title: Generation of an optimized lentiviral vector encoding a high-expression factor VIII transgene for gene therapy of hemophilia A.
abstract: We previously compared the expression of several human factor VIII (fVIII) transgene variants and demonstrated the superior expression properties of B domain-deleted porcine fVIII. Subsequently, a hybrid human/porcine fVIII molecule (HP-fVIII) comprising 91% human amino-acid sequence was engineered to maintain the high-expression characteristics of porcine fVIII. The bioengineered construct then was used effectively to treat knockout mice with hemophilia A. In the current study, we focused on optimizing self-inactivating (SIN) lentiviral vector systems by analyzing the efficacy of various lentiviral components in terms of virus production, transduction efficiency and transgene expression. Specifically, three parameters were evaluated: (1) the woodchuck hepatitis post-transcriptional regulatory element (WPRE), (2) HIV versus SIV viral vector systems and (3) various internal promoters. The inclusion of a WPRE sequence had negligible effects on viral production and HP-fVIII expression. HIV and SIV vectors were compared and found to be similar with respect to transduction efficiency in both K562s and HEK-293T cells. However, there was an enhanced expression of HP-fVIII by the SIV system, which was evident in both K562 and BHK-M cell lines. To further compare expression of HP-fVIII from an SIV-based lentiviral system, we constructed expression vectors containing the high expression transgene and a human elongation factor-1 alpha, cytomegalovirus (CMV) or phosphoglycerate kinase promoter. Expression was significantly greater from the CMV promoter, which also yielded therapeutic levels of HP-fVIII in hemophilia A mice. Based on these studies, an optimized vector contains the HP-fVIII transgene driven by a CMV internal promoter within a SIV-based lentiviral backbone lacking a WPRE.
authors.affiliation: Department of Pediatrics, Aflac Cancer Center and Blood Disorders Service, Emory Children's Center, Emory University School of Medicine, Atlanta, GA 30322, USA.
authors.forename: J M
authors.initials: JM
authors.lastname: Johnston
authors.forename: G
authors.initials: G
authors.lastname: Denning
authors.forename: C B
authors.initials: CB
authors.lastname: Doering
authors.forename: H T
authors.initials: HT
authors.lastname: Spencer
mesh_terms: D000818:Animals; D005169:Factor VIII / Q000008:administration & dosage / Q000235:genetics*; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D005822:Genetic Vectors; D057809:HEK293 Cells; D006467:Hemophilia A / Q000235:genetics / Q000628:therapy; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D051379:Mice; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000008:administration & dosage / Q000235:genetics*; D013552:Swine; D014161:Transduction, Genetic
chemical_list: D011994:Recombinant Proteins; D005169:Factor VIII