Hematopoietic stem cell mediated gene therapy for hemophilia A using high-expression factor VIII constructs

Clinical gene therapy trials for hemophilia A to date have failed due to insufficient target cell transduction or expression of coagulation factor VIII (fVIII). Numerous animal studies have documented the extreme difficulty of expressing therapeutic levels of human fVIII following transduction with recombinant viral vectors without eliciting an anti-fVIII humoral immune response. Landmark studies by Evans and Morgan first achieved tolerance to fVIII following transplantation of retrovirally transduced bone marrow cells in a hemophilia A mouse model, yet fVIII activity levels were undetectable in these mice. FVIII production is limited by inefficient protein synthesis and secretion. B-domain deleted (BDD) porcine fVIII (pfVIII) is expressed 10-14 fold higher than BDD human fVIII due to enhanced protein secretion. We hypothesize that the barrier of low transgene expression in gene therapy for hemophilia A can be overcome by using high-expression pfVIII constructs. In this study we show that sustained, curative fVIII activity levels can be achieved in hemophilia A mice following retroviral transduction and transplantation of murine hematopoietic stem cells. Stem cell antigen-1+ cells were transduced with an MSCV-BDDpfVIII oncoretroviral vector and transplanted into hemophilia A mice following nonmyeloablative conditioning. The formation of anti-BDDpfVIII inhibitory antibodies was prevented by transiently depleting T cells at the time of transplantation. In addition, we achieve sustained BDDpfVIII activity following genetically-modified hematopoietic stem cell transplantation in mice with pre-existing anti-human fVIII inhibitory antibodies, a model system for studying the population of patients with hemophilia A who have mounted a humoral immune response to infused fVIII protein product. Both non-immunized as well as pre-immunized mice were tolerant to challenge with both human fVIII and BDDpfVIII after transplantation. We then showed that the mechanism of tolerance is a T cell dependent process and that tolerance is not broken even under stringent conditions of BDDpfVIII presentation with adjuvant at levels that are normally highly immunogenic. In order to translate these findings to a clinical trial of hematopoietic stem cell gene therapy for hemophilia A, human hematopoietic cells must be able to produce fVIII at therapeutic levels. We hypothesized that sufficient fVIII expression could be achieved from human hematopoietic cells using our high-expression hybrid human-porcine fVIII constructs. Both human hematopoietic cell lines as well as primary leukocytes were transduced with a lenitviral vector encoding a genetically-engineered human-porcine fVIII transgene. Following 3 days in culture, fVIII activity was 2.4 and 0.23 U/mL, respectively, well above the 0.01 U/mL threshold for therapeutic fVIII activity. We conclude that hematopoietic stem cell gene therapy presents a potential cure for hemophilia A including patients with pre-existing anti-fVIII inhibitory antibodies.