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Discovery of An Orally Effective Factor IX-Transferrin Fusion Protein for Hemophilia B.

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.

SUBMITTER: Xie C

PROVIDER: S-EPMC6981973 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND:The prevention of bleeding with adequately sustained levels of clotting factor, after a single therapeutic intervention and without the need for further medical intervention, represents an important goal in the treatment of hemophilia. METHODS:We infused a single-stranded adeno-associated viral (AAV) vector consisting of a bioengineered capsid, liver-specific promoter and factor IX Padua (factor IX-R338L) transgene at a dose of 5×1011 vector genomes per kilogram of body weight in 10 men with hemophilia B who had factor IX coagulant activity of 2% or less of the normal value. Laboratory values, bleeding frequency, and consumption of factor IX concentrate were prospectively evaluated after vector infusion and were compared with baseline values. RESULTS:No serious adverse events occurred during or after vector infusion. Vector-derived factor IX coagulant activity was sustained in all the participants, with a mean (±SD) steady-state factor IX coagulant activity of 33.7±18.5% (range, 14 to 81). On cumulative follow-up of 492 weeks among all the participants (range of follow-up in individual participants, 28 to 78 weeks), the annualized bleeding rate was significantly reduced (mean rate, 11.1 events per year [range, 0 to 48] before vector administration vs. 0.4 events per year [range, 0 to 4] after administration; P=0.02), as was factor use (mean dose, 2908 IU per kilogram [range, 0 to 8090] before vector administration vs. 49.3 IU per kilogram [range, 0 to 376] after administration; P=0.004). A total of 8 of 10 participants did not use factor, and 9 of 10 did not have bleeds after vector administration. An asymptomatic increase in liver-enzyme levels developed in 2 participants and resolved with short-term prednisone treatment. One participant, who had substantial, advanced arthropathy at baseline, administered factor for bleeding but overall used 91% less factor than before vector infusion. CONCLUSIONS:We found sustained therapeutic expression of factor IX coagulant activity after gene transfer in 10 participants with hemophilia who received the same vector dose. Transgene-derived factor IX coagulant activity enabled the termination of baseline prophylaxis and the near elimination of bleeding and factor use. (Funded by Spark Therapeutics and Pfizer; ClinicalTrials.gov number, NCT02484092 .).

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Discovery of An Orally Effective Factor IX-Transferrin Fusion Protein for Hemophilia B.

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