Gene therapy for hemophilia A would be facilitated by development of smaller expression cassettes encoding factor VIII (FVIII) which demonstrate improved biosynthesis and/or enhanced biological properties. B-domain deleted (BDD) FVIII retains full procoagulant function and is expressed at higher levels than wild type FVIII. However a partial BDD FVIII, leaving an N-terminal 226 amino acid stretch (N6), increases in vitro secretion of FVIII tenfold compared to BDD-FVIII. In this study we tested various BDD-constructs in the context of either wild-type or codon optimised cDNA sequences expressed under control of the strong, ubiquitous Spleen Focus Forming Virus (SFFV) promoter within a self-inactivating (SIN) HIV-based lentiviral vector. Transduced 293T cells in vitro demonstrated detectable FVIII activity. Hemophilic mice treated with lentiviral vectors showed expression of FVIII activity and phenotypic correction sustained over 250 days. Importantly, codon optimised constructs achieved an unprecedented 29- to 44- fold increase in expression yielding over 200% normal human FVIII levels. Addition of B-domain sequences to BDD-FVIII did not significantly increase in vivo expression. These significant findings demonstrate that shorter FVIII constructs that can be more easily accommodated in viral vectors can result in increased therapeutic efficacy and may deliver effective gene therapy for hemophilia A.
|Number of pages||10|
|Publication status||Published - 2011|
- Gene therapy
- hemophilia A