Novel Coagulation Factor VIII Gene Therapy in a Mouse Model of Hemophilia A by Lipid-Coated Fe3O4 Nanoparticles
Abstract
:1. Introduction
2. Materials and Methods
2.1. Animals
2.2. Liver-Specific Promoter Construction
2.3. Dual Luciferase Assays
2.4. Gene Therapy Plasmid Construction and Production
2.5. Synthesis of DPPC-Fe3O4 Nanoparticles
2.6. Cell Culture and Gene Transfection
2.7. Physicochemical Analysis
2.8. Transmission Electron Microscopy (TEM)
2.9. Electrophoretic Gel Mobility Shift Assay (EMSA)
2.10. Quantitative Real-Time Reverse Transcription-PCR (qRT-PCR)
2.11. Gene Delivery via Tail Vein Injection
2.12. Flow Cytometric Analysis
2.13. Evaluation of Coagulation Phenotypic Restoration
2.14. Detection of Blood Biochemical Parameters
2.15. Histologic Section Analysis
2.16. Statistical Analysis
3. Results
3.1. Liver-Specific Promoter Activity in Different Types of Cell Lines
3.2. Characterization of DPPC-Fe3O4 Carrying Different Forms of Plasmid DNA
3.3. Characterization of Different Doses of DPPC-Fe3O4 with Circular Plasmid DNA
3.4. Evaluation of PmAlb-BDD-FVIII Circular Plasmid Expression in Mouse Hepatocytes
3.5. Coagulation Phenotypic Correction by Nanoparticle Gene Delivery
3.6. Distribution of DPPC-Fe3O4–Plasmid Nanoparticles in the Liver Tissue of Hemophilic Mice
3.7. Safety Validation after Delivery of the DPPC-Fe3O4-Plasmid Complex
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Gene Name | Primer Sequence (5′ to 3′) | Tm (°C) | Amplicon Size (bp) |
---|---|---|---|
Pα1-AT | F: 5′-ACCGAGGCACAGAGAGGTT-3′ R: 5′-GCCATTCACAAGGATACTGT-3′ | 67 | 581 |
Eα1-AT | F: 5′-CTTCAGCATCAGGCATTTTGG-3′ R: 5′-TCTCCAGAACCTCTCGCAGT-3′ | 63 | 473 |
PmAlb | F: 5′-TCACTCAAAAGAGTCCTGAA-3′ R: 5′-AGAAAGACTCGCTCTAATATAC-3′ | 57 | 1060 |
EGFP | F: 5′-GACTTCTTCAAGTCCGCCATGC-3′ R: 5′-CTCCAGCAGGACCATGTGAT-3′ | 55 | 432 |
BDD-hFVIII | F: 5′-CAGACTTTCGGAACAGAGGCA-3′ R: 5′-ATCTTTTTCCAGGTCAACATCA-3′ | 55 | 752 |
β-actin | F: 5′-CCGTCTTCCCCTCCATCGTGGG-3′ R: 5′-AGATCATTGTAGAAGGTGTGG-3′ | 68 | 199 |
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Kao, Y.-T.; Chen, Y.-T.; Fan, H.-C.; Tsai, T.-C.; Cheng, S.-N.; Lai, P.-S.; Chen, J.-K.; Chen, C.-M. Novel Coagulation Factor VIII Gene Therapy in a Mouse Model of Hemophilia A by Lipid-Coated Fe3O4 Nanoparticles. Biomedicines 2021, 9, 1116. https://doi.org/10.3390/biomedicines9091116
Kao Y-T, Chen Y-T, Fan H-C, Tsai T-C, Cheng S-N, Lai P-S, Chen J-K, Chen C-M. Novel Coagulation Factor VIII Gene Therapy in a Mouse Model of Hemophilia A by Lipid-Coated Fe3O4 Nanoparticles. Biomedicines. 2021; 9(9):1116. https://doi.org/10.3390/biomedicines9091116
Chicago/Turabian StyleKao, Yung-Tsung, Yen-Ting Chen, Hueng-Chuen Fan, Tung-Chou Tsai, Shin-Nan Cheng, Ping-Shan Lai, Jen-Kun Chen, and Chuan-Mu Chen. 2021. "Novel Coagulation Factor VIII Gene Therapy in a Mouse Model of Hemophilia A by Lipid-Coated Fe3O4 Nanoparticles" Biomedicines 9, no. 9: 1116. https://doi.org/10.3390/biomedicines9091116