title: Complete correction of hemophilia A with adeno-associated viral vectors containing a full-size expression cassette.
abstract: Hemophilia A is caused by a deficiency in the factor VIII (FVIII) gene. Constrained by limited packaging capacity, even the 4.3-kb B domain-deleted FVIII remained a challenge for delivery by a single adeno-associated viral (AAV) vector. Studies have shown that up to a 6.6-kb vector sequence may be packaged into AAV virions, which suggested an alternative strategy for hemophilia A gene therapy. To explore the usefulness of AAV vectors carrying an oversized FVIII gene, we constructed the AAV-FVIII vector under the control of a beta-actin promoter with a cytomegalovirus enhancer (CB) and a bovine growth hormone (bGH) poly(A) sequence. The CB promoter plus bGH signal was shown to be 3- to 5-fold more potent than the mini-transthyretin (TTR) promoter with a synthetic poly(A) sequence for directing FVIII expression in the liver. Despite the 5.75-kb genome size of pAAV-CB-FVIII, sufficient AAV vectors were produced for in vivo testing. Approximately 3- to 5-fold more FVIII secretion was observed in animals receiving AAV-CB-FVIII vectors than in those receiving standard-sized AAV-TTR-FVIII vectors. Both the activated partial thromboplastin time assay and the whole blood thromboelastographic analysis confirmed that AAV-FVIII vectors fully corrected the bleeding phenotype of hemophilia mice. These results suggest that AAV vectors with an oversized genome should be useful for not only hemophilia A gene therapy but also other diseases with large cDNA such as muscular dystrophy and cystic fibrosis.
authors.affiliation: Department of Pediatrics, University of Pennsylvania Medical Center and Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA.
authors.forename: Hui
authors.initials: H
authors.lastname: Lu
authors.forename: Lingxia
authors.initials: L
authors.lastname: Chen
authors.forename: Jinhui
authors.initials: J
authors.lastname: Wang
authors.forename: Bernd
authors.initials: B
authors.lastname: Huack
authors.forename: Rita
authors.initials: R
authors.lastname: Sarkar
authors.forename: Shangzhen
authors.initials: S
authors.lastname: Zhou
authors.forename: Ray
authors.initials: R
authors.lastname: Xu
authors.forename: Qiulan
authors.initials: Q
authors.lastname: Ding
authors.forename: Xuefeng
authors.initials: X
authors.lastname: Wang
authors.forename: Hongli
authors.initials: H
authors.lastname: Wang
authors.forename: Weidong
authors.initials: W
authors.lastname: Xiao
mesh_terms: D000818:Animals; D002460:Cell Line; D000229:Dependovirus; D005169:Factor VIII / Q000235:genetics*; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D006467:Hemophilia A / Q000628:therapy*; D006801:Humans; D051379:Mice; D018345:Mice, Knockout
chemical_list: D005169:Factor VIII