Minimal Essential Human Factor VIII Alterations Enhance Secretion and Gene Therapy Efficiency

Molecular Therapy: Methods & Clinical Development

Weidong Xiao¹  Jenni A. Firrman²  Carmen J. Booth³  Carol Miao⁴  Hanspeter Rottensteiner⁵  Werner Hoellriegl⁵  Birgit M. Reipert⁵  Johannes Lengler⁵  Franziska Horling⁵  Matthias Klugmann⁵  Maurus de la Rosa⁵  Friedrich Scheiflinger⁵  Barbara Konkle⁶  Biao Dong⁷  Hongying Wei⁷  Qizhao Wang⁷  Wenman Wu⁷  Andrea R. Moore⁷  Sean A. Roberts⁷  Dong Li⁷  Wenjing Cao⁷ 

[1] Corresponding author: Hanspeter Rottensteiner, PhD, Baxalta Innovations GmbH (now part of Takeda), Donau-City Str. 7, Vienna 1220, Austria.;Dairy and Functional Foods Research Unit, ARS, USDA, 600 East Mermaid Lane, Wyndmoor, PA 19038, USA;Department of Comparative Medicine, Yale University School of Medicine, 310 Cedar St., BML 330, New Haven, CT 06510, USA;Department of Medicine/Hematology, University of Washington, 1900 9th Ave, Seattle, WA 98195, USA;Drug Discovery Austria, Baxalta Innovations GmbH (now part of Takeda), Donau-City Str. 7, Vienna 1220, Austria;Seattle Children’s Research Institute, University of Washington, 1900 9th Ave, Seattle, WA 98195, USA;Sol Sherry Thrombosis Research Center, Temple University Medical School, 3400 North Broad Street, Philadelphia, PA, 19140, USA;

关键词: factor VIII;    AAV;    gene therapy;    X5;    secretion;    vector;   

DOI  :  

来源: DOAJ

【 摘 要 】

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.

【 授权许可】

Unknown