Therapeutic pathway reprogramming for metabolic liver disease

项目摘要 代谢性肝病是一个新兴的公共卫生问题。在美国， 糖尿病和肥胖相关的代谢性肝病是原位肝硬化最常见的原因。 肝移植（奥尔特），需要终身免疫抑制，并与 严重的发病率和死亡率（10年生存率60-80%）。超过两倍的病人 与可用器官相比，列出了奥尔特，说明迫切需要探索 代谢性肝病的替代治疗策略。 我们最近开发了一种新的治疗策略，称为代谢途径 重新编程这个概念基于疾病中一个关键代谢基因的缺失- 相关途径，导致代谢途径改道，导致良性疾病 表型。作为原理证明，我们专注于遗传性酪氨酸血症I型（HT-1）， 由富马酰乙酰乙酸基因（FAH）突变引起。多年来，HT-1患者 用尼替西酮治疗，尼替西酮是一种抑制羟苯丙酮酸氧化酶的药物 (HPD)是FAH上游的一个基因，导致毒性较低的可排泄catalysts的积累， 类似于相对良性的酪氨酸血症III型（HT-III）。我们假设新陈代谢 通过体细胞HPD基因缺失的途径重编程是HT-1 OLT的替代奥尔特 患者和上级优于目前的药理学方法。我们测试了 在短期（3个月）实验中， CRISPR/Cas9基因组编辑和流体动力学尾静脉注射（Pankowicz等人，Nat Commun.）。虽然我们的方法在小鼠中取得了成功，但有三个主要障碍， 临床翻译;这种疗法的长期后果，基因递送方法和 将这种序列特异性治疗转化为人类环境。 我们建议在HT-1小鼠模型中研究这些主要障碍， 利用腺相关病毒（AAV）基因治疗方法的人肝嵌合小鼠 (Aim 1a）。我们将在酪氨酸血症患者中确定AAV删除Hpd的长期益处和风险。 小鼠在最新技术水平的治疗与尼替西酮（目标1b），以及确定效率 以及在人源化小鼠中进行这种治疗的风险（目的2）。 该提案的成功执行将验证代谢的治疗应用。 在原代人类细胞中的途径重编程，并有可能建立一个新的 代谢性肝病的治疗范例。

PROJECT SUMMARY Metabolic liver disease is an emerging public health problem. In the United States, diabetes- and obesity-related metabolic liver disease is the most common cause for orthotopic liver transplantation (OLT), which requires life-long immunosuppression and is associated with substantial morbidity and mortality (10-year survival 60-80%). More than twice as many patients are listed for OLT relative to organs available, illustrating a compelling need to explore alternative treatment strategies for metabolic liver disease. We have recently developed a novel therapeutic strategy called metabolic pathway reprogramming. The concept rests on deletion of a critical metabolic gene in a disease- associated pathway, causing the metabolic pathway to be rerouted resulting in a benign disease phenotype. As a proof-of-principle, we focus on hereditary tyrosinemia type I (HT-1), which is caused by mutations of the fumarylacetoacetate gene (FAH). For many years, HT-1 patients have been treated with nitisinone, a drug that inhibits hydroxyphenylpyruvate dioxigenase (HPD), a gene upstream of FAH, and leads to accumulation of less toxic, excretable catabolites similar to the comparatively benign tyrosinemia type III (HT-III). We hypothesize that metabolic pathway reprogramming via somatic HPD gene deletion is an alternative to OLT for HT-1 patients and superior to the current pharmacological approach. We tested the concept of metabolic pathway reprogramming for HT-1 in a short-term (3 months) experiment using CRISPR/Cas9 genome editing and hydrodynamic tail vein injections (Pankowicz et al. Nat Commun.). While our approach was successful in mice, there are three major roadblocks for clinical translation; the long-term consequences of this therapy, the gene delivery method and the translation of this sequence specific therapy into the human setting. We propose to investigate these major roadblocks in the murine model of HT-1 and human liver chimeric mice utilizing a gene therapy approach with Adeno-Associated Virus (AAV) (Aim 1a). We will determine long-term benefit and risk of Hpd deletion by AAV in tyrosinemic mice over the state of the art therapy with nitisinone (Aim 1b), as well as determine efficiency and risk of such a therapy in humanized mice (Aim 2). Successful execution of this proposal will validate therapeutic applications of metabolic pathway reprogramming in primary human cells and has the potential to establish a new therapeutic paradigm for metabolic liver disease.