In Vivo Gene Editing of Muscle Stem Cells with Adeno-Associated Viral Vectors in a Mouse Model of Duchenne Muscular Dystrophy
Delivery of therapeutic transgenes with adeno-associated viral (AAV) vectors for treatment of myopathies has yielded encouraging results in animal models and early clinical studies. Although certain AAV serotypes efficiently target muscle fibers, transduction of the muscle stem cells, also known as...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2020-12-01
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| Series: | Molecular Therapy: Methods & Clinical Development |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2329050120302011 |
| _version_ | 1818664319586402304 |
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| author | Jennifer B. Kwon Adarsh R. Ettyreddy Ashish Vankara Joel D. Bohning Garth Devlin Stephen D. Hauschka Aravind Asokan Charles A. Gersbach |
| author_facet | Jennifer B. Kwon Adarsh R. Ettyreddy Ashish Vankara Joel D. Bohning Garth Devlin Stephen D. Hauschka Aravind Asokan Charles A. Gersbach |
| author_sort | Jennifer B. Kwon |
| collection | DOAJ |
| description | Delivery of therapeutic transgenes with adeno-associated viral (AAV) vectors for treatment of myopathies has yielded encouraging results in animal models and early clinical studies. Although certain AAV serotypes efficiently target muscle fibers, transduction of the muscle stem cells, also known as satellite cells, is less studied. Here, we used a Pax7nGFP;Ai9 dual reporter mouse to quantify AAV transduction events in satellite cells. We assessed a panel of AAV serotypes for satellite cell tropism in the mdx mouse model of Duchenne muscular dystrophy and observed the highest satellite cell labeling with AAV9 following local or systemic administration. Subsequently, we used AAV9 to interrogate CRISPR/Cas9-mediated gene editing of satellite cells in the Pax7nGFP;mdx mouse. We quantified the level of gene editing using a Tn5 transposon-based method for unbiased sequencing of editing outcomes at the Dmd locus. We also found that muscle-specific promoters can drive transgene expression and gene editing in satellite cells. Lastly, to demonstrate the functionality of satellite cells edited at the Dmd locus by CRISPR in vivo, we performed a transplantation experiment and observed increased dystrophin-positive fibers in the recipient mouse. Collectively, our results confirm that satellite cells are transduced by AAV and can undergo gene editing to restore the dystrophin reading frame in the mdx mouse. |
| first_indexed | 2024-12-17T05:30:51Z |
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| institution | Directory Open Access Journal |
| issn | 2329-0501 |
| language | English |
| last_indexed | 2024-12-17T05:30:51Z |
| publishDate | 2020-12-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Molecular Therapy: Methods & Clinical Development |
| spelling | doaj.art-027bf4f182644f8e99d5b59e596aa1d12022-12-21T22:01:43ZengElsevierMolecular Therapy: Methods & Clinical Development2329-05012020-12-0119320329In Vivo Gene Editing of Muscle Stem Cells with Adeno-Associated Viral Vectors in a Mouse Model of Duchenne Muscular DystrophyJennifer B. Kwon0Adarsh R. Ettyreddy1Ashish Vankara2Joel D. Bohning3Garth Devlin4Stephen D. Hauschka5Aravind Asokan6Charles A. Gersbach7University Program in Genetics and Genomics, Duke University Medical Center, Durham, NC 27710, USA; Center for Advanced Genomic Technologies, Duke University, Durham, NC 27708, USACenter for Advanced Genomic Technologies, Duke University, Durham, NC 27708, USA; Department of Biomedical Engineering, Duke University, Durham, NC 27708, USACenter for Advanced Genomic Technologies, Duke University, Durham, NC 27708, USA; Department of Biomedical Engineering, Duke University, Durham, NC 27708, USACenter for Advanced Genomic Technologies, Duke University, Durham, NC 27708, USA; Department of Biomedical Engineering, Duke University, Durham, NC 27708, USADepartment of Surgery, Duke University Medical Center, Durham, NC 27710, USADepartment of Biochemistry, University of Washington, Seattle, WA, USACenter for Advanced Genomic Technologies, Duke University, Durham, NC 27708, USA; Department of Biomedical Engineering, Duke University, Durham, NC 27708, USA; Department of Surgery, Duke University Medical Center, Durham, NC 27710, USA; Regeneration Next Initiative, Duke University Medical Center, Durham, NC 27710, USACenter for Advanced Genomic Technologies, Duke University, Durham, NC 27708, USA; Department of Biomedical Engineering, Duke University, Durham, NC 27708, USA; Department of Surgery, Duke University Medical Center, Durham, NC 27710, USA; Regeneration Next Initiative, Duke University Medical Center, Durham, NC 27710, USA; Corresponding author: Charles A. Gersbach, PhD, Department of Biomedical Engineering, Duke University, Room 1427 CIEMAS, 101 Science Drive, Box 90281, Durham, NC 27708-0281, USA.Delivery of therapeutic transgenes with adeno-associated viral (AAV) vectors for treatment of myopathies has yielded encouraging results in animal models and early clinical studies. Although certain AAV serotypes efficiently target muscle fibers, transduction of the muscle stem cells, also known as satellite cells, is less studied. Here, we used a Pax7nGFP;Ai9 dual reporter mouse to quantify AAV transduction events in satellite cells. We assessed a panel of AAV serotypes for satellite cell tropism in the mdx mouse model of Duchenne muscular dystrophy and observed the highest satellite cell labeling with AAV9 following local or systemic administration. Subsequently, we used AAV9 to interrogate CRISPR/Cas9-mediated gene editing of satellite cells in the Pax7nGFP;mdx mouse. We quantified the level of gene editing using a Tn5 transposon-based method for unbiased sequencing of editing outcomes at the Dmd locus. We also found that muscle-specific promoters can drive transgene expression and gene editing in satellite cells. Lastly, to demonstrate the functionality of satellite cells edited at the Dmd locus by CRISPR in vivo, we performed a transplantation experiment and observed increased dystrophin-positive fibers in the recipient mouse. Collectively, our results confirm that satellite cells are transduced by AAV and can undergo gene editing to restore the dystrophin reading frame in the mdx mouse.http://www.sciencedirect.com/science/article/pii/S2329050120302011dmdsatellitepax7gene-editingcrispraav |
| spellingShingle | Jennifer B. Kwon Adarsh R. Ettyreddy Ashish Vankara Joel D. Bohning Garth Devlin Stephen D. Hauschka Aravind Asokan Charles A. Gersbach In Vivo Gene Editing of Muscle Stem Cells with Adeno-Associated Viral Vectors in a Mouse Model of Duchenne Muscular Dystrophy Molecular Therapy: Methods & Clinical Development dmd satellite pax7 gene-editing crispr aav |
| title | In Vivo Gene Editing of Muscle Stem Cells with Adeno-Associated Viral Vectors in a Mouse Model of Duchenne Muscular Dystrophy |
| title_full | In Vivo Gene Editing of Muscle Stem Cells with Adeno-Associated Viral Vectors in a Mouse Model of Duchenne Muscular Dystrophy |
| title_fullStr | In Vivo Gene Editing of Muscle Stem Cells with Adeno-Associated Viral Vectors in a Mouse Model of Duchenne Muscular Dystrophy |
| title_full_unstemmed | In Vivo Gene Editing of Muscle Stem Cells with Adeno-Associated Viral Vectors in a Mouse Model of Duchenne Muscular Dystrophy |
| title_short | In Vivo Gene Editing of Muscle Stem Cells with Adeno-Associated Viral Vectors in a Mouse Model of Duchenne Muscular Dystrophy |
| title_sort | in vivo gene editing of muscle stem cells with adeno associated viral vectors in a mouse model of duchenne muscular dystrophy |
| topic | dmd satellite pax7 gene-editing crispr aav |
| url | http://www.sciencedirect.com/science/article/pii/S2329050120302011 |
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