CRISPR-mediated generation and characterization of a Gaa homozygous c.1935C>A (p.D645E) Pompe disease knock-in mouse model recapitulating human infantile onset-Pompe disease
Abstract Pompe disease, an autosomal recessive disorder caused by deficient lysosomal acid α-glucosidase (GAA), is characterized by accumulation of intra-lysosomal glycogen in skeletal and oftentimes cardiac muscle. The c.1935C>A (p.Asp645Glu) variant, the most frequent GAA pathogenic mutation in...
| Main Authors: | , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2022-12-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-022-25914-8 |
| _version_ | 1798005332153204736 |
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| author | Shih-hsin Kan Jeffrey Y. Huang Jerry Harb Allisandra Rha Nancy D. Dalton Chloe Christensen Yunghang Chan Jeremy Davis-Turak Jonathan Neumann Raymond Y. Wang |
| author_facet | Shih-hsin Kan Jeffrey Y. Huang Jerry Harb Allisandra Rha Nancy D. Dalton Chloe Christensen Yunghang Chan Jeremy Davis-Turak Jonathan Neumann Raymond Y. Wang |
| author_sort | Shih-hsin Kan |
| collection | DOAJ |
| description | Abstract Pompe disease, an autosomal recessive disorder caused by deficient lysosomal acid α-glucosidase (GAA), is characterized by accumulation of intra-lysosomal glycogen in skeletal and oftentimes cardiac muscle. The c.1935C>A (p.Asp645Glu) variant, the most frequent GAA pathogenic mutation in people of Southern Han Chinese ancestry, causes infantile-onset Pompe disease (IOPD), presenting neonatally with severe hypertrophic cardiomyopathy, profound muscle hypotonia, respiratory failure, and infantile mortality. We applied CRISPR-Cas9 homology-directed repair (HDR) using a novel dual sgRNA approach flanking the target site to generate a Gaa em1935C>A knock-in mouse model and a myoblast cell line carrying the Gaa c.1935C>A mutation. Herein we describe the molecular, biochemical, histological, physiological, and behavioral characterization of 3-month-old homozygous Gaa em1935C>A mice. Homozygous Gaa em1935C>A knock-in mice exhibited normal Gaa mRNA expression levels relative to wild-type mice, had near-abolished GAA enzymatic activity, markedly increased tissue glycogen storage, and concomitantly impaired autophagy. Three-month-old mice demonstrated skeletal muscle weakness and hypertrophic cardiomyopathy but no premature mortality. The Gaa em1935C>A knock-in mouse model recapitulates multiple salient aspects of human IOPD caused by the GAA c.1935C>A pathogenic variant. It is an ideal model to assess innovative therapies to treat IOPD, including personalized therapeutic strategies that correct pathogenic variants, restore GAA activity and produce functional phenotypes. |
| first_indexed | 2024-04-11T12:37:26Z |
| format | Article |
| id | doaj.art-23464ddf02a94fbeb809115494e48be1 |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-04-11T12:37:26Z |
| publishDate | 2022-12-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-23464ddf02a94fbeb809115494e48be12022-12-22T04:23:35ZengNature PortfolioScientific Reports2045-23222022-12-0112111310.1038/s41598-022-25914-8CRISPR-mediated generation and characterization of a Gaa homozygous c.1935C>A (p.D645E) Pompe disease knock-in mouse model recapitulating human infantile onset-Pompe diseaseShih-hsin Kan0Jeffrey Y. Huang1Jerry Harb2Allisandra Rha3Nancy D. Dalton4Chloe Christensen5Yunghang Chan6Jeremy Davis-Turak7Jonathan Neumann8Raymond Y. Wang9CHOC Children’s Research InstituteCHOC Children’s Research InstituteCHOC Children’s Research InstituteCHOC Children’s Research InstituteCHOC Children’s Research InstituteCHOC Children’s Research InstituteSchool of Medicine, New York Medical CollegeROSALINDTransgenic Mouse Facility, University of California IrvineDivision of Metabolic Disorders, CHOC Children’s SpecialistsAbstract Pompe disease, an autosomal recessive disorder caused by deficient lysosomal acid α-glucosidase (GAA), is characterized by accumulation of intra-lysosomal glycogen in skeletal and oftentimes cardiac muscle. The c.1935C>A (p.Asp645Glu) variant, the most frequent GAA pathogenic mutation in people of Southern Han Chinese ancestry, causes infantile-onset Pompe disease (IOPD), presenting neonatally with severe hypertrophic cardiomyopathy, profound muscle hypotonia, respiratory failure, and infantile mortality. We applied CRISPR-Cas9 homology-directed repair (HDR) using a novel dual sgRNA approach flanking the target site to generate a Gaa em1935C>A knock-in mouse model and a myoblast cell line carrying the Gaa c.1935C>A mutation. Herein we describe the molecular, biochemical, histological, physiological, and behavioral characterization of 3-month-old homozygous Gaa em1935C>A mice. Homozygous Gaa em1935C>A knock-in mice exhibited normal Gaa mRNA expression levels relative to wild-type mice, had near-abolished GAA enzymatic activity, markedly increased tissue glycogen storage, and concomitantly impaired autophagy. Three-month-old mice demonstrated skeletal muscle weakness and hypertrophic cardiomyopathy but no premature mortality. The Gaa em1935C>A knock-in mouse model recapitulates multiple salient aspects of human IOPD caused by the GAA c.1935C>A pathogenic variant. It is an ideal model to assess innovative therapies to treat IOPD, including personalized therapeutic strategies that correct pathogenic variants, restore GAA activity and produce functional phenotypes.https://doi.org/10.1038/s41598-022-25914-8 |
| spellingShingle | Shih-hsin Kan Jeffrey Y. Huang Jerry Harb Allisandra Rha Nancy D. Dalton Chloe Christensen Yunghang Chan Jeremy Davis-Turak Jonathan Neumann Raymond Y. Wang CRISPR-mediated generation and characterization of a Gaa homozygous c.1935C>A (p.D645E) Pompe disease knock-in mouse model recapitulating human infantile onset-Pompe disease Scientific Reports |
| title | CRISPR-mediated generation and characterization of a Gaa homozygous c.1935C>A (p.D645E) Pompe disease knock-in mouse model recapitulating human infantile onset-Pompe disease |
| title_full | CRISPR-mediated generation and characterization of a Gaa homozygous c.1935C>A (p.D645E) Pompe disease knock-in mouse model recapitulating human infantile onset-Pompe disease |
| title_fullStr | CRISPR-mediated generation and characterization of a Gaa homozygous c.1935C>A (p.D645E) Pompe disease knock-in mouse model recapitulating human infantile onset-Pompe disease |
| title_full_unstemmed | CRISPR-mediated generation and characterization of a Gaa homozygous c.1935C>A (p.D645E) Pompe disease knock-in mouse model recapitulating human infantile onset-Pompe disease |
| title_short | CRISPR-mediated generation and characterization of a Gaa homozygous c.1935C>A (p.D645E) Pompe disease knock-in mouse model recapitulating human infantile onset-Pompe disease |
| title_sort | crispr mediated generation and characterization of a gaa homozygous c 1935c a p d645e pompe disease knock in mouse model recapitulating human infantile onset pompe disease |
| url | https://doi.org/10.1038/s41598-022-25914-8 |
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