Generation of hyperfunctional recombinant human factor IX variants expressed in human cell line SK-Hep-1

Author(s)

Sousa Bomfim, Aline ^{[1, 2, 3]} ; Correa de Freitas, Marcela Cristina ^[2] ; Picanco Castro, Virginia ^[2] ; Abreu Soares Neto, Mario ^[2] ; Padua, Ricardo ^[4] ; Covas, Dimas Tadeu ^{[5, 2]} ; Sousa Russo, Elisa Maria ^{[1, 2]}

Affiliation

^[1] Univ Sao Paulo, Sch Pharmaceut Sci Ribeira Preto, Dept Clin Toxicol & Food Sci Anal, Sao Paulo - Brazil

^[2] Univ Sao Paulo, Ctr Cell Based Therapy & Reg Blood Ctr, Biotechnol Lab, Sao Paulo - Brazil

^[3] Univ Sao Paulo, Dept Clin Toxicol & Food Sci Anal, Sch Pharmaceut Sci Ribeirao Preto, Cafe Ave, Block R, Room 7, BR-14040903 Ribeirao Preto, SP - Brazil

^[4] Univ Sao Paulo, Dept Chem & Phys, Sch Pharmaceut Sci Ribeirao Preto, Sao Paulo - Brazil

^[5] Univ Sao Paulo, Dept Clin Med, Fac Med Ribeirao Preto, Sao Paulo - Brazil

• Document type: Journal article
• Source: Biotechnology Letters; v. 43, n. 1, p. 143-152, JAN 2021.
• Web of Science Citations: 0

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 mu g(-mL)), followed by FIX-LLW (1.5 mu g(-mL)) and FIX-ALL (0.9 mu g(-mL)). The expression level of FIX variants was two-five fold lower than FIX-wild-type (FIX-WT) (4.37 mu g(-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. (AU)

• FAPESP's process: 17/25364-6 - Mapping of Cooperation Networks and Technological Trends in Biotechnology Patents through Social Network Analysis
• Grantee: Geciane Silveira Porto
• Support Opportunities: Regular Research Grants
• FAPESP's process: 14/22500-8 - Identification of patent-based technological trends in red biotechnology
• Grantee: Cristiano Gonçalves Pereira
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• FAPESP's process: 13/08135-2 - CTC - Center for Cell-Based Therapy
• Grantee: Dimas Tadeu Covas
• Support Opportunities: Research Grants - Research, Innovation and Dissemination Centers - RIDC
• FAPESP's process: 16/02433-0 - Purification, formulation and preclinical testing of recombinant FVIII produced in human cells.
• Grantee: Virginia Picanço e Castro
• Support Opportunities: Regular Research Grants
• FAPESP's process: 15/13816-4 - Scientific and Technological Productivity of the Brazilian Research Infrastructures
• Grantee: Sérgio Kannebley Júnior
• Support Opportunities: Regular Research Grants
• FAPESP's process: 14/50947-7 - INCT 2014: in Stem Cell and Cell Therapy
• Grantee: Dimas Tadeu Covas
• Support Opportunities: Research Projects - Thematic Grants