Post-Transplantation Immunosuppression Facilitates Sustained Expression of Porcine Factor VIII after Reduced-Intensity Transplantation of Genetically-Modified Stem Cells.

A major barrier to successful gene therapy for hemophilia A using bone marrow cells is achieving engraftment of a sufficient number of gene-modified cells to result in therapeutic factor VIII (fVIII) levels under conditions of acceptable patient risk. Previously, we demonstrated sustained levels of a high-expression porcine (HEP)-fVIII construct, at greater than 1 unit/mL for over one year, in hemophilia A mice following myeloablative HSC transplantation (HSCT) thus overcoming the barrier of low fVIII expression (Gangadharan et al, Blood, 2006). In the current study, we tested HEP-fVIII expression after several non-myeloablative and reduced-intensity stem cell transplantation (RIST) protocols incorporating a clinically-relevant dose of genetically-modified hematopoietic stem cells transduced at low multiplicity of infections of ≤ 2. Recipient fVIII exon 16-knockout mice received 3 × 10⁵ HEP-fVIII-MSCV transduced, congenic stem cell antigen-1⁺ cells. Mice were conditioned using one of the following regimens: 5.5 Gy total body irradiation (TBI), 2 Gy TBI, 2 Gy TBI + 200 mg/kg cyclophosphamide (Ctx), 200 mg/kg Ctx, 200 mg/kg Ctx + 90 mg/kg fludarabine (Flu), 35 mg/kg busulfan (Bu), 35 mg/kg Bu + 200 mg/kg Ctx, 35 mg/kg Bu + costimulation blockade (0.5mg/day CTLA4-Ig and α-CD40L on days 0, 2 post-transplantation). Resulting donor cell engraftment, HEP-fVIII activity, and inhibitory anti-fVIII antibody levels were followed. No fVIII activity was seen in the 2 Gy, 2 Gy + Ctx, Ctx only, or Ctx + Flu cohorts. The Bu and Bu/Ctx treated animals expressed therapeutic levels of HEP-fVIII (0.05 ± 0.07 U/mL and 0.81 ± 0.37 U/mL, respectively) at 1 wk post-HSCT, but returned to baseline in all mice by 2 wks with inhibitor formation in 3 of 7 mice in the Bu group and 0 of 4 mice in the Bu/Ctx cohort. In order to suppress inhibitor formation following conditioning with Bu, recipient mice were administered a low dose course of costimulation blockade post-HSCT. Four of 10 mice in this group demonstrated therapeutic levels of HEP-fVIII activity on day 34-post HSCT (0.11 ± .09 U/mL). All animals demonstrated donor cell engraftment in peripheral blood mononuclear cells at day 23 post-HSCT (18.4 ± 12.1%) and at 34 days post-HSCT, none of the animals had detectable levels of fVIII inhibitors in a modified Bethesda assay. Additionally, we were able to identify specific hematopoietic lineages expressing HEP-fVIII in recipient mice using a novel enzyme-linked immunospot (ELISPOT) assay developed in our laboratory. These data demonstrate that it is possible to achieve sufficient fVIII expression under conditions of i) low MOI transduction with an optimized high-expression fVIII transgene, ii) mild pretransplantation conditioning, iii) limited genetically-modified HSC engraftment, and iv) mild post-HSCT immunosuppression. Furthermore, our results show that clinically-significant fVIII activity levels can be maintained without inhibitor formation suggesting that the current protocol may be on the threshold, in terms of the level of immunosuppression, of promoting tolerance to HEP-fVIII.