ALBERT

Description: We have determined the 2905 nucleotide sequence of the rhesus macaque factor IX complementary DNA (cDNA) and found it to be greater than 95% identical to that of the human factor IX cDNA. The cDNA has a large 3′ untranslated region like the human cDNA, but unlike the human cDNA has two polyadenylation sites 224 nucleotides apart that are used for transcription of the messenger RNA. The deduced amino acid sequence is greater than 97% identical to that of human factor IX, differing in only 11 of 461 amino acids in the complete precursor protein. We found a single silent polymorphism in the nucleotide sequence at the third position of the codon for asparagine at position 167 in the secreted protein (AAC/AAT). All residues subject to posttranslational modifications in the human protein are also found in the rhesus factor IX sequence. The high degree of homology between the rhesus and human factor IX proteins suggested the possibility that the human factor IX protein might be nonimmunogenic in the rhesus. We tested the immunogenicity of human factor IX in three rhesus macaques by repeated intravenous injections of monoclonal antibody–purified, plasma-derived human factor IX over the course of more than a year and assessed the recovery and half-life of the infused protein, as well as in vitro indicators of antihuman factor IX antibodies. Human factor IX recovery and half-life remained unchanged over the course of a year in the three animals studied, and aPTT mixing studies showed no evidence for neutralizing antihuman factor IX antibodies. An outbred, nonhuman primate model that permits assessment of the level and duration of factor IX expression as well as vector safety would complement the use of other (mouse and canine) hemophilia B animal models in current use for the development of gene therapy for hemophilia B.

Description: Intron-22-inversion patients express the entire Factor VIII (FVIII)-amino-acid sequence intracellularly as 2 non-secreted polypeptides and have a positive “intracellular (I)-FVIII-CRM” status. Mutations conferring a positive I-FVIII-CRM status are associated with low inhibitor risk and are pharmacogenetically relevant because inhibitor risk may be affected by the nature of the therapeutic FVIII-protein (tFVIII), the affinity of any tFVIII-derived foreign peptide (tFVIII-fp) for any HLA class-II isomer (HLA-II) comprising individual major histocompatibility complex (MHC) repertoires, and the stability of any tFVIII-fp/HLA-II complex. We hypothesize that mutations conferring a completely or substantially negative I-FVIII-CRM status are pharmacogenetically irrelevant because inhibitor risk is high with any tFVIII and individual MHC repertoire.

Description: Abstract 54 Severe aplastic anemia (SAA) is characterized by trilineage marrow hypoplasia and a paucity of hematopoietic stem cell (HSC) progenitors. SAA is treated with immunosuppressive therapy (IST) or allogeneic HSC transplantation (HSCT), with a successful outcome after either treatment in a majority of patients. However, 20–40% of patients without a suitable donor for HSCT and a suboptimal response to IST may have persistent severe thrombocytopenia. Thrombopoietin (TPO) is the principal regulator of platelet production, and it exerts its effects through binding the megakaryocyte progenitor TPO receptor mpl, which stimulates production of mature megakaryocytes and platelets. Several lines of evidence support the concept that signaling through mpl also influences expansion and maintenance of primitive HSCs and multi-potent progenitor cells. Eltrombopag, is a small molecule TPO receptor agonist that stimulates mpl, and increases platelet counts in patients with chronic immune thrombocytopenic purpura (ITP). It is approved for the treatment of chronic ITP (Promacta®). We conducted a non-randomized, pilot phase II study of eltrombopag in SAA patients who remained severely thrombocytopenic at least six months after one or more rounds of IST (clinical trials.gov identifier NCT00922883). Consecutive patients fulfilling the inclusion criteria received eltrombopag 50mg daily with dose escalation every two weeks to a maximum dose of 150mg daily. Primary end points assessed after three months of treatment were changes in peripheral blood counts (platelets, hemoglobin, absolute neutrophil counts), with hematologic response criteria defined a priori for each lineage. Secondary endpoints included the incidence of bleeding events, and health related quality of life. Patients who achieved hematologic responses were maintained on eltrombopag through an extended access protocol. We completed our planned accrual of twenty five patients, and 22 are evaluable for response to date. Median age was 45 years old (range 18–77 years), and the median time from the last course of IST was 13 months (range 6–54 months). Median follow up time was 9 months (range 1–24 months). Nine of twenty two patients (41%) achieved hematologic responses: seven of twenty-two patients (32%) achieved platelet responses with transfusion independence for eight weeks or greater; six patients had improved hemoglobin levels after starting treatment (mean hemoglobin increase of 3.8 g/dL) and 4 patients who were previously dependent on red blood cell transfusions have achieved transfusion-independence. Five neutropenic patients had increased neutrophil counts after treatment with eltrombopag (mean increase 660 cells/uL). Plasma TPO levels did not predict for hematologic response to eltrombopag. Serial bone marrow biopsies performed on patients with hematologic responses demonstrated normalization of trilineage hematopoiesis and cellularity in three of four responders receiving a year or more of therapy, with no increase in reticulin fibrosis (Figure 1). These results represent the first evidence that TPO stimulation can expand the HSC pool in humans, with clinically meaningful trilineage hematologic improvements in patients with SAA, resulting in transfusion-independence and improved quality of life with a simple daily oral regimen. Updated response data on the full 25 patients will be presented at the Society's meeting. Disclosures: Off Label Use: Eltrombopag for severe aplastic anemia.

Description: Nitric oxide (NO) is generated by serial reduction pathway of nitrate and nitrite or by activity of endogenous nitric oxide synthase (NOS), and modulates platelet function and hemostasis. We have shown by aggregometry and flow cytometric analysis of P-selectin that nitrite in the presence of erythrocytes inhibits platelet aggregation and activation after its reduction to NO at low oxygen tension [PLoS One. 2012; 7: e30380. 10.1371/journal.pone.0030380]. We then investigated how nitrite may affect overall clotting processes by regulating platelet function using thrombelastography (TEG), a method used to assess platelet function, fibrin clot formation, and fibrinolysis in blood and/or plasma. We measured the effect of NO donors and nitrite on three TEG parameters, reaction time (R, time to initial fibrin formation), α angle (velocity of clot growth) and maximum amplitude (MA, clot strength), in healthy volunteers. The NO donor (DEANONOate) inhibited all three TEG parameters in response to two independent platelet activators (ADP and AA), in platelet rich plasma (PRP) or whole blood diluted with plasma to yield 20% hematocrit (Hct) to model blood in the microcirculation in vivo. At DEANONOate concentrations ranging from 0-1 μM, R values were progressively prolonged from 1.9 to 3.6 (p=0.008), α angle was decreased from 17.8 to 9.5 (p=0.01) and maximum clot amplitude was reduced from 9.0 to 4.4 (p=0.001) in 20% Hct with ADP stimulation. In contrast, nitrite did not affect clotting parameters in PRP, but exhibited moderate inhibitory effects in 20% Hct at concentrations from 0-10 μM; R values were slightly prolonged from 1.6 to 2.7 (p=0.12), α angle was decreased from 21.8 to 12.8 (p=0.07) and maximum clot amplitude was reduced from 11.0 to 5.0 (p=0.02). This inhibitory effect of nitrite on clotting was greatly enhanced under hypoxic conditions (blood pO2 46.5±11.6 mmHg); R values from 1.4 to 4.0 (p=0.003), α angle from 21.7 to 7.8 (p=0.002), and maximum clot amplitude from 10.0 to 3.4 (p=0.0003). These results suggest that the nitrite effect may be greatest in the microcirculation and be important in differences between arterial and venous clotting. In conclusion, our results show TEG parameters indicate NO inhibition of platelet-mediated blood clotting and that the physiological effect of factors which determine NO bioavailability, such as reduction of blood and tissue nitrite, could be used to predict hemostasis. Disclosures: Schechter: National Institutes of Health: Dr. Alan Schechter is listed as a co-inventor on several patents issued to the National Institutes of Health for the use of nitrite salts for the treatment of cardiovascular diseases., Dr. Alan Schechter is listed as a co-inventor on several patents issued to the National Institutes of Health for the use of nitrite salts for the treatment of cardiovascular diseases. Patents & Royalties.

Description: Abstract 4631 A 44 year old African-American asplenic male with sickle cell disease (HbSS) underwent allogeneic HSCT in September 2009. Upon engraftment, the patient had 97% donor myeloid, and 30% donor lymphoid chimerism, and he was free of his sickle cell disease with HbA 97%, HbA2 2.9%,and HbF 96%). Concomitantly, the patient's endogenous FVIII rose to 〉100% of normal by day 22, and remained normal after completion of three doses of rituximab and a complete taper of prednisone over the next two months. The patient had an H2 FVIII haplotype sequence, whereas his donor was heterozygous for the FVIII H1 and H2 haplotypes. A study of stored samples showed that at the onset of mild soft tissue hemorrhage the Bethesda inhibitor titer against a recombinant FVIII with H2 haplotype sequence (Recombinate) was higher (4.2 BIAU) than that against H1 (Kogenate) or “mixed” haplotypes derived from pooled plasma (1.9 BIAU). However, by the time of his compartment syndrome hemorrhage the differential reactivity of neutralizing titers to the H2 haplotype FVIII was not as pronounced (5.2 BIAU for H2 haplotype vs. 4–4.5 BIAU for all other haplotypes/products), suggesting that any initial specificity for the H2 FVIII haplotype had been diminished. No inhibition of OBI-1 porcine factor VIII was seen prior to treatment, or in the 90 day period after treatment. Interestingly, FVIII binding antibodies could be detected prior to the viral infection preceding the overt clinical inhibitor and soft tissue hemorrhage. In summary we demonstrate use of recombinant porcine FVIII in a patient with a compartment syndrome due to acquired hemophilia, and elimination of the inhibitor, while preserving the transplant that corrected his sickle cell disease. Disclosures: Lee: Inspiration Biopharmaceuticals Inc: Employment.

Description: The feasibility, safety, and efficacy of liver-directed gene transfer was evaluated in 5 male macaques (aged 2.5 to 6.5 years) by using a recombinant adeno-associated viral (rAAV) vector (rAAV-2 CAGG-hFIX) that had previously mediated persistent therapeutic expression of human factor IX (hFIX; 6%-10% of physiologic levels) in murine models. A dose of 4 × 1012 vector genomes (vgs)/kg of body weight was administered through the hepatic artery or portal vein. Persistence of the rAAV vgs as circular monomers and dimers and high-molecular-weight concatamers was documented in liver tissue by Southern blot analysis for periods of up to 1 year. Vector particles were present in plasma, urine, or saliva for several days after infusion (as shown by polymerase chain reaction analysis), and the vgs were detected in spleen tissue at low copy numbers. An enzyme-linked immunosorption assay capable of detecting between 1% and 25% of normal levels of hFIX in rhesus plasma was developed by using hyperimmune serum from a rhesus monkey that had received an adenoviral vector encoding hFIX. Two macaques having 3 and 40 rAAV genome equivalents/cell, respectively, in liver tissue had 4% and 8% of normal physiologic plasma levels of hFIX, respectively. A level of hFIX that was 3% of normal levels was transiently detected in one other macaque, which had a genome copy number of 25 before abrogation by a neutralizing antibody (inhibitor) to hFIX. This nonhuman-primate model will be useful in further evaluation and development of rAAV vectors for gene therapy of hemophilia B.

Description: We used a first-generation adenovirus vector (AVC3FIX5) to assess whether human factor IX could be expressed and detected in the rhesus macaque, which we have shown does not make high-titer antibodies to human factor IX protein. Three animals received 1 × 1010to 1 × 1011 plaque-forming units per kilogram by intravenous injection. Human factor IX was present within 24 hours of vector administration and peaked 4 days later at 4,000 ng/mL in the high-dose recipient, and lower levels were seen in the intermediate-dose recipient. No human factor IX was detected in the low-dose recipient's plasma. Serum cytokine analysis and early hypoferremia suggested a dose-dependent acute-phase response to the vector. Human factor IX was detectable in rhesus plasma for 2 to 3 weeks for the high- and intermediate-dose recipients, but disappeared concomitant with high-titer antihuman factor IX antibody development. There was substantial, dose-dependent, dose-limiting liver toxicity that was manifest as elevated serum transaminase levels, hyperbilirubinemia, hypoalbuminemia, and prolongation of clotting times. Of particular interest was prolongation of the thrombin clotting time, an indicator of decreased fibrinogen or fibrinogen dysfunction. All evidence of liver toxicity resolved except for persistent hypofibrinogenemia in the high-dose recipient, indicating possible permanent liver damage. Our data suggest a narrow therapeutic window for first-generation adenovirus-mediated gene transfer. The development of antihuman factor IX antibodies and abnormalities of fibrinogen in the rhesus macaque is of concern for application of adenovirus (or other viral) vectors to hemophilia gene therapy.

Description: Abstract 218 Introduction: Inhibitor antibodies to FVIII develop in ∼20% of patients with severe hemophilia A, and are the most important adverse events associated with FVIII replacement therapy. Mutations in the FVIII gene are the major determinant of inhibitor risk, but variations in immune response genes, such as IL10 or TNFα, (Astermark, et al, 2006a, 2006b) may also confer risk of inhibitor development and variations in the CTLA4 gene may protect against inhibitors (Astermark et al, 2006c). Specific Objective: Using a case-control study design we sought to confirm and extend previous observations of inhibitor risk modifying genes seen in family studies. Candidate genes were selected based on previous clinical or animal studies and included cytokines involved in the TH1/TH2 immune responses, and genes thought to decrease demand for FVIII therapy (e.g., FV Leiden and prothrombin 20210 polymorphisms). Materials and Methods: We used the CEU population data in the HapMap database to select haplotype tagging SNPs in IL1α/β, IL2, IL4, IL6, IL10, IL12A/B, IL13, IL17A, IL22, TNFα, CTLA4, interferon-γ, TGFβ, zinc α-2 glycoprotein I, IL1RN, and the FVIII gene, as well as the FV Leiden and prothrombin 20210 gene polymorphisms. A total of 366 tagging SNPs were selected with a goal of covering the entire coding and regulatory regions of the genes (spanning from 20kb 5' to 10kb 3' of the gene's coding sequence), resulting in 100% coverage of potential haplotypes (r2 = 1.0). DNA was purified from 915 Caucasian, severe hemophilia A patients (282 inhibitor cases and 633 non-inhibitor controls) who participated in the Multicenter Hemophilia Cohort Studies I & II. Subjects were classified as having an inhibitor to FVIII on the basis of at least one inhibitor titer ≥1.0 Bethesda unit. SNP genotypes were determined by Sequenom MALDI-TOF spectroscopy. Haplotype frequencies were estimated using expectation maximization (EM) algorithm, and generalized linear models (GLM) were used to assess the marginal effect of SNPs and haplotypes on FVIII inhibitor development risk after adjusting for HIV infection and HCV persistence (prevalence in controls, 46% and 77%, respectively). Results: Of the 366 SNPs, 298 (81%) had unambiguous genotypes in 〉80% of the subjects and consequently qualified to the association analyses. Significant associations were seen between loci in the IL10, IL12A, IL1, IL2, TNFα, & IL17A genes and inhibitor development. Particularly, individuals carrying an 8 SNP haplotype located ∼10 kb 5' to the IL10 initiation start site were at higher risk to develop inhibitors than individuals with the most common haplotype (OR:1.54, 95% CI:1.15–2.06, p-value = 0.004). This haplotype covers a region that includes the IL10G microsatellite previously studied by Astermark et al (2006a), as well as the nearby IL10R microsatellite. Interestingly, the effect of this haplotype on FVIII inhibitor development was larger among HIV+ subjects in our study (OR: 1.81, 95% CI: 1.18–2.77 vs. OR: 1.28, 95% CI: 0.85–1.93 for HIV+ and HIV- subjects, respectively). A similar phenomenon was seen with haplotypes in the IL12A and IL2 genes. Additional haplotypes in genes for IL1α, IL1β, TNFα, and IL17A significantly increased or decreased risk for inhibitor development. No association was seen between FVIII haplotypes and inhibitors in this group of Caucasian subjects with hemophilia A, in contrast to the findings in African-Americans by Viel et al (2009). Also, no significant effect on inhibitor risk was seen for the factor V Leiden or prothrombin 20210 polymorphisms. Conclusions and Future Directions: Our large case-control study confirms the findings in family studies that IL10 and TNFα genotypes confer risk for inhibitor development in Caucasians with severe hemophilia A, which differed by HIV status and were of lower magnitude than seen previously in family studies. We identified four other genes in which certain haplotypes alter inhibitor risk. More work is needed to identify inhibitor associations in non-Caucasian populations and to corroborate and more specifically define our novel associations in Caucasians. Moreover, whole genome association and other studies should be considered to identify additional modifier genes and potential targets for intervention to prevent inhibitors. Disclosures: No relevant conflicts of interest to declare.

Description: We have reported a case of acquired hemophilia A in our series of sickle cell disease patients treated with hematopoietic stem cell transplantation (HSCT) using HLA matched sibling donors, non-myeloablative conditioning, and sirolimus immunosuppression for GVHD prevention (Lozier et al, Haemophilia 2013). Subsequently we encountered another case in the 48 patients now transplanted. The objective of our transplant regimen is to establish a stable engraftment that will correct the sickle cell disease, with minimal morbidity. As a result of the non-myeloablative conditioning, our HSCT recipients typically display mixed chimerism between donor and recipient after HSCT with a post-HSCT immune system derived from both donor and recipient. We investigated the prevalence of FVIII-binding antibodies by ELISA in HSCT recipients prior to and after HSCT, and sought to determine if prior exposure to blood products, differences in FVIII haplotypes, and/or ethnicity may predispose to FVIII inhibitors. We measured IgG antibodies that bind FVIII by ELISA in 30 of our 48 transplanted subjects and donors for whom plasma, serum, and DNA were available, before and after HSCT. Published evidence suggests 3-19% of normal blood donors have detectable FVIII binding antibodies when tested in an ELISA format (Krudysz-Amblo et al, Blood 2009; Whelan et al, Blood 2012). We were surprised to find that 22 of 30 recipients (73%) had measurable titers of ≥ 1:50 prior to HSCT. We also assessed their sibling donors as a control group and found 13 of 24 evaluable donors had titers ≥ 1:50 (54%). Measurement of antibodies to FVIII in African-American blood donors at NIH showed 8 of 13 (62%) to have ≥ 1:50 titers, in contrast to only 1 of 20 Caucasian (5%) NIH blood donors tested (P 〈 0.05). This suggests African-Americans are more likely to form antibodies to FVIII than Caucasians, and might explain the two cases of acquired hemophilia A in our HSCT study. This has not been reported in allogeneic HSCT studies, though 2 out of 155 autologous HSCT patients with autoimmune diseases were reported to have acquired hemophilia after HSCT (Loh et al, Blood 2007). Of 8 patients with titers 50 units of RBCs prior to transplant (12.5%), while 9 of 22 with titers ≥ 1:50 prior to transplant (41%) recalled transfusion with 〉 50 units of RBCs prior to transplant (P = 0.14, NS). Six haplotypes of the FVIII protein are known, and empiric data suggests uncommon haplotypes more prevalent in African-Americans may be associated with higher rates of FVIII inhibitors in African-Americans with hemophilia A after FVIII treatment (Viel et al, NEJM 2009), and rare FVIII polymorphisms are associated with acquired hemophilia A in the Caucasian population as well (Tiede et al, Ann Hematol 2010). We ascertained FVIII haplotypes in HSCT recipients and their donors to see if mismatches between donor and recipient may be associated with FVIII antibodies or inhibitors. Of 30 patients studied, there were 27 for whom DNA data permitted us to infer haplotypes. Fourteen recipients were identical matches to their donors, of whom one (7%) had an increase in titer from baseline after transplant; none developed an inhibitor. Of 15 recipients with mismatches in FVIII haplotypes with their donors, 3 had increases from baseline titers after transplant (20%), including the two FVIII inhibitor patients (P = 0.45, NS). Our study shows that African-Americans have a higher prevalence of low-titer/subclinical anti-human FVIII antibodies compared to Caucasians which may explain subsequent FVIII inhibitors in the setting of hematopoietic stem cell HSCT. Neither greater recalled exposure to RBC transfusions pre-HSCT nor mismatch of FVIII haplotypes with donor explain the risk of inhibitor development. Disclosures No relevant conflicts of interest to declare.