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Development of a porcine model of phenylketonuria with a humanized R408W mutation for gene editing

Phenylketonuria (PKU) is a metabolic disorder whereby phenylalanine metabolism is deficient due to allelic variations in the gene for phenylalanine hydroxylase (PAH). There is no cure for PKU other than orthotopic liver transplantation, and the standard of care for patients is limited to dietary res...

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Autores principales: Kaiser, Robert A., Carlson, Daniel F., Allen, Kari L., Webster, Dennis A., VanLith, Caitlin J., Nicolas, Clara T., Hillin, Lori G., Yu, Yue, Kaiser, Catherine W., Wahoff, William R., Hickey, Raymond D., Watson, Adrienne L., Winn, Shelley R., Thöny, Beat, Kern, Douglas R., Harding, Cary O., Lillegard, Joseph B.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7833140/
https://www.ncbi.nlm.nih.gov/pubmed/33493163
http://dx.doi.org/10.1371/journal.pone.0245831
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author Kaiser, Robert A.
Carlson, Daniel F.
Allen, Kari L.
Webster, Dennis A.
VanLith, Caitlin J.
Nicolas, Clara T.
Hillin, Lori G.
Yu, Yue
Kaiser, Catherine W.
Wahoff, William R.
Hickey, Raymond D.
Watson, Adrienne L.
Winn, Shelley R.
Thöny, Beat
Kern, Douglas R.
Harding, Cary O.
Lillegard, Joseph B.
author_facet Kaiser, Robert A.
Carlson, Daniel F.
Allen, Kari L.
Webster, Dennis A.
VanLith, Caitlin J.
Nicolas, Clara T.
Hillin, Lori G.
Yu, Yue
Kaiser, Catherine W.
Wahoff, William R.
Hickey, Raymond D.
Watson, Adrienne L.
Winn, Shelley R.
Thöny, Beat
Kern, Douglas R.
Harding, Cary O.
Lillegard, Joseph B.
author_sort Kaiser, Robert A.
collection PubMed
description Phenylketonuria (PKU) is a metabolic disorder whereby phenylalanine metabolism is deficient due to allelic variations in the gene for phenylalanine hydroxylase (PAH). There is no cure for PKU other than orthotopic liver transplantation, and the standard of care for patients is limited to dietary restrictions and key amino acid supplementation. Therefore, Pah was edited in pig fibroblasts for the generation of PKU clone piglets that harbor a common and severe human mutation, R408W. Additionally, the proximal region to the mutation was further humanized by introducing 5 single nucleotide polymorphisms (SNPs) to allow for development of gene editing machinery that could be translated directly from the pig model to human PKU patients that harbor at least one classic R408W allele. Resulting piglets were hypopigmented (a single Ossabaw piglet) and had low birthweight (all piglets). The piglets had similar levels of PAH expression, but no detectable enzymatic activity, consistent with the human phenotype. The piglets were fragile and required extensive neonatal care to prevent failure to thrive and early demise. Phenylalanine levels rose sharply when dietary Phe was unrestricted but could be rapidly reduced with a low Phe diet. Fibroblasts isolated from R408W piglets show susceptibility to correction using CRISPR or TALEN, with subsequent homology-directed recombination to correct Pah. This pig model of PKU provides a powerful new tool for development of all classes of therapeutic candidates to treat or cure PKU, as well as unique value for proof-of-concept studies for in vivo human gene editing platforms in the context of this humanized PKU allele.
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spelling pubmed-78331402021-01-26 Development of a porcine model of phenylketonuria with a humanized R408W mutation for gene editing Kaiser, Robert A. Carlson, Daniel F. Allen, Kari L. Webster, Dennis A. VanLith, Caitlin J. Nicolas, Clara T. Hillin, Lori G. Yu, Yue Kaiser, Catherine W. Wahoff, William R. Hickey, Raymond D. Watson, Adrienne L. Winn, Shelley R. Thöny, Beat Kern, Douglas R. Harding, Cary O. Lillegard, Joseph B. PLoS One Research Article Phenylketonuria (PKU) is a metabolic disorder whereby phenylalanine metabolism is deficient due to allelic variations in the gene for phenylalanine hydroxylase (PAH). There is no cure for PKU other than orthotopic liver transplantation, and the standard of care for patients is limited to dietary restrictions and key amino acid supplementation. Therefore, Pah was edited in pig fibroblasts for the generation of PKU clone piglets that harbor a common and severe human mutation, R408W. Additionally, the proximal region to the mutation was further humanized by introducing 5 single nucleotide polymorphisms (SNPs) to allow for development of gene editing machinery that could be translated directly from the pig model to human PKU patients that harbor at least one classic R408W allele. Resulting piglets were hypopigmented (a single Ossabaw piglet) and had low birthweight (all piglets). The piglets had similar levels of PAH expression, but no detectable enzymatic activity, consistent with the human phenotype. The piglets were fragile and required extensive neonatal care to prevent failure to thrive and early demise. Phenylalanine levels rose sharply when dietary Phe was unrestricted but could be rapidly reduced with a low Phe diet. Fibroblasts isolated from R408W piglets show susceptibility to correction using CRISPR or TALEN, with subsequent homology-directed recombination to correct Pah. This pig model of PKU provides a powerful new tool for development of all classes of therapeutic candidates to treat or cure PKU, as well as unique value for proof-of-concept studies for in vivo human gene editing platforms in the context of this humanized PKU allele. Public Library of Science 2021-01-25 /pmc/articles/PMC7833140/ /pubmed/33493163 http://dx.doi.org/10.1371/journal.pone.0245831 Text en © 2021 Kaiser et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Kaiser, Robert A.
Carlson, Daniel F.
Allen, Kari L.
Webster, Dennis A.
VanLith, Caitlin J.
Nicolas, Clara T.
Hillin, Lori G.
Yu, Yue
Kaiser, Catherine W.
Wahoff, William R.
Hickey, Raymond D.
Watson, Adrienne L.
Winn, Shelley R.
Thöny, Beat
Kern, Douglas R.
Harding, Cary O.
Lillegard, Joseph B.
Development of a porcine model of phenylketonuria with a humanized R408W mutation for gene editing
title Development of a porcine model of phenylketonuria with a humanized R408W mutation for gene editing
title_full Development of a porcine model of phenylketonuria with a humanized R408W mutation for gene editing
title_fullStr Development of a porcine model of phenylketonuria with a humanized R408W mutation for gene editing
title_full_unstemmed Development of a porcine model of phenylketonuria with a humanized R408W mutation for gene editing
title_short Development of a porcine model of phenylketonuria with a humanized R408W mutation for gene editing
title_sort development of a porcine model of phenylketonuria with a humanized r408w mutation for gene editing
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7833140/
https://www.ncbi.nlm.nih.gov/pubmed/33493163
http://dx.doi.org/10.1371/journal.pone.0245831
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