Improved liver function and regeneration with A20

描述（申请人提供）：肝细胞坏死和凋亡是与肝损伤相关的关键病理特征。肝细胞凋亡是病毒性肝炎、缺血性肝损伤、败血症、胆汁淤积以及暴露于肝毒性物质（如乙醇、对乙酰氨基酚和细胞抑制药物）的结果。大量肝细胞凋亡和坏死导致暴发性肝衰竭（FHF）。诊断为FHF的患者中只有14%通过药物治疗得以康复。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。肝脏独特的再生能力平衡了这种令人沮丧的局面。肝细胞复制导致手术、病毒性或化学性肝丧失后1-2周肝功能和肿块的完全恢复。我们提出保护肝细胞免于凋亡和促进其增殖是两种可能对FHF有益的策略。我们的初步数据表明，A20促进肝细胞增殖和抗凋亡。A20是肝细胞对损伤生理反应的一部分。A20在肝细胞中通过TNF和LPS等促炎刺激上调，并起保护TNF介导的细胞凋亡的作用。在半乳糖胺和LPS （D-gal/LPS）模型中，A20基因转移到小鼠肝脏可保护毒性FHF的致死性。腺病毒介导的A20在BALB/c小鼠肝脏中的表达在给予D-gal/LPS后获得89%的存活率，而对照组小鼠的存活率为15-20%。通过凝血酶原时间评估，表达A20的小鼠肝功能维持正常，而对照组则出现严重出血。肝脏中A20的表达保护了与次全肝切除（87%）相关的致死性。在这个模型中，切除87%的肝肿块导致100%的死亡率。相比之下，60%的表达A20的小鼠在87%的LR中存活下来，并且通过肝脏中增殖细胞核抗原（PCNA）阳性细胞核的数量来评估其再生能力。这些结果证明A20是加速肝脏再生和促进肝细胞存活和功能的关键基因，即使在面临极端代谢需求时也是如此。这些令人鼓舞的结果促使提出了这项建议。我们的具体目标是i)在体外解剖（1)肝细胞中A20抗凋亡和(2)促增殖功能的分子基础；ii）确认使用A20进行肝定向基因治疗将对FHF的毒性、fas介导和手术实验模型产生有益影响。从基础科学的角度来看，提出的体外工作将解决A20对肝细胞凋亡、活化和增殖相关转录因子和基因表达的影响。这将揭示我们对肝细胞生物学的理解中的许多未知因素，并可能导致新的治疗靶点的发现。从治疗的角度来看，验证A20在小鼠体内FHF模型中的有益作用应该为将该方法扩展到非人灵长类动物的FHF模型以及潜在的临床应用奠定基础。用于基因转移的新型更安全、组织特异性的病毒载体的产生，以及向细胞递送蛋白质的非病毒手段的发展，将促进基于A20的治疗方法的临床转化。

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.