CRISPR/nCas9-Edited CD34+ Cells Rescue Mucopolysaccharidosis IVA Fibroblasts Phenotype
Abstract
1. Introduction
2. Results
2.1. CRISPR/nCas9-Based Genome Editing System Efficiently Preserves the Self-Renewal and Differentiation Potential of CD34+ Cells
2.2. Therapeutic Efficacy of CRISPR/nCas9-Edited HSCs in MPS IVA Fibroblasts Co-Culture
2.3. Reduction of Oxidative Stress in MPS IVA Fibroblasts
2.4. Decreased Levels of Pro-Inflammatory Markers in MPS IVA Fibroblasts
3. Discussion
4. Materials and Methods
4.1. Cell Culture and Expansion
4.2. Donor Template
4.3. CRISPR/nCas9 Editing Protocol
4.4. Donor Template Integration at the AAVS1 Locus
4.5. Stemness Evaluation
4.5.1. CD Markes
4.5.2. Colony-Forming Unit (CFU)
4.5.3. Proliferation and Cell Cycle Analysis
4.6. GALNS Activity Assays
4.7. Lysosomal Accumulation
4.7.1. Lysosomal Mass Evaluation
4.7.2. Analysis of Mono-Sulfated Keratan Sulfate
4.8. Oxidative Profile Assessment
4.8.1. Reactive Oxygen Species (ROS)
4.8.2. Mitochondrial-Derived Reactive Oxygen Species (mtROS)
4.9. Mitochondrial Mass Detection
4.10. Apoptosis Ratio
4.11. Pro-Inflammatory Profile Evaluation
5. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
Abbreviations
MPS | Mucopolysaccharidosis |
LSD | Lysosomal storage disease |
GAGs | Glycosaminoglycans |
KS | Keratan sulfate |
C6S | Chondroitin 6-sulfate |
GALNS | N-acetylgalactosamine-6-sulfatase |
GT | Gene therapy |
ERT | Enzyme replacement therapy |
HSC | Hematopoietic stem cell |
HSCT | Hematopoietic stem cell transplantation |
GVHD | Graft versus host disease |
PC | Pharmacological chaperones |
SDT | Substrate degradation therapy |
WT | Wild type |
EGFP | Enhanced green fluorescent protein |
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Herreno-Pachón, A.M.; Leal, A.F.; Khan, S.; Alméciga-Díaz, C.J.; Tomatsu, S. CRISPR/nCas9-Edited CD34+ Cells Rescue Mucopolysaccharidosis IVA Fibroblasts Phenotype. Int. J. Mol. Sci. 2025, 26, 4334. https://doi.org/10.3390/ijms26094334
Herreno-Pachón AM, Leal AF, Khan S, Alméciga-Díaz CJ, Tomatsu S. CRISPR/nCas9-Edited CD34+ Cells Rescue Mucopolysaccharidosis IVA Fibroblasts Phenotype. International Journal of Molecular Sciences. 2025; 26(9):4334. https://doi.org/10.3390/ijms26094334
Chicago/Turabian StyleHerreno-Pachón, Angélica María, Andrés Felipe Leal, Shaukat Khan, Carlos Javier Alméciga-Díaz, and Shunji Tomatsu. 2025. "CRISPR/nCas9-Edited CD34+ Cells Rescue Mucopolysaccharidosis IVA Fibroblasts Phenotype" International Journal of Molecular Sciences 26, no. 9: 4334. https://doi.org/10.3390/ijms26094334
APA StyleHerreno-Pachón, A. M., Leal, A. F., Khan, S., Alméciga-Díaz, C. J., & Tomatsu, S. (2025). CRISPR/nCas9-Edited CD34+ Cells Rescue Mucopolysaccharidosis IVA Fibroblasts Phenotype. International Journal of Molecular Sciences, 26(9), 4334. https://doi.org/10.3390/ijms26094334