Novel Human Factor VIII Variant with Impaired Intracellular Processing Exhibits Enhanced In Vivo Efficacy

Abstract During intracellular processing, B-domain deleted human factor VIII (hFVIII-BDD) undergoes proteolysis at multiple sites with the most predominant cleavage at the furin recognition site (1645-RHQR-1648) at the carboxy-terminus of the B-domain. This cleavage gives rise to two polypeptide chains, the 90 kDa heavy chain (HC) and the 80 kDa light chain (LC), that form a heterodimer which becomes the major secreted form of the protein. In contrast, B-domain deleted canine factor VIII (cFVIII-BDD) is secreted primarily in the uncleaved single chain (SC) form, exhibits higher activity and shows increased expression in the gene therapy setting. To investigate the difference in the amino acid sequence at the furin recognition site between cFVIII-BDD (1645-HHQR-1648) and hFVIII-BDD, our previous work generated and characterized a series of hFVIII-BDD furin site deletion variants. Our studies demonstrated that hFVIII- BDD-del1645-1647 (Δ3) variant is secreted predominantly as SC, with a two-fold increase in activity and improved secretion over hFVIII-BDD. N-terminal sequencing of the hFVIII LC revealed that while hFVIII-BDD is cleaved after R1648 and S1657, cleavage of Δ3 occurs exclusively after S1657. Amino acid sequence analysis revealed differences at this site between hFVIII (S1657/D1658) and cFVIII (P1657/E1658). We hypothesized that modifications to this downstream cleavage site in hFVIII-BDD may further decrease cleavage efficiency, leading to a higher portion of SC, and further increase protein expression after gene therapy. We generated a new variant where this downstream cleavage sequence was modified to that of cFVIII, S1657P/D1658E, in combination with our previously described Δ3 variant. hFVIII-BDD, hFVIII-Δ3, and hFVIII-Δ3-S1657P/D1658E were purified using ion exchange chromatography. Interestingly, optical densitometry analysis of all purified proteins on a reducing SDS-PAGE gel revealed that hFVIII-Δ3-S1657P/D1658E was expressed almost entirely as a SC (91.7% ± 5.1%), similar to what was observed with cFVIII (86.8% ± 1.0%). hFVIII-Δ3 and hFVIII-BDD were 56.5% ± 5.7% and 24.3% ± 0.6% SC, respectively. Once activated by thrombin, hFVIII-Δ3-S1657P/D1658E yielded the same expected species as hFVIII-Δ3 and hFVIII-BDD. In a one-stage aPTT assay, hFVIII-Δ3-S1657P/D1658E had activity comparable to hFVIII-Δ3 and hFVIII-BDD. In the two-stage aPTT assay, hFVIII-Δ3 and hFVIII-Δ3-S1657P/D1658E exhibited a two-fold increase in activity over hFVIII-BDD. Thus, hFVIII-Δ3-S1657P/D1658E was secreted primarily in the SC form and had higher activity similar to the hFVIII-Δ3 variant in vitro. In the setting of gene-based therapeutics, strategies to increase hFVIII expression provide a platform for reducing the vector dose required to achieve therapeutically relevant FVIII levels. To investigate the efficacy of this variant in vivo, the hFVIII-Δ3-S1657P/D1658E variant was introduced into an adeno-associated viral vector serotype 8 expressing the wild type hFVIII-BDD cDNA sequence (AAV8-hAAT-hFVIII-BDD) for liver-targeted delivery. The vector was delivered to immune deficient hemophilia A mice (5x1011 vector genomes/mouse) (n=4/group). At 12 weeks post-vector administration, the hFVIII-Δ3-S1657P/D1658E had a 4.5-fold increase in protein expression over hFVIII-BDD, a significant improvement over hFVIII-Δ3, which was two-fold higher than hFVIII-BDD. Altogether, the data supports that the modifications at the downstream cleavage site at residue S1657 and D1658, in combination with a furin evading variant in hFVIII-BDD, further hinders cleavage during intracellular processing. This unexpectedly leads to additional enhancement of hFVIII-BDD expression in the gene therapy setting. Not only might the studies of these variants improve our understanding of the intracellular processing of hFVIII-BDD, but they also provide a novel approach to increase hFVIII expression that will allow the use of a lower vector dose, improving the safety of gene-based therapeutics. Disclosures Sabatino: Spark Therapeutics: Research Funding.