Gene therapy of alcoholic liver disease

摘要 甘油三酯（TG）代谢异常是脂肪肝、动脉粥样硬化， 肥胖和糖尿病，所有这些都是世界范围内的主要临床问题。ApoAV是一个新的成员， 它是载脂蛋白家族的一员，仅在肝脏中合成。一个经常被忽视的事实是， 人血浆中apoAV的浓度极低（114-258 ng/mL），比人血浆中apoAV的浓度低1，000 - 10，000倍。 apoB 100和apoAI的表达。多年来，研究人员一直对一个非常低的 apoAV的循环水平可以对人和动物的血浆TG稳态产生如此深远的影响。 因为约20%的apoAV分泌到循环中，所以其在肝脏中的浓度显著高于 在血浆中。我们的初步数据表明apoAV在调节肝脏TG中起着关键作用 乙醇破坏肝脏TG稳态，并加剧TG的积累。 肝脏中过量的TG，导致apoAV敲除（KO）中更严重的酒精性肝病（ALD） 野生型（WT）小鼠。我们进一步发现，乙醇在一定剂量下抑制apoAV的表达- 在小鼠原代肝细胞中的依赖性方式。慢性和酗酒的乙醇喂养（即，该研究所 模型）持续4周导致肝脏脂肪变性的显著和快速发展，逐渐从 在apoAV KO小鼠中，单纯性脂肪变性变为酒精性脂肪性肝炎，然后变为肝纤维化，但WT小鼠没有。更 重要的是，乙醇显著增加肝脏溶血磷脂酰胆碱（lysoPC）的浓度， 脂肪酸代谢物，通过增强其在apoAV KO小鼠中的生物合成。最近的一项人类研究发现， lysoPC是过量饮酒的一种新的重要生物标志物;然而，其在发病机制中的重要作用 目前，ALD还不清楚。相比之下，人APOAV基因在小鼠中的转基因表达可以保护小鼠免受 酒精性肝损伤我们的研究结果表明，apoAV的缺乏是一个重要的危险因素， ALD和apoAV是预防和治疗ALD的新靶点。因此，我们的初步结果 支持腺相关病毒2/8（AAV 2/8）介导的人类基因转移的假设， APOAV在乙醇喂养的小鼠中保护免于ALD。在常用的病毒载体中，AAV产生 最低的免疫应答，并且即使在野生型状态下也是非致病性的，以及是最合适的病毒 用于治疗应用的载体。AAV 2/8（即，包装AAV-2基因组的AAV-8假型 AAV-8衣壳）显示出高效的肝脏基因转移。因此，AAV 2/8是一种很有前途的方法， ALD的基因治疗我们将在两个具体目标中检验这一假设：首先，我们将调查 APOAV的过表达在用AAV 2/8转导的小鼠中保护乙醇诱导的肝脂肪变性。 APOAV。第二，我们将探索AAV 2/8-APOAV治疗是否可以预防乙醇触发的进展。 从单纯性脂肪变性到酒精性脂肪性肝炎。拟议的研究是创新的 为ALD的基因治疗提供了一种有效的策略。

Abstract Abnormalities in triglyceride (TG) metabolism are a critical risk factor for fatty liver disease, atherosclerosis, obesity and diabetes, all of which are major clinical problems worldwide. ApoAV is a new member of the apolipoprotein family and is synthesized exclusively in the liver. A fact that is often neglected is that the concentration of apoAV in human plasma is extremely low (114-258 ng/mL), 1,000 to 10,000-fold lower than that of apoB100 and apoAI, respectively. It has fascinated investigators for many years how a very low circulating level of apoAV can exert such a profound effect on plasma TG homeostasis in humans and animals. Because ~20% of apoAV is secreted into the circulation, its concentration is significantly higher in the liver than in plasma. Our preliminary data show that apoAV plays a critical role in regulating hepatic TG metabolism, and ethanol disrupts hepatic TG homeostasis and exacerbates the accumulation of excess TG in the liver, leading to more severe alcoholic liver disease (ALD) in apoAV knockout (KO) mice than in wild-type (WT) mice. We further found that ethanol inhibits expression of apoAV in a dose- dependent manner in the mouse primary hepatocytes. Chronic and binge ethanol feeding (i.e., the NIAAA model) for 4 weeks leads to a significant and rapid development of liver steatosis, gradually evolving from simple steatosis to alcoholic steatohepatitis and then to liver fibrosis in apoAV KO, but not WT mice. More importantly, ethanol significantly increases hepatic concentrations of lysophosphatidylcholine (lysoPC), a major fatty acid metabolite, by enhancing its biosynthesis in apoAV KO mice. A recent human study has found that lysoPC is a new and important biomarker for excess alcohol intake; however, its vital role in the pathogenesis of ALD is still unknown. By contrast, transgenic expression of the human APOAV gene in mice protects against ethanol-induced liver injury. Our results demonstrate that the absence of apoAV is an important risk factor for ALD and apoAV is a novel target for the prevention and the treatment of ALD. Thus, our preliminary results support the hypothesis that adeno-associated-virus 2/8 (AAV2/8)-mediated gene transfer of the human APOAV protects against ALD in ethanol-fed mice. Of the commonly used viral vectors, AAV produces the lowest immune response and is non-pathogenic even in the wild-type state, as well as is the most suitable viral vector for therapeutic applications. AAV2/8 (i.e., AAV-8 pseudotypes in which AAV-2 genomes are packaged into AAV-8 capsids) displays highly efficient liver gene transfer. Therefore, AAV2/8 is a promising method for gene therapy of ALD. We will test the hypothesis in two specific aims: First, we will investigate whether overexpression of APOAV protects against ethanol-induced liver steatosis in mice transduced with AAV2/8- APOAV. Second, we will explore whether AAV2/8-APOAV treatment prevents ethanol-triggered progression from simple steatosis to alcoholic steatohepatitis through lysoPC in mice. The proposed studies are innovative because this project may provide an efficacious strategy for the gene therapy of ALD.