Discovery of an Orally Effective Factor IX-Transferrin Fusion Protein for Hemophilia B
Abstract
:1. Introduction
2. Results
2.1. Construction, Preparation and Quantification of FIX Fusion Proteins
2.1.1. FIX-Tf Expression Constructs and Fusion Protein Expression
2.1.2. Purification of Recombinant Fusion Protein by Size Exclusion Chromatography
2.2. In vitro Activity Studies
2.2.1. Apical-to-Basolateral Transcytosis of BeneFIX® and FIX–Tf Fusion Proteins Across Caco-2 Cell Monolayers
2.2.2. In vitro Tf Receptor binding activity of the FIX–Tf fusion proteins
2.2.3. Activated Thromboplastin (aPTT) Time Assay for in Vitro Clotting Activities of Fusion Proteins
2.3. In Vivo Studies
In Vivo Efficacy in Hemophilia B Mice
3. Discussion
4. Materials and Methods
4.1. Construction, Preparation and Quantification of FIX Fusion Proteins
4.1.1. Cloning of Recombinant FIX Fusion Proteins
4.1.2. Expression of Fusion Proteins
4.1.3. Characterization of the Fusion Proteins by SDS-PAGE and Western Blot Analysis
4.1.4. Purification of Recombinant FIX Fusion Protein by Size Exclusion HPLC
4.1.5. Human FIX ELISA Assay
4.2. In Vitro Activity Studies
4.2.1. TfR-Mediated Apical-to-Basolateral Transcytosis of Fusion Proteins Across the Caco-2 Cell Monolayer
4.2.2. TfR Binding Affinity of Fusion Proteins
4.2.3. Activated Thromboplastin (aPTT) Time Assay
4.3. In Vivo Studies
4.3.1. Efficacy Study Using a Tail-Bleeding Mouse Model of Hemophilia B
4.3.2. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
References
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Sample Designation | Expression of FIX:Ag in One 150 cm2 Dish (IU/Dish) |
---|---|
rFIX-Tf/G2 | 5.81 |
rFIX-Tf/G5 | 4.04 |
rFIX-Tf/A2 | 4.50 |
rFIX-Tf/A5 | 4.78 |
rFIX-Tf/Dithi | 1.04 |
rFIX-Tf/SVSQ | 1.82 |
rFIX-Tf | 1.76 |
Sample Designation | FIX Clotting Activity/OD280–320 (IU/OD) |
---|---|
rFIX-Tf | 0.075 |
rFIX-Tf/G2 | 0.199 |
rFIX-Tf/G5 | 0.185 |
rFIX-Tf/A2 | 0.096 |
rFIX-Tf/A5 | 0.094 |
rFIX-Tf/Dithi | 0.073 |
rFIX-Tf/SVSQ | 0.118 |
Sample Designation | Linker Type | Linker Sequence * |
---|---|---|
rFIX-Tf | Dipeptide | LE |
rFIX-Tf/G2 | Non-cleavable | (GGGGS)2 + LE |
rFIX-Tf/G5 | Non-cleavable | (GGGGS)5 + LE |
rFIX-Tf/A2 | Non-cleavable | A(EAAAK)2A + LE |
rFIX-Tf/A5 | Non-cleavable | A(EAAAK)5A + LE |
rFIX-Tf/Dithi | Cleavable | Dithiocyclopeptide + LE |
rFIX-Tf/SVSQ | Cleavable | SVSQTSKLTRAETVFPDVDGS + LE |
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Xie, C.; Wang, Z.; Su, Y.; Wang, J.; Shen, W.-C. Discovery of an Orally Effective Factor IX-Transferrin Fusion Protein for Hemophilia B. Int. J. Mol. Sci. 2020, 21, 21. https://doi.org/10.3390/ijms21010021
Xie C, Wang Z, Su Y, Wang J, Shen W-C. Discovery of an Orally Effective Factor IX-Transferrin Fusion Protein for Hemophilia B. International Journal of Molecular Sciences. 2020; 21(1):21. https://doi.org/10.3390/ijms21010021
Chicago/Turabian StyleXie, Chen, Zhijun Wang, Yang Su, Jeffrey Wang, and Wei-Chiang Shen. 2020. "Discovery of an Orally Effective Factor IX-Transferrin Fusion Protein for Hemophilia B" International Journal of Molecular Sciences 21, no. 1: 21. https://doi.org/10.3390/ijms21010021