In utero gene editing to cure a metabolic liver disease

子宫内基因编辑治疗代谢性肝病

• 批准号: 10550192
• 负责人: William H. Peranteau
• 金额: $ 73.74万
• 依托单位: CHILDREN'S HOSP OF PHILADELPHIA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-02-01 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10550192
• 关键词: Adenine; Adult; Albumins; Birth; CRISPR/Cas technology; Cause of Death; Cell Line; Cells; Childhood; Clustered Regularly Interspaced Short Palindromic Repeats; Cytosine; DNA Double Strand Break; DNA Repair; Data; Development; Dioxygenases; Disease; Enzymes; Fetal Development; Fetal Liver; Fetal Weight; Fetus; Fumarylacetoacetase; Gene Mutation; Gene Silencing; Genes; Genetic Diseases; Genome; Goals; Growth; Guanine; Guide RNA; Health; Hepatocyte; Human; Human Cell Line; Human Engineering; Hydrolase; Immune; Immune Tolerance; Immunologics; In Vitro; Inbred BALB C Mice; Knowledge; Life; Liver; Liver Failure; Liver diseases; Mediating; Metabolic; Mission; Modeling; Morbidity - disease rate; Mus; Mutation; Nonhomologous DNA End Joining; Nonsense Mutation; Onset of illness; Organ; Other Genetics; Pathology; Pathway interactions; Patients; Pharmaceutical Preparations; Phenotype; Primary carcinoma of the liver cells; Proliferating; Property; Public Health; Publishing; RNA Splicing; Research; Resistance; Risk; Safety; Site; Technology; Testing; Thymine; Transplantation; Tyrosine; Tyrosinemias; United States National Institutes of Health; Viral; Work; base; base editing; base editor; disability; disease-causing mutation; endonuclease; fetal; gallium arsenide; humanized mouse; improved outcome; in utero; in vivo; in vivo Model; innovation; insertion/deletion mutation; knockout gene; lipid nanoparticle; liver cell proliferation; liver transplantation; mortality; mouse model; mutation correction; novel; novel therapeutic intervention; patient subsets; perinatal period; postnatal; prenatal; repaired; safety and feasibility; stem cells; success; therapeutic gene

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.

项目摘要 代谢性肝脏疾病是小儿肝移植的第二常见指示。遗传 I型酪氨酸血症（HT1）是一种代谢性肝病，是由FAH基因突变引起的，导致缺乏症 在粉刺乙酸水解酶（FAH）中，酪氨酸分解代谢途径中的最后一个酶。 HT1可能导致死亡 在生命的头几个月内，并且在年龄中期患有肝癌（HCC）的风险增加。肝 移植是HT1的唯一治疗方法。虽然用硝酸酮终身治疗抑制羟基苯基丙酮酸 FAH上游的双加氧酶（HPD）有改善的结局，一些患者对硝酸酮有抗性，并且 尽管有药物，但仍会发生HCC和肝衰竭。这是迫切需要制定新的策略 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 治疗遗传疾病。基础编辑，一种不引入双链DNA的CRIS剪辑方法 断裂是一种潜在的安全机制，可以使基因沉默或纠正突变，而不是CRISPR介导的 非动力学的最终连接和同源指导修复（HDR）。在子宫内基因编辑有可能 通过利用胎儿特性 - 小小的大小，免疫学不成熟，提高编辑效率， 增生剂细胞的抽象 - 并在出生前治疗疾病和不可逆的发作 病理。该提案的总体目的是通过子宫基础编辑和HDR来治疗HT1。我们的中心 假设是固有的胎儿特性将使体内基础编辑有效，并拯救HDR HT1小鼠中的致命表型，以及专注于治疗HT1的基础编辑将有效地在人源化中起作用 型号。我们的假设基于我们的初步数据，其中我们1）通过病毒有效地靶向胎儿肝脏 和非病毒方法，2）沉默HPD基因并通过产前碱基挽救HT1小鼠表型 编辑，3）识别针对人类HPD基因进行沉默的指南RNA通过基础编辑，4）营救 通过基础编辑的HT1表型纠正成年小鼠的FAH突变。我们对这些研究的理由是 他们将确定HT1作为代谢肝的模型的产前基因编辑的安全性和可行性 疾病。为了实现我们的目标，我们将追求以下目标：1）通过产前基础使HPD基因沉默 编辑以治愈HT1小鼠表型并评估人体化小鼠模型中的HPD碱基编辑， 2）通过HT1小鼠中的产前基础编辑和在工程人类细胞中的体外纠正FAH突变 线，以及3）比较产前和产后CRISPR介导的核酸内切酶的效率和安全性 - 自由HDR及其营救HT1表型的能力。我们的研究在小说的产前时机上具有创新性 HT1的CRISPR和非CRISPR基因编辑方法以及人源化的HT1基础编辑 型号。这项工作的重大贡献将是支持一种可能的产前基因编辑方法 产生一种治愈HT1的一弹性长期治疗，可以扩展以治疗其他遗传疾病。