A porcine model of phenylketonuria generated by CRISPR/Cas9 genome editing
Open Access
- 15 October 2020
- journal article
- research article
- Published by American Society for Clinical Investigation in JCI Insight
- Vol. 5 (20)
- https://doi.org/10.1172/jci.insight.141523
Abstract
Phenylalanine hydroxylase–deficient (PAH-deficient) phenylketonuria (PKU) results in systemic hyperphenylalaninemia, leading to neurotoxicity with severe developmental disabilities. Dietary phenylalanine (Phe) restriction prevents the most deleterious effects of hyperphenylalaninemia, but adherence to diet is poor in adult and adolescent patients, resulting in characteristic neurobehavioral phenotypes. Thus, an urgent need exists for new treatments. Additionally, rodent models of PKU do not adequately reflect neurocognitive phenotypes, and thus there is a need for improved animal models. To this end, we have developed PAH-null pigs. After selection of optimal CRISPR/Cas9 genome-editing reagents by using an in vitro cell model, zygote injection of 2 sgRNAs and Cas9 mRNA demonstrated deletions in preimplantation embryos, with embryo transfer to a surrogate leading to 2 founder animals. One pig was heterozygous for a PAH exon 6 deletion allele, while the other was compound heterozygous for deletions of exon 6 and of exons 6–7. The affected pig exhibited hyperphenylalaninemia (2000–5000 μM) that was treatable by dietary Phe restriction, consistent with classical PKU, along with juvenile growth retardation, hypopigmentation, ventriculomegaly, and decreased brain gray matter volume. In conclusion, we have established a large-animal preclinical model of PKU to investigate pathophysiology and to assess new therapeutic interventions.Funding Information
- NIH/NINDS (R21NS099450)
- NIH/NIAID (U42OD011140)
This publication has 78 references indexed in Scilit:
- Efficient nonmeiotic allele introgression in livestock using custom endonucleasesProceedings of the National Academy of Sciences of the United States of America, 2013
- Analyses of pig genomes provide insight into porcine demography and evolutionNature, 2012
- Brain Growth of the Domestic Pig (Sus scrofa) from 2 to 24 Weeks of Age: A Longitudinal MRI StudyDevelopmental Neuroscience, 2012
- Adult phenylketonuria outcome and managementMolecular Genetics and Metabolism, 2011
- Diagnosis, classification, and genetics of phenylketonuria and tetrahydrobiopterin (BH4) deficienciesMolecular Genetics and Metabolism, 2011
- Different Strokes for Different Folks: The Rich Diversity of Animal Models of Focal Cerebral IschemiaJournal of Cerebral Blood Flow & Metabolism, 2010
- The Development of Lung Disease in Cystic Fibrosis PigsScience Translational Medicine, 2010
- Disruption of the CFTR Gene Produces a Model of Cystic Fibrosis in Newborn PigsScience, 2008
- Mathematical Analysis of Copy Number Variation in a DNA Sample Using Digital PCR on a Nanofluidic DevicePLOS ONE, 2008
- Complete correction of hyperphenylalaninemia following liver-directed, recombinant AAV2/8 vector-mediated gene therapy in murine phenylketonuriaGene Therapy, 2005