Improved liver function and regeneration with A20

描述（由申请方提供）：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、脓毒症、胆汁淤积的特征，也是暴露于肝毒性物质如乙醇、对乙酰氨基酚和细胞生长抑制药物的结果。暴发性肝功能衰竭（FHF）是由肝细胞大量凋亡和坏死引起的。只有14%的FHF患者通过药物治疗康复。原位肝移植（奥尔特）极大地改善了这些患者的命运（49%接受奥尔特），但37%在等待奥尔特时死亡。肝脏再生的独特能力平衡了这种悲观的景象。肝细胞复制导致手术、病毒或化学性肝损失后1-2周肝功能和质量完全恢复。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影响FHF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是肝细胞对损伤的生理反应的一部分。A20在肝细胞中被促炎刺激物（包括TNF和LPS）上调，并起到保护免于TNF介导的细胞凋亡的作用。A20基因转移到小鼠肝脏保护免受半乳糖胺和LPS（D-gal/LPS）毒性FHF模型的致死性。腺病毒介导的A20在BALB/c小鼠肝脏中的表达在给予D-gal/LPS后产生89%的存活率，而对照小鼠中的存活率为15-20%。表达A20的小鼠通过凝血酶原时间评估维持正常的肝功能，而对照组患有严重的出血素质。A20在肝脏中的表达保护免于与次全（87%）肝切除（LR）相关的致死性。在该模型中，切除87%的肝脏质量导致100%的致死率。相比之下，>60%的表达A20的小鼠在87%LR中存活，并且表现出增加的再生能力，如通过肝脏中PCNA（增殖细胞核抗原）阳性核的数量所评估的。这些结果使A20成为一个关键基因，参与加速肝再生和促进肝细胞存活和功能，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一建议。我们的具体目标是i）在体外剖析A20在肝细胞中的（1）抗凋亡和（2）促增殖功能的分子基础，和ii）证实使用A20的肝定向基因治疗将有益地影响FHF的毒性、FAS介导和手术实验模型。从基础科学的角度来看，提出的体外工作将解决A20对转录因子和参与肝细胞凋亡，激活和增殖的基因表达的影响。这将揭开我们对肝细胞生物学理解中的许多未知，并可能导致发现新的治疗靶点。从治疗的角度来看，A20在FHF小鼠体内模型中的有益作用的验证应该为将这种方法扩展到非人灵长类动物的FHF模型和潜在的临床应用奠定基础。用于基因转移的新型更安全和组织特异性病毒载体的产生以及蛋白质递送至细胞的非病毒手段的开发将促进基于A20的疗法的临床转化。

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.