High-Capacity Adenoviral Vectors Permit Robust and Versatile Testing of <i>DMD</i> Gene Repair Tools and Strategies in Human Cells
Duchenne muscular dystrophy (DMD) is a fatal X-linked muscle wasting disorder arising from mutations in the ~2.4 Mb dystrophin-encoding <i>DMD</i> gene. RNA-guided CRISPR-Cas9 nucleases (RGNs) are opening new DMD therapeutic routes whose bottlenecks include delivering sizable RGN complex...
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MDPI AG
2020-04-01
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| Series: | Cells |
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| Online Access: | https://www.mdpi.com/2073-4409/9/4/869 |
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| author | Marcella Brescia Josephine M. Janssen Jin Liu Manuel A. F. V. Gonçalves |
| author_facet | Marcella Brescia Josephine M. Janssen Jin Liu Manuel A. F. V. Gonçalves |
| author_sort | Marcella Brescia |
| collection | DOAJ |
| description | Duchenne muscular dystrophy (DMD) is a fatal X-linked muscle wasting disorder arising from mutations in the ~2.4 Mb dystrophin-encoding <i>DMD</i> gene. RNA-guided CRISPR-Cas9 nucleases (RGNs) are opening new DMD therapeutic routes whose bottlenecks include delivering sizable RGN complexes for assessing their effects on human genomes and testing ex vivo and in vivo <i>DMD</i>-correcting strategies. Here, high-capacity adenoviral vectors (HC-AdVs) encoding single or dual high-specificity RGNs with optimized components were investigated for permanently repairing defective <i>DMD</i> alleles either through exon 51-targeted indel formation or major mutational hotspot excision (>500 kb), respectively. Firstly, we establish that, at high doses, third-generation HC-AdVs lacking all viral genes are significantly less cytotoxic than second-generation adenoviral vectors deleted in <i>E1</i> and <i>E2A</i>. Secondly, we demonstrate that genetically retargeted HC-AdVs can correct up to 42% ± 13% of defective <i>DMD</i> alleles in muscle cell populations through targeted removal of the major mutational hotspot, in which over 60% of frame-shifting large deletions locate. Both <i>DMD</i> gene repair strategies tested readily led to the detection of Becker-like dystrophins in unselected muscle cell populations, leading to the restoration of β-dystroglycan at the plasmalemma of differentiated muscle cells. Hence, HC-AdVs permit the effective assessment of <i>DMD</i> gene-editing tools and strategies in dystrophin-defective human cells while broadening the gamut of <i>DMD</i>-correcting agents. |
| first_indexed | 2024-03-10T20:43:18Z |
| format | Article |
| id | doaj.art-c5a32dfddf4a4d22a011e3bee605c29b |
| institution | Directory Open Access Journal |
| issn | 2073-4409 |
| language | English |
| last_indexed | 2024-03-10T20:43:18Z |
| publishDate | 2020-04-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Cells |
| spelling | doaj.art-c5a32dfddf4a4d22a011e3bee605c29b2023-11-19T20:33:40ZengMDPI AGCells2073-44092020-04-019486910.3390/cells9040869High-Capacity Adenoviral Vectors Permit Robust and Versatile Testing of <i>DMD</i> Gene Repair Tools and Strategies in Human CellsMarcella Brescia0Josephine M. Janssen1Jin Liu2Manuel A. F. V. Gonçalves3Department of Cell and Chemical Biology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, The NetherlandsDepartment of Cell and Chemical Biology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, The NetherlandsDepartment of Cell and Chemical Biology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, The NetherlandsDepartment of Cell and Chemical Biology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, The NetherlandsDuchenne muscular dystrophy (DMD) is a fatal X-linked muscle wasting disorder arising from mutations in the ~2.4 Mb dystrophin-encoding <i>DMD</i> gene. RNA-guided CRISPR-Cas9 nucleases (RGNs) are opening new DMD therapeutic routes whose bottlenecks include delivering sizable RGN complexes for assessing their effects on human genomes and testing ex vivo and in vivo <i>DMD</i>-correcting strategies. Here, high-capacity adenoviral vectors (HC-AdVs) encoding single or dual high-specificity RGNs with optimized components were investigated for permanently repairing defective <i>DMD</i> alleles either through exon 51-targeted indel formation or major mutational hotspot excision (>500 kb), respectively. Firstly, we establish that, at high doses, third-generation HC-AdVs lacking all viral genes are significantly less cytotoxic than second-generation adenoviral vectors deleted in <i>E1</i> and <i>E2A</i>. Secondly, we demonstrate that genetically retargeted HC-AdVs can correct up to 42% ± 13% of defective <i>DMD</i> alleles in muscle cell populations through targeted removal of the major mutational hotspot, in which over 60% of frame-shifting large deletions locate. Both <i>DMD</i> gene repair strategies tested readily led to the detection of Becker-like dystrophins in unselected muscle cell populations, leading to the restoration of β-dystroglycan at the plasmalemma of differentiated muscle cells. Hence, HC-AdVs permit the effective assessment of <i>DMD</i> gene-editing tools and strategies in dystrophin-defective human cells while broadening the gamut of <i>DMD</i>-correcting agents.https://www.mdpi.com/2073-4409/9/4/869gene editinggene repairCRISPR-Cas9multiplexinghigh-specificity nucleaseshigh-capacity adenoviral vectors |
| spellingShingle | Marcella Brescia Josephine M. Janssen Jin Liu Manuel A. F. V. Gonçalves High-Capacity Adenoviral Vectors Permit Robust and Versatile Testing of <i>DMD</i> Gene Repair Tools and Strategies in Human Cells Cells gene editing gene repair CRISPR-Cas9 multiplexing high-specificity nucleases high-capacity adenoviral vectors |
| title | High-Capacity Adenoviral Vectors Permit Robust and Versatile Testing of <i>DMD</i> Gene Repair Tools and Strategies in Human Cells |
| title_full | High-Capacity Adenoviral Vectors Permit Robust and Versatile Testing of <i>DMD</i> Gene Repair Tools and Strategies in Human Cells |
| title_fullStr | High-Capacity Adenoviral Vectors Permit Robust and Versatile Testing of <i>DMD</i> Gene Repair Tools and Strategies in Human Cells |
| title_full_unstemmed | High-Capacity Adenoviral Vectors Permit Robust and Versatile Testing of <i>DMD</i> Gene Repair Tools and Strategies in Human Cells |
| title_short | High-Capacity Adenoviral Vectors Permit Robust and Versatile Testing of <i>DMD</i> Gene Repair Tools and Strategies in Human Cells |
| title_sort | high capacity adenoviral vectors permit robust and versatile testing of i dmd i gene repair tools and strategies in human cells |
| topic | gene editing gene repair CRISPR-Cas9 multiplexing high-specificity nucleases high-capacity adenoviral vectors |
| url | https://www.mdpi.com/2073-4409/9/4/869 |
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