| Hemophilia A is an excellent candidate for gene therapy because it can be cured by the transfer of a single gene into any cell with access to the bloodstream. Unfortunately, the success of gene therapy has been hampered by low and transient transgene expression, the risk of insertional mutagenesis, and the frequent induction of an immune response. We hypothesized that the development of a safer viral vector incorporating a high-expressing fVIII transgene for the transduction and transplantation of hematopoietic stem cells can overcome the barriers of low and transient expression with a decreased risk of insertional mutagenesis. With respect to the development of a high-expressing transgene, a number of human, porcine, and human/porcine chimeric transgenes were developed for the purpose of determining which transgene exhibits the highest fVIII expression following lentiviral-mediated hematopoietic stem cell transduction and transplantation in a mouse model of hemophilia A. We discovered that porcine fVIII and one chimeric fVIII transgene express at significantly higher levels than any of the human constructs. We also demonstrated sustained fVIII expression in hemophilic mice following lentiviral-mediated transduction and transplantation of gene-modified hematopoietic stem cells. To evaluate the ability of a lentivirus to mediate lineage-specific fVIII expression in gene-modified bone marrow cells, self-inactivating lentiviral vectors were developed to express porcine fVIII under the control of the beta-globin promoter and locus control region. We demonstrated high-level fVIII expression following lentiviral mediated gene-transfer into a myelogenous leukemic cell line, as well as from gene-modified hematopoietic cells in transplanted mice. Although we confirmed this lentiviral-mediated gene transfer platform is capable of GFP and fVIII expression in mice, hematopoietic stem cell transduction efficiency was limiting. Transduction efficiency was lower with the porcine fVIII-containing lentivirus compared to the GFP-containing lentivirus. Together, these studies demonstrate that a porcine or human/porcine chimeric transgene is able to mediate high level fVIII expression, that erythroid-specific fVIII expression is possible, and that hematopoietic stem cell transduction is the limiting factor in the future success of this erythroid-specific lentiviral-mediated gene transfer system. |
