Abstract: Abstract —Gangliosides, sialic acid–containing glycosphingolipids, accumulate in atherosclerotic vessels. Their role in the pathogenesis of atherosclerosis is unknown. Gangliosides isolated from tumor cells promote collagen-stimulated platelet aggregation and ATP secretion and enhance platelet adhesion to immobilized collagen. These activities are all mediated by ganglioside effects on the platelet integrin collagen receptor α 2 β 1 . Therefore, we hypothesized that gangliosides isolated from atherosclerotic plaques would enhance platelet adhesion to immobilized collagen, a major component of the subendothelial matrix of blood vessels. Furthermore, we questioned whether this effect of atherosclerotic gangliosides might play a role in the pathogenesis of atherosclerosis. To test this hypothesis, we isolated the gangliosides from postmortem aortas of patients with extensive atherosclerotic disease and examined their effects on platelet adhesion. Samples of aortic tissue taken from areas involved with atherosclerotic plaque demonstrated accumulation of gangliosides (64.9±6.5 nmol/g wet weight) compared with gangliosides isolated from control normal aortic tissue taken from children who died of noncardiac causes (NAGs; 21.1±6.4 nmol/g wet weight). Interestingly, samples of tissue taken from diseased aortas but from areas not involved with gross plaque formation also demonstrated ganglioside accumulation (47.6±12.8 nmol/g wet weight). Next, the activity of each of these gangliosides on platelet adhesion to immobilized type I collagen was studied. Atherosclerotic aortic gangliosides (AAGs) as well as those isolated from grossly unaffected areas of the same aorta (UAGs) both increased platelet adhesion compared with control NAGs (OD 570 , 0.37±0.11 and 0.29±0.14 versus 0.16±0.07, respectively; P 〈 0.01 and P 〈 0.05, respectively). These OD 570 values corresponded to 9×10 5 , 8×10 4 , and 6×10 3 platelets per well after preincubation with 5 μmol/L AAG, UAG, and NAG, respectively. Increased adhesion was observed after preincubation with as little as 0.5 μmol/L AAG, and maximal adhesion was seen at 2.5 μmol/L, with a plateau extending to the highest concentration tested, 10 μmol/L. The effect of AAGs on platelet adhesion to collagen was abrogated by incubation of treated platelets with F-17 anti-α 2 monoclonal antibody (OD 570 , 0.13±0.02). Finally, the effects of the major individual gangliosides isolated from atherosclerotic tissues, G M3 and G D3 , were tested. G M3 increased adhesion to collagen (OD 570 , 0.415±0.06) as did G D3 (0.31±0.08). Similar to that of AAGs, the effect of both molecules was blocked by F-17 (0.09±0.04 and 0.13±0.06, respectively). These experiments demonstrate that accumulated atherosclerotic gangliosides promote platelet adhesion to collagen, the major component of the subendothelial matrix. Furthermore, this activity is mediated by an effect of the gangliosides on the collagen-binding integrin α 2 β 1 . This activity may provide a mechanism for the development of platelet thrombi at sites where atherosclerotic gangliosides accumulate and help to explain the role of platelets in the process of atherosclerotic disease progression.

Abstract: Abstract 3374 Patients with hemophilia A and factor VIII inhibitors are at increased risk for repetitive, poorly controlled joint bleeding and progression to end-stage joint disease. The investigator-initiated, prospective, randomized, crossover Prophylaxis with Factor Eight Inhibitor Bypassing Activity (Pro-FEIBA) study showed that prophylaxis with activated prothrombin complex concentrates (aPCC) infused at a target dose of 85 U/kg (±15%) on 3 nonconsecutive days per week safely reduced the frequency of hemarthroses and target joint bleeding by 61% and 72%, respectively (P 〈 0.001), as compared with aPCC on-demand therapy (target dose 85 U/kg [±15%]). A majority of patients (62%) had ≥50% reduction in bleeding during the prophylaxis period. Deemed “good responders,” these patients missed significantly fewer days from school or work and experienced improved health-related quality of life while receiving prophylactic infusions of aPCC. Methods: To determine whether aPCC prophylaxis in good responders had a favorable impact on joint mobility, we evaluated joint range of motion (ROM) data obtained at baseline, after the prophylaxis period, and after the on-demand therapy period for 17 patients for whom complete information was available. Ten of the patients (59%) were good responders to aPCC, and 7 (41%) were not. We assessed ROM measures in the ankles, knees, and elbows; the number of target joints; joint contractures at baseline; and the global ROM joint score at baseline. The following system was used to calculate the ROM score: 0 = no loss of ROM, 1 = 〈 10% ROM loss, 2 = 10–33% ROM loss, 3 = 34–50% ROM loss, and 4 = 〉 50% ROM loss. We determined whether the good responders experienced any improvement in joint function during the prophylaxis period compared with patients who did not have a significant reduction in bleeding during prophylaxis. Results: Mean age, global ROM score at baseline, and the number of flexion contractures at baseline were similar in both groups (see Table). The number of joints that had target joint bleeding (defined as 〉 3 bleeds in a 6-month period) was also similar in the 2 groups. Reductions in overall bleeding and target joint bleeding were significantly greater during the prophylaxis period in the good responder group. In these patients, the change in ROM during prophylaxis approached (but did not reach) statistical significance. ROM in 100% of the good responders stabilized or improved during prophylaxis versus 42.9% of the patients who did not have a good response to aPCC prophylaxis (P = 0.015). Discussion: In this patient population who had significant joint damage at baseline, good responders to aPCC prophylaxis showed improvement in or stabilization of joint ROM during the prophylaxis period. This finding suggests that prophylaxis may be even more helpful in preserving ROM when used in younger patients with healthier joints at baseline. Additionally, it further supports the need for a clinical trial in young patients with inhibitors, who could potentially achieve the greatest joint preservation benefits from aPCC prophylaxis. Disclosures: Leissinger: Baxter Healthcare: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Off Label Use: Prophylactic use of Feiba. Gringeri:Biotest: Research Funding; Grifols: Honoraria, Research Funding; CSL Behring: Honoraria, Research Funding; Baxter: Honoraria, Research Funding; Kedrion: Research Funding. Valentino:BAXTER: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Abstract: Background: Retrospective reviews of hemophilia therapy correlate normal joint X-ray (XR) and physical exam (PE) with early institution of routine infusions of FVIII dosed to prevent bleeding. Although the National Hemophilia Foundation recommended prophylaxis for all persons with severe hemophilia in 1995, fewer than half of affected persons in the US have adopted prophylaxis due to cost, effort, lack of perceived need and complications. Objective: The “Joint Outcome Study” was constructed as a multi-center, open-label, two-arm, prospective, randomized clinical trial to compare full prophylaxis with an intensive treatment regimen for joint hemorrhage. Methods: A regimen of every other day infusions of FVIII at 25 U/kg to prevent hemorrhage (prophylaxis, P) was compared with intensive therapy using ≥ 3 infusions totaling ≥ 80 U/kg FVIII at the time of each joint hemorrhage to minimize joint damage (enhanced episodic, EE). Primary outcome was the proportion of boys in each arm with bone or cartilage damage on XR or magnetic resonance imaging (MR) of index joints (elbows, knees, and ankles). Outcomes were judged independently by two research radiologists who were blinded to treatment arm and bleed history, with a third for discrepant readings. Other outcomes included joint function on a physical exam scale validated for young children (PE), # of joint hemorrhages and factor utilization. Boys were followed from entry between 12 and 30 months until age 6 years. At entry all children had normal joints on XR and MR and had no more than two hemorrhages into any one joint. Results: Sixty-five boys were randomized to P (32) or EE (33). The study has been completed. While adjudication of XR and MR outcomes is still ongoing, clinical study results show the following: Children on P consumed more FVIII (mean 163 vs 47 infusions/year, P & lt; 0.001) and had fewer joint hemorrhages per year (0.47 vs 4.9, p & lt; 0.001) than boys on EE. PE scores for the six index joints were lower on the P arm compared with EE (mean score 4.7 vs. 8.6, p & lt; 0.01). Conclusion: This first randomized clinical trial of prophylaxis in young children with FVIII deficiency showed improved joint function by age 6 years in children on early every other day prophylaxis in comparison to an aggressive program of multiple infusions administered promptly at the time of joint hemorrhage. Joint structural outcome confirmation will be based on MR and XR.

Abstract: Abstract 27 Antibodies (inhibitors) to exogenously administered factor VIII (FVIII) develop in as many as 30 – 40% of patients with severe hemophilia A. Patients with persistent inhibitors are at increased risk of serious, poorly controlled bleeding which results in significant morbidity and in some cases early death. Rituximab, a chimeric murine/human monoclonal antibody directed against CD20, suppresses circulating and tissue B cells and pre-B cells and has been used in the treatment of a variety of autoimmune and alloimmune disorders. The primary objective of this NHLBI Transfusion Medicine Hemostasis network-sponsored study was to evaluate the role of rituximab as an approach to inhibit the production of FVIII antibodies in patients with severe congenital hemophilia A and high titer inhibitors. Methods: This was a prospective, multi-institution, single-arm, open-label Phase II trial. Eligible subjects were males over 18 months of age with severe hemophilia A and a history of an inhibitor ≥5 Bethesda units (BU). Individuals who were HIV positive, undergoing immune tolerance, or receiving immune modulating therapies were excluded. Following a challenge dose of recombinant FVIII (rAHF-PFM) at 50 IU/kg, and evidence of an inhibitor titer ≥ 5 BU at 5 – 14 days after the challenge dose, subjects received rituximab 375 mg/m2 weekly for 4 weeks. Starting two weeks after the fourth rituximab treatment, inhibitor titers were drawn every 4 weeks. A major response was defined as a fall in the inhibitor titer to 〈 5 BU at any time up to and including week 22, with the titer remaining 〈 5 BU following re-challenge with FVIII. A minor response was defined as inhibitor falling to 〈 5 BU at any time up to and including week 22, with the anamnestic peak following re-challenge with FVIII between 5–10 BU and less than 50% of the original anamnestic peak. The null hypothesis was that no more than 5% of subjects treated with rituximab would be major responders. Results: A total of 23 subjects were enrolled; 21 received the initial FVIII challenge. Of these, 4 subjects did not meet the criteria to receive rituximab treatment, and 1 subject withdrew consent. A total of 16 subjects received at least one dose of rituximab and are included in this analysis. The median age was 14 y (range 4 – 38 y). Three subjects (18.8%) had a major response. If the null hypothesis were true, the probability of 3 or more major responses in 16 subjects would be 0.043, so the null hypothesis was rejected. One subject (6.2%) had a minor response to treatment. All 4 responders and 8 non-responders had a baseline inhibitor titer 〈 20 BU, resulting in a response rate of 33% in that group vs. 0% in the 4 subjects with a baseline inhibitor titer ≥ 20 BU. Discussion: Infusion of rituximab 375 mg/m2 once per week for four weeks was effective in reducing the anamnestic inhibitor response in 25% of severe hemophilia A subjects with inhibitors who were not receiving concurrent immune tolerance therapy. Those who responded tended to have lower baseline inhibitor levels compared to the group that did not meet the criteria for response. This Phase II study, designed as a proof of concept, demonstrated that rituximab may be useful in lowering inhibitor levels and anamnesis in some patients with inhibitors, but that the effect as a solo treatment strategy is modest, and possibly restricted to patients with inhibitor titers under 20 BU. Further studies are indicated to determine the role of rituximab as an adjunctive therapy in immune tolerization strategies. Acknowledgments: The authors acknowledge the support of Genentech for the provision of rituximab and partial financial support for the study. The authors also acknowledge Baxter Healthcare Corporation for donating the recombinant FVIII used in the trial. Disclosures: Leissinger: Baxter: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Off Label Use: Rituximab, a drug approved for use in treating lymphoma, was studied for its efficacy in suppressing inhibitors against factor VIII in patients with hemophilia and high titer inhibitors. Kruse-Jarres:Baxter: Consultancy, Honoraria; Bayer:; Griforls: Consultancy, Honoraria; Inspiration: Consultancy, Honoraria; NovoNordisk: Consultancy, Honoraria. Konkle:Baxter Corporation: Consultancy, Research Funding; Bayer Corp: Consultancy; Inspiration Biopharmaceuticals: Research Funding; CSL Behring: Consultancy, Membership on an entity's Board of Directors or advisory committees. Neufeld:Genentech: Research Funding; Baxter: Research Funding; Bayer: Research Funding. Bennett:Biogen IDEC: Honoraria. Valentino:Baxter Bioscience, Bayer Healthcare, GTC Biotherapeutics, NovoNordisk, Pfizer, CSL Behring, Inspiration Bioscience, and Biogen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Abstract: Gene transfer for hemophilia A offers the potential for a one-time disease altering treatment, eliminating the risk of bleeds while freeing patients from the burden of lifelong chronic therapy. SPK-8011 consists of a bioengineered AAV capsid expressing B domain-deleted factor VIII (FVIII) under the control of a liver-specific promoter. In pre-clinical studies, we showed a dose-dependent increase in circulating FVIII levels in non-human primates infused with SPK-8011. We conducted a Phase I/II study of SPK-8011 in 12 men (ages 18-52 years) with severe (n=11) or moderately severe (n=1) hemophilia A. Prior to gene therapy, 8/12 subjects were on prophylaxis, and 4/12 received on-demand treatment. Subjects were enrolled in 1 of 3 dose cohorts, 5E11 vg/kg (n=2), 1E12(n=3), or 2E12(N=7). Safety analysis showed no inhibitor formation. A single serious adverse event (SAE) was reported, associated with an immune response to AAV capsid characterized by simultaneous decline in FVIII, transaminase elevation peaking at Grade 2, and development of positive IFN-g ELISPOTs to capsid was observed beginning at week 6.5 after vector infusion. The asymptomatic transaminase elevation did not respond promptly to initiation of oral steroids and the subject received two infusions of IV methylprednisolone in hospital, thereby fulfilling SAE criteria. The SAE has resolved. All vector doses led to expression of FVIII levels adequate to prevent bleeding and allow cessation of prophylaxis. Across the 12 subjects at 3 doses, there was a 97% reduction in annualized bleeding rate (ABR), and a 97% reduction in annualized infusion rate (AIR). In the 5E11 dose cohort, mean FVIII levels beginning 12 weeks post vector infusion are 13%, with no bleeding events, no elevated transaminase levels, no use of steroids, and stable FVIII expression out to 66 weeks (ongoing). In the 1E12 dose cohort, mean FVIII levels are 15% beginning at 12 weeks post-infusion and stable out to 46 weeks (ongoing). The first subject in the 1E12 dose infused a single dose of factor concentrate for a spontaneous joint bleed at day 159, and the second received multiple infusions for a traumatic bleed beginning at day 195. Declining FVIII levels triggered initiation of a course of tapering steroids in both subjects, at 12 and 7 weeks post vector infusion respectively, which led to stabilization of FVIII levels. The third subject has had no bleeding and did not receive factor infusions or steroids. In the 2E12 (highest) dose cohort, 5/7 subjects currently have FVIII levels 16-49%; their mean FVIII level beginning 12 weeks post-infusion is 30%. No bleeds have been reported among these subjects beginning 4 weeks post vector infusion. Additionally, 5/7 subjects in the 2E12 dose cohort received a course of steroids, initiated at 6-11 weeks post vector infusion, for one or more of the following: declining FVIII levels, rise in ALT above subject baseline, or elevated IFN-g ELISPOTs to AAV capsid. Steroid initiation normalized ALT levels and extinguished the ELISPOT signal in all cases; 2 subjects showed limited stabilization of FVIII levels, which fell to 〈 6% likely due to the immune response. For one of these, no bleeds have been reported through 12 weeks of follow up; the other has had 4 bleeds through 37 weeks of observation. Our data indicate that the kinetics of SPK-8011 expression are similar to those observed with investigational SPK-9001 for hemophilia B. All subjects demonstrated durable transgene expression for up to 66 weeks post vector administration (data cutoff 7/13/18). On cumulative follow up of 345 weeks, SPK-8011 demonstrated a favorable safety profile with no evidence of FVIII inhibitor formation, a single SAE, and 2/12 subjects who experienced ALT elevation above the upper limit of normal that resolved with steroid initiation. Data from the 5E11 (lowest) dose cohort are consistent with published natural history data indicating FVIII:C 12% is adequate to prevent spontaneous bleeding events. Given that 2 subjects in the 2E12 dose cohort lost some FVIII expression, which then stabilized on steroids, and 5/7 subjects in this cohort required steroids, prophylactic steroids may be warranted. We conclude that infusion of SPK-8011 in 12 subjects with severe or moderately severe hemophilia A resulted in safe, durable, dose-dependent FVIII expression resulting in an excellent preliminary efficacy profile with an overall 97% reduction in ABR and AIR. Disclosures High: Spark Therapeutics: Employment, Equity Ownership, Patents & Royalties. George:University of Pennsylvania: Equity Ownership; Pfizer: Consultancy. Ragni:CSL Behring: Research Funding; Alnylam: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sangamo: Research Funding; Shire: Research Funding; Biomarin: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novo Nordisk: Research Funding; Bioverativ: Consultancy, Research Funding; MOGAM: Membership on an entity's Board of Directors or advisory committees; SPARK: Consultancy, Research Funding. Croteau:Novo Nordisk: Consultancy; Octapharma: Consultancy, Honoraria, Research Funding; Pfizer: Research Funding; Spark Therapeutics: Research Funding; Tremeau Pharmaceuticals: Consultancy; Genetech: Consultancy, Research Funding; CSL-Behring: Consultancy; Catalyst Biosciences: Consultancy; Bioveritiv: Consultancy; Biomarin: Consultancy; Bayer: Consultancy; Baxalta/Shire: Consultancy, Research Funding. Joseney-Antoine:Spark Therapeutics: Employment. Macdougall:Spark Therapeutics: Employment. Tompkins:Spark Therapeutics: Employment. Hait:Spark Therapeutics: Employment. Couto:Spark Therapeutics: Employment. Bassiri:Spark Therapeutics: Employment. Valentino:Spark Therapeutics: Employment. Carr:Spark Therapeutics: Employment. Hui:Spark Therapeutics: Employment. Wachtel:Spark Therapeutics: Employment. Takefman:Spark Therapeutics: Employment. Mingozzi:Spark Therapeutics, Inc.: Employment. Anguela:Spark Therapeutics, Inc.: Employment. Reape:Spark Therapeutics: Employment.

Abstract: Hemophilia is a rare congenital bleeding disorder that is due to the deficiency of blood coagulation factor VIII or IX. Recurrent musculoskeletal bleeding is common and bleeding into joints results in a chronic inflammatory condition termed hemophilic synovitis. This destructive process is characterized by hemosiderin deposition in the superficial and deeper layers of the synovial membrane as well as a proliferation of synovial fibroblasts and vascular cells. The hyperplastic synovium and neovascular changes are reminiscent of the histopathologic appearance observed in malignant tissues. Indeed, the benign hyperplastic synovium in patients with hemophilia displays similar invasive and destructive behaviors suggesting the possibility of analogous disturbances in growth control and locally invasive mechanisms. Iron plays a role in malignant cell growth, local invasion, and tumor progression, possibly due to changes in the expression of the proto-oncogene, c-myc. We hypothesized that iron plays a similar role in hemophilic synovitis. To explore this hypothesis, we investigated the in vitro effects of iron on the proliferation of a primary, human synovial fibroblast cell (HSFC) line and the involvement of c-myc in this process. We also examined the role of ceramide, a sphingolipid capable of inducing apoptosis in this model system. HSFC proliferation was increased in a dose-dependent fashion and c-myc expression was enhanced by ferric citrate compared to sodium citrate control. Ceramide prevented both the iron-induced increases in HSFC proliferation andc-myc expression. These results indicate that iron probably plays a role in the proliferative changes observed in hemophilic joint disease and that aberrant expression of c-myc may underlie the iron effects. Furthermore, these results suggest that there may be a therapeutic role for ceramide in reversing these changes.