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Principal Investigator: William H. Peranteau
Organization: CHILDREN'S HOSP OF PHILADELPHIA
Fiscal Year: 2023
Award: $737,418
Funding agency: National Institute of Diabetes and Digestive and Kidney Diseases
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.
Terms: <1H-Purin-6-amine><21+ years old><Adenine><Adult><Adult Human><Albumins><BALB C Mouse><BALB/c><Birth><CRISPR><CRISPR approach><CRISPR based approach><CRISPR method><CRISPR methodology><CRISPR technique><CRISPR technology><CRISPR tools><CRISPR-CAS-9><CRISPR-based method><CRISPR-based technique><CRISPR-based technology><CRISPR-based tool><CRISPR/CAS approach><CRISPR/Cas method><CRISPR/Cas system><CRISPR/Cas technology><CRISPR/Cas9><CRISPR/Cas9 technology><Cas nuclease technology><Cause of Death><Cell Body><Cell Line><CellLine><Cells><Childhood><Clustered Regularly Interspaced Short Palindromic Repeats><Clustered Regularly Interspaced Short Palindromic Repeats approach><Clustered Regularly Interspaced Short Palindromic Repeats method><Clustered Regularly Interspaced Short Palindromic Repeats methodology><Clustered Regularly Interspaced Short Palindromic Repeats technique><Clustered Regularly Interspaced Short Palindromic Repeats technology><Cytosine><DNA Damage Repair><DNA Double Strand Break><DNA Repair><Data><Developing fetus><Development><Dioxygenases><Disease><Disorder><Drugs><Enzyme Gene><Enzymes><Fetal Body Weight><Fetal Development><Fetal Liver><Fetal Weight><Fetus><Fumarylacetoacetase><GaAs><Gene Alteration><Gene Inactivation><Gene Mutation><Gene Silencing><Generalized Growth><Genes><Genetic Alteration><Genetic Change><Genetic Diseases><Genetic defect><Genome><Goals><Growth><Guanine><Guide RNA><Health><Hepatic Cells><Hepatic Disorder><Hepatic Failure><Hepatic Parenchymal Cell><Hepatic Transplantation><Hepatocarcinoma><Hepatocellular Carcinoma><Hepatocellular cancer><Hepatocyte><Hepatoma><Hereditary Tyrosinemias><Human><Human Cell Line><Human Engineering><Hydrolase><Hydrolase Family Gene><Hydrolase Gene><Immune><Immune Tolerance><Immunes><Immunochemical Immunologic><Immunologic><Immunologic Tolerance><Immunological><Immunologically><Immunologics><In Vitro><Inbred BALB C Mice><Knowledge><Life><Liver><Liver Cells><Liver Cells Carcinoma><Liver Failure><Liver Grafting><Liver Transplant><Liver diseases><Mediating><Medication><Metabolic><Mice><Mice Mammals><Mission><Modeling><Modern Man><Morbidity><Morbidity - disease rate><Murine><Mus><Mutation><NHEJ><NIH><National Institutes of Health><Non-Homologous End Joining><Non-homologous DNA End Joining><Nonhomologous DNA End Joining><Nonhomologous End Joining><Nonsense Mutation><Onset of illness><Organ><Other Genetics><Parturition><Pathology><Pathway interactions><Patients><Pharmaceutic Preparations><Pharmaceutical Preparations><Phenotype><Primary carcinoma of the liver cells><Progenitor Cells><Proliferating><Property><Public Health><Publishing><RNA Splicing><Research><Resistance><Risk><Safety><Site><Splicing><Strains Cell Lines><Technology><Testing><Thymine><Tissue Growth><Transplantation><Tyrosine><Tyrosinemias><United States National Institutes of Health><Unscheduled DNA Synthesis><Viral><Vitamin B4><Work><adulthood><base><base editing><base editor><bases><cultured cell line><developmental><disability><disease onset><disease-causing mutation><disorder onset><drug/agent><endonuclease><fetal><fumarylacetoacetate fumarylhydrolase><fumarylacetoacetate hydrolase><gRNA><gallium arsenide><gene therapeutics><gene-based therapeutic><gene-based therapeutics><genes therapeutic><genes therapeutics><genetic condition><genetic disorder><genome mutation><hepatic body system><hepatic cell proliferation><hepatic cellular proliferation><hepatic disease><hepatic organ system><hepatocyte cell proliferation><hepatocyte cellular proliferation><hepatocyte proliferation><hepatopathy><humanized mice><humanized mouse><immune system tolerance><immune unresponsiveness><immunological paralysis><improved outcome><in utero><in vivo><in vivo Model><indel><innovate><innovation><innovative><insertion-deletion><insertion-deletion mutation><insertion/deletion><insertion/deletion mutation><knockout gene><lipid based nanoparticle><lipid nanoparticle><liver carcinoma><liver cell proliferation><liver cellular proliferation><liver disorder><liver transplantation><mortality><mouse model><murine model><mutation correction><new therapeutic approach><new therapeutic intervention><new therapeutic strategies><new therapy approaches><new treatment approach><new treatment strategy><non-sense mutation><novel><novel therapeutic approach><novel therapeutic intervention><novel therapeutic strategies><novel therapy approach><ontogeny><pathway><patient subgroups><patient subpopulations><patient subsets><patient subtypes><pediatric><perinatal period><perinatal phase><postnatal><prenatal><repair><repaired><resistant><safety and feasibility><stem cells><success><therapeutic gene><transcriptional silencing><transplant><unborn>