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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....

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Autores principales: Leal, Andrés Felipe, Cifuentes, Javier, Quezada, Valentina, Benincore-Flórez, Eliana, Cruz, Juan Carlos, Reyes, Luis Humberto, Espejo-Mojica, Angela Johana, Alméciga-Díaz, Carlos Javier
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
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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.
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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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