Efficient CRISPR/Cas9 Nickase-mediated Genome Editing in an in Vitro Model of Mucopolysaccharidosis IVA

Overview

Journal Gene Ther

Date 2022 May 17

PMID 35581402

Authors

Andres Felipe Leal

Carlos Javier Almeciga-Diaz

Affiliations

Soon will be listed here.

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.

Citing Articles

CRISPR/Cas9 technology in the modeling of and treatment of mucopolysaccharidosis.

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PMID: 39044769 PMC: 11263496. DOI: 10.1016/j.bbrep.2024.101771.

Current Strategies for Increasing Knock-In Efficiency in CRISPR/Cas9-Based Approaches.

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PMID: 38473704 PMC: 10931195. DOI: 10.3390/ijms25052456.

Iron oxide-coupled CRISPR-nCas9-based genome editing assessment in mucopolysaccharidosis IVA mice.

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PMID: 38107675 PMC: 10724691. DOI: 10.1016/j.omtm.2023.101153.

Mucopolysaccharidosis IVA: Current Disease Models and Drawbacks.

Leal A, Almeciga-Diaz C, Tomatsu S Int J Mol Sci. 2023; 24(22).

PMID: 38003337 PMC: 10671113. DOI: 10.3390/ijms242216148.

Genome Editing Tools for Lysosomal Storage Disorders.

Gonzalez E, Nader H, Siebert M, Suarez D, Almeciga-Diaz C, Baldo G Adv Exp Med Biol. 2023; 1429:127-155.

PMID: 37486520 DOI: 10.1007/978-3-031-33325-5_8.

References

Rivera-Colon Y, Schutsky E, Kita A, Garman S . The structure of human GALNS reveals the molecular basis for mucopolysaccharidosis IV A. J Mol Biol. 2012; 423(5):736-51. PMC: 3472114. DOI: 10.1016/j.jmb.2012.08.020. View

Gomez A, Garcia-Robles R, Suarez-Obando F . [Estimation of the mucopolysaccharidoses frequencies and cluster analysis in the Colombian provinces of Cundinamarca and Boyacá]. Biomedica. 2013; 32(4):602-9. DOI: 10.1590/S0120-41572012000400015. View

Puentes-Tellez M, Lerma-Barbosa P, Garzon-Jaramillo R, Suarez D, Espejo-Mojica A, Guevara J . A perspective on research, diagnosis, and management of lysosomal storage disorders in Colombia. Heliyon. 2020; 6(3):e03635. PMC: 7113438. DOI: 10.1016/j.heliyon.2020.e03635. View

Cleary M, Davison J, Gould R, Geberhiwot T, Hughes D, Mercer J . Impact of long-term elosulfase alfa treatment on clinical and patient-reported outcomes in patients with mucopolysaccharidosis type IVA: results from a Managed Access Agreement in England. Orphanet J Rare Dis. 2021; 16(1):38. PMC: 7818902. DOI: 10.1186/s13023-021-01675-x. View

Leal A, Espejo-Mojica A, Sanchez O, Ramirez C, Reyes L, Cruz J . Lysosomal storage diseases: current therapies and future alternatives. J Mol Med (Berl). 2020; 98(7):931-946. DOI: 10.1007/s00109-020-01935-6. View

Akyol M, Alden T, Amartino H, Ashworth J, Belani K, Berger K . Recommendations for the management of MPS IVA: systematic evidence- and consensus-based guidance. Orphanet J Rare Dis. 2019; 14(1):137. PMC: 6567385. DOI: 10.1186/s13023-019-1074-9. View

Sawamoto K, Stapleton M, Almeciga-Diaz C, Espejo-Mojica A, Losada J, Suarez D . Therapeutic Options for Mucopolysaccharidoses: Current and Emerging Treatments. Drugs. 2019; 79(10):1103-1134. DOI: 10.1007/s40265-019-01147-4. View

Donida B, Marchetti D, Jacques C, Ribas G, Deon M, Manini P . Oxidative profile exhibited by Mucopolysaccharidosis type IVA patients at diagnosis: Increased keratan urinary levels. Mol Genet Metab Rep. 2017; 11:46-53. PMC: 5408501. DOI: 10.1016/j.ymgmr.2017.04.005. View

Donida B, Marchetti D, Biancini G, Deon M, Manini P, Da Rosa H . Oxidative stress and inflammation in mucopolysaccharidosis type IVA patients treated with enzyme replacement therapy. Biochim Biophys Acta. 2015; 1852(5):1012-9. DOI: 10.1016/j.bbadis.2015.02.004. View

10.

Schweighardt B, Tompkins T, Lau K, Jesaitis L, Qi Y, Musson D . Immunogenicity of Elosulfase Alfa, an Enzyme Replacement Therapy in Patients With Morquio A Syndrome: Results From MOR-004, a Phase III Trial. Clin Ther. 2014; 37(5):1012-1021.e6. DOI: 10.1016/j.clinthera.2014.11.005. View

11.

Long B, Tompkins T, Decker C, Jesaitis L, Khan S, Slasor P . Long-term Immunogenicity of Elosulfase Alfa in the Treatment of Morquio A Syndrome: Results From MOR-005, a Phase III Extension Study. Clin Ther. 2016; 39(1):118-129.e3. DOI: 10.1016/j.clinthera.2016.11.017. View

12.

Olarte-Avellaneda S, Cepeda Del Castillo J, Rojas-Rodriguez A, Sanchez O, Rodriguez-Lopez A, Suarez Garcia D . Bromocriptine as a Novel Pharmacological Chaperone for Mucopolysaccharidosis IV A. ACS Med Chem Lett. 2020; 11(7):1377-1385. PMC: 7356376. DOI: 10.1021/acsmedchemlett.0c00042. View

13.

Almeciga-Diaz C, Hidalgo O, Olarte-Avellaneda S, Rodriguez-Lopez A, Guzman E, Garzon R . Identification of Ezetimibe and Pranlukast as Pharmacological Chaperones for the Treatment of the Rare Disease Mucopolysaccharidosis Type IVA. J Med Chem. 2019; 62(13):6175-6189. PMC: 11292729. DOI: 10.1021/acs.jmedchem.9b00428. View

14.

Almeciga-Diaz C, Barrera L . Design and applications of gene therapy vectors for mucopolysaccharidosis in Colombia. Gene Ther. 2019; 27(1-2):104-107. DOI: 10.1038/s41434-019-0086-3. View

15.

Biffi A . Gene therapy for lysosomal storage disorders: a good start. Hum Mol Genet. 2015; 25(R1):R65-75. DOI: 10.1093/hmg/ddv457. View

16.

Puentes-Tellez M, Sanchez O, Rojas-Rodriguez F, Benincore-Florez E, Barbosa H, Almeciga Diaz C . Evaluation of HIV-1 derived lentiviral vectors as transductors of Mucopolysaccharidosis type IV a fibroblasts. Gene. 2021; 780:145527. DOI: 10.1016/j.gene.2021.145527. View

17.

Almeciga-Diaz C, Montano A, Barrera L, Tomatsu S . Tailoring the AAV2 capsid vector for bone-targeting. Pediatr Res. 2018; 84(4):545-551. PMC: 6266866. DOI: 10.1038/s41390-018-0095-8. View

18.

Gutierrez M, Garcia-Vallejo F, Tomatsu S, Ceron F, Almeciga-Diaz C, Dominguez M . [Construction of an adenoassociated, viral derived, expression vector to correct the genetic defect in Morquio A disease]. Biomedica. 2008; 28(3):448-59. View

20.

Sawamoto K, Karumuthil-Melethil S, Khan S, Stapleton M, Bruder J, Danos O . Liver-Targeted AAV8 Gene Therapy Ameliorates Skeletal and Cardiovascular Pathology in a Mucopolysaccharidosis IVA Murine Model. Mol Ther Methods Clin Dev. 2020; 18:50-61. PMC: 7301175. DOI: 10.1016/j.omtm.2020.05.015. View

21.

Bertolin J, Sanchez V, Ribera A, Jaen M, Garcia M, Pujol A . Treatment of skeletal and non-skeletal alterations of Mucopolysaccharidosis type IVA by AAV-mediated gene therapy. Nat Commun. 2021; 12(1):5343. PMC: 8429698. DOI: 10.1038/s41467-021-25697-y. View