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Assessment of different promoters in lentiviral vectors for expression of the N-acetyl-galactosamine-6-sulfate sulfatase gene

Journal of Human Genetics volume 70, pages 463–473 (2025)Cite this article

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Abstract

Mucopolysaccharidosis IVA (MPS IVA) is caused by pathogenic variants in the GALNS gene encoding N-acetylgalactosamine-6-sulfate sulfatase (GALNS) enzyme, leading to glycosaminoglycan (GAG) accumulation in multiple tissues, resulting in progressive skeletal dysplasia and poor quality of life. There is currently no effective treatment for this skeletal disease. This study proposes a novel lentiviral vector (LV)-based gene therapy that produces and secretes the active GALNS enzyme at supraphysiologic levels within the cells. LVs carrying the native GALNS encoding sequence (cDNA) were made under three different promoters: CBh, COL2A1, and CD11b. Moreover, we designed LVs carrying the native GALNS cDNA tagged with D8 octapeptide under the CD11b promoter and a human codon-optimized GALNS cDNA under the CBh promoter, respectively. Transduced HEK293 cells, HepG2 cells, and MPS IVA fibroblasts and chondrocytes were cultured for 8 and 30 days, and the media were collected every three days. The enzyme activity, GAG levels, and vector copy numbers (VCNs) in these cells and media were analyzed. LV with the COL2A1 promoter produced the highest enzyme activity in HEK293, HepG2, MPS IVA fibroblasts, and chondrocytes, followed by LV with the CBh promoter. VCNs were higher in MPS IVA fibroblasts treated with LV-CBh-hGALNS and in HepG2 cells treated with LV-CD11b-hGALNS than in HEK293 cells. Accumulated GAGs were normalized to wild-type levels by the LV gene therapy, especially with CBh and COL2A1 promoters. These findings, if further validated, could significantly impact the treatment of MPS IVA, offering a more effective and feasible treatment option.

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Fig. 1: Intracellular enzyme activity of MPS IVA fibroblasts treated under different MOIs with LVs.
Fig. 2: Enzyme activity in the culture media of fibroblasts.
Fig. 3: Therapeutic efficacy of LVs in MPS IVA chondrocytes treated with different LVs at the MOI of 20.
Fig. 4: GAG levels in MPS IVA fibroblasts and chondrocytes at MOI 20.
Fig. 5: Vector copy numbers at MOIs of 5, 10, 15, and 20.
Fig. 6: Immunofluorescence screening of EGFP fluorescence in MPS IVA fibroblasts at the increasing MOIs (5, 10, 15, and 20).

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Acknowledgements

BC was supported by the National MPS Society (ID 4892) and the Turkish Ministry of Education (YLSY2017-Doctoral Scholarship). This work was also supported by grants from the Austrian MPS society, A Cure for Robert, Inc., The Carol Ann Foundation, Angelo R. Cali & Mary V. Cali Family Foundation, Inc., The Vain and Harry Fish Foundation, Inc., The Bennett Foundation, Jacob Randall Foundation, and Nemours Funds. ST was supported by an Institutional Development Award from the Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health (NICHD) (1R01HD102545-01A1).

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Authors and Affiliations

  1. Department of Biological Sciences, University of Delaware, Newark, DE, USA

    Betul Celik

  2. Nemours Children’s Health, Wilmington, DE, USA

    Betul Celik, Andrés Felipe Leal, Shaukat Khan & Shunji Tomatsu

  3. Institute for the Study of Inborn Errors of Metabolism, Faculty of Science, Pontificia Universidad Javeriana, Bogotá, DC, Colombia

    Andrés Felipe Leal

  4. Department of Pediatrics, Graduate School of Medicine, Gifu University, Gifu, Japan

    Shunji Tomatsu

  5. Department of Pediatrics, Thomas Jefferson University, Philadelphia, PA, USA

    Shunji Tomatsu

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  1. Betul Celik
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  2. Andrés Felipe Leal
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Contributions

Conceptualization, BC; methodology, BC; software, BC; validation, BC, AFL, SK, and ST; formal analysis, BC; investigation, BC, AFL, SK; resources, ST; data curation, BC, and ST; writing—original draft preparation, BC; writing—review and editing, BC; visualization, BC; supervision, ST; project administration, BC, and ST; funding acquisition, ST. All authors have read and agreed to the published version of the manuscript.

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Correspondence to Shunji Tomatsu.

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Celik, B., Leal, A.F., Khan, S. et al. Assessment of different promoters in lentiviral vectors for expression of the N-acetyl-galactosamine-6-sulfate sulfatase gene. J Hum Genet 70, 463–473 (2025). https://doi.org/10.1038/s10038-025-01353-x

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