CRISPR-Mediated In Situ Introduction or Integration of F9-Padua in Human iPSCs for Gene Therapy of Hemophilia B
Abstract
:1. Introduction
2. Results
2.1. Introduction of the F9-Padua Mutation into hiPSCs
2.2. Generation and Characterization of HB-hiPSCs
2.3. Integration of Modified Full-Length F9 cDNA with the F9-Padua Mutation in HB-hiPSCs
2.4. Derivation of Hepatocytes from Edited Clones
2.5. Validation of the Hyperactivity of FIX-Padua Proteins Produced by Hepatocytes from the F9-Padua hiPSCs Model and Integrated HB-hiPSCs
3. Discussion
4. Materials and Methods
4.1. Plasmid Construction
4.2. Generation of HB-hiPSCs from Renal Tubular Epithelial Cells
4.3. Alkaline Phosphatase (ALP) Staining
4.4. Karyotyping
4.5. Teratoma Formation Experiments
4.6. Cell Culture
4.7. HEK293T Transfection
4.8. T7E1 Cleavage Assay
4.9. hiPSCs Nucleofection and Genotyping
4.10. Off-Target Analysis
4.11. Immunofluorescence Analysis
4.12. Hepatocyte Differentiation
4.13. qRT-PCR Analysis
4.14. ICG Uptake Assay
4.15. PAS Staining
4.16. FIX Coagulation Activity Test
4.17. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Tang, Q.; Hu, Z.; Zhao, J.; Zhou, T.; Tang, S.; Wang, P.; Xiao, R.; Chen, Y.; Wu, L.; Zhou, M.; et al. CRISPR-Mediated In Situ Introduction or Integration of F9-Padua in Human iPSCs for Gene Therapy of Hemophilia B. Int. J. Mol. Sci. 2023, 24, 9013. https://doi.org/10.3390/ijms24109013
Tang Q, Hu Z, Zhao J, Zhou T, Tang S, Wang P, Xiao R, Chen Y, Wu L, Zhou M, et al. CRISPR-Mediated In Situ Introduction or Integration of F9-Padua in Human iPSCs for Gene Therapy of Hemophilia B. International Journal of Molecular Sciences. 2023; 24(10):9013. https://doi.org/10.3390/ijms24109013
Chicago/Turabian StyleTang, Qiyu, Zhiqing Hu, Junya Zhao, Tao Zhou, Shuqing Tang, Peiyun Wang, Rou Xiao, Yan Chen, Lingqian Wu, Miaojin Zhou, and et al. 2023. "CRISPR-Mediated In Situ Introduction or Integration of F9-Padua in Human iPSCs for Gene Therapy of Hemophilia B" International Journal of Molecular Sciences 24, no. 10: 9013. https://doi.org/10.3390/ijms24109013