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ApoAV的新成员 载脂蛋白家族，仅在肝脏中合成。一个经常被忽视的事实是， 人血浆中载脂蛋白病毒的浓度极低(114-258 ng/毫升)，比 ApoB100和apoAI分别为。多年来一直让调查人员着迷的是一个多么低的 载脂蛋白AV的循环水平可以对人和动物的血浆甘油三酯稳态产生如此深远的影响。 由于~20%的载脂蛋白病毒被分泌到循环中，其在肝脏中的浓度明显高于 在血浆中。我们的初步数据表明apoAV在调节肝脏甘油三酯方面起着关键作用 代谢，而乙醇破坏肝脏甘油三酯的动态平衡，并加剧 肝脏中过多的甘油三酯，导致载脂蛋白AV基因敲除(KO)中更严重的酒精性肝病(ALD) 小鼠比野生型(WT)小鼠更容易感染。我们进一步发现，乙醇在一定剂量下抑制apoAV的表达。 小鼠原代肝细胞的依赖方式。长期大量饮酒(即NIAAA 模型)4周导致肝脏脂肪变性显著而快速发展，逐渐从 在apoAV KO小鼠中，单纯性脂肪变性转变为酒精性脂肪性肝炎，然后转变为肝纤维化，但不是WT小鼠。更多 重要的是，乙醇显著增加肝脏溶血磷脂酰胆碱(LysoPC)的浓度，这是一种主要的 脂肪酸代谢物，通过促进其在载脂蛋白ApoAV KO小鼠的生物合成。最近的一项人类研究发现， 溶血磷脂酶是一种新的重要的过量饮酒的生物标志物；然而，它在发病机制中起着至关重要的作用 阿尔茨海默病的病因尚不清楚。相比之下，人APOAV基因在小鼠体内的转基因表达可保护小鼠免受 酒精性肝损伤。我们的结果表明apoAV的缺失是一个重要的危险因素。 ALD和apoAV是防治ALD的新靶点。因此，我们的初步结果 支持腺相关病毒2/8(AAV2/8)介导的人 APOAV对乙醇喂养的小鼠的ALD具有保护作用。在常用的病毒载体中，AAV产生 免疫反应最低，即使在野生型状态下也是非致病性的，也是最适合的病毒 用于治疗应用的载体。AAV2/8(即，包装AAV-2基因组的AAV-8伪型 AAV-8衣壳蛋白)显示了高效的肝脏基因转移。因此，AAV2/8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上检验这一假设：第一，我们将调查 过表达APOAV对AAV2/8-转基因小鼠酒精性肝脂肪变性的保护作用 无人驾驶飞机。第二，我们将探索AAV2/8-APOAV治疗是否能阻止乙醇引发的进展 从单纯性脂肪变性到酒精性脂肪性肝炎。建议的研究具有创新性。 因为本项目可能为ALD的基因治疗提供一种有效的策略。

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.