In utero gene editing to cure a metabolic liver disease

子宫内基因编辑治疗代谢性肝病

基本信息

批准号：
10093033
负责人：
William H. Peranteau
金额：
$ 73.76万
依托单位：
CHILDREN'S HOSP OF PHILADELPHIA
依托单位国家：
美国
项目类别：
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/10093033
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 Knock-out 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 Perinatal 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 disability endonuclease fetal gallium arsenide humanized mouse improved outcome in utero in vivo in vivo Model innovation insertion/deletion mutation lipid nanoparticle liver transplantation mortality mouse model mutation correction novel patient subsets postnatal prenatal repaired safety and feasibility stem cell proliferation stem cells success therapeutic gene viral nanoparticle

项目摘要

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.

项目概要代谢性肝病是儿科遗传性肝移植的第二常见适应症。 I 型酪氨酸血症 (HT1) 是一种代谢性肝病，由 FAH 基因突变导致缺乏延胡索酰乙酰乙酸水解酶 (FAH) 是酪氨酸分解代谢途径中的最后一种酶，可导致死亡。在出生后的最初几个月内，到儿童中期患肝细胞癌 (HCC) 的风险会增加。移植是治疗 HT1 的唯一方法，尽管需要终身接受尼替西农治疗以抑制羟苯丙酮酸。 FAH 上游的双加氧酶 (HPD) 改善了预后，一些患者对尼替西农有耐药性，并且尽管使用了该药物，仍然发生了肝癌和肝衰竭，因此迫切需要制定新的策略来治疗。 CRISPR-Cas9 基因编辑为治疗 HT1 和其他代谢性肝病提供了前所未有的机会。治疗遗传疾病，这是一种不引入双链 DNA 的 CRISPR 编辑方法。断裂，是一种比 CRISPR 介导的潜在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）有潜力。通过利用胎儿特性来提高编辑效率——体积小、免疫不成熟、丰富的增殖祖细胞——并在出生前和不可逆转的发作前治疗疾病该编辑提案的总体目标是通过子宫内基础和 HDR 治愈 HT1。假设胎儿的内在特性将允许有效的体内碱基编辑和 HDR 来拯救 HT1 小鼠的致死表型，并且专注于治疗 HT1 的碱基编辑将在人源化中有效地发挥作用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 基因并通过产前基础拯救 HT1 小鼠表型编辑，3) 识别靶向人类 HPD 基因的向导 RNA，通过碱基编辑进行沉默，以及 4) 拯救通过碱基编辑纠正成年小鼠的 HT1 表型，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为了实现我们的目标，我们将追求以下目标：1）通过产前碱基沉默 Hpd 基因。编辑以治愈 HT1 小鼠表型并评估体内人源化小鼠模型中的 HPD 碱基编辑， 2) 通过 HT1 小鼠的产前碱基编辑和体外工程化的人类细胞纠正 FAH 突变线，3) 比较产前和产后 CRISPR 介导的和核酸内切酶的效率和安全性游离 HDR 及其拯救 HT1 表型的能力我们的研究在新颖的产前时机方面具有创新性。 HT1的CRISPR和非CRISPR基因编辑方法以及HT1碱基编辑在人源化中的研究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疗法，可以治愈 HT1，并且可以扩展到治疗其他遗传性疾病。