Cao, WenjingDong, BiaoHorling, FranziskaFirrman, Jenni A.Lengler, JohannesKlugmann, Matthiasde la Rosa, MaurusWu, WenmanWang, QizhaoWei, HongyingMoore, Andrea R.Roberts, Sean A.Booth, Carmen J.Hoellriegl, WernerLi, DongKonkle, BarbaraMiao, CarolReipert, Birgit M.Scheiflinger, FriedrichRottensteiner, HanspeterXiao, Weidong2022-04-202022-04-202020-10-22Cao W, Dong B, Horling F, et al. Minimal Essential Human Factor VIII Alterations Enhance Secretion and Gene Therapy Efficiency. Mol Ther Methods Clin Dev. 2020;19:486-495. Published 2020 Oct 22. doi:10.1016/j.omtm.2020.10.013https://hdl.handle.net/1805/28607One important limitation for achieving therapeutic expression of human factor VIII (FVIII) in hemophilia A gene therapy is inefficient secretion of the FVIII protein. Substitution of five amino acids in the A1 domain of human FVIII with the corresponding porcine FVIII residues generated a secretion-enhanced human FVIII variant termed B-domain-deleted (BDD)-FVIII-X5 that resulted in 8-fold higher FVIII activity levels in the supernatant of an in vitro cell-based assay system than seen with unmodified human BDD-FVIII. Analysis of purified recombinant BDD-FVIII-X5 and BDD-FVIII revealed similar specific activities for both proteins, indicating that the effect of the X5 alteration is confined to increased FVIII secretion. Intravenous delivery in FVIII-deficient mice of liver-targeted adeno-associated virus (AAV) vectors designed to express BDD-FVIII-X5 or BDD-FVIII achieved substantially higher plasma FVIII activity levels for BDD-FVIII-X5, even when highly efficient codon-optimized F8 nucleotide sequences were employed. A comprehensive immunogenicity assessment using in vitro stimulation assays and various in vivo preclinical models of hemophilia A demonstrated that the BDD-FVIII-X5 variant does not exhibit an increased immunogenicity risk compared to BDD-FVIII. In conclusion, BDD-FVIII-X5 is an effective FVIII variant molecule that can be further developed for use in gene- and protein-based therapeutics for patients with hemophilia A.en-USAttribution-NonCommercial-NoDerivatives 4.0 InternationalFactor VIIIAAVGene therapyX5SecretionVectorArticle