Hemophilia B Patients Research Articles

Molecular genetic diagnosis by next-generation sequencing in a cohort of Mexican patients with haemophilia and report of novel variants

IntroductionMolecular analysis in haemophilia is currently used in the diagnosis, treatment and prognosis of this disease. Hispanic populations in Latin America have been of interest to researchers due to the reportedly high prevalence of inhibitors in these patients. AimTo perform next-generation sequencing (NGS) in a cohort of Mexican patients with HA and HB and correlate with clinical phenotypes. MethodsPatients with Haemophilia A (HA) or haemophilia B (HB), were evaluated using NGS with an Ion AmpliSeq Custom Panel. Odds ratios (ORs) for associations between F8 variants and inhibitors were obtained. ResultsA total of 85 patients (60 with HA and 25 with HB) were included. Pathogenic variants in F8 were found in 93.3% of HA patients and in F9 in 96% of HB patients. Twelve novel potentially pathogenic variants were found. Inhibitors were observed in 20% of patients with severe HA. Four patients clinically diagnosed with HA were negative for F8 variants. ConclusionOverall detection rate of pathogenic variants in F8 and F9 genes was 94.6%. We identified 12 non previously reported variants and pathogenic variants in other coagulation related genes. Molecular diagnosis of HA and HB permits better options for management, assessment and genetic counseling.

Identification of the Profile of the Patients with Hemophilia B Eligible for Treatment with Nonacog Alfa Once-Weekly

This study aimed to identify the characteristics of patients with hemophilia B eligible for once-weekly treatment with Nonacog alfa. Methods: A survey was conducted in 14 Hemophilia (HCs) of Italy. These centers were given a questionnaire consisting of ten closed multiple-choice questions. The centers were asked: (a) the percentages of their hemophilia B (HB) patients undergoing replacement therapy, “On-demand”, or weekly prophylaxis, (b) the criteria guiding the monitoring of patients, the advantages according to the age of patients, and (c) the obstacles to prophylaxis. The percentage of patients receiving “On-demand” (OD) treatment or continuous prophylaxis (prophy) differed depending on patient age and the severity of the disease. Only 57% of HCs provided “On-demand” therapy to the mild HB patients, about 93% to moderate ones, of whom 43% on prophylaxis. About 78% of patients <6 years old, were on treatment in 9 out of 14 HCs, by prophylaxis 66.7% and 33.3% by On-demand. In the 6–18 age group, 90.1% of HCs treated HB patients with prophylaxis, 42.8% in the 18–30 age range. On-demand treatment was the therapy of choice in 61.5% of HCs for patients aged 30–65 years. In total, 64% of the HCs assigned the maximum score to bleeding frequency, especially in the <6 and 6–18 age groups. Bleeding severity was also taken into significant consideration, particularly in subjects up to 30 years old. The scores regarding venous access were distributed relatively evenly throughout all age groups. The majority of the centers attributed a medium-high score to treatment compliance, especially in the 6–65 age range. In actuality, 55% of HCs attributed pro-thrombotic comorbidity a low score in the 18–30 age group, whereas 81% gave pro-hemorrhagic comorbidity a high rating in patients aged >65 years old. Many centers assigned a medium-high score to the baseline concentration of FIX level at diagnosis in all age groups. Most HCs attributed a medium-high score to type of genetic mutation in the younger age groups. As for socio-cultural barriers and quality of life, the majority of respondents gave a medium-high score in all age groups. For periodic monitoring of patients receiving continuous prophylaxis, 59% of the centers reported using clinical assessment. With regard to prophylaxis administration method, the majority of hemophiliacs were given infusions twice weekly, while as regards to the dose of FIX concentrate delivered, 50% of the centers reported administering prophylaxis once-weekly at a dose ranging from 5–100 IU/kg in 10–50% of HB patients. Thus, 93% of the centers reported using a dose of 25–50 IU/kg for twice-weekly prophylaxis in 6–100% of the patients. The majority of centers (86%) believe that, in a program of early primary prevention, once-weekly treatment with nonacog alfa may represent an alternative strategy to dose escalation. The results show that patients with mild hemophilia, with functional musculoskeletal status and difficulties with venous access, are candidates for once-weekly prophylaxis with nonacog alfa. For such patients, this regimen can improve treatment compliance and quality of life.

Efficacy and Safety in 15 Hemophilia B Patients Treated with the AAV Gene Therapy Vector Fidanacogene Elaparvovec and Followed for at Least 1 Year

Background: Adeno-Associated Virus (AAV) based liver transduction has emerged as a potentially viable gene therapy approach for the treatment of hemophilia patients. Fidanacogene elaparvovec (previously SPK-9001) is a hepatotropic bioengineered AAV based vector that delivers a high activity factor IX (FIX) transgene driven by a liver specific promoter. The Phase 1/2a development consists of a dosing study where patients are followed for 52 weeks post vector infusion followed by a long-term follow-up study for an additional 5 years. Data on the first 10 patients were previously published and demonstrated safe and sustained expression of a high activity FIX protein with an associated decreased requirement for exogenous factor administration and markedly reduced annualized bleeding rate. Here we present data on 15 patients infused with fidanacogene elaparvovec with ≥ 1 year of follow-up, which represents the largest cohort of Hemophilia B (HB) patients treated with the same vector at the same dose. Methods: Fifteen (15) adult HB patients were infused with 5 x 1011 vg/kg of fidanacogene elaparvovec and followed for at least 1 year as part of the Phase 1/2a dosing study. FIX activity (FIX:C) levels were measured using a one-stage assay. Endpoints include: Safety and tolerability, steady-state activity calculated as the geometric mean of all observed FIX:C activity levels from week 12 through week 52; annualized bleeding rate (ABR) prior to and 52 weeks after vector infusion; T cell response to fidanacogene elaparvovec capsid and transgene monitored post-infusion using an interferon-γ enzyme-linked immunospot (ELISpot) assay. Results: Three of fifteen patients were treated with corticosteroids for elevations in hepatic transaminases of which 2 were positive for capsid reactive T cells by interferon-γ ELISpot. There were otherwise no treatment related adverse events. The mean post-infusion steady-state FIX:C was 22.9%±9.9% at 1 year post vector infusion as measured in a central laboratory by one-stage assay utilizing Actin-FSL. Mean ABR during the first 52 weeks following fidanacogene elaparvovec infusion was 0.4±1.1 compared to 8.9±14.0 in the 52 weeks preceding infusion (p<0.001). Twelve (12) out of 15 patients reported zero bleeds in the 52 weeks post-vector infusion. Five of 15 subjects infused factor for a total of 20 infusions. Additional follow-up data will be presented for all patients enrolled in the long-term follow-up study. Conclusions: Fidanacogene elaparvovec was well tolerated in 15 patients with no serious adverse events. Data for all patients at 52 weeks post-infusion demonstrated a marked reduction in bleeding frequency and exogenous FIX use. All hepatic transaminase elevations responded to treatment with corticosteroids. Collectively, to date, this represents the largest cohort of HB patients treated with the same AAV based gene therapy and at the lowest dose. Treatment has been efficacious for all patients with manageable immune responses when present. These data support progression to a pivotal Phase 3 study at the dose evaluated. Disclosures George: University of Pennyslvania: Employment; Avrobio: Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy. Sullivan:Octapharma: Consultancy, Other: Advisory Board. Rasko:bluebird bio: Honoraria; Celgene: Honoraria; Novartis: Honoraria; FSHD Global Research Foundation: Membership on an entity's Board of Directors or advisory committees; Rarecyte: Consultancy, Equity Ownership; Gene Technology Technical Advisory, Australian Government: Other: Advisory committee; GSK: Honoraria; Takeda: Honoraria; Cynata: Honoraria; Genea: Equity Ownership; Cure The Future Foundation: Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria; Pfizer: Honoraria; Spark: Honoraria; Imago: Consultancy; Advisory Committee on Biologics, Australian Government: Other: Advisory Committee; NHMRC Mitochondrial Donation Expert Working Committee: Other: Advisory Committee; Australian Cancer Research Scientific Advisory Board: Membership on an entity's Board of Directors or advisory committees. Giermasz:Genentech/Roche: Consultancy, Other: Research, Speakers Bureau; uniQure: Consultancy, Other: Research; Bioverativ/Sanofi: Consultancy, Speakers Bureau; BioMarin: Consultancy, Other: Research; Sangamo: Other: Research. Samelson-Jones:The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania: Employment. Ducore:Bayer: Consultancy, Honoraria, Other: speaker (not bureau); Spark Therapeutics: Research Funding; Shire: Consultancy, Honoraria; Octapharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; HEMA Biologics: Consultancy, Honoraria; BioMarin: Research Funding; Bioverativ: Research Funding. Teitel:BioMarin: Consultancy; Bayer: Consultancy, Research Funding; Shire: Consultancy; Pfizer: Consultancy, Research Funding; Novo Nordisk: Consultancy; Octapharma: Consultancy; CSL Behring: Consultancy. McGuinn:Biogen: Research Funding; Roche/Genetech: Research Funding; Spark: Research Funding; Shire/Baxalta: Consultancy, Research Funding. Wright:Solid Biosciences: Consultancy; Yposkesi: Other: Senior Advisor, SAB; LogicBio Therapeutics: Other: Member, SAB; Memorial Sloan Kettering Cancer Center: Consultancy; Agilis Biotherapeutics: Consultancy; Axovant Sciences: Other: Chief Technology Officer, Gene Therapies; Akous Therapeutics: Consultancy; National Institutes of Health: Consultancy; Leland Stanford Junior University: Consultancy; Wright Biologics: Other; Sanofi Genzyme: Consultancy; Spark Therapeutics: Consultancy, Other: co-founder, Chief Technology Advisor/Officer, Member, SAB; Adrenas Therapeutics: Other: Member, SAB; Ambys Medicines: Consultancy; CEVEC Pharmaceuticl: Other: Member, SAB. Anguela:Spark Therapeutics: Employment, Equity Ownership, Patents & Royalties. High:Spark Therapeutics: Employment, Equity Ownership, Patents & Royalties. Rybin:Pfizer: Employment. Murphy:Pfizer Inc.: Employment. Rupon:Pfizer: Employment.

Spontaneous Bleeding and Poor Bleeding Response with Extended Half-Life Factor IX Products: A Survey of Select US and Canadian Hemophilia Treatment Centers

Background: Factor IX (FIX) has distinct pharmacokinetic properties compared to factor VIII including significant distribution to the extravascular space. Extravascular distribution and binding to type IV collagen is important in hemostasis but not readily measureable in clinical practice for patients with hemophilia B receiving factor products. We previously reported data regarding use of EHL-FIX products in a cohort of patients who demonstrated issues with spontaneous bleeding and poorly controlled bleeding events; we now report data from an expanded cohort including performance of all EHL-FIX products available in US and Canada. Aims: To characterize the use and performance of EHL-FIX in clinical practice at six hemophilia treatment centers (HTCs). Methods: An electronic survey regarding center specific use of EHL-FIX amongst patients with severe hemophilia B (HB) was sent in summer 2019, including 4 previously surveyed centers and 2 additional centers. Providers were asked if patients utilizing EHL-FIX for prophylaxis had experienced 1) spontaneous/minimally traumatic bleeding events at factor levels >10% or 2) poorly controlled bleeding events requiring more frequent/higher doses of EHL-FIX than anticipated in addition to patterns of EHL-FIX product switching. Results: Surveyed HTCs cared for 90 patients with HB including 67 (74%) who utilized EHL-FIX, including 26 (39%) recombinant factor IX (FIX) albumin fusion protein (rFIX-FP), 37 (55%) recombinant factor IX Fc fusion protein (rFIXFc), and 4 (6%) received glycopegylated recombinant FIX (rFIX-GP). All patients had severe hemophilia B with the exception of one smaller center also contributing data regarding moderate HB patients on prophylaxis. All centers reported having patients with unexpected spontaneous/minimally traumatic bleeding and poorly controlled bleeding which did not seem to be dependent on age (median age 14.5 years, range 1.4-44). This occurred in 18 patients on prophylaxis, including 16 of 26 (62%) patients using rFIX-FP, and 2 of 4 (50%) of patients using rFIX-GP. Conclusions: Although plasma FIX activity levels have driven prophylaxis and bleed management decisions, clinical experience suggests novel properties of EHL-FIX may impact hemostasis. Although achieving seemingly adequate FIX plasma troughs (>10%), limited clinical experience suggests patients with SHB may have a differential response to EHL-FIX, noted in our cohort with FIX-FP and rFIX-GP. Successful bleed prevention or control in SHB may be predicted by the distribution of FIX in circulation and extravascular space, and the presence of FIX in tissues at time of injury. These data demonstrate the importance of real-world monitoring of efficacy of new FIX products and suggest the need for more robust mechanisms to understand the hemostatic performance of products. Disclosures Malec: Bayer: Honoraria; Spark: Honoraria; CSL: Honoraria; Sanofi: Consultancy, Honoraria, Speakers Bureau; Takeda: Honoraria. Croteau:Novo Nordisk: Consultancy, Honoraria, Research Funding; Shire: Consultancy, Honoraria; Bayer: Consultancy, Honoraria; Bayer: Consultancy, Honoraria; Octapharma: Honoraria; Octapharma: Honoraria; Genentech: Consultancy, Honoraria; Pfizer: Research Funding; Spark Therapeutics: Research Funding; Novo Nordisk: Consultancy, Honoraria, Research Funding; Spark Therapeutics: Research Funding; Pfizer: Research Funding; Genentech: Consultancy, Honoraria; CSL Behring: Consultancy, Honoraria; CSL Behring: Consultancy, Honoraria; Shire: Consultancy, Honoraria. Callaghan:Biomarin, Bioverativ, Grifols, Kedrion, Pfizer, Roche/Genentech, Shire, and Spark Therapeutics: Consultancy; Alnylum: Equity Ownership; Bayer: Consultancy, Speakers Bureau; Takeda: Consultancy, Research Funding; Sanofi: Consultancy; Global Blood Therapeutics: Consultancy; Novonordisk: Consultancy, Speakers Bureau; Octapharma: Consultancy; Pfizer: Research Funding; Roche: Research Funding; Shire/Takeda: Speakers Bureau; Roche/Genentech: Speakers Bureau. Matino:Bayer: Honoraria, Research Funding; Sobi: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Roche: Research Funding; Bioviiix: Honoraria; Sanofi: Honoraria. Friedman:CSL: Consultancy; Bayer: Consultancy; Genentech: Consultancy; Instrumentation Laboratory: Consultancy; Siemens: Consultancy. Sidonio:Grifols: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genetech: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda-Shire: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bioverativ: Membership on an entity's Board of Directors or advisory committees, Research Funding; Octapharma: Membership on an entity's Board of Directors or advisory committees, Research Funding; Uniqure: Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees; Biomarin: Membership on an entity's Board of Directors or advisory committees; Kedrion: Research Funding.

New data from the Italian National Register of Congenital Coagulopathies, 2016 Annual Survey.

In Italy, the National Register of Congenital Coagulopathies (NRCC) collects epidemiological and therapeutic data from patients affected by haemophilia A (HA), haemophilia B (HB), von Willebrand's disease (vWD) and other rare coagulation disorders. Here we present data from the 2016 annual survey. Data are provided by the Italian Haemophilia Centres, on a voluntary basis. Information flows from every Centre to a web-based platform of the Italian Association of Haemophilia Centres, shared with the Italian National Institute of Health, in accordance with current privacy laws. Patients are classified by diagnosis, disease severity, age, gender and treatment-related complications. In 2016, the total number of patients with congenital coagulopathies in the NRCC was 10,360: 39.8% of these patients had HA, 31.5% had vWD, 8.5% had HB, and 20.2% had less common factor deficiencies. The overall prevalence of HA and HB was 13.9/100,000 males and 3.0/100,000 males, respectively. The overall prevalence of vWD was 5.4/100,000 inhabitants. During 2016, 126 patients had current alloantibodies to factor VIII (FVIII) or factor IX (FIX) and were under treatment with bypassing agents and/or immune tolerance induction. Overall, 388 patients with a history of alloantibodies were recorded in the NRCC of whom 337 with severe HA and 12 with severe HB. Coagulation factor use, evaluated from treatment plans, was approximately 451,000,000 IU of FVIII for HA patients (7.5 IU/inhabitant), and approximately 53,000,000 IU of FIX for HB patients (0.9 IU/inhabitant). The prevalences of HA and HB fall within the ranges reported in more developed countries; the consumption of FVIII and FIX was in line with that of other European countries (France, United Kingdom) and Canada. The NRCC, with its bleeding disorder dataset, is a helpful tool for shaping public health policies, as well as planning clinical and epidemiological research projects.

Tissue factor pathway inhibitor is the main determinant of thrombin generation in haemophilic patients.

The thrombin generation (TG) assay evaluates haemostatic balance, which is influenced by the levels of many coagulation factors and inhibitors. Our objective was to identify the determinant factors of TG in haemophilia A (HA) and haemophilia B (HB) patients and to compare them to those in healthy controls. Coagulation factor and inhibitor levels, and TG, were measured in platelet-poor plasma from 40 patients with HA, 32 patients with HB and 40 healthy subjects. Data were analysed using multiple regression models. In HA patients, factor VIII was a positive determinant of endogenous thrombin potential (ETP) and peak, whereas tissue factor pathway inhibitor (TFPI) and factor V were negative determinants of ETP and peak. In HB patients, FIX was a positive determinant of ETP and peak, FVII being a positive determinant of peak. Antithrombin and protein S (PS) were negative determinants of ETP while FX was a negative determinant of peak. Above all, in HB patients, TFPI was a negative determinant of ETP and peak. In healthy subjects, FVIII was a positive determinant of ETP and peak, whereas FX and protein S were negative determinants of these parameters. TFPI was not a negative determinant of either peak or ETP. In haemophilic patients, the determinant factors of TG are all implicated in FXa generation and inhibition, the crucial determinant factor being TFPI whatever the type of haemophilia, A or B. These findings contribute to the rationale that recently place TFPI as a target for innovative therapies of haemophilia.

The characteristics and spectrum of F9 mutations in Chinese sporadic haemophilia B pedigrees.

Sporadic haemophilia B (HB) without obvious familial history poses challenges for genetic diagnosis and counselling. To identify the F9 variants in sporadic HB patients and probe the origin of these de novo mutations. A total of 294 unrelated HB pedigrees sought genetic diagnosis were analysed in this single-centre study. The F9 gene was analysed by direct sequencing, and AccuCopy technique was adopted to screen for gene copy number variations. Six short tandem repeats approximal or within F9 gene were applied for linkage analysis. Mosaicism of sequence variant was determined by ddNTP Primer Extension method. Sporadic HB patients constituted 36% (61/294) of cases enrolled in current study. The sporadic and familial HB patients shared similar spectrum of F9 variants, with single nucleotide substitution as predominant form of disease-causing mutation and no mutation prone hotspot sites, including CpG dinucleotide sequences, had been identified. Majority of the mothers of sporadic HB patients were F9 mutation carriers (70%, 43/61), and most of them (95%, 41/43) had the inherited bleeding trait traced back to maternal grandfathers. Although most de novo mutations occur in germ cells, 2 maternal grandfathers, who had somatic mosaic mutations of F9, were also revealed to be the source of genetic variations identified in patients. In our cohort, FIX inhibitor incidence was 1%, developed only in patients carrying null mutations. The diversity of F9 genetic variants and possible mosaicism of de novo mutation demand extensive study and more cautious in genetic counselling of sporadic HB.

“Prevalence of Inhibitors in Hemophilia Patients and its Clinical Implications”: A Study of 276 Patients in Western India

Introduction: Hemophilia is an X-linked congenital bleeding disorder caused by a deficiency of coagulation factor VIII (FVIII) in hemophilia A (HA) or factor IX (FIX) in hemophilia B (HB). Accurate diagnosis of hemophilia by factor assay to demonstrate deficiency of FVIII or FIX is essential for appropriate management. Inhibitor development results in partial or complete lack of the efficacy of replacement therapy, and it makes the management of patients more difficult with an increased risk of morbidity, serious bleeding, and disability, resulting in a substantial impact on patient's quality of life and health-care costs, compared to patients without inhibitors. Aims and Objectives: To assess the incidence of inhibitor development in HA and HB patients along with its consequences. Materials and Methods: The present study was carried out at a tertiary care teaching hospital in Western India. A total of 276 patients of hemophilia were included in the study. FVIII, FIX, and inhibitor screening were carried out in all patients sample as routine testing. Patients who were found positive in inhibitor screening were further evaluated for quantitative assay (Bethesda assay). Results: Out of total 276 patients, 243 patients of HA and 33 patients of HB were observed. The incidence of inhibitor development is 20.57% in HA and 6.06% in HB. The maximum number of patients and maximum number of inhibitors was between the age group of 11 and 30 years. There was more number of patients with severe disease as compared to mild and moderate forms. The concentration of inhibitor >5 BU was seen in 76% of HA patients and 100% of HB patients with inhibitor. Sixty-one patients came for follow-up. In three patients, inhibitor disappeared. The incidence of complications was more in patients who had developed inhibitor which increases the cost of treatment and increases the social suffering of the patients. Conclusion: Inhibitor development affect the severity and treatment of the disease significantly and there by increases the suffering and cost to the patient.

Inhibitors among patients with hemophilia in Basra, Iraq - A single center experience.

Inhibitor formation is a major complication of hemophilia treatment because it interferes with the clinical response to factor replacement and causes significant morbidity. This cross-sectional study was conducted to assess the presence and frequency of inhibitors among registered person with hemophilia and to identify risk factors associated with inhibitor development. A total of 143 hemophilics, 118 with hemophilia A (HA) and 25 with hemophilia B (HB), were enrolled for the study. Participant's clinical data were obtained through patient's medical records. Factor VIII and IX levels and the presence of inhibitors were assessed using a fully automated coagulometer. From the results of a Bethesda assay, patients were divided into those with high titers (≥5 BU) and those with low titers (<5 BU). The patient's age ranged from 1 to 67 years with median of 13.8 years. Inhibitors were detected in 18.6% and none of HA and HB patients, respectively. Of the 22 patients with HA and inhibitors, 18 (82%) had high titer inhibitors. The frequency of inhibitors was significantly higher among patients with severe hemophilia, a history of early exposure (≤3 months) to factor VIII concentrate, and family histories of autoimmune disease and immune system challenges (P < 0.05). The independent risk factors associated with inhibitor development were severe hemophilia (95% CIs = 1.02-55.6, OR = 7.5) and immune system challenges (95% CIs = 1.14-5.99, OR = 2.6). Inhibitors were common among HA patients, and both severe HA and immune system challenges (surgery and trauma) are independent risk factors for inhibitor development.

Platelet-Targeted Hyperfunctional Factor IX (FIX) Gene Therapy for Hemophilia B Mice with Pre-Existing Anti-FIX Immunity

Hemophilia B (HB) is a prime model for gene therapy. While data from current clinical trials using AAV-mediated liver-targeted FIX gene therapy are very encouraging, this protocol can only be applied to adults without liver disease or anti-AAV antibodies. Thus, developing another gene therapy protocol is desired. Our previous studies have demonstrated that platelet-targeted FIX expression driven by the platelet-specific αIIb promoter (2bF9) restores hemostasis and induces immune tolerance in HB mice (Chen et al. Mol Ther 2014). To improve the efficacy, we used a codon-optimized hyperfunctional FIX Padua (2bCoF9R338L) to replace the normal FIX expression cassette. We showed a 5.8-fold higher platelet-FIX antigen (plt-F9:Ag) and a 28-fold activity (plt-F9:C) levels in HB mice (the non-inhibitor model), respectively, in the 2bCoF9R338L group compared to the 2bF9 group. Here we evaluate 2bCoF9R338L gene therapy in HB mice with preexisting anti-F9 immunity (the inhibitor model). Both donor and recipient HB mice were immunized with rhF9 in the presence of Incomplete Freund's Adjuvant to induce inhibitor development. Plt-F9 expression was introduced by 2bCoF9R338L lentivirus transduction of Sca-1+ cells followed by syngeneic transplantation into primed recipients preconditioned with either a lethal 11Gy or a sub-lethal 6.6Gy total body irradiation. Animals were analyzed starting at 4 weeks after transplantation.FACS analysis showed that there was 21.4 ± 11.4% (n = 6) transduced platelets in the 11Gy group, which was not significantly different compared to the 6.6Gy group (12.5 ± 10.9%, n = 7) or the 6.6Gy non-inhibitor model (17.7 ± 11.7%, n = 4). Plt-F9:C levels in the 11Gy and 6.6Gy groups were 18.68 ± 8.61 and 9.17 ± 12.22 mU/108 platelets, respectively. Plt-F9:Ag levels in the two groups were 3.96 ± 1.57 and 1.70 ± 1.64 mU/108 platelets, respectively, which were not significantly different compared to that obtained in the non-inhibitor model. To determine if plt-F9 is γ-carboxylated, BaSO4 precipitation was performed on platelet lysates from 2bCoR338L-transduced recipients. Diluted platelet lysate samples were treated with or without BaSO4 , and the remaining unprecipitated FIX (meaning un-γ-carboxylated) in supernatants was determined by FIX ELISA. No F9:Ag remained in the supernatants of BaSO4-treated platelet lysates, suggesting that FIX can fully undergo functional γ-carboxylation in platelets.Of note, no anaphylaxis occurred in primed HB mice after platelet gene therapy. The inhibitor titers declined to undetectable with time in recipients after treatment with a half-life (t1/2) of inhibitor reduction in the 11Gy group of 49.2 ± 3.9 days, which was significantly shorter than in the 6.6Gy group (82.6 ± 32.6 days) and in the untransduced 6.6Gy control group (77.7 ± 8.7 days), indicating that plt-F9 does not trigger a memory immune response in primed F9null mice. Since our previous studies have shown that the clinical efficacy of plt-F9 is limited in the presence of anti-F9 inhibitors (Zhang et al. Blood 2010), we did not assess the bleeding phenotype in recipients until inhibitor titers dropped to an undetectable level. When a 6-hour tail bleeding test was used to assess the clinical efficacy of 2bCoF9R338L gene therapy, the bleeding time in the 11Gy and 6.6Gy groups was 2.5 ± 1.3 and 2.5 ± 1.1 hours, respectively, which was similar to that obtained in the wild-type (WT) control (1.7 ± 0.9 hours). In contrast, none of the F9null mice clotted within 6 hours. The remaining hemoglobin levels after the test were 79.5 ± 15.5% and 64.7 ±23.4% in the 11Gy and 6.6Gy groups, respectively, which were not significantly different compared to the WT group (70.6 ± 13.9%), but significantly higher than in the F9null control (38.8 ± 6.7%). We used ROTEM to analyze the whole blood clotting time (WBCT). WBCT in the 2bCoF9R338L-transduced group was significantly shorter than in the F9null control group (1328 ± 236 vs. 2426 ± 476 sec, respectively), confirming that hemostasis was improved in 2bCoF9R338L transduced recipients.Together, our data demonstrated that 2bCoF9R338L gene therapy could efficiently introduce sustained levels of plt-F9 in HB mice even with pre-existing immunity, resulting in antibody eradication and phenotypic correction when inhibitors diminished, suggesting that 2bCoF9R338L gene therapy can be a promising protocol for all HB patients, including patients with inhibitors. DisclosuresNo relevant conflicts of interest to declare.

Adeno-Associated Mediated Gene Transfer for Hemophilia B:8 Year Follow up and Impact of Removing “Empty Viral Particles” on Safety and Efficacy of Gene Transfer

▪Background: We have previously shown that a single intravenous administration of a self-complementary adeno-associated virus (scAAV) vector containing a codon-optimised factor IX gene, under control of a synthetic liver specific promoter and pseudotyped with serotype 8 capsid, (scAAV2/8-LP1-hFIXco) resulted in a dose-dependent increase in plasma FIX levels in all 10 enrolled severe hemophilia B (HB) patients (ClinicalTrials.gov:NCT00979238; Nathwani et al 2011). FIX activity was stably maintained for at least 3 years (Nathwani et al 2014) but concerns over FIX expression declining over time remain. This is because AAV-mediated transgene expression is mediated mainly by episomally retained viral genomes, which may be lost with natural hepatocyte turn-over. The only vector-associated adverse event was an asymptomatic rise in liver enzymes associated with a decline in FIX levels, occurring within 3 months of gene transfer in two-thirds of the patients treated at a dose of 2x1012 vector genomes(vg)/kg. Liver enzymes normalized with corticosteroids without complete loss of transgene expression. There was no long-lasting toxicity over a period of 3 years but further follow-up is required. The vector preparation used contained an excess of empty capsids, which lacked a full-length viral genome, and are therefore, non-functional but capable of provoking an immune response against transduced hepatocytes. Therefore, a new clinical preparation of scAAV2/8-LP1-hFIXco was manufactured from which most of the empty particles were removed by caesium chloride density centrifugation in the hope that this would reduce the risk of hepatotoxicity. We report on the evaluation of this new vector preparation in severe HB patients and provide an update on up to 8 years follow-up of our original cohort of patients.Methods: Ten subjects were recruited in 2010-2012 to the initial dose-escalation/extension study arm, which entailed a single intravenous infusion of scAAV2/8-LP1-hFIXco (full: empty capsid ratio ~1:10) at a dose of either 2x1011vg/kg, 6x1011vg/kg or 2x1012vg/kg. Two severe HB patients (FIX <1%) were enrolled into the first and mid-dose cohorts, with six patients treated at the high dose. In a follow-on study arm, two severe HB subjects received a dose of 2x1012vg/kg of the new scAAV2/8-LP1-hFIXco preparation (full: empty capsid ratio 1:3) whilst the next 2 patients were treated at a dose of 5x1012vg/kg. In both arms, vector was administered without prophylactic immunosuppression but corticosteroids (starting at 60mg/day) were commenced if liver enzymes increased to ≥2 fold over baseline levels after gene transfer.Results: Transgenic FIX activity levels have remained stable in all 10 subjects treated in the initial dose escalation/extension arm over a median follow-up of 6.7±1.0 years with mean levels in the three dose cohorts at the time of reporting of 1.9±0.6, 2.3±0.3 and 5.1±1.4 IU/l respectively. Over this period, annual FIX concentrate usage has dropped by 66% and annual bleed rate has declined by 82% when compared to pre-gene therapy levels. No late toxicity was observed. Neutralising antibodies to FIX were not detected in any patient but anti-AAV8 capsid-specific antibody levels persisted at high titres in 9 of 10 patients. In patients treated with the new preparation of scAAV2/8-LP1-hFIXco (median follow up = 2.1±1.4 years), mean FIX activity in the 2x1012vg/kg dose cohort was 2.6±0.7 IU/l. This is lower than observed previously at this dose level, but the difference is not statistically significant. Mean steady state FIX levels in the 5x1012vg/kg cohort were 17±5 IU/l. FIX antigen to activity ratio was 1:1. Elevation of serum alanine aminotransferase was observed in 3 of 4 patients treated with the new vector preparation, requiring treatment with corticosteroids.Conclusion: This is the first report to demonstrate stable therapeutic expression of FIX in patients with severe HB over a period of 8 years following systemic administration of scAAV2/8-LP1-hFIXco without late toxicities. We show for the first time that reducing the capsid load by removing empty AAV capsids does not appear to reduce the incidence of hepatotoxicity in patients with severe HB suggesting that other factors are involved in the aetiology of this process. DisclosuresNathwani:Freeline: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; BioMarin: Consultancy, Patents & Royalties; UniQure: Patents & Royalties. Tuddenham:BioMarin: Consultancy, Patents & Royalties; Freeline: Consultancy. Chowdary:Freeline: Consultancy; Baxalta (Shire): Honoraria, Membership on an entity's Board of Directors or advisory committees; Bayer: Honoraria; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Biogen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; CSL Behring: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Swedish Orphan Biovitrum AB (Sobi): Honoraria. McIntosh:Freeline: Consultancy. Recht:Biogen: Research Funding; Shire: Research Funding; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees, Research Funding; Kedrion: Membership on an entity's Board of Directors or advisory committees.

In Vivo Enrichment of Genetically Manipulated Platelets for Murine Haemophilia B Gene Therapy

Hemophilia B (HB) is an X chromosome-linked bleeding disorder resulting from a deficiency of factor FIX (FIX). Our previous studies have demonstrated that platelet-targeted FIX gene therapy can provide sustained therapeutic levels of FIX expression in platelets without inhibitory antibody (inhibitor) development, indicating platelet gene therapy could be a promising strategy for the treatment of HB patients. In this study, we aimed to enhance platelet-FIX expression in HB mice with MGMT P140K-mediated in vivo selection of hematopoietic stem cells (HSCs) under a nonmyeloablative preconditioning. We constructed a novel lentiviral vector (2bF9/MGMT LV), which harbors dual genes, the FIX gene driven by the αIIb promoter (2bF9) and the drug-resistance MGMT P140K gene. Platelet-FIX expression was introduced in nonmyeloablative preconditioned HB mice after 2bF9/MGMT LV-mediated HSC transduction and transplantation. After 8 weeks of bone marrow reconstitution, recipients were administered with O6-benzylguanine/1, 3-bis-2-chloroethyl-1- nitrosourea (BG/BCNU) once every 4 weeks for a total of 3 treatments. Animals were analyzed starting at 4 weeks after transplantation by PCR, quantitative real-time PCR (qPCR), FIX assays, inhibitor assays, and tail bleeding test.PCR results showed that 2bF9 proviral DNA was detected in all recipients that received 2bF9/MGMT LV-transduced HSCs. Quantitative PCR results confirmed that 2bF9/MGMT LV-transduced cells were effectively enriched after each round of BG/BCNU selective treatment in vivo. The average copy number of the 2bF9 cassette per cell after three BG/BCNU treatments was 0.50 ± 0.04, which was significantly higher than pre-BG/BCNU treatment (0.13 ± 0.01). There was an approximately 2.9-fold higher FIX antigen (FIX:Ag) (4.11 ± 0.76 vs. 1.43 ± 0.06 mU/108 platelets) and a 3.7-fold FIX activity (FIX:C) (2.56 ± 0.50 vs. 0.69 ± 0.04 mU/108 platelets) level, respectively, post-treatment compared to pre-treatment. There was a small amount of FIX:Ag detected in the 2bF9/MGMT LV-transduced recipient plasma. When we normalized FIX:Ag levels to total whole blood FIX content, the results demonstrated that 96.32 ± 0.32% of whole blood FIX in the BG/BCNU treated animals was stored in platelets. To assess whether the bleeding phenotype was rescued in HB mice after receiving 2bF9/MGMT LV-transduced HSCs, we used a 6-hour tail bleeding test. All 2bF9/MGMT LV-transduced recipients stopped bleeding within 6 hours with a clotting time of 2.6 ± 0.5 hours and a remaining hemoglobin level of 65.1 ± 4.9%, which were not significantly different from those of the wild-type control group (1.7 ± 0.9 hours and 70.6 ± 13.9%). In contrast, none of the FIXnull mice stopped bleeding within 6 hours with a remaining hemoglobin level of 38.8 ± 6.7%.Notably, none of the recipients developed anti-FIX inhibitory antibodies as measured by a modified Bethesda assay. To investigate whether immune tolerance was induced in 2bF9/MGMT LV-transduced recipients, all the recipients were challenged with 200U/kg recombinant human FIX (rhFIX) in the presence of Incomplete Freund's adjuvant (IFA) at 9 months after transplantation. Only one out of four recipients developed a low titer of inhibitors (1.3 BU/ml). In contrast, all of the HB controls developed inhibitors ranging from 17-120 BU/ml after the same challenge (n = 4). When we used the ELISA assay for the quantification of total anti-FIX IgG antibodies, low titer antibodies were found in the recipients after they were challenged with rhFIX plus IFA. However, the antibody levels in the HB control mice under the same conditioning were 213-fold higher than those developed in 2bF9 MGMT LV-transduced recipients.In conclusion, our data demonstrated that using the MGMT-mediated drug-selection system in 2bF9 gene therapy can significantly enhance therapeutic platelet-FIX expression, resulting in sustained phenotypic correction and immune suppression in HB mice. Our studies suggest that platelet-targeted FIX gene therapy together with in vivo enrichment of engineered cells may provide a promising strategy for the treatment of HB patients. DisclosuresNo relevant conflicts of interest to declare.

Joint status in Spanish haemophilia B patients assessed using the Haemophilia Early Arthropathy Detection with Ultrasound (HEAD-US) score.

The use of musculoskeletal ultrasound (MSK-US) following protocols for haemophilic arthropathy and the Haemophilia Early Arthropathy Detection with Ultrasound (HEAD-US) score can help standardize monitoring in haemophilia. This study evaluated the joint status (elbows, knees and ankles) of patients with haemophilia B (HB) in Spain using MSK-US and the HEAD-US score. Haemophilia B patients ≥14years old were included in this observational, multicentre, cross-sectional study, regardless of their clinical condition, HB severity and treatment received. Two blinded observers were involved in image acquisition and scoring in each centre. Eighty-two patients from 12 centres were enrolled: 27% mild HB, 23% moderate, 50% severe HB. Mean age was 38.9±16.4years, 60% were treated on demand (OD) and 40% were on prophylaxis. HEAD-US was zero in all joints in 28.6% OD patients and 36.4% on prophylaxis. Mean scores significantly worsened with HB severity, except for the left knee. Patients on primary and secondary prophylaxis had significantly better joint health vs OD patients in all joints, except the right ankle. Among OD patients, those with severe disease presented significantly worse scores in all HEAD-US items related to permanent damage. Joint status of HB patients in Spain is influenced by severity and treatment modality, related to the development of arthropathy, which appears prevalent in OD patients with severe HB. Routine assessment with an imaging tool such as ultrasound and HEAD-US system may help to improve joint health by personalizing and adjusting treatment in this population.

Mutational Profiles of F8 and F9 in a Cohort of Haemophilia A and Haemophilia B Patients in the Multi-ethnic Malaysian Population.

BackgroundHaemophilia A (HA) and Haemophilia B (HB) are X-linked blood disorders that are caused by various mutations in the factor VIII (F8) and factor IX (F9) genes respectively. Identification of mutations is essential as some of the mutations are associated with the development of inhibitors. This study is the first comprehensive study of the F8 mutational profile in Malaysia.Materials and methodsWe analysed 100 unrelated HA and 15 unrelated HB patients for genetic alterations in the F8 and F9 genes by using the long-range PCR, DNA sequencing, and the multiplex-ligation-dependent probe amplification assays. The prediction software was used to confirm the effects of these mutations on factor VIII and IX proteins.Results44 (53%) of the severe HA patients were positive for F8 intron 22 inversion, and three (3.6%) were positive for intron one inversion. There were 22 novel mutations in F8, including missense (8), frameshift (9), splice site (3), large deletion (1) and nonsense (1) mutations. In HB patients, four novel mutations were identified including the splice site (1), small deletion (1), large deletion (1) and missense (1) mutation.DiscussionThe mutational spectrum of F8 in Malaysian patients is heterogeneous, with a slightly higher frequency of intron 22 inversion in these severe HA patients when compared to other Asian populations. Identification of these mutational profiles in F8 and F9 genes among Malaysian patients will provide a useful reference for the early detection and diagnosis of HA and HB in the Malaysian population.

Real-world comparative analysis of bleeding complications and health-related quality of life in patients with haemophilia A and haemophilia B.

Clinical severity and impact of haemophilia on quality of life have been generally considered to be lower for haemophilia B (HB) compared with haemophilia A (HA) patients. To compare annual bleeding rate (ABR), target joint development and health-related quality of life (HRQoL) between adult (≥18years) severe HA and HB patients using recent data from the Cost of Haemophilia in Europe: a Socioeconomic Survey (CHESS) study. Multivariate generalized linear models (GLM) were constructed to assess the relationship between haemophilia type, ABR, HRQoL (derived from EQ-5D index scores) and the presence of target joints while controlling for covariates. Of the 1225 patients included, 77% (n=949) had HA and 23% (n=278) had HB. Of the 514 patients who completed the EQ-5D, 78% (n=405) had HA, and 22% (n=110) had HB. Unadjusted mean ABR was 3.79 in HA and 4.60 in HB. The presence of ≥1 target joint was reported in 59% and 54% of patients with HA and HB, respectively. Unadjusted mean EQ-5D index score was 0.78 in HA and 0.76 in HB. Haemophilia type was not a significant predictor of ABR, target joints or HRQoL when adjusted for confounding factors such as BMI, age and replacement therapy regimen. Data suggest comparable ABR, incidence of target joints and HRQoL between patients with HB and HA indicating comparable clinical severity and disease impact on patient quality of life.

Recurrent F8 and F9 gene variants result from a founder effect in two large French haemophilia cohorts.

Haemophilia A (HA) and haemophilia B (HB) are X-linked recessive diseases, caused by a large number of pathogenic variants in the F8 and F9 genes. With the exception of introns 22 and 1 inversions which are frequent in severe HA cases, about 2000 unique variants in F8 and 1000 in F9 have been described in databases and their recurrence remains limited. During routine analysis, we identified two recurrent missense variants, the F8 gene c.1244C>T, p.Ala415Val variant in 27 HA patients and the F9 gene c.835G>A, p.Ala279Thr variant in 34 HB patients, in two groups of haemophiliac patients from two different regions of France. We aimed to identify whether these variants result from a founder effect. We performed haplotype reconstruction after analysis of extragenic and intragenic polymorphic markers. The ESTIAGE programme was used to estimate the age of the variant. We identified a common ancestral haplotype HA1, in all the HA patients sharing the p.Ala415Val variant, and HB1 for 22 of 34 HB patients sharing the p.Ala279Thr variant. The estimated time of occurrence of the founder variant was between the 13th and 17th century (95% CI: 16 to 29 generations) for the F8 variant and between the 3rd and the 11th century for the F9 variant (95% CI: 44 to 72 generations). This study supports a founder effect for these two variants in the two largest reported cohorts of haemophilia patients with an identical variant. These pathogenic variants are among the three most early reported variants in haemophilia.

High resolution melting for F9 gene mutation analysis in patients with haemophilia B.

Since 2001, we have used conformation sensitive gel electrophoresis (CSGE) as our first choice for F9 gene mutation screening, leading to the identification of about 300 mutations causing haemophilia B (HB). To circumvent the disadvantages of CSGE, we recently evaluated high-resolution melting analysis (HRM), which represents the next-generation mutation scanning technology. In order to explore and validate HRM as a new screening method, we analysed 26 HB patients with previous CSGE-detected mutations, 22 patients with CSGE-undetectable mutations and 13 HB patients who had not been previously investigated. All 61 investigated samples, including the previously investigated and not previously investigated samples, proved to be HRM-positive, with the new screening method showing good efficiency and higher sensitivity than the previously used method. Mixing normal and unknown DNA to generate heterozygote conditions proved an excellent strategy to push the detection performance to its maximum. Mutation scanning by HRM analysis seems to be ideal in our context because it is rapid, cheap and capable of detecting the vast majority of mutations in HB patients. Nevertheless, to improve the detection ability of this scanning technology, it is recommended to start with a good strategy, based on good quality samples and optimised polymerase chain reaction amplification parameters, especially regarding primers and length of the amplicons.

Australian multicentre study of current real-world prophylaxis practice in severe and moderate haemophilia A and B.

With the emergence of novel treatment products for haemophilia and an increasing focus on the benefits of pharmacokinetic driven individualized prophylaxis, robust national data with regard to current patterns of factor consumption and adherence are required. To characterize current Australian practice with regard to use of prophylactic clotting factor infusions in patients with moderate or severe haemophilia A (HA) and haemophilia B (HB). This was a retrospective, non-interventional study utilizing Australian Bleeding Disorder Registry (ABDR) data collected over a 12month period. Registered and consented patients with moderate or severe HA or HB without inhibitors were included. A total of 718 HA (551 severe, 167 moderate) and 166 HB (87 severe, 79 moderate) patients were included. Regular prophylaxis was prescribed in 453 patients (82%) with severe HA, 42 patients (25%) with moderate HA, 66 patients (75%) with severe HB and 11 patients (14%) with moderate HB. Near universal prophylaxis was achieved in the paediatric subgroup. The mean weekly dose of factor VIII in severe HA was 84 international units/kg/wk (IU/kg/wk) vs 71 IU/kg/wk of factor IX in severe HB. Most patients on prophylaxis were treated ≥3times/wk (HA) or 2times/wk (HB). Non-adherence peaked in the 20-29year age group. Older individuals on regular prophylaxis used more factor than was expected for their prescribed regimen. Prophylaxis rates in severe haemophilia are comparable with other developed nations. The benefit of a national registry is demonstrable. Furthermore research into the underlying reasons for non-compliance in young adults with haemophilia is required.

Mutations causing hemophilia B in Algeria: Identification of two novel mutations of the factor 9 gene

Abla Z, Mouloud Y, Hejer El, Emna G, Abdi Meriem A, Ouarhlent Yamina O, Naouel S. 2018. Mutations causing hemophilia B in Algeria: Identification of two novel mutations of the factor 9 gene. Biodiversitas 19: 52-58. Hemophilia B (HB) (also known as Christmas disease; Christmas is the family name of the first patient.) is an X linked recessive hemorrhagic disorder caused by mutations in factor 9 (F9: is used for the gene) gene that leads to deficient or defective coagulation factor IX (FIX: is used for the protein). The variable phenotype of HB results from wide range of mutations affecting the F9 gene. Our study was aimed at molecular analysis of HB to identify the causative mutation in known patients with HB in a part of Algeria. For genotyping, polymerase chain reaction (PCR) and direct sequencing have been applied to all the essential regions of the F9 gene from 39 Algerian HB patients belonging to 13 unrelated families. We identified 10 different mutations. The identified mutations included 1 duplication and 9 substitutions. In total 9 point mutations were identified, of which 5 are located in exon 8, the hotspot region in the F9 gene. Among the 10 mutations, 2 are novel and not deposited in database sites nor described in recently published articles. The results of this study emphasize the heterogeneity of HB. In summary, our preliminary results will be used to build an Algerian mutation database which would facilitate genetic counseling.

Inhibitors in Hemophilia: Treatment Challenges and Novel Options.

Hemophilia A (HA) and hemophilia B (HB) are rare congenital severe bleeding disorders, that may be controlled by proper administration of adequate prophylaxis with factor VIII (FVIII), and factor IX (FIX) concentrates, respectively, to prevent joint damage due to recurrent bleeding. However, approximately 30% of patients develop inhibitory antibodies that render factor replacement therapy ineffective. Due to the high variability of patients' bleeding tendency, there is a need to "individually tailor treatment" for this unique group of patients. While replacement therapy with FVIII or FIX can be used for treating HA or HB patients with low responding inhibitors, hemophilia patients with high-responding inhibitors are treated with bypassing agents. Unfortunately, the Bethesda assay applied for inhibitor measurement in most laboratories does not fully predict either bleeding tendency or therapy response. Immune tolerance induction (ITI) may eradicate most inhibitors, yet treatment is challenging during bleeding episodes. The role of bypassing agents and their various treatment strategies still deserves attention. Recently, new nonreplacement therapies have emerged for patients with hemophilia including patients with inhibitors. Adequate monitoring of bypassing therapy and of the new nonreplacement therapies in inhibitor patients is extremely challenging, thus global hemostatic assays are increasingly used to assess clot formation. This review aims to summarize the current treatment and monitoring challenges for inhibitor patients; in this perspective, we will discuss our institutional approach for optimal decision-making and individual therapy tailoring.