In utero gene editing to cure a metabolic liver disease

项目总结 代谢性肝病是儿童肝移植的第二大适应症。遗传性 酪氨酸血症I型(HT1)是一种由FAH基因突变引起的代谢性肝病。 在富马酸乙酰乙酸酯水解酶(FAH)中，酪氨酸分解代谢途径中的最后一种酶。HT1可致人死亡 在生命的头几个月内，到童年中期，患肝细胞癌的风险增加。肝 移植是HT1的唯一治愈方法。尽管终生服用尼替西酮抑制羟基苯丙酮酸 FAH上游的双加氧酶(HPD)可改善预后，一些患者对尼替西酮耐药，以及 尽管使用了这种药物，肝细胞癌和肝功能衰竭还是发生了。因此，迫切需要制定新的战略，以 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遇 来治疗遗传性疾病。碱基编辑，一种不引入双链DNA的CRISPR编辑方法 与CRISPR介导的机制相比，Break是一种潜在的更安全的沉默基因或纠正突变的机制 非同源末端连接和同源定向修复(HDR)。在子宫内进行基因编辑有可能 通过利用胎儿的特性提高编辑效率-体积小，免疫不成熟， 丰富的增殖祖细胞--在出生前治疗疾病和不可逆转的发病 病理学。这项建议的总体目标是通过宫内编辑和HDR治愈HT1。我们的中央 假设胎儿固有的特性将允许有效的活体基础编辑和HDR来拯救 HT1小鼠致死表型，而以治疗HT1为重点的基础编辑将在人源化的情况下有效地工作 模特们。我们的假设基于我们的初步数据，在这些数据中，我们1)通过病毒有效地靶向胎肝 和非病毒途径，2)沉默HpD基因，通过产前基础挽救HT1小鼠的表型 编辑，3)通过碱基编辑识别靶向人类HPD基因的引导RNA以沉默，以及4)拯救 通过碱基编辑纠正成年小鼠的Fah突变的HT1表型。我们进行这些研究的理由是 他们将建立HT1作为代谢性肝脏模型的产前基因编辑的安全性和可行性 疾病。为了达到我们的目标，我们将追求以下目标：1)通过产前基础沉默hpd基因 编辑以治愈HT1小鼠的表型并评估体内人源化小鼠模型中的HPD碱基编辑， 2)在HT1小鼠和体外工程人类细胞中，通过产前碱基编辑来纠正FAH突变 3)比较出生前和出生后CRISPR介导的核酸内切酶的效率和安全性。 游离HDR及其挽救HT1表型的能力。我们的研究在小说的产前计时方面是创新的 HT1的CRISPR和非CRISPR基因编辑方法及人源化HT1碱基编辑的研究 模特们。这项工作的重大贡献将是支持一种产前基因编辑方法，这种方法可以 产生一种一次性的长期疗法，治愈HT1，并可以扩大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.