mRNA-mediated delivery of gene editing tools to human primary muscle stem cells
Muscular dystrophies are approximately 50 devastating, untreatable monogenic diseases leading to progressive muscle degeneration and atrophy. Gene correction of transplantable cells using CRISPR/Cas9-based tools is a realistic scenario for autologous cell replacement therapies to restore organ funct...
| Main Authors: | , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2022-06-01
|
| Series: | Molecular Therapy: Nucleic Acids |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2162253122000440 |
| _version_ | 1828858684120236032 |
|---|---|
| author | Christian Stadelmann Silvia Di Francescantonio Andreas Marg Stefanie Müthel Simone Spuler Helena Escobar |
| author_facet | Christian Stadelmann Silvia Di Francescantonio Andreas Marg Stefanie Müthel Simone Spuler Helena Escobar |
| author_sort | Christian Stadelmann |
| collection | DOAJ |
| description | Muscular dystrophies are approximately 50 devastating, untreatable monogenic diseases leading to progressive muscle degeneration and atrophy. Gene correction of transplantable cells using CRISPR/Cas9-based tools is a realistic scenario for autologous cell replacement therapies to restore organ function in many genetic disorders. However, muscle stem cells have so far lagged behind due to the absence of methods to isolate and propagate them and their susceptibility to extensive ex vivo manipulations. Here, we show that mRNA-based delivery of SpCas9 and an adenine base editor results in up to >90% efficient genome editing in human muscle stem cells from many donors regardless of age and gender and without any enrichment step. Using NCAM1 as an endogenous reporter locus expressed by all muscle stem cells and whose knockout does not affect cell fitness, we show that cells edited with mRNA fully retain their myogenic marker signature, proliferation capacity, and functional attributes. Moreover, mRNA-based delivery of a base editor led to the highly efficient repair of a muscular dystrophy-causing SGCA mutation in a single selection-free step. In summary, our work establishes mRNA-mediated delivery of CRISPR/Cas9-based tools as a promising and universal approach for taking gene edited muscle stem cells into clinical application to treat muscle disease. |
| first_indexed | 2024-12-13T02:01:33Z |
| format | Article |
| id | doaj.art-bca5f4941def4db8a722074c437a2bbb |
| institution | Directory Open Access Journal |
| issn | 2162-2531 |
| language | English |
| last_indexed | 2024-12-13T02:01:33Z |
| publishDate | 2022-06-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Molecular Therapy: Nucleic Acids |
| spelling | doaj.art-bca5f4941def4db8a722074c437a2bbb2022-12-22T00:03:15ZengElsevierMolecular Therapy: Nucleic Acids2162-25312022-06-01284757mRNA-mediated delivery of gene editing tools to human primary muscle stem cellsChristian Stadelmann0Silvia Di Francescantonio1Andreas Marg2Stefanie Müthel3Simone Spuler4Helena Escobar5Experimental and Clinical Research Center, A Cooperation Between the Max Delbrück Center for Molecular Medicine in the Helmholtz Association and the Charité – Universitätsmedizin Berlin, 13125 Berlin, Germany; Charité – Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Experimental and Clinical Research Center, Charité Campus Buch, Lindenberger Weg 80, 13125 Berlin, Germany; Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), 13125 Berlin, Germany; Department of Biology, Chemistry, and Pharmacy, Freie Universität Berlin, 14195 Berlin, GermanyExperimental and Clinical Research Center, A Cooperation Between the Max Delbrück Center for Molecular Medicine in the Helmholtz Association and the Charité – Universitätsmedizin Berlin, 13125 Berlin, Germany; Charité – Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Experimental and Clinical Research Center, Charité Campus Buch, Lindenberger Weg 80, 13125 Berlin, Germany; Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), 13125 Berlin, GermanyExperimental and Clinical Research Center, A Cooperation Between the Max Delbrück Center for Molecular Medicine in the Helmholtz Association and the Charité – Universitätsmedizin Berlin, 13125 Berlin, Germany; Charité – Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Experimental and Clinical Research Center, Charité Campus Buch, Lindenberger Weg 80, 13125 Berlin, Germany; Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), 13125 Berlin, GermanyExperimental and Clinical Research Center, A Cooperation Between the Max Delbrück Center for Molecular Medicine in the Helmholtz Association and the Charité – Universitätsmedizin Berlin, 13125 Berlin, Germany; Charité – Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Experimental and Clinical Research Center, Charité Campus Buch, Lindenberger Weg 80, 13125 Berlin, Germany; Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), 13125 Berlin, GermanyExperimental and Clinical Research Center, A Cooperation Between the Max Delbrück Center for Molecular Medicine in the Helmholtz Association and the Charité – Universitätsmedizin Berlin, 13125 Berlin, Germany; Charité – Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Experimental and Clinical Research Center, Charité Campus Buch, Lindenberger Weg 80, 13125 Berlin, Germany; Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), 13125 Berlin, Germany; Berlin Institute of Health at Charité – Universitätsmedizin Berlin, 10178 Berlin, Germany; Corresponding author. Simone Spuler, MD, Experimental and Clinical Research Center, A Cooperation Between the Max Delbrück Center for Molecular Medicine in the Helmholtz Association and the Charité – Universitätsmedizin Berlin, 13125 Berlin, Germany.Experimental and Clinical Research Center, A Cooperation Between the Max Delbrück Center for Molecular Medicine in the Helmholtz Association and the Charité – Universitätsmedizin Berlin, 13125 Berlin, Germany; Charité – Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Experimental and Clinical Research Center, Charité Campus Buch, Lindenberger Weg 80, 13125 Berlin, Germany; Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), 13125 Berlin, Germany; Corresponding author. Helena Escobar, PhD, Experimental and Clinical Research Center, A Cooperation Between the Max Delbrück Center for Molecular Medicine in the Helmholtz Association and the Charité – Universitätsmedizin Berlin, 13125 Berlin, Germany.Muscular dystrophies are approximately 50 devastating, untreatable monogenic diseases leading to progressive muscle degeneration and atrophy. Gene correction of transplantable cells using CRISPR/Cas9-based tools is a realistic scenario for autologous cell replacement therapies to restore organ function in many genetic disorders. However, muscle stem cells have so far lagged behind due to the absence of methods to isolate and propagate them and their susceptibility to extensive ex vivo manipulations. Here, we show that mRNA-based delivery of SpCas9 and an adenine base editor results in up to >90% efficient genome editing in human muscle stem cells from many donors regardless of age and gender and without any enrichment step. Using NCAM1 as an endogenous reporter locus expressed by all muscle stem cells and whose knockout does not affect cell fitness, we show that cells edited with mRNA fully retain their myogenic marker signature, proliferation capacity, and functional attributes. Moreover, mRNA-based delivery of a base editor led to the highly efficient repair of a muscular dystrophy-causing SGCA mutation in a single selection-free step. In summary, our work establishes mRNA-mediated delivery of CRISPR/Cas9-based tools as a promising and universal approach for taking gene edited muscle stem cells into clinical application to treat muscle disease.http://www.sciencedirect.com/science/article/pii/S2162253122000440MT: DNA editinghuman muscle stem cellsCRISPR/Cas9gene editingbase editingmuscular dystrophy |
| spellingShingle | Christian Stadelmann Silvia Di Francescantonio Andreas Marg Stefanie Müthel Simone Spuler Helena Escobar mRNA-mediated delivery of gene editing tools to human primary muscle stem cells Molecular Therapy: Nucleic Acids MT: DNA editing human muscle stem cells CRISPR/Cas9 gene editing base editing muscular dystrophy |
| title | mRNA-mediated delivery of gene editing tools to human primary muscle stem cells |
| title_full | mRNA-mediated delivery of gene editing tools to human primary muscle stem cells |
| title_fullStr | mRNA-mediated delivery of gene editing tools to human primary muscle stem cells |
| title_full_unstemmed | mRNA-mediated delivery of gene editing tools to human primary muscle stem cells |
| title_short | mRNA-mediated delivery of gene editing tools to human primary muscle stem cells |
| title_sort | mrna mediated delivery of gene editing tools to human primary muscle stem cells |
| topic | MT: DNA editing human muscle stem cells CRISPR/Cas9 gene editing base editing muscular dystrophy |
| url | http://www.sciencedirect.com/science/article/pii/S2162253122000440 |
| work_keys_str_mv | AT christianstadelmann mrnamediateddeliveryofgeneeditingtoolstohumanprimarymusclestemcells AT silviadifrancescantonio mrnamediateddeliveryofgeneeditingtoolstohumanprimarymusclestemcells AT andreasmarg mrnamediateddeliveryofgeneeditingtoolstohumanprimarymusclestemcells AT stefaniemuthel mrnamediateddeliveryofgeneeditingtoolstohumanprimarymusclestemcells AT simonespuler mrnamediateddeliveryofgeneeditingtoolstohumanprimarymusclestemcells AT helenaescobar mrnamediateddeliveryofgeneeditingtoolstohumanprimarymusclestemcells |