title: Bioengineering of coagulation factor VIII for improved secretion.
abstract: Factor VIII (FVIII) functions as a cofactor within the intrinsic pathway of blood coagulation. Quantitative or qualitative deficiencies of FVIII result in the inherited bleeding disorder hemophilia A. Expression of FVIII (domain structure A1-A2-B-A3-C1-C2) in heterologous mammalian systems is 2 to 3 orders of magnitude less efficient compared with other proteins of similar size compromising recombinant FVIII production and gene therapy strategies. FVIII expression is limited by unstable mRNA, interaction with endoplasmic reticulum (ER) chaperones, and a requirement for facilitated ER to Golgi transport through interaction with the mannose-binding lectin LMAN1. Bioengineering strategies can overcome each of these limitations. B-domain-deleted (BDD)-FVIII yields higher mRNA levels, and targeted point mutations within the A1 domain reduce interaction with the ER chaperone immunoglobulin-binding protein. In order to increase ER to Golgi transport we engineered several asparagine-linked oligosaccharides within a short B-domain spacer within BDD-FVIII. A bioengineered FVIII incorporating all of these elements was secreted 15- to 25-fold more efficiently than full-length FVIII both in vitro and in vivo. FVIII bioengineered for improved secretion will significantly increase potential for success in gene therapy strategies for hemophilia A as well as improve recombinant FVIII production in cell culture manufacturing or transgenic animals.
authors.affiliation: Department of Pediatrics, Howard Hughes Medical Institute, University of Michigan Medical Center, Ann Arbor, MI 48109, USA.
authors.forename: Hongzhi Z
authors.initials: HZ
authors.lastname: Miao
authors.forename: Nongnuch
authors.initials: N
authors.lastname: Sirachainan
authors.forename: Lisa
authors.initials: L
authors.lastname: Palmer
authors.forename: Phillip
authors.initials: P
authors.lastname: Kucab
authors.forename: Michael A
authors.initials: MA
authors.lastname: Cunningham
authors.forename: Randal J
authors.initials: RJ
authors.lastname: Kaufman
authors.forename: Steven W
authors.initials: SW
authors.lastname: Pipe
mesh_terms: D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D006224:Cricetinae; D005169:Factor VIII / Q000235:genetics* / Q000378:metabolism*; D015316:Genetic Therapy / Q000379:methods; D014644:Genetic Variation; D005822:Genetic Vectors / Q000008:administration & dosage / Q000627:therapeutic use; D006031:Glycosylation; D006467:Hemophilia A / Q000628:therapy; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D008822:Mice, Transgenic; D015202:Protein Engineering / Q000379:methods*; D021381:Protein Transport / Q000235:genetics; D014162:Transfection
chemical_list: D005169:Factor VIII