In utero gene editing to cure a metabolic liver disease

项目摘要 代谢性肝病是儿科肝移植的第二大常见适应症。遗传性 酪氨酸血症I型（HT1）是由FAH基因突变引起的代谢性肝病， 延胡索酰乙酰乙酸水解酶（FAH），酪氨酸分解代谢途径中的最后一种酶。HT1可导致死亡 在生命的最初几个月内，并且在儿童中期患肝细胞癌（HCC）的风险增加。肝 移植是治疗HT1的唯一方法。虽然用尼替西酮终身治疗以抑制羟苯丙酮酸 FAH上游的双加氧酶（HPD）改善了结局，一些患者对尼替西酮耐药， 尽管有药物，但仍发生了HCC和肝功能衰竭。因此，迫切需要制定新的战略， 治疗HT1和其他代谢性肝病。CRISPR-Cas9基因编辑提供了前所未有的机会 来治疗遗传疾病碱基编辑，一种不引入双链DNA的CRISPR编辑方法 与CRISPR介导的突变相比， 非同源末端连接和同源定向修复（HDR）。子宫内基因编辑有可能 通过利用胎儿特性-小尺寸、免疫不成熟 丰富的增殖祖细胞-和治疗出生前的疾病和不可逆的发病 病理该提案的总体目标是通过子宫内碱基编辑和HDR治愈HT1。我们的中央 假设是，内在胎儿特性将允许有效的基于体内的编辑和HDR来拯救胎儿， HT1小鼠中致死表型，而专注于治疗HT1的碱基编辑将在人源化小鼠中有效地起作用。 模型我们的假设是基于我们的初步数据，其中我们1）有效地通过病毒靶向胎儿肝脏 和非病毒方法，2）沉默Hpd基因并通过产前碱基突变拯救HT1小鼠表型 编辑，3）鉴定靶向人HPD基因的向导RNA以用于通过碱基编辑沉默，以及4）拯救人HPD基因， HT1表型通过碱基编辑来校正成年小鼠中的Fah突变。我们进行这些研究的理由是， 他们将建立HT1产前基因编辑作为代谢肝脏模型的安全性和可行性 疾病为了达到这一目的，我们将从以下几个方面着手：1）通过产前碱基沉默Hpd基因 编辑以治愈HT1小鼠表型并在体内人源化小鼠模型中评估HPD碱基编辑， 2)在HT1小鼠中和体外在工程化人细胞中通过产前碱基编辑校正FAH突变 以及3）比较产前和产后CRISPR介导的和核酸内切酶- 游离HDR和它们拯救HT1表型的能力。我们的研究是创新的产前时间的小说 HT1的CRISPR和非CRISPR基因编辑方法以及人源化细胞中HT1碱基编辑的研究 模型这项工作的重大贡献将是支持产前基因编辑方法， 产生一种一次性的长期治疗方法，可以治愈HT1，并可以扩展到治疗其他遗传疾病。

PROJECT SUMMARY Metabolic liver diseases are the second most common indication for a pediatric liver transplant. Hereditary tyrosinemia type I (HT1) is a metabolic liver disease that results from FAH gene mutations causing a deficiency in fumarylacetoacetate hydrolase (FAH), the last enzyme in the tyrosine catabolic pathway. HT1 can cause death within the first months of life and has an increased risk of hepatocellular cancer (HCC) by mid-childhood. Liver transplant is the only cure for HT1. Although lifelong treatment with nitisinone to inhibit hydroxyphenylpyruvate dioxygenase (HPD) upstream of FAH has improved outcomes, some patients are resistant to nitisinone, and HCC and liver failure have occurred despite the drug. Thus, there is a critical need to develop new strategies to treat HT1 and other metabolic liver diseases. CRISPR-Cas9 gene editing offers an unprecedented opportunity to treat genetic diseases. Base editing, a CRISPR editing approach that does not introduce double-strand DNA breaks, is a potentially safer mechanism to silence a gene or correct a mutation than CRISPR-mediated nonhomologous end-joining and homology-directed repair (HDR). In utero gene editing has the potential to increase editing efficiency by taking advantage of fetal properties–small size, immunologic immaturity, abundance of proliferative progenitor cells–and treat a disease prior to birth and the onset of irreversible pathology. The overall objective of this proposal is to cure HT1 via in utero base editing and HDR. Our central hypotheses are that intrinsic fetal properties will allow for efficient in vivo base editing and HDR to rescue the lethal phenotype in HT1 mice, and that base editing, focused on treating HT1, will work efficiently in humanized models. Our hypotheses are based on our preliminary data in which we 1) efficiently target the fetal liver via viral and nonviral approaches, 2) silence the Hpd gene and rescue the HT1 mouse phenotype via prenatal base editing, 3) identify guide RNAs targeting the human HPD gene for silencing via base editing, and 4) rescue the HT1 phenotype via base editing to correct the Fah mutation in adult mice. Our rationale for these studies is that they will establish the safety and feasibility of prenatal gene editing for HT1 as a model for metabolic liver diseases. To attain our objective, we will pursue the following aims: 1) silence the Hpd gene via prenatal base editing to cure the HT1 mouse phenotype and evaluate HPD base editing in humanized mouse models in vivo, 2) correct the FAH mutation via prenatal base editing in the HT1 mouse and in vitro in an engineered human cell line, and 3) compare the efficiency and safety of prenatal and postnatal CRISPR-mediated and endonuclease- free HDR and their ability to rescue the HT1 phenotype. Our research is innovative in the prenatal timing of novel CRISPR and non-CRISPR gene editing approaches for HT1 and the study of HT1 base editing in humanized models. The significant contribution of this work will be to support a prenatal gene editing approach that could yield a one-shot, long-term therapy that cures HT1 and which could be expanded to treat other genetic disorders.