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.

Public Health Relevance

The proposed research is relevant to public health because there is an unmet need to develop new treatment approaches for patients with hereditary tyrosinemia type I (HT1) and other metabolic liver diseases which are a leading indication for liver transplants and cause significant morbidity and mortality. In this project, we seek to use gene editing, including its prenatal application, to treat HT1 as a model for other genetic metabolic liver diseases. Thus, the proposed research is relevant to the part of the NIH?s mission that seeks to develop fundamental knowledge to enhance health, lengthen life and reduce illness and disability.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Special Emphasis Panel (ZRG1)
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Doo, Edward
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Children's Hospital of Philadelphia
United States
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