CRISPR/Cas9 generated knockout mice lacking phenylalanine hydroxylase protein as a novel preclinical model for human phenylketonuria
Abstract Phenylketonuria (PKU) is an autosomal recessive inborn error of l-phenylalanine (Phe) metabolism. It is caused by a partial or complete deficiency of the enzyme phenylalanine hydroxylase (PAH), which is necessary for conversion of Phe to tyrosine (Tyr). This metabolic error results in build...
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2021
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oai:doaj.org-article:85cf103dd353470493e9b182fec30ee32021-12-02T14:23:05ZCRISPR/Cas9 generated knockout mice lacking phenylalanine hydroxylase protein as a novel preclinical model for human phenylketonuria10.1038/s41598-021-86663-82045-2322https://doaj.org/article/85cf103dd353470493e9b182fec30ee32021-03-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-86663-8https://doaj.org/toc/2045-2322Abstract Phenylketonuria (PKU) is an autosomal recessive inborn error of l-phenylalanine (Phe) metabolism. It is caused by a partial or complete deficiency of the enzyme phenylalanine hydroxylase (PAH), which is necessary for conversion of Phe to tyrosine (Tyr). This metabolic error results in buildup of Phe and reduction of Tyr concentration in blood and in the brain, leading to neurological disease and intellectual deficits. Patients exhibit retarded body growth, hypopigmentation, hypocholesterolemia and low levels of neurotransmitters. Here we report first attempt at creating a homozygous Pah knock-out (KO) (Hom) mouse model, which was developed in the C57BL/6 J strain using CRISPR/Cas9 where codon 7 (GAG) in Pah gene was changed to a stop codon TAG. We investigated 2 to 6-month-old, male, Hom mice using comprehensive behavioral and biochemical assays, MRI and histopathology. Age and sex-matched heterozygous Pah-KO (Het) mice were used as control mice, as they exhibit enough PAH enzyme activity to provide Phe and Tyr levels comparable to the wild-type mice. Overall, our findings demonstrate that 6-month-old, male Hom mice completely lack PAH enzyme, exhibit significantly higher blood and brain Phe levels, lower levels of brain Tyr and neurotransmitters along with lower myelin content and have significant behavioral deficit. These mice exhibit phenotypes that closely resemble PKU patients such as retarded body growth, cutaneous hypopigmentation, and hypocholesterolemia when compared to the age- and sex-matched Het mice. Altogether, biochemical, behavioral, and pathologic features of this novel mouse model suggest that it can be used as a reliable translational tool for PKU preclinical research and drug development.Kuldeep SinghCathleen S. CornellRobert JacksonMostafa KabiriMichael PhippsMitul DesaiRobert FogleXiaoyou YingGulbenk Anarat-CappillinoSarah GellerJennifer JohnsonErrin RobertsKatie MalleyTim DevlinMatthew DeRisoPatricia BertheletteYao V. ZhangSusan RyanSrinivas RaoBeth L. ThurbergDinesh S. BangariSirkka Kyostio-MooreNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-14 (2021) |
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Medicine R Science Q |
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Medicine R Science Q Kuldeep Singh Cathleen S. Cornell Robert Jackson Mostafa Kabiri Michael Phipps Mitul Desai Robert Fogle Xiaoyou Ying Gulbenk Anarat-Cappillino Sarah Geller Jennifer Johnson Errin Roberts Katie Malley Tim Devlin Matthew DeRiso Patricia Berthelette Yao V. Zhang Susan Ryan Srinivas Rao Beth L. Thurberg Dinesh S. Bangari Sirkka Kyostio-Moore CRISPR/Cas9 generated knockout mice lacking phenylalanine hydroxylase protein as a novel preclinical model for human phenylketonuria |
| description |
Abstract Phenylketonuria (PKU) is an autosomal recessive inborn error of l-phenylalanine (Phe) metabolism. It is caused by a partial or complete deficiency of the enzyme phenylalanine hydroxylase (PAH), which is necessary for conversion of Phe to tyrosine (Tyr). This metabolic error results in buildup of Phe and reduction of Tyr concentration in blood and in the brain, leading to neurological disease and intellectual deficits. Patients exhibit retarded body growth, hypopigmentation, hypocholesterolemia and low levels of neurotransmitters. Here we report first attempt at creating a homozygous Pah knock-out (KO) (Hom) mouse model, which was developed in the C57BL/6 J strain using CRISPR/Cas9 where codon 7 (GAG) in Pah gene was changed to a stop codon TAG. We investigated 2 to 6-month-old, male, Hom mice using comprehensive behavioral and biochemical assays, MRI and histopathology. Age and sex-matched heterozygous Pah-KO (Het) mice were used as control mice, as they exhibit enough PAH enzyme activity to provide Phe and Tyr levels comparable to the wild-type mice. Overall, our findings demonstrate that 6-month-old, male Hom mice completely lack PAH enzyme, exhibit significantly higher blood and brain Phe levels, lower levels of brain Tyr and neurotransmitters along with lower myelin content and have significant behavioral deficit. These mice exhibit phenotypes that closely resemble PKU patients such as retarded body growth, cutaneous hypopigmentation, and hypocholesterolemia when compared to the age- and sex-matched Het mice. Altogether, biochemical, behavioral, and pathologic features of this novel mouse model suggest that it can be used as a reliable translational tool for PKU preclinical research and drug development. |
| format |
article |
| author |
Kuldeep Singh Cathleen S. Cornell Robert Jackson Mostafa Kabiri Michael Phipps Mitul Desai Robert Fogle Xiaoyou Ying Gulbenk Anarat-Cappillino Sarah Geller Jennifer Johnson Errin Roberts Katie Malley Tim Devlin Matthew DeRiso Patricia Berthelette Yao V. Zhang Susan Ryan Srinivas Rao Beth L. Thurberg Dinesh S. Bangari Sirkka Kyostio-Moore |
| author_facet |
Kuldeep Singh Cathleen S. Cornell Robert Jackson Mostafa Kabiri Michael Phipps Mitul Desai Robert Fogle Xiaoyou Ying Gulbenk Anarat-Cappillino Sarah Geller Jennifer Johnson Errin Roberts Katie Malley Tim Devlin Matthew DeRiso Patricia Berthelette Yao V. Zhang Susan Ryan Srinivas Rao Beth L. Thurberg Dinesh S. Bangari Sirkka Kyostio-Moore |
| author_sort |
Kuldeep Singh |
| title |
CRISPR/Cas9 generated knockout mice lacking phenylalanine hydroxylase protein as a novel preclinical model for human phenylketonuria |
| title_short |
CRISPR/Cas9 generated knockout mice lacking phenylalanine hydroxylase protein as a novel preclinical model for human phenylketonuria |
| title_full |
CRISPR/Cas9 generated knockout mice lacking phenylalanine hydroxylase protein as a novel preclinical model for human phenylketonuria |
| title_fullStr |
CRISPR/Cas9 generated knockout mice lacking phenylalanine hydroxylase protein as a novel preclinical model for human phenylketonuria |
| title_full_unstemmed |
CRISPR/Cas9 generated knockout mice lacking phenylalanine hydroxylase protein as a novel preclinical model for human phenylketonuria |
| title_sort |
crispr/cas9 generated knockout mice lacking phenylalanine hydroxylase protein as a novel preclinical model for human phenylketonuria |
| publisher |
Nature Portfolio |
| publishDate |
2021 |
| url |
https://doaj.org/article/85cf103dd353470493e9b182fec30ee3 |
| work_keys_str_mv |
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