Circumventing furin enhances factor VIII biological activity and ameliorates bleeding phenotypes in hemophilia models

Abstract

Processing by the proprotein convertase furin is believed to be critical for the biological activity of multiple proteins involved in hemostasis, including coagulation factor VIII (FVIII). This belief prompted the retention of the furin recognition motif (amino acids 1645-1648) in the design of B-domain-deleted FVIII (FVIII-BDD) products in current clinical use and in the drug development pipeline, as well as in experimental FVIII gene therapy strategies. Here, we report that processing by furin is in fact deleterious to FVIII-BDD secretion and procoagulant activity. Inhibition of furin increases the secretion and decreases the intracellular retention of FVIII-BDD protein in mammalian cells. Our new variant (FVIII-ΔF), in which this recognition motif is removed, efficiently circumvents furin. FVIII-ΔF demonstrates increased recombinant protein yields, enhanced clotting activity, and higher circulating FVIII levels after adeno-associated viral vector-based liver gene therapy in a murine model of severe hemophilia A (HA) compared with FVIII-BDD. Moreover, we observed an amelioration of the bleeding phenotype in severe HA dogs with sustained therapeutic FVIII levels after FVIII-ΔF gene therapy at a lower vector dose than previously employed in this model. The immunogenicity of FVIII-ΔF did not differ from that of FVIII-BDD as a protein or a gene therapeutic. Thus, contrary to previous suppositions, FVIII variants that can avoid furin processing are likely to have enhanced translational potential for HA therapy.

Correction of bleeding in experimental severe hemophilia A by systemic delivery of factor VIII-encoding mRNA

“…Importantly, the levels of light chain may be under-estimated owing to the short in vivo half-life of the light chain alone. Future experiments will indicate whether the use of mRNA encoding FVIII lacking the furin cleavage site 39,40 between the heavy and light chains, or FVIII mutants with increased FVIII A2 subunit stability 41 yields FVIII with improved specific activity. An alternative and non-exclusive explanation for the poor specific activity of the endogenously produced FVIII is the fact that the TransIT® used to formulate the mRNA does not target particular cell types.…”

“…Further work will indicate whether targeting mRNA delivery to hepatocytes or to endothelial cells using improved lipid nanoparticle-based formulating agents, 50 using mRNA encoding single chain or A2 variant stable FVIII 39,40,41 and including miRNA target sequences to prevent off-target expression in hematopoietic cells 51 are plausible strategies to improve the specific activity and reduce the immunogenicity of the endogenously produced molecule. For quantitation of the FVIII light chain in mouse plasma (which represents both the active and inactive levels of FVIII), plates were coated with the human anti-C2 monoclonal IgG BO2C11 (a kind gift from Prof Saint-Remy and Jacquemin, KUL, Leuven, Belgium) and the light chain was recognized with biotinylated EHS-8.…”

Potential limits of AAV‐based gene therapy with the use of new transgenes expressing factor IX fusion proteins

“…There are reports of mutations within FIX or factor VIII (FVIII) genes leading to changes in protein configuration and poor secretion from the producer cells . The role of PACE/furin expression could also be considered as a potential limiting underlying mechanism for low hFIX‐fusion protein secretion . It is intriguing to speculate whether similar observations are made during the large‐scale manufacture of these proteins as recombinant concentrates, though it is possible that changes in secretion pattern of hFIX‐fusion protein may not be detected if the producer cells are lysed prior to purification of the hFIX‐fusion protein.…”

Clinical advances in gene therapy updates on clinical trials of gene therapy in haemophilia

“…The development of a codon‐optimized human FVIII cDNA in which a variant of a B‐domain deleted replaces the whole B domain with the liver‐specific promoter has improved expression efficacy and packaging in an AAV vector . Increased FVIII expression has been reported in in‐vivo gene expression experiments conducted in mice and non‐human primates …”