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Haemophilia B (HB) is less well studied than haemophilia A (HA); despite similarities between the two inherited bleeding disorders, important differences remain that require further research.B-Natural is a multi-centre, prospective, observational study of HB, designed to increase understanding of clinical manifestations, treatment, quality-of-life (QoL), inhibitor development, immune tolerance induction (ITI) outcome, renal function and create a biorepository for future investigations.Participants include sibling pairs/groups without a current/history of inhibitors and singletons or siblings with a current/history of inhibitors followed for six months. Demographics, medical, social history and treatment were recorded. A physical examination including joint range of motion (ROM) was performed; QoL was assessed. Samples were collected for F9 gene mutation, HLA typing, non-inhibitory antibodies and renal function testing.Twenty-four centres enrolled 224 individuals from 107 families including 29 with current/history of inhibitors. Of these, 68, 30.4%, had severe (1% FIX level of normal); 114, 50.9%, moderate (1%-5%); and 42, 18.8%, mild (5-40%) disease. At enrolment, 53.1% had 50 + exposure days to exogenous FIX. Comparison of joint scores showed significant (P .05) differences between those with severe (with/without inhibitors), and those with moderate/mild disease. The majority with severe disease, 80.0% with current/history of inhibitors and 64.3% of those without, were treated with prophylaxis.B-Natural provides data supporting an increased understanding of HB and its impact throughout life. The need for optimal disease control to normalize physical and psychosocial outcomes is underscored, and further analyses will contribute to an increased understanding of critical issues in HB.

BACKGROUND Congenital factor X deficiency (FXD) is a rare bleeding disorder that often presents with severe bleeding in the neonatal period. Long-term prophylaxis with infusions of FX-containing products is recommended in patients with FXD and a personal or family history of severe bleeding. A plasma-derived FX concentrate (pdFX) is approved for on-demand and prophylactic therapy in adults and children with FXD. The safety and efficacy of pdFX has been demonstrated in patients

Introduction: The B-Natural study is a multicentre, multinational, observational study of haemophilia B (HB) designed to increase understanding of clinical manifestations, treatment and quality of life (QoL). Aim: To characterise and compare QoL in HB across disease severity groups and individuals with inhibitors to identify gaps in treatment. Methods: A total of 224 individuals from 107 families were enrolled from a total of 24 centres in North America (n = 16), Europe (n = 7) and Asia (n = 1). Of these, 68 (30.4%) subjects had severe (5–

Introduction: Inhibitors develop less frequently in haemophilia B (HB) than haemophilia A (HA). However, when present, the success of tolerization by immune tolerance induction (ITI) therapy is lower and the risk of complications higher. Aim: To evaluate the use and outcome of ITI in patients with HB and inhibitors. Methods: Subjects include singletons or siblings with a current/history of inhibitors enrolled in B-Natural—an observational study designed to increase understanding of clinical management of patients with HB. Patients were followed for 6 months and information on demographics, medical and social history, and treatment were recorded. Results: Twenty-nine patients with severe HB and inhibitors were enrolled in 24 centres. Twenty-two underwent one or more courses of ITI with or without immune suppression. Eight patients (36.4%) were successfully tolerized after the first course of ITI. One of these successes (12.5%) experienced allergic manifestations, whereas the corresponding number for the 10 treatment failures was five (50%). One of seven (14.2%) patients with large deletions and three of eight (37.5%) with nonsense mutations were tolerized at the first attempt, and all patients experiencing nephrosis either failed or were on-going. At study end, 11 (50%) were considered successfully tolerized after one or more ITI courses, three were unsuccessful, and eight were still undergoing treatment. Conclusion: Our data underscore the possibilities and difficulties of achieving tolerization in patients with HB with inhibitors. The type of mutation and complications appear to correlate with ITI outcome, but more accurate definitions of successful ITI are warranted.

Congenital dyserythropoietic anemias (CDAs) are characterized by ineffective erythropoiesis and distinctive erythroblast abnormalities; the diagnosis is often missed or delayed due to significant phenotypic heterogeneity. We established the CDA Registry of North America (CDAR) to study the natural history of CDA and create a biorepository to investigate the pathobiology of this heterogeneous disease. Seven of 47 patients enrolled so far on CDAR have CDA-I due to biallelic CDAN1 mutations. They all presented with perinatal anemia and required transfusions during infancy. Anemia spontaneously improved during infancy in three patients; two became transfusion-independent rapidly after starting interferon-α(2); and two remain transfusion-dependent at last follow-up at ages 5 and 30 y.o. One of the transfusion-dependent patients underwent splenectomy at 11 y.o due to misdiagnosis and returned to medical attention at 27 y.o with severe hemolytic anemia and pulmonary hypertension. All patients developed iron overload even without transfusions; four were treated with chelation. Genetic testing allowed for more rapid and accurate diagnosis; the median age of confirmed diagnosis in our cohort was 3 y.o compared to 17.3 y.o historically. In conclusion, CDAR provides an organized research network for multidisciplinary clinical and research collaboration to conduct natural history and biologic studies in CDA.

Adeno-associated virus (AAV) vectors have been successfully used in patients with bleeding disorders and blindness. For human liver targeting, two major factors restrict effective AAV transduction after systemic administration of AAV vectors: human hepatocyte tropism and neutralizing antibodies (Nabs). In this study, we attempted to isolate AAV variants with the ability to transduce human hepatocytes and escape Nabs using a directed evolution approach in vivo. After four cycles of selection, 14 AAV capsid mutants were identified from a capsid shuffling library selected in the presence of human Intravenous Immunoglobulin (IVIG) and isolated from human hepatocytes xenografted into chimeric mice. AAV neutralization assays using IVIG showed that most of the mutants showed the Nab escape pattern in a manner similar to that of AAV8 or AAV9 and better than that of other AAV serotypes. Different mutants displayed varying capacities to escape Nab activity from individual serum samples collected from healthy subjects or hemophilia patients. The mutant AAV LP2-10 was found in 12 colonies out of 25, which was composed of capsids from AAV serotypes 2, 6, 8, and 9, with VP3 subunits derived from AAV8 swapped with AAV6 from residues 261 to 272. The mutant AAV LP2-10 manifested a higher ability than that of other serotypes to escape Nabs in IVIG and most human serum samples. After injection of AAV vectors encoding a self-complementary GFP cassette into chimeric mice, LP2-10 transduced human hepatocytes with efficiency similar to that of AAV8. In summary, AAV mutants can be isolated in humanized mice with both human hepatocyte tropism and the ability to evade Nab activity., Graphical Abstract, AAV mutant vectors were identified from a capsid shuffling library selected in the presence of human IVIG and isolated from human hepatocytes xenografted into chimeric mice. LP2-10, whose VP3 subunits were derived from AAV8 and AAV6, manifested the highest ability to escape Nabs in IVIG and most human serum samples.

Adeno-associated virus (AAV) vectors have been successfully applied in hemophilia clinical trials. However, this approach is limited to patients without AAV-neutralizing antibodies (NAbs). In this study, we explored the feasibility of AAV re-administration in hemophilia A dogs treated initially 8 years ago with AAV8.canine FVIII. After the re-administration in two NAb-negative dogs with AAV8 vectors carrying human factor VIII (hFVIII), along with the proteasome inhibitor bortezomib, we observed a phenotypic improvement in both dogs that persisted in one dog. Phenotypic improvement disappeared at 59 days after re-administration in the other dog, and specific cytotoxic T lymphocytes (CTLs) to the capsid were detected at day 17, but not to hFVIII. hFVIII inhibitors were observed at day 59 and gradually increased. Mechanistic studies demonstrated an increase in pro-inflammatory cytokines, a decrease in immunomodulatory cytokines, as well as lower Tregs after re-administration. These results suggest that hFVIII inhibitor development may contribute to the therapeutic failure via immune response activation. Interestingly, it takes about 30–50 days for AAV NAb titers to decrease by half. Collectively, this study suggests that re-administration of the same AAV serotype after long-term follow-up is feasible and that the study of AAV NAb kinetics will provide important information for predicating the efficacy of re-administration. Keywords: AAV, hemophilia, neutralizing antibodies, NAbs, re-administration, human factor VIII, hFVIII, hFVIII inhibitor

The development of inhibitory autoantibodies to the infused clotting factor VIII (FVIII) is a major complication for severe hemophilia A management. Novel therapy options for hemophilia have significantly progressed in the last decade, and a gene therapy cure for hemophilia is becoming a reality. However, mechanistic studies of FVIII autoantibodies (FVIII inhibitors) have lagged behind and remain a challenge for both protein replacement and gene therapy. FVIII inhibitor formation is assumed to be a classical T cell–dependent immune response in which cytokines/chemokines play an important role. The study of cytokine profile changes during FVIII inhibitor development may be helpful to understand the mechanism of inhibitor development and to explore potential novel approaches that will minimize the risk. After FVIII–/– mice were treated with intravenous administration of an adeno-associated virus 8 vector encoding human FVIII, FVIII expression peaked at week 2 (W2), and FVIII inhibitor was thoroughly develop...

Introduction High-titre factor VIII (FVIII) inhibitors complicate peri-operative haemostasis. Recombinant porcine FVIII (r-pFVIII) may provide an alternative haemostatic agent for high-risk procedures and allow FVIII activity monitoring. Aim Devise an effective haemostatic plan for repair of a progressively symptomatic aortic coarctation in a 5-year-old male with immune tolerance induction (ITI) refractory high-titre FVIII inhibitors. Methods Preprocedure human FVIII inhibitor titre was 58 Bethesda Units mL−1 (BU) and cross-reacted to neutralize porcine FVIII at 30 BU. Daily ITI with plasma-derived FVIII concentrate was supplemented with anti-B-cell and anti-plasma cell immunotherapy to reduce FVIII inhibitor titres. Potential haemostatic agents were evaluated in comparative ex vivo thrombin generation assays (TGA). Results Four weeks after immunosuppression, human and porcine inhibitor titres declined to 16 and 2 BU respectively. TGA with r-pFVIII was less robust than with activated prothrombin complex concentrate (aPCC); however, r-pFVIII was selected for cardiac surgery to secure the ability to assay FVIII levels throughout this high-bleeding risk procedure. Haemostasis with r-pFVIII was excellent; initial trough FVIII activity levels ranged from 0.81–1.17 IU mL−1. On postoperative day 3, peak and trough levels markedly declined suggesting a rising porcine inhibitor titre. Postprocedure prophylaxis was transitioned to aPCC, informed by TGA. Conclusions R-pFVIII provided effective peri-procedural haemostasis with no adverse events. Rapid neutralization of r-pFVIII after the first 60 hours, despite intensive immune suppression, accentuates the importance of careful monitoring. Use of TGA can support bypassing agent selection for convalescence. The comparative cost of r-pFVIII may limit its use to high morbidity clinical scenarios.

Introduction Development of an inhibitor to Factor IX is a potentially life-threatening complication that occurs in 2-4% of severe hemophilia B patients. Attempt to eradicate these neutralizing alloantibodies with immune tolerance induction (ITI) regimens using prolonged repeated exposure to FIX fails to induce tolerance in ~70% of cases, and is limited by severe complications including FIX hypersensitivity and nephrotic syndrome. Cases of FIX ITI with immunosuppressive drugs have been reported infrequently. In light of increased understanding of the role of active T lymphocyte regulation of clotting factor-directed B lymphocyte-mediated humoral immunity, an approach combining immune modulating agents and frequent FIX infusions was reported by Beutel, et al (Haemostaseologie, 2009; 2014). We report a multi-institutional retrospective experience with combination immune modulation therapy (CIT), including B cell suppression with anti-CD20 monoclonal antibody Rituximab and T cell modulation using mycophenylate mofetil (MMF) with FIX ITI in 11 males with severe congenital hemophilia B. Methods The originally published regimen of Beutel, et al involves a 50 day course including FIX 100 IU/kg twice daily, rituximab 375 mg/m2 x 4 doses, mycophenolate (MMF) 300 mg/m2/dose daily, dexamethasone 6 mg/m2/dose pulses and IVIG 1 g/kg x 6 doses. Following individual Institutional Review Board approval, data on 11 patients was contributed by the INPH investigators and via outreach internationally and collected retrospectively using a uniform data collection form. Cases were included in the CIT series if the combination of FIX, rituximab and MMF was used. Approaches varied in the concomitant use of IVIG and pulse corticosteroids (dexamethasone or prednisone). Results CIT was the first immune tolerance therapy for 9/11 patients. Patients were 14-222 months of age at time of CIT, with a median time from inhibitor diagnosis to CIT 20 (range 0-207) months. Median historical peak inhibitor titer prior to initiation of CIT was 3.2 (range 1-42) BU/ml. Inhibitor titers at the initiation of CIT ranged from undetectable to 7 BU/ml. Eighty-onepercent of patients had a history of FIX hypersensitivity reaction. Prior to initiation of therapy, 27% of the patients underwent FIX desensitization during the initial course. The patients received FIX BID (9/11 patients) or QD (2/11 patients), 4 doses of rituximab 375 mg/m2, MMF for a minimum of 49 days (varying duration from 49-1247 days). IVIG was infused in 10/11 patients, with most receiving 3-7 doses during each course of CIT. 6/11 patients received between 2-10 courses of pulse corticosteroids during a CIT course. Each course of CIT achieved disappearance of the titer of FIX inhibitor at a median time of 1 month (range 1-41 months) Hypersensitivity reactions did not limit the courses of CIT, but did recur with inhibitor recurrence in 1 patient. Recurrence occurred in 6/11 patients at a median time of 11 months from time of CIT, in some cases soon after documented B cell recovery with a median inhibitor titer of 1.8 (range 0.7-7) BU/ml. In cases of recurrence, a negative inhibitor titer was achieved again in 1 patient by increasing FIX dose and in 4 patients who received additional courses of CIT. Repeat recurrence was seen in these patients following repeat CIT, although at low titer with a median of 1 BU/ml (range 1-4), allowing ongoing management with FIX in 3 of the 4. Overall, patients have had a median follow-up of 61 months following CIT, 9/11 patients are currently managed with factor IX for prophylaxis and bleeding, while 2/11 use bypassing agents. The most common complication was hospitalization for central venous catheter-related bacterial infection that occurred from < 1 to > 12 months after the use of rituximab in 3/11 patients. Nephrotic syndrome occurred in 3/11 patients. In each case, nephrotic syndrome responded to corticosteroids or re-initiation of CIT and FIX could be resumed. Conclusions FIX ITI with CIT targeting both B and T cells in hemophilia B is an effective means of achieving a negative inhibitor titer with tolerable safety in this series, allowing return to use of FIX for hemostasis and prophylaxis in most patients. Although recurrence is common and longer follow up is needed, when compared to the experience with using FIX alone, outcomes appear to be improved using CIT with FIX ITI as an initial approach. Disclosures Abajas: Bayer: Honoraria; CSL Limited: Honoraria. Allen:Shire: Employment, Equity Ownership. Oldenburg:Pfizer: Honoraria, Research Funding; Biogen: Honoraria, Research Funding; CSL Behring: Honoraria, Research Funding; Grifols: Honoraria, Research Funding; Octapharma: Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; Shire: Honoraria, Research Funding; Chugai: Honoraria, Research Funding; Biotest: Honoraria, Research Funding; Roche: Honoraria, Research Funding; Swedish Orphan Biovitrum: Honoraria, Research Funding; Novo Nordisk: Honoraria, Research Funding.

Introduction and Rationale: The development of alloantibodies to factor VIII remains a challenging and significant complication in the treatment of patients with congenital hemophilia A (HA). Inhibitor development is more common in severe HA than in non-severe HA (NSHA). Incidence of inhibitors in NSHA is approximately 3-13%, and even with attempted immune tolerance induction and immunosuppression, inhibitors have been reported to persist long-term in about 40% of NSHA patients (Hay 1998). Inhibitors in patients with NSHA can have variable kinetics, causing clinical characteristics similar to patients with acquired hemophilia A (AHA), with observable type 2 kinetics and inhibition of exogenous and endogenous FVIII activity (d'Oiron et al. 2006). Optimal treatment regimens have yet to be established for these patients, in terms of inhibitor eradication and choice of agent for control of bleeding episodes, with known thrombotic side effects when using bypassing agents, including in pediatric patients (d'Oiron et al. 2006). It has been observed as well that the efficacy of bypassing agents, such as activated prothrombin complex concentrates (aPCC) and recombinant activated factor VII (rFVIII), is diminished in the presence of an inhibitor (Rocino et al. 2017). Challenge with aPCC or rFVIII can also induce inhibitor recurrence in patients who have had previous resolution (Giuffrida et al. 2008). Recombinant porcine FVIII (rpFVIII) is an alternative therapy, which by virtue of its incomplete homology in the A2 and C2 domains of the FVIII molecule, has decreased reactivity with anti-human FVIII (hFVIII) inhibitors (Mulliez et al. 2014). rpFVIII has demonstrated efficacy in patients with severe HA with inhibitors, as well as in acquired HA (AHA) (Kempton et al. 2012; Kruse-Jarres et al. 2015). However, experience is limited for use in patients with NSHA and inhibitors. We report successful bleed treatment with rpFVIII of 3 adult patients and 1 child with NSHA who developed inhibitors at the University of North Carolina, with excellent hemostatic efficacy and no adverse treatment-related effects. Methods: We analyzed clinical, pharmacy, and laboratory data from 4 patients with NSHA who developed inhibitors treated with rpFVIII after failure of or contraindication to bypassing agents at the University of North Carolina. Investigational review board approval was obtained for our data collection and analysis. Results: Average baseline FVIII activity was 9.5%, with 3 of our 4 patients having a FVIII activity of Conclusions: rpFVIII appears to be a safe and effective therapy in patients with NSHA who develop inhibitors. Our patients treated with immunosuppression in addition to rpFVIII, similar to how we treat patients with AHA. Of note, we achieved effective hemostasis in our 3 adult patients at much lower than FDA-recommended doses for AHA by using our previously established dosing algorithm for rp FVIII (Martin et al. 2016; Fig 1). Our pediatric patient required much more rpFVIII for effective hemostasis. This patient also had a much higher hBIA, and was being treated after the failure of multiple bypassing agents. However, once it seemed inhibitor binding sites were saturated after frequent higher rpFVIII doses, FVIII recovery was achieved, with concomitant decrease in doses required. FVIII activity was an effective means of monitoring efficacy of rpFVIII use, as in severe HA and AHA. One adult patient in particular had a very good clinical response to rpFVIII, requiring only 1 dose in the first 24 hours, with doses as low as 19 U/kg given daily required for bleeding control. Disclosures Abajas: CSL Limited: Honoraria; Bayer: Honoraria. Moll:Stago Diagnostics: Consultancy. Ma:CSL: Consultancy; Novo Nordisk: Consultancy; Shire: Honoraria, Research Funding; CVS: Honoraria.

Gene therapy refers to any therapy in which the therapeutic entity is a nucleic acid. Gene therapy most commonly delivers a DNA sequence, the transcription of which produces a protein to perform a therapeutic function. Other approaches have delivered RNA, short interfering RNA (siRNA), RNA aptamers, nucleases (e.g., to knock out host cell genes or to facilitate “editing” of genomic DNA), or sequences to express antigens that produce specific immunization. Considered most broadly, there are two approaches to introduce therapeutic genes. The first is to directly introduce the nucleic acid sequences of interest into the target tissue in vivo. Transfer of the nucleic acid may or may not employ a delivery vehicle, referred to as a vector. Naked DNA or RNA transfer is in general inefficient unless mediated by a virus or other vector. In nature, viruses have evolved mechanisms to enter cells and deliver their viral DNA (or RNA) to the nucleus of cells, where they hijack the host cell’s machinery to transcribe and translate the virus’ genetic payload. Dozens of viruses have been investigated as potential gene delivery vectors. The second approach is to introduce the gene of interest ex vivo into cells (whether allogeneic or autologous) in culture, expand if desired the cells that express the gene efficiently, and then introduce the cells with their transgenic payload into the host. The goal of correcting hemophilia, the most common severe bleeding disorder, has been approached using multiple variations of in vivo vectors and ex vivo cell-based strategies over the last quarter century, ultimately leading to partial correction of hemophilia B in a human clinical trial (St. Louis and Verma 1988; Palmer et al. 1989; Nathwani et al. 2011).