title: Long-term expression of human coagulation factor VIII in a tolerant mouse model using the φC31 integrase system.
abstract: We generated a mouse model for hemophilia A that combines a homozygous knockout for murine factor VIII (FVIII) and a homozygous addition of a mutant human FVIII (hFVIII). The resulting mouse, having no detectable FVIII protein or activity and tolerant to hFVIII, is useful for evaluating FVIII gene-therapy protocols. This model was used to develop an effective gene-therapy strategy using the φC31 integrase to mediate permanent genomic integration of an hFVIII cDNA deleted for the B-domain. Various plasmids encoding φC31 integrase and hFVIII were delivered to the livers of these mice by using hydrodynamic tail-vein injection. Long-term expression of therapeutic levels of hFVIII was observed over a 6-month time course when an intron was included in the hFVIII expression cassette and wild-type φC31 integrase was used. A second dose of the hFVIII and integrase plasmids resulted in higher long-term hFVIII levels, indicating that incremental doses were beneficial and that a second dose of φC31 integrase was tolerated. We observed a significant decrease in the bleeding time after a tail-clip challenge in mice treated with plasmids expressing hFVIII and φC31 integrase. Genomic integration of the hFVIII expression plasmid was demonstrated by junction PCR at a known hotspot for integration in mouse liver. The φC31 integrase system provided a nonviral method to achieve long-term FVIII gene therapy in a relevant mouse model of hemophilia A.
authors.affiliation: Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305-5120, USA.
authors.forename: Christopher L
authors.initials: CL
authors.lastname: Chavez
authors.forename: Annahita
authors.initials: A
authors.lastname: Keravala
authors.forename: Jacqueline N
authors.initials: JN
authors.lastname: Chu
authors.forename: Alfonso P
authors.initials: AP
authors.lastname: Farruggio
authors.forename: Vanessa E
authors.initials: VE
authors.lastname: Cuéllar
authors.forename: Jan
authors.initials: J
authors.lastname: Voorberg
authors.forename: Michele P
authors.initials: MP
authors.lastname: Calos
mesh_terms: D000818:Animals; D004195:Disease Models, Animal; D005169:Factor VIII / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D015316:Genetic Therapy; D006467:Hemophilia A / Q000097:blood / Q000235:genetics / Q000628:therapy*; D006801:Humans; D019426:Integrases / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D014162:Transfection
chemical_list: D005169:Factor VIII; D019426:Integrases
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