title: Directed engineering of a high-expression chimeric transgene as a strategy for gene therapy of hemophilia A.
abstract: Human coagulation factor VIII (fVIII) is inefficiently biosynthesized in vitro and has proven difficult to express at therapeutic levels using available clinical gene-transfer technologies. Recently, we showed that a porcine and certain hybrid human/porcine fVIII transgenes demonstrate up to 100-fold greater expression than human fVIII. In this study, we extend these results to describe the use of a humanized, high-expression, hybrid human/porcine fVIII transgene that is 89% identical to human fVIII and was delivered by lentiviral vectors (LVs) to hematopoietic stem cells for gene therapy of hemophilia A. Recombinant human immunodeficiency virus-based vectors encoding the fVIII chimera efficiently transduced human embryonic kidney (HEK)-293T cells. Cells transduced with hybrid human/porcine fVIII encoding vectors expressed fVIII at levels 6- to 100-fold greater than cells transduced with vectors encoding human fVIII. Transplantation of transduced hematopoietic stem and progenitor cells into hemophilia A mice resulted in long-term fVIII expression at therapeutic levels despite <5% genetically modified blood mononuclear cells. Furthermore, the simian immunodeficiency virus (SIV) -derived vector effectively transduced the human hematopoietic cell lines K562, EU1, U.937, and Jurkat as well as the nonhematopoietic cell lines, HEK-293T and HeLa. All cell lines expressed hybrid human/porcine fVIII, albeit at varying levels with the K562 cells expressing the highest level of the hematopoietic cell lines. From these studies, we conclude that humanized high-expression hybrid fVIII transgenes can be utilized in gene therapy applications for hemophilia A to significantly increase fVIII expression levels compared to what has been previously achieved.
authors.affiliation: Aflac Cancer Center and Blood Disorders Service, Department of Pediatrics, Emory University, Atlanta, Georgia 30322, USA. cdoerin@emory.edu
authors.forename: Christopher B
authors.initials: CB
authors.lastname: Doering
authors.forename: Gabriela
authors.initials: G
authors.lastname: Denning
authors.forename: Kerry
authors.initials: K
authors.lastname: Dooriss
authors.forename: Bagirath
authors.initials: B
authors.lastname: Gangadharan
authors.forename: Jennifer M
authors.initials: JM
authors.lastname: Johnston
authors.forename: Keith W
authors.initials: KW
authors.lastname: Kerstann
authors.forename: David A
authors.initials: DA
authors.lastname: McCarty
authors.forename: H Trent
authors.initials: HT
authors.lastname: Spencer
mesh_terms: D000818:Animals; D002460:Cell Line; D004195:Disease Models, Animal; D005169:Factor VIII / Q000235:genetics; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D006467:Hemophilia A / Q000628:therapy*; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D051379:Mice; D013552:Swine; D019076:Transgenes / Q000235:genetics*
chemical_list: D005169:Factor VIII