title: piggyBac-mediated phenotypic correction of factor VIII deficiency.
abstract: Hemophilia A, caused by a deficiency in factor VIII (FVIII), is the most severe inherited bleeding disorder. Hemophilia A is an attractive gene therapy candidate because even small increases in FVIII levels will positively alter the phenotype. While several vectors are under investigation, gene addition from an integrated transgene offers the possibility of long term expression. We engineered the DNA transposon-based vector, piggyBac (PB), to carry a codon-optimized B-domain deleted human FVIII cDNA. Evaluation of gene transfer efficiency in FVIII null mice demonstrated that PB containing the FVIII cDNA, delivered via hydrodynamic injection to immunocompetent hemophilia mice, conferred persistent gene expression, attaining mean FVIII activity of approximately 60% with 3/19 developing inhibitors. In addition to efficacious expression, a goal of gene transfer-based therapies is to develop vectors with low toxicity. To assess endoplasmic reticulum stress in hepatocytes stably expressing the transgene, we evaluated levels of ER stress markers via qPCR and found no evidence of cell stress. To evaluate phenotypic correction, a tail clip assay performed at the end of the study revealed reduced blood loss. These data demonstrate that PB can be used to achieve sustained FVIII expression and long-term therapeutic benefit in a mouse model.
authors.affiliation: Center for Gene Therapy of Cystic Fibrosis and Other Genetic Diseases, Carver College of Medicine, University of Iowa , Iowa City, Iowa, USA ; Stead Family Department of Pediatrics, Carver College of Medicine, University of Iowa , Iowa City, Iowa, USA.
authors.forename: Janice M
authors.initials: JM
authors.lastname: Staber
authors.affiliation: Stead Family Department of Pediatrics, Carver College of Medicine, University of Iowa , Iowa City, Iowa, USA.
authors.forename: Molly J
authors.initials: MJ
authors.lastname: Pollpeter
authors.affiliation: Department of Anatomy and Cell Biology, Carver College of Medicine, University of Iowa , Iowa City, Iowa, USA.
authors.forename: Angela
authors.initials: A
authors.lastname: Arensdorf
authors.affiliation: Center for Gene Therapy of Cystic Fibrosis and Other Genetic Diseases, Carver College of Medicine, University of Iowa , Iowa City, Iowa, USA ; Stead Family Department of Pediatrics, Carver College of Medicine, University of Iowa , Iowa City, Iowa, USA.
authors.forename: Patrick L
authors.initials: PL
authors.lastname: Sinn
authors.affiliation: Department of Anatomy and Cell Biology, Carver College of Medicine, University of Iowa , Iowa City, Iowa, USA.
authors.forename: D Thomas
authors.initials: DT
authors.lastname: Rutkowski
authors.affiliation: Center for Gene Therapy of Cystic Fibrosis and Other Genetic Diseases, Carver College of Medicine, University of Iowa , Iowa City, Iowa, USA ; Stead Family Department of Pediatrics, Carver College of Medicine, University of Iowa , Iowa City, Iowa, USA ; Interdisciplinary Graduate Program in Genetics, Carver College of Medicine, University of Iowa , Iowa City, Iowa, USA.
authors.forename: Paul B
authors.initials: PB
authors.lastname: McCray