Abstract
Objective
To develop recombinant factor IX (FIX) variants with augmented clotting activity.
Results
We generated three new variants, FIX-YKALW, FIX-ALL and FIX-LLW, expressed in SK-Hep-1 cells and characterized in vitro and in vivo. FIX-YKALW showed the highest antigen expression level among the variants (2.17 µg−mL), followed by FIX-LLW (1.5 µg−mL) and FIX-ALL (0.9 µg−mL). The expression level of FIX variants was two–five fold lower than FIX-wild-type (FIX-WT) (4.37 µg−mL). However, the biological activities of FIX variants were 15–31 times greater than FIX-WT in the chromogenic assay. Moreover, the new variants FIX-YKALW, FIX-LLW and FIX-ALL also presented higher specific activity than FIX-WT (17, 20 and 29-fold higher, respectively). FIX variants demonstrated a better clotting time than FIX-WT. In hemophilia B mice, we observed that FIX-YKALW promoted hemostatic protection.
Conclusion
We have developed three improved FIX proteins with potential for use in protein replacement therapy for hemophilia B.
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Abbreviations
- A:
-
Alanine
- E:
-
Glutamic acid
- G:
-
Glycine
- K:
-
Lysine
- L:
-
Leucine
- R:
-
Arginine
- S:
-
Serine
- V:
-
Valine
- W:
-
Tryptophan
- Y:
-
Tyrosine
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Acknowledgements
The authors are grateful to the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Capes, Conselho Nacional de Desenvolvimento Científico e Tecnológico (Grant No. 142406/2016-3) – CNPq, the National Institute of Science and Technology in Stem Cells and Cell Therapy – INCTC, Centro de Pesquisa, Inovação e Difusão – CPID and Fundação de Amparo à Pesquisa do Estado de São Paulo (Grant Nos. 2013/08135-2, 2014/50947-7, 2015/13816-4, 2016/02433-0, 2017/25364-6 and 2014/22500-8) – FAPESP and Professor Dr. Luis Alexandre Pedro de Freitas from University of São Paulo who contributed to statistical data analysis.
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Sousa Bomfim, A., Corrêa de Freitas, M.C., Picanço Castro, V. et al. Generation of hyperfunctional recombinant human factor IX variants expressed in human cell line SK-Hep-1. Biotechnol Lett 43, 143–152 (2021). https://doi.org/10.1007/s10529-020-03040-7
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DOI: https://doi.org/10.1007/s10529-020-03040-7