Therapeutic pathway reprogramming for metabolic liver disease

项目总结 代谢性肝病是一个新出现的公共卫生问题。在美国， 糖尿病和肥胖相关的代谢性肝病是原位移植最常见的原因 肝移植(OLT)，这需要终身免疫抑制，并与 相当高的发病率和死亡率(10年存活率60-80%)。两倍多的病人 列出了与可用器官相关的OLT，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。这一概念基于疾病中关键代谢基因的缺失-- 相关途径，导致代谢途径改变路线，导致良性疾病 表型。作为一个原则证明，我们关注遗传性酪氨酸血症I型(HT-1)，它是 由富马酸乙酰乙酸酯基因(FAH)突变引起。多年来，HT-1患者 已经接受了烟替酮的治疗，这是一种抑制羟苯丙酮酸二氧酶的药物 (Hpd)，FAH的上游基因，并导致毒性较低、可排泄的分解代谢产物的积累 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基因缺失重新编程通路是治疗HT-1的OLT的替代方案 并优于目前的药理治疗方法。我们测试了这一概念 一项短期(3个月)实验中对HT-1代谢途径的重新编程 CRISPR/Cas9基因组编辑和流体动力尾静脉注射(Pankowicz等人纳特 通信。)。虽然我们的方法在小鼠身上是成功的，但有三个主要的障碍 临床翻译；这种疗法的长期后果，基因输送方法和 将这一序列特异性疗法转化为人类环境。 我们建议在HT-1小鼠模型中研究这些主要障碍和 用腺相关病毒进行基因治疗的人肝嵌合小鼠 (目标1a)。我们将确定AAV对酪氨酸血症患者HPD缺失的长期益处和风险 尼替松对小鼠最新治疗水平的影响(目标1b)，以及测定效率 以及在人源化小鼠中使用这种疗法的风险(目标2)。 这项提案的成功执行将验证新陈代谢的治疗应用 在原代人类细胞中重新编程通路，并有可能建立新的 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.