CRISPR/nCas9-Based Genome Editing on GM2 Gangliosidoses Fibroblasts via Non-Viral Vectors
The gangliosidoses GM2 are a group of pathologies mainly affecting the central nervous system due to the impaired GM2 ganglioside degradation inside the lysosome. Under physiological conditions, GM2 ganglioside is catabolized by the β-hexosaminidase A in a GM2 activator protein-dependent mechanism....
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MDPI AG
2022-09-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/23/18/10672 |
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| author | Andrés Felipe Leal Javier Cifuentes Valentina Quezada Eliana Benincore-Flórez Juan Carlos Cruz Luis Humberto Reyes Angela Johana Espejo-Mojica Carlos Javier Alméciga-Díaz |
| author_facet | Andrés Felipe Leal Javier Cifuentes Valentina Quezada Eliana Benincore-Flórez Juan Carlos Cruz Luis Humberto Reyes Angela Johana Espejo-Mojica Carlos Javier Alméciga-Díaz |
| author_sort | Andrés Felipe Leal |
| collection | DOAJ |
| description | The gangliosidoses GM2 are a group of pathologies mainly affecting the central nervous system due to the impaired GM2 ganglioside degradation inside the lysosome. Under physiological conditions, GM2 ganglioside is catabolized by the β-hexosaminidase A in a GM2 activator protein-dependent mechanism. In contrast, uncharged substrates such as globosides and some glycosaminoglycans can be hydrolyzed by the β-hexosaminidase B. Monogenic mutations on <i>HEXA</i>, <i>HEXB</i>, or <i>GM2A</i> genes arise in the Tay–Sachs (TSD), Sandhoff (SD), and AB variant diseases, respectively. In this work, we validated a CRISPR/Cas9-based gene editing strategy that relies on a Cas9 nickase (nCas9) as a potential approach for treating GM2 gangliosidoses using in vitro models for TSD and SD. The nCas9 contains a mutation in the catalytic RuvC domain but maintains the active HNH domain, which reduces potential off-target effects. Liposomes (LPs)- and novel magnetoliposomes (MLPs)-based vectors were used to deliver the CRISPR/nCas9 system. When LPs were used as a vector, positive outcomes were observed for the β-hexosaminidase activity, glycosaminoglycans levels, lysosome mass, and oxidative stress. In the case of MLPs, a high cytocompatibility and transfection ratio was observed, with a slight increase in the β-hexosaminidase activity and significant oxidative stress recovery in both TSD and SD cells. These results show the remarkable potential of CRISPR/nCas9 as a new alternative for treating GM2 gangliosidoses, as well as the superior performance of non-viral vectors in enhancing the potency of this therapeutic approach. |
| first_indexed | 2024-03-09T23:44:28Z |
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| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-09T23:44:28Z |
| publishDate | 2022-09-01 |
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| spelling | doaj.art-0fd634efe2bf484eae27400e11735bfb2023-11-23T16:46:15ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672022-09-0123181067210.3390/ijms231810672CRISPR/nCas9-Based Genome Editing on GM2 Gangliosidoses Fibroblasts via Non-Viral VectorsAndrés Felipe Leal0Javier Cifuentes1Valentina Quezada2Eliana Benincore-Flórez3Juan Carlos Cruz4Luis Humberto Reyes5Angela Johana Espejo-Mojica6Carlos Javier Alméciga-Díaz7Institute for the Study of Inborn Errors of Metabolism, Faculty of Science, Pontificia Universidad Javeriana, Bogotá 110231, ColombiaDepartment of Biomedical Engineering, Universidad de los Andes, Bogotá 111711, ColombiaDepartment of Biomedical Engineering, Universidad de los Andes, Bogotá 111711, ColombiaInstitute for the Study of Inborn Errors of Metabolism, Faculty of Science, Pontificia Universidad Javeriana, Bogotá 110231, ColombiaDepartment of Biomedical Engineering, Universidad de los Andes, Bogotá 111711, ColombiaGrupo de Diseño de Productos y Procesos (GDPP), Department of Chemical and Food Engineering, Universidad de los Andes, Bogotá 111711, ColombiaInstitute for the Study of Inborn Errors of Metabolism, Faculty of Science, Pontificia Universidad Javeriana, Bogotá 110231, ColombiaInstitute for the Study of Inborn Errors of Metabolism, Faculty of Science, Pontificia Universidad Javeriana, Bogotá 110231, ColombiaThe gangliosidoses GM2 are a group of pathologies mainly affecting the central nervous system due to the impaired GM2 ganglioside degradation inside the lysosome. Under physiological conditions, GM2 ganglioside is catabolized by the β-hexosaminidase A in a GM2 activator protein-dependent mechanism. In contrast, uncharged substrates such as globosides and some glycosaminoglycans can be hydrolyzed by the β-hexosaminidase B. Monogenic mutations on <i>HEXA</i>, <i>HEXB</i>, or <i>GM2A</i> genes arise in the Tay–Sachs (TSD), Sandhoff (SD), and AB variant diseases, respectively. In this work, we validated a CRISPR/Cas9-based gene editing strategy that relies on a Cas9 nickase (nCas9) as a potential approach for treating GM2 gangliosidoses using in vitro models for TSD and SD. The nCas9 contains a mutation in the catalytic RuvC domain but maintains the active HNH domain, which reduces potential off-target effects. Liposomes (LPs)- and novel magnetoliposomes (MLPs)-based vectors were used to deliver the CRISPR/nCas9 system. When LPs were used as a vector, positive outcomes were observed for the β-hexosaminidase activity, glycosaminoglycans levels, lysosome mass, and oxidative stress. In the case of MLPs, a high cytocompatibility and transfection ratio was observed, with a slight increase in the β-hexosaminidase activity and significant oxidative stress recovery in both TSD and SD cells. These results show the remarkable potential of CRISPR/nCas9 as a new alternative for treating GM2 gangliosidoses, as well as the superior performance of non-viral vectors in enhancing the potency of this therapeutic approach.https://www.mdpi.com/1422-0067/23/18/10672CRISPR/nCas9genome editingGM2 gangliosidosesnon-viral vectorsSandhoffTay–Sachs |
| spellingShingle | Andrés Felipe Leal Javier Cifuentes Valentina Quezada Eliana Benincore-Flórez Juan Carlos Cruz Luis Humberto Reyes Angela Johana Espejo-Mojica Carlos Javier Alméciga-Díaz CRISPR/nCas9-Based Genome Editing on GM2 Gangliosidoses Fibroblasts via Non-Viral Vectors International Journal of Molecular Sciences CRISPR/nCas9 genome editing GM2 gangliosidoses non-viral vectors Sandhoff Tay–Sachs |
| title | CRISPR/nCas9-Based Genome Editing on GM2 Gangliosidoses Fibroblasts via Non-Viral Vectors |
| title_full | CRISPR/nCas9-Based Genome Editing on GM2 Gangliosidoses Fibroblasts via Non-Viral Vectors |
| title_fullStr | CRISPR/nCas9-Based Genome Editing on GM2 Gangliosidoses Fibroblasts via Non-Viral Vectors |
| title_full_unstemmed | CRISPR/nCas9-Based Genome Editing on GM2 Gangliosidoses Fibroblasts via Non-Viral Vectors |
| title_short | CRISPR/nCas9-Based Genome Editing on GM2 Gangliosidoses Fibroblasts via Non-Viral Vectors |
| title_sort | crispr ncas9 based genome editing on gm2 gangliosidoses fibroblasts via non viral vectors |
| topic | CRISPR/nCas9 genome editing GM2 gangliosidoses non-viral vectors Sandhoff Tay–Sachs |
| url | https://www.mdpi.com/1422-0067/23/18/10672 |
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