殖并具有抗凋亡作用。A20是肝细胞对损伤的生理反应的一部分。A20在包括肿瘤坏死因子和脂多糖在内的促炎刺激下在肝细胞中表达上调，并具有保护肿瘤坏死因子介导的细胞凋亡的作用。将A20基因转移到小鼠肝脏可以保护小鼠免受氨基半乳糖和脂多糖(D-Gal/LPS)毒性流行性出血热模型的致死。腺病毒介导的A20在BALB/c小鼠肝脏中的表达在给药后存活率为89%，而对照组为15%-20%。根据凝血酶原时间的评估，表达A20的小鼠维持正常的肝功能，而对照组的小鼠出现严重出血。A20在肝脏中的表达可以保护肝脏免受肝大部切除(87%)所致的死亡。在这个模型中，切除87%的肝脏肿块会导致100%的死亡率。相比之下，表达A20的小鼠中有60%的小鼠存活了87%的LR，并通过肝脏中增殖细胞核(增殖细胞核抗原)阳性核的数量来评估再生能力的增强。这些结果使A20有资格成为一个关键基因，参与促进肝脏再生和促进肝细胞存活和功能，即使在面临极端的代谢需求时也是如此。这些令人鼓舞的结果促使提交了这项提案。我们的具体目标是：1)在体外解剖A20在肝细胞中抗凋亡和促增殖作用的分子基础；2)证实使用A20进行肝脏导向基因治疗将有益于FHF毒性、Fas介导和外科实验模型的影响。从基础科学的观点来看，这项体外工作将解决A20对转录因子和涉及肝细胞凋亡、激活和增殖的基因表达的影响。这应该会揭示我们对肝细胞生物学的许多未知之处，并可能导致发现新的治疗靶点。从治疗的角度来看，A20在小鼠体内FHF模型中的有益效果的验证应该为将该方法扩展到非人类灵长类动物的FHF模型以及潜在的临床应用奠定基础。用于基因转移的新的更安全和组织特异性的病毒载体的产生，以及非病毒蛋白输送到细胞的方法的发展，将促进基于A20的治疗的临床翻译。

DESCRIPTION (provided by applicant): Necrosis and apoptosis of hepatocytes are critical pathologic features associated with liver injury. Hepatocyte apoptosis is a feature of viral hepatitis, ischemic liver injury, sepsis, cholestasis, and a result of exposure to hepatotoxic substances such as ethanol, acetaminophen and cytostatic drugs. Massive hepatocyte apoptosis and necrosis result in fulminant hepatic failure (FHF). Only 14% of patients diagnosed with FHF recover with medical therapy. Orthotopic liver transplantation (OLT) has dramatically improved the fate of these patients (49% undergo OLT), yet 37% die while awaiting OLT. This gloomy picture is balanced by the unique capacity of the liver to regenerate. Hepatocyte replication leads to a full recovery of liver function and mass 1-2 weeks following surgical, viral or chemical hepatic loss. We propose that protecting hepatocytes from apoptosis and promoting their proliferation are two strategies that could beneficially impact FHF. Our preliminary data demonstrate that A20 promotes hepatocyte proliferation and is anti-apoptotic. A20 is part of the physiologic response of hepatocytes to injury. A20 is upregulated in hepatocytes by pro-inflammatory stimuli including TNF and LPS and functions to protect from TNF mediated apoptosis. Gene transfer of A20 to mice livers protects from lethality in the galactosamine and LPS (D-gal/LPS) model of toxic FHF. Adenovirus mediated expression of A20 in livers of BALB/c mice yields an 89% survival rate following administration of D-gal/LPS as compared to 15-20% in control mice. Mice expressing A20 maintain normal liver function as assessed by prothrombin time while controls suffer from a severe bleeding diathesis. Expression of A20 in the liver protects from lethality associated with a subtotal (87%) liver resection (LR). In this model, resection of 87% of the liver mass results in 100% lethality. In contrast, >60% of mice expressing A20 survive the 87% LR and demonstrate increased regenerative capacity as assessed by the number of PCNA (proliferating cell nuclear antigen) positive nuclei in the liver. These results qualify A20 as a critical gene involved in accelerating liver regeneration and promoting hepatocyte survival and function, even when facing extreme metabolic demands. These encouraging results prompted the submission of this proposal. Our specific aims are i) to dissect, in vitro, the molecular basis of the (1) anti-apoptotic and (2) pro-proliferative function of A20 in hepatocytes and ii) to confirm that liver directed gene therapy using A20 will beneficially impact upon toxic, FAS-mediated and surgical experimental models of FHF. From a basic science standpoint, the in vitro work proposed will address the effect of A20 upon transcription factors and expression of genes involved in apoptosis, activation and proliferation of hepatocytes. This should unveil many unknowns in our understanding of hepatocyte biology and could lead to the discovery of novel therapeutic targets. From a therapeutic standpoint, validation of the beneficial effect of A20 in the murine in vivo models of FHF should set the basis for extending this approach to models of FHF in non human primates and potentially to clinical applications. The generation of novel safer and tissue specific viral vectors for gene transfer and the development of non-viral means of protein delivery to cells will facilitate clinical translation of A20 based therapies.