title: Efficient adenoviral vector transduction and expression of functional human factor VIII in cultured primary human hepatocytes.
abstract: Hemophilia A is a severe bleeding disorder caused by a deficiency in blood coagulation factor VIII (FVIII). Adenoviral vectors containing a potent human FVIII expression cassette encoding a truncated FVIII cDNA were developed that mediated sustained FVIII expression in normal and haemophiliac mice and complete phenotypic correction of the bleeding disorder in haemophiliac mice and dogs (Connelly and Kaleko, Haemophilia, 1998; 4: 380-8). Here, we evaluated two E1/E2a/E3-deleted adenoviral vectors encoding human FVIII, one containing the full-length cDNA and the second containing a truncated cDNA lacking the B-domain. Viral vectors encoding the human full-length FVIII cDNA have not been described previously. Hepatocyte transduction was efficient and dose dependent, ranging from 50% to 100%. High levels of functional FVIII were secreted from transduced cells at amounts up to 6000 mU-1 10(6)cells-1 60 h. B-domain deleted FVIII was expressed at levels at least 8-fold higher than the full-length FVIII protein, whereas FVIII RNA levels were similar with both vectors. These data provide the first demonstration of FVIII adenoviral vector function in primary human cells and verify the potential clinical utility of adenoviral vectors for the treatment of haemophilia A.
authors.affiliation: Genetic Therapy Inc., A Novartis Company, 938 Clopper Road, Gaithersburg, MD 20878, USA.
authors.forename: J L
authors.initials: JL
authors.lastname: Andrews
authors.forename: L
authors.initials: L
authors.lastname: Weaver
authors.forename: M
authors.initials: M
authors.lastname: Kaleko
authors.forename: S
authors.initials: S
authors.lastname: Connelly
mesh_terms: D000256:Adenoviridae; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics / Q000627:therapeutic use; D004285:Dogs; D005169:Factor VIII / Q000096:biosynthesis / Q000235:genetics*; D015870:Gene Expression; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D005822:Genetic Vectors; D006467:Hemophilia A / Q000235:genetics* / Q000378:metabolism / Q000628:therapy*; D006801:Humans; D008099:Liver / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data
chemical_list: D018076:DNA, Complementary; D005169:Factor VIII