Hematopoietic Stem Cells Encoding Porcine Factor VIII Induce Pro-coagulant Activity in Hemophilia A Mice with Pre-existing Factor VIII Immunity
Authors: Christopher B Doering, Bagirath Gangadharan, Hillary Z Dukart, H Trent Spencer
Journal: Molecular Therapy (2007)

The development of inhibitory antibodies directed against factor VIII (fVIII) remains the most significant clinical complication associated with the treatment of hemophilia A. Recently, we demonstrated that transplantation of genetically modified hematopoietic stem cells containing a high-expression porcine fVIII transgene promoted sustained high-level fVIII expression in naïve hemophilia A mice. In the current study, a similar gene transfer strategy was tested in hemophilia A mice harboring clinically significant anti–human factor VIII (anti-hfVIII) inhibitory antibody titers. Although the majority of mice contained circulating antibodies that cross-reacted with and inhibited porcine fVIII activity, transplantation of genetically modified hematopoietic stem cells containing a porcine fVIII transgene into myeloablated hemophilia A mice induced high-level fVIII activity. Furthermore, anti-hfVIII antibody titers steadily declined throughout the course of the study. However, non-myeloablative transplantation conditioning resulted in only partial success. No correlation between pre-transplantation antibody titers and post-transplantation fVIII activity levels or donor cell engraftment was observed. These data suggest that hematopoietic stem cell transplantation–based gene therapy incorporating a high-expression porcine fVIII transgene can be utilized successfully to treat hemophilia A patients harboring anti-hfVIII inhibitors. The development of inhibitory antibodies directed against factor VIII (fVIII) remains the most significant clinical complication associated with the treatment of hemophilia A. Recently, we demonstrated that transplantation of genetically modified hematopoietic stem cells containing a high-expression porcine fVIII transgene promoted sustained high-level fVIII expression in naïve hemophilia A mice. In the current study, a similar gene transfer strategy was tested in hemophilia A mice harboring clinically significant anti–human factor VIII (anti-hfVIII) inhibitory antibody titers. Although the majority of mice contained circulating antibodies that cross-reacted with and inhibited porcine fVIII activity, transplantation of genetically modified hematopoietic stem cells containing a porcine fVIII transgene into myeloablated hemophilia A mice induced high-level fVIII activity. Furthermore, anti-hfVIII antibody titers steadily declined throughout the course of the study. However, non-myeloablative transplantation conditioning resulted in only partial success. No correlation between pre-transplantation antibody titers and post-transplantation fVIII activity levels or donor cell engraftment was observed. These data suggest that hematopoietic stem cell transplantation–based gene therapy incorporating a high-expression porcine fVIII transgene can be utilized successfully to treat hemophilia A patients harboring anti-hfVIII inhibitors. IntroductionHemophilia A is a hereditary bleeding disorder caused by genetic mutations that disrupt the function or inhibit the biosynthesis of coagulation factor VIII (fVIII). Treatment of hemophilia A has seen significant advances over the past several decades and patients are now routinely treated at home by regular (prophylactic) infusion of plasma-derived or recombinant human fVIII (hfVIII) products to maintain therapeutic levels of circulating fVIII. However, approximately 20–30% of all severe hemophiliacs develop humoral immune responses to hfVIII, which generally present during the first 2 years of treatment (10–15 days of product exposure) and frequently render the patient unresponsive to future treatment with hfVIII products (for review, see ref. 1Lollar P Pathogenic antibodies to coagulation factors. Part one: factor VIII and factor IX.J Thromb Haemost. 2004; 2: 1082-1095Crossref PubMed Scopus (112) Google Scholar). Furthermore, a small percentage of non-hemophiliacs develop autoimmune responses to hfVIII that can lead to a severe bleeding diathesis. In addition to being difficult and expensive to treat clinically, inhibitor patients are at increased risk for bleeding-related morbidity and mortality.Acute treatment for high-titer inhibitor patients includes the use of fVIII-bypassing agents, e.g., prothrombin complex concentrates, activated prothrombin complex concentrates, or recombinant activated factor VII, all of which can provide short-term hemostasis, but are not amenable to long-term use owing to (i) financial cost, (ii) lack of efficacy in chronic arthropathy, and (iii) the difficulty of using these agents in a prophylactic manner. A distinct form of long-term, as opposed to acute, treatment for inhibitor patients involves frequent high-dose hfVIII infusion, termed immune tolerance induction. Although the mechanism of action is not understood, immune tolerance induction effectively eradicates inhibitory antibodies in 60–80% of inhibitor patients, but it is extremely expensive and the cost often exceeds $1,000,000 in hfVIII product alone.2Nilsson IM Berntorp E Zettervall O Induction of immune tolerance in patients with hemophilia and antibodies to factor VIII by combined treatment with intravenous IgG, cyclophosphamide, and factor VIII.N Engl J Med. 1988; 318: 947-950Crossref PubMed Scopus (253) Google Scholar Historically, inhibitor patients also have received treatment for acute bleeding episodes by infusion of plasma-derived porcine fVIII. This treatment was effective because of the general lack of inhibitory reactivity of anti-hfVIII antibodies to porcine fVIII.3Hay CR Lozier JN Lee CA Laffan M Tradati F Santagostino E et al.Safety profile of porcine factor VIII and its use as hospital and home-therapy for patients with haemophilia-A and inhibitors: the results of an international survey.Thromb Haemost. 1996; 75: 25-29PubMed Google Scholar However, currently, there is not a porcine fVIII product available owing to the discovery of viral DNA and particulate contamination in plasma-derived porcine fVIII products.4Heneine W Switzer WM Soucie JM Evatt BL Shanmugam V Sosales GV et al.Evidence of porcine endogenous retroviruses in porcine factor VIII and evaluation of transmission to recipients with hemophilia.J Infect Dis. 2001; 183: 648-652Crossref PubMed Scopus (35) Google Scholar,5Soucie JM Erdman DD Evatt BL Anderson LJ Torok TJ El-Jamil M et al.Investigation of porcine parvovirus among persons with hemophilia receiving Hyate:C porcine factor VIII concentrate.Transfusion. 2000; 40: 708-711Crossref PubMed Scopus (30) Google Scholar,6Takefman DM Wong S Maudru T Peden K Wilson CA Detection and characterization of porcine endogenous retrovirus in porcine plasma and porcine factor VIII.J Virol. 2001; 75: 4551-4557Crossref PubMed Scopus (35) Google Scholar Therefore, investigation into novel treatments for patients with fVIII inhibitors is warranted.Hematopoietic stem cell transplantation (HSCT) is being tested clinically as a treatment option for a variety of human autoimmune disorders, including multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, and Crohn disease.7Burt RK Georganas C Schroeder J Traynor A Stefka J Schuening F et al.Autologous hematopoietic stem cell transplantation in refractory rheumatoid arthritis: sustained response in two of four patients.Arthritis Rheum. 1999; 42: 2281-2285Crossref PubMed Scopus (73) Google Scholar,8Burt RK Traynor A Oyama Y Craig R High-dose immune suppression and autologous hematopoietic stem cell transplantation in refractory Crohn disease.Blood. 2003; 101: 2064-2066Crossref PubMed Scopus (87) Google Scholar,9Burt RK Traynor A Ramsey-Goldman R Hematopoietic stem-cell transplantation for systemic lupus erythematosus.N Engl J Med. 1997; 337: 1777-1778Crossref PubMed Scopus (70) Google Scholar,10Burt RK Traynor A Statkute L Barr WG Rosa R Schroeder J et al.Nonmyeloablative hematopoietic stem cell transplantation for systemic lupus erythematosus.JAMA. 2006; 295: 527-535Crossref PubMed Scopus (253) Google Scholar,11Burt RK Traynor AE Cohen B Karlin KH Davis FA Stefoski D et al.T cell-depleted autologous hematopoietic stem cell transplantation for multiple sclerosis: report on the first three patients.Bone Marrow Transplant. 1998; 21: 537-541Crossref PubMed Scopus (94) Google Scholar Furthermore, HSCT combined with gene therapy has been shown in animal models to facilitate the introduction of and tolerance to neoantigens.12Gangadharan B Parker ET Ide LM Spencer HT Doering CB High-level expression of porcine factor VIII from genetically modified bone marrow-derived stem cells.Blood. 2006; 107: 3859-3864Crossref PubMed Scopus (46) Google Scholar,13Moayeri M Ramezani A Morgan RA Hawley TS Hawley RG Sustained phenotypic correction of hemophilia a mice following oncoretroviral-mediated expression of a bioengineered human factor VIII gene in long-term hematopoietic repopulating cells.Mol Ther. 2004; 10: 892-902Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar,14Bagley J Tian C Sachs DH Iacomini J Induction of T-cell tolerance to an MHC class I alloantigen by gene therapy.Blood. 2002; 99: 4394-4399Crossref PubMed Scopus (67) Google Scholar,15Xu B Haviernik P Wolfraim LA Bunting KD Scott DW Bone marrow transplantation combined with gene therapy to induce antigen-specific tolerance and ameliorate EAE.Mol Ther. 2006; 13: 42-48Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar,16Bracy JL Iacomini J Induction of B-cell tolerance by retroviral gene therapy.Blood. 2000; 96: 3008-3015PubMed Google Scholar In a previous study, we demonstrated that transplantation of genetically modified hematopoietic stem cells (HSCs) containing a high-expression porcine (HEP)-fVIII transgene into hemophilia A mice resulted in the production of high levels of circulating fVIII activity.12Gangadharan B Parker ET Ide LM Spencer HT Doering CB High-level expression of porcine factor VIII from genetically modified bone marrow-derived stem cells.Blood. 2006; 107: 3859-3864Crossref PubMed Scopus (46) Google Scholar The mice used in the previous study were naïve to both hfVIII and HEP-fVIII before transplantation and none of the recipient mice developed anti-fVIII antibodies after HSCT gene therapy. The ability of HSCT gene therapy to (i) reset host immune tolerance, (ii) introduce tolerance to neo-antigens, and (iii) simultaneously provide a long-term source of fVIII production represents a potentially powerful clinical tool for the treatment of refractory anti-fVIII inhibitors. The current study was designed to evaluate the effectiveness of HEP-fVIII HSCT gene therapy in hemophilia A mice pre-immunized with fVIII.ResultsInduction and characterization of the anti-fVIII immune responseTo induce humoral immune responses to hfVIII, hemophilia A mice were injected intravenously with recombinant hfVIII (10 U/injection) on a weekly basis for 4 consecutive weeks. Using this immunization protocol, all mice developed anti-hfVIII inhibitory antibodies. Furthermore, a significant correlation between the anti-hfVIII enzyme-linked immunosorbent assay (ELISA) and Bethesda (inhibitory) titers was observed (P < 0.001) (Figure 1a). Plasma samples also were analyzed for the presence of antibodies that cross-reacted with HEP-fVIII. With the exception of two animals, all plasma samples analyzed contained anti-HEP-fVIII cross-reactive antibodies by ELISA, and 5 of 16 displayed detectable anti-HEP-fVIII inhibitory activity by Bethesda assay. Again, a significant correlation was observed between the individual anti-hfVIII and cross-reactive anti-HEP-fVIII ELISA titers (P = 0.007) (Figure 1b). The median cross-reactivity, (anti-HEP-fVIII titer/anti-hfVIII titer) × 100%, observed using the ELISA and Bethesda titers was 10 and 14%, respectively. The reduced antigenicity displayed toward HEP-fVIII compared with hfVIII is consistent with previous observations made in hemophilia A inhibitor patients where the median cross-reactivity with porcine fVIII was reported to be 15%.3Hay CR Lozier JN Lee CA Laffan M Tradati F Santagostino E et al.Safety profile of porcine factor VIII and its use as hospital and home-therapy for patients with haemophilia-A and inhibitors: the results of an international survey.Thromb Haemost. 1996; 75: 25-29PubMed Google Scholar Data for the individual animals in this study are provided in Supplementary Table S1.Myeloablative HEP-fVIII HSCT-based gene therapyStem cell antigen-1–positive (sca-1+) cells (a population containing HSCs and other progenitor cells) were isolated from bone marrow obtained from transgenic mice constitutively expressing enhanced green fluorescence protein (EGFP) and transduced ex vivo with ecotropic-pseudotyped recombinant murine stem cell retrovirus (MSCV) carrying a HEP-fVIII transgene. Before transplantation, mice were divided into two cohorts containing similar numbers of mice with low (≤10), mid (11–100), or high (≥101) anti-hfVIII Bethesda (inhibitory) titers. Cohort 1 (n = 11) was pre-conditioned with 11 Gy total body irradiation (TBI) (myeloablative), and cohort 2 (n = 7) was pre-conditioned with 5.5 Gy TBI (non-myeloablative). Immediately after TBI, mice in both cohorts received a transplant of 3 × 105 HEP-fVIII-MSCV-transduced sca-1+ cells. Overall, cohort 1 mice displayed high-level donor cell engraftment in peripheral blood mononuclear cells (PBMCs) at 81 ± 22% (mean ± sample SD) 12 weeks after HSCT (Figure 2a and c). Six weeks after HSCT, donor cell engraftment levels began to decrease in two animals. In one animal (ID 52R), donor cell engraftment dropped to 60% of total PBMCs at 12 weeks after HSCT then increased to 87% at the time of killing (36 weeks after HSCT). Donor cell engraftment levels in the second animal (ID 52L) continued to decrease throughout the course of the experiment to 19% at 36 weeks after HSCT.Figure 2Donor cell engraftment and circulating factor VIII (fVIII) activity levels after myeloablative hematopoietic stem cell transplantation (HSCT) gene therapy. (a–d) Recipient animals (n = 11) were pre-conditioned with a myeloablative dose of total body irradiation (TBI) (11 Gy) before receiving a transplant of high-expression porcine–factor VIII–murine stem cell retrovirus (HEP-fVIII-MSCV)–transduced stem cell antigen-1–positive (sca-1+) cells. (a, c) Donor cell engraftment was monitored by flow cytometry analysis of enhanced green fluorescent protein (EGFP+) donor cells at several time points after transplantation. (b, d) Plasma fVIII activity levels were measured by chromogenic fVIII assay at each time point. The animals represented in c and d were re-challenged with human (h)-fVIII (10 U/injection) on days 139 and 154 after HSCT (indicated by arrows). Identical symbols are used to represent each individual animal in a and b or c and d.View Large Image Figure ViewerDownload Hi-res image Download (PPT)All cohort 1 animals expressed therapeutic amounts of HEP-fVIII, with a mean activity level of 3.6 ± 1.3 U/ml at 19 weeks after HSCT (Figure 2b and d). For reference, 1 U of fVIII activity represents the amount present in 1 ml of pooled normal human plasma, and severe hemophilia A is defined by <0.01 U/ml. Achievement of fVIII activity levels ≥0.05 U/ml generally is considered sufficient to convert the bleeding phenotype from severe to mild. Of note, the animal (ID 52L) that demonstrated decreased donor cell engraftment (19%) retained circulating fVIII activity levels >1 U/ml throughout the course of the study, with a terminal activity level of 1.76 U/ml at 36 weeks after HSCT.To determine whether re-exposure to hfVIII would result in a rapid secondary immune response, which could affect the expression of HEP-fVIII or the persistence of genetically modified cells, we challenged 5 of the 11 animals in cohort 1 with two intravenous infusions of hfVIII (10 U/injection) on weeks 20 and 22 after HSCT (Figure 2c and d). No change in donor cell engraftment was observed after either injection. However, all five mice demonstrated a transient decrease in circulating fVIII activity after the first and/or second injection of hfVIII. In four of five mice, fVIII activity levels returned to the respective pre-challenge levels by 36 weeks after HSCT (14 weeks after the last hfVIII challenge).At 36 weeks after HSCT, the five cohort 1 animals that were not re-challenged with hfVIII were killed and bone marrow and spleen were harvested and analyzed for donor cell engraftment, proviral genome copy number, and HEP-fVIII RNA levels (Table 1). Overall, the donor cell engraftment levels were similar among all three tissues. The level of gene marking in bone marrow and spleen displayed marked inter-animal variability, ranging from 0.04 to 2 proviral genomes per diploid genome equivalent. No correlation was observed between the proviral copy numbers and circulating fVIII activity levels similar to what was observed in our previous study.12Gangadharan B Parker ET Ide LM Spencer HT Doering CB High-level expression of porcine factor VIII from genetically modified bone marrow-derived stem cells.Blood. 2006; 107: 3859-3864Crossref PubMed Scopus (46) Google Scholar However, a significant correlation was observed between proviral copy number and HEP-fVIII mRNA levels in both the bone marrow and spleen (P = 0.014).Table 1Gene-marking, fVIII transcript and donor cell engraftment levels following myeloablative hematopoietic stem cell transplantation gene therapyBone marrowSpleenPBMCCohort IDProviral copy numberaProviral genomes per diploid genome equivalent;HEP transcriptsbHEP-fVIII RNA transcripts per cell equivalent.% Donor cell engraftmentProviral copy numberaProviral genomes per diploid genome equivalent;HEP transcriptsbHEP-fVIII RNA transcripts per cell equivalent.% Donor cell engraftment% Donor cell engraftment1–47L1.52717980.8116492961–49L1.34442981.5842890961–52N0.0424970.1720288961–52L0.1167110.128623191–52R1.81932742.032,5628987Abbreviations: HEP, high-expression porcine; PBMC, peripheral blood mononuclear cells.a Proviral genomes per diploid genome equivalent;b HEP-fVIII RNA transcripts per cell equivalent. Open table in a new tab Non-myeloablative HEP-fVIII HSCT-based gene therapyCohort 2 animals displayed lower-level donor cell engraftment at 6.5 ± 6% (range 0.7–13%) in PBMCs at 12 weeks after HSCT (Figure 3a). As a positive control for donor cell engraftment, a separate cohort of animals (cohort 3, n = 3) pre-immunized with hfVIII was pre-conditioned with 5.5 Gy TBI before receiving a transplant of 3 × 105 mock-transduced sca-1+ cells. Cohort 3 animals displayed significantly higher donor cell engraftment (36 ± 17%, P = 0.017) than did the cohort 2 mice at 12 weeks after HSCT. However, it is possible that contaminants transferred from the viral producer cells, which would not have been exposed to the mock-transduced cells, could have negatively affected the engraftment of transduced cells as compared with the mock-transduced cells. At day 9 after HSCT, cohort 2 animals displayed transient high-level plasma fVIII activity at 1 ± 0.6 U/ml (Figure 3b). However, in six of seven animals, fVIII activity levels returned to baseline (≤0.01 U/ml) by day 28 after HSCT, and in four of seven animals, fVIII activity remained below the level of detection throughout the remainder of the experiment. The remaining three animals displayed 0.22 ± 0.3 (range 0.04–0.57) U/ml plasma fVIII activity at 12 weeks after HSCT and showed a trend toward greater donor PBMC engraftment at 12.8 ± 0.2% versus1.8 ± 1.3% (P = 0.057).Figure 3Donor cell engraftment and circulating factor VIII (fVIII) activity levels after non-myeloablative hematopoietic stem cell transplantation (HSCT) gene therapy. Recipient mice (n = 7) were pre-conditioned with a non-myeloablative dose of total body irradiation (TBI) (5.5 Gy)before receiving a transplant of high-expression porcine–factor VIII–murine stem cell retrovirus (HEP-fVIII-MSCV)–transduced stem cell antigen-1–positive (sca-1+) cells. (a) Donor cell engraftment and (b) plasma fVIII activity levels were determined as described in Figure 2a and b. Identical symbols are used to represent each individual animal in a and b.View Large Image Figure ViewerDownload Hi-res image Download (PPT)One method to overcome low levels of engraftment is to increase the size of the transplant (i.e., infuse more donor cells). Since some of the animals in cohort 2 did express therapeutic levels of HEP-fVIII, it was possible that the cell dose used (3 × 105 sca-1+ cells) was at a threshold required to produce donor cell engraftment in this pre-immunization model. To test this hypothesis, we transplanted two additional cohorts of pre-immunized hemophilia A mice with either 3 × 105 (cohort 4) or 6 × 105 (cohort 5) transduced sca-1+ cells under non-myeloablative TBI conditioning (Supplementary Figure S1a–d and Supplementary Table S2). Similar results were obtained for the animals in cohorts 4 and 5, with three of five animals in each cohort demonstrating sustained therapeutic fVIII levels at 14 weeks after HSCT.Analysis of the anti-fVIII immune response during HSCT-based gene therapyA dose of 5.5 Gy TBI, although sublethal, eliminates approximately 80–90% of circulating PBMCs in hemophilia A mice, and 11 Gy TBI reduces the PBMC cell count to undetectable levels within 24–48 hours (data not shown). To examine the effect of TBI on circulating antibody levels, we measured anti-hfVIII immunoglobulin (Ig) by ELISA at several time points before and after HSCT. Cohort 1 mice demonstrated a steady decrease in ELISA titer from before HSCT to 19 weeks after HSCT (Figure 4a). Using a two-parameter single exponential decay model, the calculated anti-hfVIII antibody half-life was 16 days. This value is similar to that observed by Ahmed and colleagues (12 days) after lymphocytic choriomeningitis virus–immune serum transfer into naïve mice.17Slifka MK Antia R Whitmire JK Ahmed R Humoral immunity due to long-lived plasma cells.Immunity. 1998; 8: 363-372Abstract Full Text Full Text PDF PubMed Scopus (936) Google Scholar In contrast, no significant decrease in anti-hfVIII or anti-HEP-fVIII ELISA titers was observed until week 9 in the cohort 2 animals that received non-myeloablative TBI (Figure 4b and c).Figure 4Effect of total body irradiation (TBI) and hematopoietic stem cell transplantation (HSCT) gene therapy on anti-factor VIII (anti-fVIII) immune response and donor cell engraftment. Anti-hfVIII immunoglobulin (Ig) levels (solid lines) were measured by enzyme-linked immunosorbent assay (ELISA) at several time points after (a) 11 or (b) 5.5 Gy TBI. (c) Anti-high-expression porcine–factor VIII (Anti-HEP-fVIII) ELISA titers also were measured for cohort 2 mice at multiple time points after HSCT. The closed circles and associated error bars in a, b, and c indicate the means and sample SD for the anti-fVIII ELISA titers at each time point. The solid line in a represents a single exponential, two-parameter regression fit of the data. (d) Cohort 1 and 2 animals are designated by closed and open circles, respectively. Pre-HSCT fVIII cross-reactive antibody titers were determined by ELISA and are shown on the z-axis. Donor cell engraftment and fVIII activity at 12 weeks after HSCT were determined as described in Figures 2 and 3.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Before HSCT, many of the hfVIII-immunized animals possessed antibodies that cross-reacted with HEP-fVIII (Figure 1b). A priori, an attractive prediction is that the level of HEP-fVIII cross-reactive antibodies negatively correlates with the engraftment of HEP-fVIII-expressing genetically modified HSCs owing to the presence of activated T and B cells with fVIII specificity. However, no correlation was observed between pre-HSCT anti-HEP-fVIII ELISA titers and total donor cell engraftment (Figure 4d).DiscussionCellular and humoral immunity are significant obstacles to the development of improved fVIII protein replacement products as well as gene transfer–based therapeutic applications for hemophilia A (for review see ref. 18Herzog RW Dobryzynski E Rawle FE Lillicrap D Gomez-Vargas A Hortelana G et al.Immune implications of gene therapy for hemophilia.Semin Thromb Hemost. 2004; 30: 215-226Crossref PubMed Scopus (38) Google Scholar). The goal of the current study was to determine the effect that pre-existing anti-hfVIII immunity has on HEP-fVIII/HSCT gene therapy using a murine hemophilia A model system. Several laboratories have utilized this murine model to study the immune response to fVIII.19Qian J Borovok M Bi L Kazazian Jr, HH Hoyer LW Inhibitor antibody development and T cell response to human factor VIII in murine hemophilia A.Thromb Haemost. 1999; 81: 240-244PubMed Google Scholar,20Reipert BM Ahmad RU Turecek PL Schwarz HP Characterization of antibodies induced by human factor VIII in a murine knockout model of hemophilia A.Thromb Haemost. 2000; 84: 826-832PubMed Google Scholar,21Sasgary M Ahmad RU Schwarz HP Turecek PL Reipert BM Single cell analysis of factor VIII-specific T cells in hemophilic mice after treatment with human factor VIII.Thromb Haemost. 2002; 87: 266-272PubMed Google Scholar However, the majority of these studies have been directed toward understanding the molecular processes involved in anti-fVIII immunity and developing strategies to reduce the risk of generating a primary immune response to fVIII.22Qian J Collins M Sharpe AH Hoyer LW Prevention and treatment of factor VIII inhibitors in murine hemophilia A.Blood. 2000; 95: 1324-1329PubMed Google Scholar,23Barrow RT Healey JF Gailani D Scandella D Lollar P Reduction of the antigenicity of factor VIII toward complex inhibitory antibody plasmas using multiply-substituted hybrid human/porcine factor VIII molecules.Blood. 2000; 95: 564-568PubMed Google Scholar,24Parker ET Healey JF Barrow RT Craddock HN Lollar P Reduction of the inhibitory antibody response to human factor VIII in hemophilia A mice by mutagenesis of the A2 domain B-cell epitope.Blood. 2004; 104: 704-710Crossref PubMed Scopus (44) Google Scholar,25Miao CH Ye P Thompson AR Rawlings DJ Ochs HD Immunomodulation of transgene responses following naked DNA transfer of human factor VIII into hemophilia A mice.Blood. 2006; 108: 19-27Crossref PubMed Scopus (52) Google Scholar Until recently, few pre-clinical studies have focused on the treatment of animals with pre-existing immunity to fVIII. Reipert and colleagues demonstrated that at high concentrations hfVIII irreversibly inhibits the re-stimulation of fVIII-specific memory B cells in vitro and in vivo,26Hausl C Ahmad RU Sasgary M Doering CB Lollar P Richter G et al.High-dose factor VIII inhibits factor VIII-specific memory B cells in hemophilia A with factor VIII inhibitors.Blood. 2005; 106: 3415-3422Crossref PubMed Scopus (95) Google Scholar which provides a plausible mechanism for the success of immune tolerance induction in hemophilia A inhibitor patients. Gene transfer–based strategies also are being investigated for the treatment of inhibitor patients. Scott and co-workers have shown that expression of chimeric proteins containing fusions of antigenic hfVIII polypeptides to Ig scaffolds by genetically modified B cells reduces the anti-fVIII antibody titers directed toward the fVIII polypeptides contained in the chimeric transgene products.27Lei TC Scott DW Induction of tolerance to factor VIII inhibitors by gene therapy with immunodominant A2 and C2 domains presented by B cells as Ig fusion proteins.Blood. 2005; 105: 4865-4870Crossref PubMed Scopus (142) Google Scholar Furthermore, a study by Montgomery and colleagues demonstrated a therapeutic benefit of transgenic, platelet-stored fVIII in mice with clinically significant anti-hfVIII inhibitor titers.28Shi Q De Angioletti M Donahue RE Notaro R Luzzatto L Dunbar CE Factor VIII ectopically targeted to platelets is therapeutic in hemophilia A with high-titer inhibitory antibodies.J Clin Invest. 2006; 116: 1974-1982Crossref PubMed Scopus (140) Google Scholar Although fVIII activity could not be measured directly in plasma samples, mice containing transgenic platelet-stored hfVIII and harboring anti-hfVIII inhibitors demonstrated increased survival after hemostatic challenge by tail transection. These results suggest that platelet-derived fVIII is protected from anti-fVIII inhibitors until release after platelet activation at sites of vessel injury. Although each of these gene therapy approaches provided evidence for pre-clinical improvement, neither study utilized nucleic acid transfer to demonstrate a cure for both the immune response and the underlying bleeding disorder.We provide evidence that HSCT-based gene therapy incorporating a HEP-fVIII transgene can be utilized to cure hemophilia A mice harboring inhibitor levels that would be considered high titer (>5 Bethesda U/ml) and refractory to acute treatment with hfVIII. The fVIII activity and donor engraftment levels achieved in myeloablated pre-immunized animals were similar to those achieved previously in naïve animals12Gangadharan B Parker ET Ide LM Spencer HT Doering CB High-level expression of porcine factor VIII from genetically modified bone marrow-derived stem cells.Blood. 2006; 107: 3859-3864Crossref PubMed Scopus (46) Google Scholar despite the presence of fVIII-reactive antibodies during the period of donor cell engraftment. The observation that anti-fVIII ELISA titers decreased at a rate similar to t