Browsing by Author "Lengler, Johannes"

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    Minimal Essential Human Factor VIII Alterations Enhance Secretion and Gene Therapy Efficiency
    (Elsevier, 2020-10-22) Cao, Wenjing; Dong, Biao; Horling, Franziska; Firrman, Jenni A.; Lengler, Johannes; Klugmann, Matthias; de la Rosa, Maurus; Wu, Wenman; Wang, Qizhao; Wei, Hongying; Moore, Andrea R.; Roberts, Sean A.; Booth, Carmen J.; Hoellriegl, Werner; Li, Dong; Konkle, Barbara; Miao, Carol; Reipert, Birgit M.; Scheiflinger, Friedrich; Rottensteiner, Hanspeter; Xiao, Weidong; Pediatrics, School of Medicine
    One 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.