title: Enhanced factor VIII heavy chain for gene therapy of hemophilia A.
abstract: Hemophilia A gene therapy using recombinant adenovirus-associated virus (AAV) vectors has been hampered by the size of the factor VIII (FVIII) cDNA. Previously, splitting the FVIII coding sequence into a heavy-chain (HC) fragment and a light-chain (LC) fragment for dual recombinant AAV vector delivery has been successfully explored. However, the main disadvantage of this approach is a "chain imbalance" problem in which LC secretion is approximately 1-2 logs higher than that of HC, and therefore, the majority of protein synthesized is nonfunctional. To improve HC secretion, we constructed alternate FVIII HCs based on our observation that LC facilitates HC secretion. To our surprise, most of the new HC molecules exhibited enhanced expression over the traditional HC molecule (HC(745)). The optimized HC mutein, HC(HL), including additional acidic-region-3 (ar3) sequences, exhibited three- to fivefold higher activity in both enzyme-linked immunosorbent assay (ELISA) and activated partial thromboplastin time (aPTT) assay in in vitro testing. Further characterization suggested ar3 sequences increased HC secretion, rather than promoting HC synthesis. Intravenous delivery of AAV8-HC(HL)+AAV8-LC or AAV8-HC(745)+AAV8-LC achieved phenotypic correction in hemophilia A mice. Mice receiving AAV8-HC(HL)+AAV8-LC achieved three- to fourfold higher HC expression than AAV8-HC(745)+AAV8-LC, consistent with the FVIII functional assays. HC(HL) should be substituted for HC(745) in a dual AAV vector strategy due to its enhanced expression.
authors.affiliation: Department of Pediatrics, University of Pennsylvania Medical Center and The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA.
authors.forename: Lingxia
authors.initials: L
authors.lastname: Chen
authors.forename: Hui
authors.initials: H
authors.lastname: Lu
authors.forename: Jinhui
authors.initials: J
authors.lastname: Wang
authors.forename: Rita
authors.initials: R
authors.lastname: Sarkar
authors.forename: Xiao
authors.initials: X
authors.lastname: Yang
authors.forename: Hongli
authors.initials: H
authors.lastname: Wang
authors.forename: Katherine A
authors.initials: KA
authors.lastname: High
authors.forename: Weidong
authors.initials: W
authors.lastname: Xiao
mesh_terms: D000143:Acids / Q000737:chemistry; D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D000941:Antigens / Q000276:immunology; D002460:Cell Line; D005169:Factor VIII / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D015316:Genetic Therapy; D006467:Hemophilia A / Q000235:genetics* / Q000276:immunology / Q000378:metabolism / Q000628:therapy*; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D009154:Mutation / Q000235:genetics; D010641:Phenotype
chemical_list: D000143:Acids; D000941:Antigens; D005169:Factor VIII