Improved liver function and regeneration with A20

Beth Israel Deaconess Medical Center, Boston MA

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Abstract

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.