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Efficient CRISPR/Cas9 nickase-mediated genome editing in an in vitro model of mucopolysaccharidosis IVA

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Abstract

Mucopolysaccharidosis IVA (MPS IVA) is a lysosomal storage disorder (LSD) caused by mutations in gene encoding for GALNS enzyme. Lack of GALNS activity leads to the accumulation of glycosaminoglycans (GAGs) keratan sulfate and chondroitin 6-sulfate. Although enzyme replacement therapy has been approved since 2014 for MPS IVA, still there is an unmet medical need to have improved therapies for this disorder. CRISPR/Cas9-based gene therapy has been tested for several LSDs with encouraging findings, but to date it has not been assayed on MPS IVA. In this work, we validated for the first time the use of CRISPR/Cas9, using a Cas9 nickase, for the knock-in of an expression cassette containing GALNS cDNA in an in vitro model of MPS IVA. The results showed the successful homologous recombination of the expression cassette into the AAVS1 locus, as well as a long-term increase in GALNS activity reaching up to 40% of wild-type levels. We also observed normalization of lysosomal mass, total GAGs, and oxidative stress, which are some of the major findings regarding the pathophysiological events in MPS IVA. These results represent a proof-of-concept of the use of CRISPR/Cas9 nickase strategy for the development of a novel therapeutic alternative for MPS IVA.

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Fig. 1: Evaluation of the nCas9 double nickase-mediated cut.
Fig. 2: Molecular validation of HR and transgene expression.
Fig. 3: Long-term edition on MPS IVA fibroblast.
Fig. 4: Phenotype correction of MPS IVA biomarkers.

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Data availability

Data generated or analyzed during this study are available from the corresponding author on reasonable request.

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Acknowledgements

We thank Dr. Paola Lasso and the Biomedics Science Unit, at the Faculty of Science at Pontificia Universidad Javeriana, for their assistance during flow cytometry experiments.

Funding

CJAD was suppoted by Ministerio de Ciencia, Tecnología e Innovación, Colombia (Contract 120380763212, ID 8352), Pontificia Universidad Javeriana (ID 20289), and the National MPS Society (ID 9509). AFL recived doctoral scholarship from Pontificia Universidad Javeriana and was supported by Suministros Clínicos ISLA S.A.S.

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  1. Institute for the Study of Inborn Errors of Metabolism, Faculty of Science, Pontificia Universidad Javeriana, Bogotá D.C., 110231, Colombia

    Andrés Felipe Leal & Carlos Javier Alméciga-Díaz

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  1. Andrés Felipe Leal
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AFL was responsible for conducting the search, extracting and analyzing data, and interpreting results. CJAD contributed to experiments planning, data analysis and interpretation, and provided feedback.

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Correspondence to Carlos Javier Alméciga-Díaz.

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Leal, A.F., Alméciga-Díaz, C.J. Efficient CRISPR/Cas9 nickase-mediated genome editing in an in vitro model of mucopolysaccharidosis IVA. Gene Ther 30, 107–114 (2023). https://doi.org/10.1038/s41434-022-00344-3

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