Functional aspects of factor VIII expression after transplantation of genetically-modified hematopoietic stem cells for hemophilia A.

BACKGROUND: Major complications with respect to the development of gene therapy treatments for hemophilia A include low factor VIII (fVIII) expression and humoral immune responses resulting in inhibitory anti-fVIII antibodies. We previously achieved sustained curative fVIII activity levels in hemophilia A mice after nonmyeloablative transplantation of genetically-modified hematopoietic stem cells (HSCs) encoding a B-domain deleted porcine fVIII (BDDpfVIII) transgene with no evidence of an immune response. METHODS: Mouse HSCs were transduced using MSCV-based recombinant virus encoding BDDpfVIII and transplanted into hemophilia A mice. Transplanted mice were followed for donor cell engraftment, fVIII expression and activity, and generation of anti-fVIII immune response. RESULTS: We now show that: (i) the protein expressed by hematopoietic cells has a specific activity similar to that of purified protein; (ii) BDDpfVIII expressed from hematopoietic cells effectively induces thrombus formation, which is shown using a new method of in vivo analysis of fVIII function; (iii) naïve and pre-immunized mice receiving HSC gene therapy are nonresponsive to challenges with recombinant human fVIII; (iv) nonresponsiveness is not broken after stringent challenges with BDDpfVIII; and (v) T cells from these mice are unresponsive to BDDpfVIII presentation. Furthermore, stem cells isolated from donors with high titer anti-human fVIII antibodies show no defects in donor cell engraftment or the ability to express BDDpfVIII. CONCLUSIONS: These results demonstrate that HSC gene therapy can be an effective alternative treatment for individuals with hemophilia A and may benefit patients by inducing immunological nonresponsiveness to fVIII replacement products.

Transduction of murine hematopoietic stem cells and in vivo selection of gene-modified cells.

Hematopoietic stem cells (HSCs) were among the first targets of genetic manipulation for the purpose of treating human diseases. The translational aspects of the first human clinical trials were based on results obtained using the mouse as an experimental model. Murine studies have shown that the major limitations of HSC gene therapy are similar to those encountered when using non-hematopoietic cells as targets and include (1) an inability to genetically modify sufficient numbers of target cells, (2) the loss of transgene function over time, and (3) potential complications due to vector integration. With continued improvements in transduction protocols, murine HSC transduction and transplantation are now routine with transduction efficiencies >50% easily achievable and even >90% feasible. However, attaining high-level engraftment of gene-modified cells after transplantation is still problematic. Basic transduction conditions entail cytokine stimulation of HSC populations, such as stem cell antigen-1 positive (Sca-1(+)) cells isolated from bone marrow, in serum-free media followed by multiple additions of recombinant retrovirus. Analysis of peripheral blood 12 weeks post transplantation of transduced cells into lethally irradiated recipients shows genetic marking in all hematopoietic lineages. Transduction of HSCs is then confirmed by transplanting bone marrow cells harvested from primary transplant recipients into lethally irradiated secondary recipients. Analysis of these mice shows that recombinant retroviruses transduce murine HSCs efficiently and stably and that the genetically modified cells are capable of completely repopulating the hematopoietic system.

Hematopoietic stem-cell gene therapy of hemophilia A incorporating a porcine factor VIII transgene and nonmyeloablative conditioning regimens.

Insufficient expression of factor VIII (fVIII) is a major hurdle in the development of successful nucleic acid treatments for hemophilia. However, we recently showed that under myeloablative and reduced-intensity total body irradiation (TBI) conditioning, transplantation of hematopoietic stem cells (HSCs) transduced with recombinant retroviruses containing B domain-deleted porcine fVIII (BDDpfVIII) sequences provides curative fVIII levels in a hemophilia A mouse model. In the current study, we tested BDDpfVIII activity after nonmyeloablative conditioning with busulfan, cyclophosphamide, or fludarabine and immunosuppressive agents CTLA4-Ig + anti-CD40L or anti-(murine)thymocyte serum (ATS). ATS is similar in action to anti-(human)thymocyte globulin (ATG), which is used clinically with busulfan in bone marrow transplantations to increase donor cell engraftment. Mice conditioned with busulfan + ATS and that received a transplant of BDDpfVIII-transduced stem-cell antigen 1-positive cells exhibited moderate levels of donor cell chimerism (between 20% and 60%) and achieved sustained fVIII levels more than 1 U/mL. Similar results were observed in mice preimmunized with human fVIII and conditioned with 5 Gy TBI + ATS or busulfan + ATS. These data demonstrate that it is possible to achieve sufficient fVIII expression after transplantation of BDDpfVIII-transduced HSCs following low-toxicity pretransplantation conditioning with targeted immunosuppression, potentially even in the context of preexisting inhibitors.

High-level expression of porcine factor VIII from genetically modified bone marrow-derived stem cells.

Clinical success for gene therapy of hemophilia A will be judged by achievement of sustained, therapeutic levels of coagulation factor VIII (fVIII). Previous clinical trials have suffered from transient, subtherapeutic expression of human fVIII transgenes. Porcine fVIII contains sequence elements that enable more efficient biosynthesis than human fVIII due to enhanced posttranslational transit through the secretory pathway. In this study, we evaluated ex vivo retroviral gene transfer of a high-expression porcine fVIII transgene into bone marrow-derived stromal and hematopoietic stem/progenitor cells (MSCs and HSCs, respectively) and transplantation into genetically immunocompetent hemophilia A mice. Both MSCs and HSCs demonstrated high-level expression of porcine fVIII in vivo. However, following transplantation of gene-modified MSCs, fVIII activity levels rapidly returned to baseline due to the formation of anti-porcine fVIII-neutralizing antibodies. Alternatively, transplantation of HSCs into myeloablated and nonmyeloablated hemophilia A mice resulted in high-level fVIII expression despite low-level hematopoietic reconstitution by gene-modified cells. FVIII expression was sustained beyond 10 months, indicating that immunologic tolerance to porcine fVIII was achieved. Furthermore, transplantation of bone marrow from primary recipients into naive secondary recipients resulted in sustained, high-level fVIII expression demonstrating successful genetic modification and engraftment of HSCs.