Abstract
Hemophilia B is a severe blood clotting disorder caused by the deficiency of factor IX (FIX). FIX is not bioavailable when given orally due to poor stability and permeability in the gastrointestinal tract. The feasibility of fusing FIX with transferrin (Tf) to enhance the oral bioavailability of FIX is explored. Seven recombinant fusion proteins (rFIX-Tf) with different linkers were constructed and expressed in HEK293 cells and characterized by in vitro transcytosis and transferrin receptor (TfR) binding assay in Caco-2 cells and a one-stage clotting assay. The in vivo efficacy study was performed using a tail-bleeding model in hemophilia B mice. Fusion proteins rFIX-Tf/G2 and rFIX-Tf/SVSQ were most permeable and showed a specific binding ability to TfR in Caco-2 cells. Both proteins retained FIX activity in clotting generation. The in vivo efficacy study showed that both proteins by intravenous injection significantly reduced blood loss. Most significantly, rFIX-Tf/G2 demonstrated anti-bleeding activity when administered orally. Our results showed that the fusion protein technique with Tf could be potentially used for oral delivery of FIX and the linker between FIX and Tf in the fusion protein is crucial. rFIX-Tf/G2 appears to be the most promising fusion protein as potential oral therapeutics for hemophilia B.
Highlights
Coagulation factor IX (FIX, Christmas factor), encoded by the gene located on the X chromosome (Xq27.1-q27.2), is a serine protease essential for hemostasis with X-linked recessive inheritance [1]
Under non-reducing conditions, SDS-PAGE analysis demonstrated that FIX and Tf expressed as a single, dominant band with about 90% abundance and about 130 kDa molecular weight corresponding to the fusion protein, which is the sum of FIX (57 kDa) and Tf (79 kDa) (Figure 1A)
FIX was fused with Tf, and the feasibility of using transferrin as a carrier for oral delivery of recombinant FIX (rFIX) was investigated
Summary
Coagulation factor IX (FIX, Christmas factor), encoded by the gene located on the X chromosome (Xq27.1-q27.2), is a serine protease essential for hemostasis with X-linked recessive inheritance [1]. It was discovered in 1952 after a young boy named Stephen Christmas was found having hemophilia due to the lack of this factor [2]. It is converted to the active form (FIXa) with two chains linked by a disulfide bond via minor proteolysis by either factor IXa (contact pathway) or factor VIIa (tissue factor pathway) [5]. In the presence of calcium ions, membrane phospholipids and a factor VIII cofactor, FIXa, activates the coagulation cascade by converting factor X to factor Xa
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