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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....
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9505638/ https://www.ncbi.nlm.nih.gov/pubmed/36142595 http://dx.doi.org/10.3390/ijms231810672 |
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author | Leal, Andrés Felipe Cifuentes, Javier Quezada, Valentina Benincore-Flórez, Eliana Cruz, Juan Carlos Reyes, Luis Humberto Espejo-Mojica, Angela Johana Alméciga-Díaz, Carlos Javier |
author_facet | Leal, Andrés Felipe Cifuentes, Javier Quezada, Valentina Benincore-Flórez, Eliana Cruz, Juan Carlos Reyes, Luis Humberto Espejo-Mojica, Angela Johana Alméciga-Díaz, Carlos Javier |
author_sort | Leal, Andrés Felipe |
collection | PubMed |
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 HEXA, HEXB, or GM2A 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. |
format | Online Article Text |
id | pubmed-9505638 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-95056382022-09-24 CRISPR/nCas9-Based Genome Editing on GM2 Gangliosidoses Fibroblasts via Non-Viral Vectors Leal, Andrés Felipe Cifuentes, Javier Quezada, Valentina Benincore-Flórez, Eliana Cruz, Juan Carlos Reyes, Luis Humberto Espejo-Mojica, Angela Johana Alméciga-Díaz, Carlos Javier Int J Mol Sci Article 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 HEXA, HEXB, or GM2A 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. MDPI 2022-09-14 /pmc/articles/PMC9505638/ /pubmed/36142595 http://dx.doi.org/10.3390/ijms231810672 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Leal, Andrés Felipe Cifuentes, Javier Quezada, Valentina Benincore-Flórez, Eliana Cruz, Juan Carlos Reyes, Luis Humberto Espejo-Mojica, Angela Johana Alméciga-Díaz, Carlos Javier CRISPR/nCas9-Based Genome Editing on GM2 Gangliosidoses Fibroblasts via Non-Viral Vectors |
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 | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9505638/ https://www.ncbi.nlm.nih.gov/pubmed/36142595 http://dx.doi.org/10.3390/ijms231810672 |
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