Current and emerging gene therapies for haemophilia A and B.

INTRODUCTION: After decades of stumbling clinical development, the first gene therapies for haemophilia A and B have been commercialized and have normalized factor (F)VIII and factor (F)IX levels in some individuals in the long term. Several other clinical programs testing adeno-associated viral (AAV) vector gene therapy are at various stages of clinical testing. DISCUSSION: Multiyear follow-up in phase 1/2 and 3 studies showed long-term and sometimes curative but widely variable and unpredictable efficacy. Liver toxicities, mostly low-grade, occur in the 1st year in at least some individuals in all haemophilia A and B trials and are poorly understood. Wide variability and unpredictability of outcome and slow decline of FVIII levels are a major disadvantage because immune responses to AAV vectors preclude repeat dosing, which otherwise could improve suboptimal or restore declining expression, while overexpression may predispose to thrombosis. Long-term safety outcomes will need lifelong monitoring because AAV vectors infused at high doses integrate into chromosomes at rates that raise questions about potential oncogenicity and necessitate vigilance. Alternative gene transfer systems employing gene editing and/or non-viral vectors are under development and promise to overcome some limitations of the current state of the art for both haemophilia A and B. CONCLUSIONS: AAV gene therapies for haemophilia have now become new treatment options but not universal cures. AAV is a powerful but imperfect gene transfer platform. Biobetter FVIII transgenes may help solve some problems plaguing gene therapy for haemophilia A. Addressing variability and unpredictability of efficacy, and delivery of gene therapy to ineligible patient subgroups may require different gene transfer systems, most of which are not ready for clinical translation yet but bring innovations needed to overcome the current limitations of gene therapy.

Efanesoctocog Alfa Prophylaxis for Patients with Severe Hemophilia A.

BACKGROUND: Efanesoctocog alfa provides high sustained factor VIII activity by overcoming the von Willebrand factor-imposed half-life ceiling. The efficacy, safety, and pharmacokinetics of efanesoctocog alfa for prophylaxis and treatment of bleeding episodes in previously treated patients with severe hemophilia A are unclear. METHODS: We conducted a phase 3 study involving patients 12 years of age or older with severe hemophilia A. In group A, patients received once-weekly prophylaxis with efanesoctocog alfa (50 IU per kilogram of body weight) for 52 weeks. In group B, patients received on-demand treatment with efanesoctocog alfa for 26 weeks, followed by once-weekly prophylaxis with efanesoctocog alfa for 26 weeks. The primary end point was the mean annualized bleeding rate in group A; the key secondary end point was an intrapatient comparison of the annualized bleeding rate during prophylaxis in group A with the rate during prestudy factor VIII prophylaxis. Additional end points included treatment of bleeding episodes, safety, pharmacokinetics, and changes in physical health, pain, and joint health. RESULTS: In group A (133 patients), the median annualized bleeding rate was 0 (interquartile range, 0 to 1.04), and the estimated mean annualized bleeding rate was 0.71 (95% confidence interval [CI], 0.52 to 0.97). The mean annualized bleeding rate decreased from 2.96 (95% CI, 2.00 to 4.37) to 0.69 (95% CI, 0.43 to 1.11), a finding that showed superiority over prestudy factor VIII prophylaxis (P<0.001). A total of 26 patients were enrolled in group B. In the overall population, nearly all bleeding episodes (97%) resolved with one injection of efanesoctocog alfa. Weekly prophylaxis with efanesoctocog alfa provided mean factor VIII activity of more than 40 IU per deciliter for the majority of the week and of 15 IU per deciliter at day 7. Prophylaxis with efanesoctocog alfa for 52 weeks (group A) improved physical health (P<0.001), pain intensity (P = 0.03), and joint health (P = 0.01). In the overall study population, efanesoctocog alfa had an acceptable side-effect profile, and the development of inhibitors to factor VIII was not detected. CONCLUSIONS: In patients with severe hemophilia A, once-weekly efanesoctocog alfa provided superior bleeding prevention to prestudy prophylaxis, normal to near-normal factor VIII activity, and improvements in physical health, pain, and joint health. (Funded by Sanofi and Sobi; XTEND-1 ClinicalTrials.gov number, NCT04161495.).

Differences in wild-type- and R338L-tenase complex formation are at the root of R338L-factor IX assay discrepancies.

Adeno-associated virus (AAV) gene therapy has the potential to functionally cure hemophilia B by restoring factor (F)IX concentrations into the normal range. Next-generation AAV therapies express a naturally occurring gain-of-function FIX variant, FIX-Padua (R338L-FIX), that increases FIX activity (FIX:C) by approximately eightfold compared with wild-type FIX (FIX-WT). Previous studies have shown that R338L-FIX activity varies dramatically across different clinical FIX:C assays, which complicates the monitoring and management of patients. To better understand mechanisms that contribute to R338L-FIX assay discrepancies, we characterized the performance of R338L-FIX in 13 1-stage clotting assays (OSAs) and 2 chromogenic substrate assays (CSAs) in a global field study. This study produced the largest R338L-FIX assay dataset to date and confirmed that clinical FIX:C assay results vary over threefold. Both phospholipid and activating reagents play a role in OSA discrepancies. CSA generated the most divergent FIX:C results. Manipulation of FIX:C CSA kits demonstrated that specific activity gains for R338L-FIX were most profound at lower FIX:C concentrations and that these effects were enhanced during the early phases of FXa generation. Supplementing FX into CSA had the effect of dampening FIX-WT activity relative to R338L-FIX activity, suggesting that FX impairs WT tenase formation to a greater extent than R338L-FIX tenase. Our data describe the scale of R338L-FIX assay discrepancies and provide insights into the causative mechanisms that will help establish best practices for the measurement of R338L-FIX activity in patients after gene therapy.

Managing surgery in hemophilia with recombinant factor VIII Fc and factor IX Fc: Data on safety and effectiveness from phase 3 pivotal studies.

Background: Surgical procedures impose hemostatic risk to people with hemophilia, which may be minimized by optimal factor (F) replacement therapy. Methods: This analysis evaluates the efficacy and safety of extended half-life factor replacement recombinant FVIII and FIX Fc fusion proteins (rFVIIIFc and rFIXFc) during surgery in phase 3 pivotal (A-LONG/Kids A-LONG and B-LONG/Kids B-LONG) and extension (ASPIRE and B-YOND) studies. Dosing regimens were determined by investigators. Injection frequency, dosing, blood loss, transfusions, and hemostatic response were assessed. Results: Forty-five major (n = 31 subjects) and 90 minor (n = 70 subjects) procedures were performed in hemophilia A; 35 major (n = 22) and 62 minor (n = 37) procedures were performed in hemophilia B. Unilateral knee arthroplasty was the most common major orthopedic procedure (hemophilia A: n = 15/34; hemophilia B: n = 8/24). On the day of surgery, median total dose in adults/adolescents was 81 IU/kg for rFVIIIFc and 144 IU/kg for rFIXFc; most major procedures required ≤2 injections (including loading dose). Through days 1-14, most major procedures had ≤1 injection/day. Hemostasis was rated excellent (rFVIIIFc: n = 39/42; rFIXFc: n = 29/33) or good (n = 3/42; n = 4/33) in evaluable major surgeries, with blood loss comparable with subjects without hemophilia. Most minor procedures in adults/adolescents required one injection on the day of surgery, including median loading dose of 51 IU/kg (rFVIIIFc) and 80 IU/kg (rFIXFc). No major treatment-related safety concerns were identified. No subjects developed inhibitors or serious vascular thromboembolic events. Conclusions: rFVIIIFc and rFIXFc were efficacious and well tolerated for the management of perioperative hemostasis across a wide spectrum of major and minor surgeries in hemophilia.

Phase 1-2 Trial of AAVS3 Gene Therapy in Patients with Hemophilia B.

BACKGROUND: FLT180a (verbrinacogene setparvovec) is a liver-directed adeno-associated virus (AAV) gene therapy that uses a synthetic capsid and a gain-of-function protein to normalize factor IX levels in patients with hemophilia B. METHODS: In this multicenter, open-label, phase 1-2 trial, we assessed the safety and efficacy of varying doses of FLT180a in patients with severe or moderately severe hemophilia B (factor IX level, ≤2% of normal value). All the patients received glucocorticoids with or without tacrolimus for immunosuppression to decrease the risk of vector-related immune responses. After 26 weeks, patients were enrolled in a long-term follow-up study. The primary end points were safety and efficacy, as assessed by factor IX levels at week 26. RESULTS: Ten patients received one of four FLT180a doses of vector genomes (vg) per kilogram of body weight: 3.84×1011 vg, 6.40×1011 vg, 8.32×1011 vg, or 1.28×1012 vg. After receiving the infusion, all the patients had dose-dependent increases in factor IX levels. At a median follow-up of 27.2 months (range, 19.1 to 42.4), sustained factor IX activity was observed in all the patients except one, who resumed factor IX prophylaxis. As of the data-cutoff date (September 20, 2021), five patients had normal factor IX levels (range, 51 to 78%), three patients had levels from 23 to 43%, and one had a level of 260%. Of the reported adverse events, approximately 10% were related to FLT180a and 24% to immunosuppression. Increases in liver aminotransferase levels were the most common FLT180a-related adverse events. Late increases in aminotransferase levels occurred in patients who had received prolonged tacrolimus beyond the glucocorticoid taper. A serious adverse event of arteriovenous fistula thrombosis occurred in the patient with high factor IX levels. CONCLUSIONS: Sustained factor IX levels in the normal range were observed with low doses of FLT180a but necessitated immunosuppression with glucocorticoids with or without tacrolimus. (Funded by Freeline Therapeutics; ClinicalTrials.gov numbers, NCT03369444 and NCT03641703; EudraCT numbers, 2017-000852-24 and 2017-005080-40.).

Gene therapy: Practical aspects of implementation.

The first wave of gene therapies for haemophilia submitted for regulatory review utilize a liver-directed approach in which a functional gene copy of factor VIII (FVIII) or factor IX (FIX) is packaged inside a recombinant adeno-associated viral vector (rAAV). Following a single treatment event, these particles are taken up into liver cells, where the rAAV uncoats and delivers the DNA to the nucleus of the cell, where genetic elements that accompany the gene allow for efficient expression and secretion of FVIII or FIX protein into the plasma. An immune response to the vector capsid has been manifest by elevations in common liver enzymes that must be diligently followed postinfusion for weeks and months afterward and if signs of toxicity appear, will trigger a course of immunosuppression. Despite this, the studies have shown that this works in the great majority of individuals and the immunosuppression course is either avoided or short-lived for many. Optimal outcomes in the haemophilia population will be dependent on proper screening assessment and maintenance of liver health prior to consideration of gene therapy, close short-term follow up and implementation of immunomodulatory strategies to identify and manage liver toxicity and preserve durable transgene expression. This review proposes best practices to assist clinical teams with overcoming the challenges this platform of therapy poses to the traditional clinical care models and infrastructure within the haemophilia treatment centres (HTCs) who will be coordinating the patient's journey through this potentially transformative therapy.

Multidisciplinary Team Care of Patients with Hemophilic Arthropathy: A Qualitative Assessment of Contemporary Practice in the UK and Canada : Canada/UK: MDT Practices for Hemophilia.

We used a structured interview to explore approaches to comprehensive hemophilia and arthropathy care among 24 healthcare professionals (HCPs) from multidisciplinary teams (MDTs) in Canada and the UK. Represented MDTs typically comprise a hematologist, nurse, physiotherapist, and sometimes an orthopedic surgeon; pediatric (and some adult) MDTs also include a social worker/psychologist. HCPs emphasized the centrality of a team approach, facilitated through MDT meetings and involvement of all MDT members in patient care. In both countries, nurses and physiotherapists play critical, multifaceted roles. Respondents agreed that MDTs are crucial for successful transitioning, which can be facilitated by close collaboration between pediatric and adult MDTs, even when they are not co-located. Physiotherapists are instrumental in providing non-pharmacological pain relief. Hematologists or physiotherapists typically make orthopedic referrals, with the nurse, physiotherapist and hematologist working together in patient preparation for (and follow-up after) surgery. MDT best practices include a non-hierarchical team approach, ensuring that all MDT members know all patients, and regular MDT meetings. Together, these real-life insights from the MDT perspective emphasize the value of the MDT approach in comprehensive hemophilia care.

Clinical outcomes after joint surgery in patients on turoctocog alfa pegol (N8-GP) prophylaxis: A post hoc analysis.

INTRODUCTION: Joint damage in haemophilia often requires surgical correction. However, the surgery effect on bleeding rates and other clinical joint outcomes can be unclear. AIM: To investigate the effects of joint surgery on joint annualized bleeding rates (JABRs) and physical health outcomes in patients with haemophilia A undergoing N8-GP prophylaxis. METHODS: Patients in the pathfinder 2 trial received N8-GP prophylaxis, enrolling in the pathfinder 3 trial for indicated surgery. Patients returned to pathfinder 2 post-surgery, continuing N8-GP prophylaxis until end-of-trial. JABRs were calculated from bleeding across all joints for pre-surgery (immediately before surgery) and post-surgery (to pathfinder 2 study end) periods. Joint-health-related outcomes were derived from patient records. RESULTS: Data (41 joint surgeries; n = 30) were analysed statistically using datamining and descriptively. Pre-surgery mean JABR was higher in patients who later were operated than in 146 non-operated patients (p = .004). In operated patients, mean JABR decreased from 1.33 pre-surgery to .37 post-surgery (p = .011). In all but three patients, JABR improved or remained the same post-surgery. In the three patients whose JABR remained at one (all with multiple joint arthropathy), post-surgery bleeds were mostly at non-operated sites. Two of the three patients whose JABR increased post-surgery had undergone surgery for reasons unlikely to improve JABR. Mobility parameters often improved in patients whose JABR remained at zero. CONCLUSION: Patients with haemophilia treated with N8-GP prophylaxis benefit from surgeries. However, this analysis could not differentiate the relative contributions of surgical interventions and prophylactic treatment to the improvement of JABR.

Delivery of AAV-based gene therapy through haemophilia centres-A need for re-evaluation of infrastructure and comprehensive care: A Joint publication of EAHAD and EHC.

INTRODUCTION: Adeno-associated virus (AAV)-based gene therapy for haemophilia presents a challenge to the existing structure of haemophilia centres and requires a rethink of current collaboration and information exchange with the aim of ensuring a system that is fit-for-purpose for advanced therapies to maximise benefits and minimise risks. In Europe, a certification process based on the number of patients and facilities is offered to the haemophilia centres by European Haemophilia Network (EUHANET). AIM AND METHODS: This joint European Association for Haemophilia and Allied Disorders (EAHAD) and European Haemophilia Consortium (EHC) publication describes criteria for centres participating in gene therapy care that require a reassessment of the infrastructure of comprehensive care and provides an outlook on how these criteria can be implemented in the future work of haemophilia centres. RESULTS: The core definition of a haemophilia treatment centre remains, but additional roles could be implemented. A modifiable 'hub-and-spoke' model addresses all aspects associated with gene therapy, including preparation and administration of the gene therapy product, determination of coagulation and immunological parameters, joint score and function, and liver health. This will also include the strategy on how to follow-up patients for a long-term safety and efficacy surveillance. CONCLUSION: We propose a modifiable, networked 'hub and spoke' model with a long term safety and efficacy surveillance system. This approach will be progressively developed with the goal of making haemophilia centres better qualified to deliver gene therapy and to make gene therapy accessible to all persons with haemophilia, irrespective of their country or centre of origin.

Validation of a perioperative population factor VIII pharmacokinetic model with a large cohort of pediatric hemophilia a patients.

AIMS: Population pharmacokinetic (PK) models are increasingly applied to perform individualized dosing of factor VIII (FVIII) concentrates in haemophilia A patients. To guarantee accurate performance of a population PK model in dose individualization, validation studies are of importance. However, external validation of population PK models requires independent data sets and is, therefore, seldomly performed. Therefore, this study aimed to validate a previously published population PK model for FVIII concentrates administrated perioperatively. METHODS: A previously published population PK model for FVIII concentrate during surgery was validated using independent data from 87 children with severe haemophilia A with a median (range) age of 2.6 years (0.03-15.2) and body weight of 14 kg (4-57). First, the predictive performance of the previous model was evaluated with MAP Bayesian analysis using NONMEM v7.4. Subsequently, the model parameters were (re)estimated using a combined dataset consisting of the previous modelling data and the data available for the external validation. RESULTS: The previous model underpredicted the measured FVIII levels with a median of 0.17 IU mL-1 . Combining the new, independent and original data, a dataset comprising 206 patients with a mean age of 7.8 years (0.03-77.6) and body weight of 30 kg (4-111) was obtained. Population PK modelling provided estimates for CL, V1, V2, and Q: 171 mL h-1  68 kg-1 , 2930 mL 68 kg-1 , 1810 mL 68 kg-1 , and 172 mL h-1  68 kg-1 , respectively. This model adequately described all collected FVIII levels, with a slight median overprediction of 0.02 IU mL-1 . CONCLUSIONS: This study emphasizes the importance of external validation of population PK models using real-life data.

Turoctocog alfa pegol provides effective management for major and minor surgical procedures in patients across all age groups with severe haemophilia A: Full data set from the pathfinder 3 and 5 phase III trials.

INTRODUCTION: Turoctocog alfa pegol is a glycoPEGylated recombinant factor VIII (FVIII) with an extended half-life developed for prophylaxis, treatment of bleeds and perioperative management in patients with haemophilia A. AIM: Evaluate the efficacy and safety of turoctocog alfa pegol treatment for major and minor surgeries in the pathfinder 3 and 5 phase III trials. METHODS: Adults/adolescents aged ≥12 years with severe haemophilia A (FVIII <1%) received perioperative turoctocog alfa pegol treatment planned to achieve FVIII activity levels >80% during major surgery (pathfinder 3). The primary end point was haemostatic efficacy during surgery; secondary end points were blood loss, haemostatic effect postsurgery, consumption, transfusions, safety and health economics. Children (0-11 years) undergoing minor surgeries received 20-75 IU/kg turoctocog alfa pegol at Investigator's discretion (pathfinder 5). RESULTS: pathfinder 3 included 35 patients undergoing 49 major surgeries. Haemostasis was successful in 47/49 (95.9%) surgeries; two had moderate haemostatic responses. Median (mean) blood loss during major surgery was 75 (322.6) mL. Four bleeds were reported postsurgery; three were successfully treated with turoctocog alfa pegol (one was not evaluated). On the day of surgery, overall mean (median) dose was 75.5 (74.5) IU/kg and mean (median) number of doses was 1.7 (2.0). Five procedures required 11 transfusions on the day of surgery or days 1-6. No safety concerns or inhibitors were identified. Forty-five minor surgeries in 23 children were performed without complications. CONCLUSION: Turoctocog alfa pegol was effective for perioperative haemostatic management of major and minor surgeries in patients across age groups with severe haemophilia A.

SHP656, a polysialylated recombinant factor VIII (PSA-rFVIII): First-in-human study evaluating safety, tolerability and pharmacokinetics in patients with severe haemophilia A.

INTRODUCTION: SHP656 is the first factor VIII (FVIII) product developed using polysialylation (PSA) technology, in which full-length recombinant (r) FVIII (anti-haemophilic factor [recombinant]) is conjugated with a 20 kDa PSA polymer. AIM: To compare the safety, immunogenicity and pharmacokinetics of SHP656 vs the parent rFVIII (octocog alfa) after single infusions of 25-75 IU/kg in patients with severe haemophilia A (FVIII activity <1%). METHODS: Multinational, phase 1, prospective, open-label, two-period, fixed-sequence, dose-escalation trial (clinicaltrials.gov NCT02716194). Patients received single doses of rFVIII and then SHP656 sequentially at the same dose: 25 ± 3 IU/kg (Cohort 1), 50 ± 5 IU/kg (Cohort 2) and 75 ± 5 IU/kg (Cohort 3). RESULTS: Forty patients received rFVIII: 11 in Cohort 1, 16 in Cohort 2 and 13 in Cohort 3. Two patients withdrew before receiving SHP656, leaving 38 patients who completed the study and received both treatments. No treatment-related adverse events (AEs), serious AEs, deaths, study withdrawals, thrombotic events or allergic reactions were reported; and no significant treatment-related changes in laboratory parameters or vital signs. No patients developed FVIII inhibitors or antibodies to PSA. FVIII activity was significantly prolonged following SHP656 administration vs rFVIII with an approximately 1.5-fold extension in mean residence time (P < .05). Exposure increased proportional to the SHP656 dose over the 25-75 IU/kg dose range. CONCLUSION: Polysialylation of rFVIII confers a half-life extension similar to that of approved extended half-life products that use either PEGylation or Fc fusion technology and was not associated with any treatment-related adverse events.

Retrospective Review of a Prothrombin Complex Concentrate (Beriplex P/N) for the Management of Perioperative Bleeding Unrelated to Oral Anticoagulation.

A multicenter, retrospective, observational study of 4-factor prothrombin complex concentrate (PCC) and/or fresh frozen plasma (FFP) use within routine clinical care unrelated to vitamin K antagonists was conducted. The PCC was administered preprocedure for correction of coagulopathy (prophylactic cohort) and treatment of bleeding postsurgery (treatment cohort). Of the 445 patients included, 40 were in the prophylactic cohort (PCC alone [n = 16], PCC and FFP [n = 5], FFP alone [n = 19]) and 405 were in the treatment cohort (PCC alone [n = 228], PCC and FFP [n = 123], FFP alone [n = 54]). Cardiovascular surgery was the most common setting. PCC doses ranged between 500 and 5000 IU. Effectiveness (assessed retrospectively) was reported as effective in 93.0% in the PCC-only group (95% confidence interval, 89.1% to 95.9%), 78.9% (70.8% to 85.6%) with PCC and FFP, and 86.3% (76.2% to 93.2%) with FFP alone. In the treatment cohort, international normalized ratio was significantly reduced in all 3 groups. In patients who received PCC, the rate of thromboembolic events (1.9%) was below rates in the literature for similar procedures. PCCs offer a potential alternative to FFP in the management of perioperative bleeding unrelated to oral anticoagulant therapy.

Therapeutic and routine prophylactic properties of rFactor VIII Fc (efraloctocog alfa, Eloctate®) in hemophilia A.

rFVIIIFc (efraloctocog alfa, Eloctate®) is an extended half-life (EHL) factor VIII licensed for use in patients with hemophilia A for prophylaxis and treatment of bleeding and surgical episodes. Pharmacokinetic studies in adults have shown a mean 1.5-fold increase in half-life compared to full-length factor VIII. When compared to adults, the half-life is decreased by 8% in adolescents between 12 and 17 years, by 18% in children 6 to <12 years, and by 33% in children between the ages of 2 and <6 years. There is a considerable interindividual variation in the prolongation of the half-life particularly in children and across the age groups, the range extending from no increase to a 2.5-fold increase. In addition to age, von willebrand factor (VWF) antigen level has demonstrated a significant impact on rFVIIIFc half-life, with higher VWF levels associated with greater prolongation of half-life. The pivotal and pediatric clinical trials have demonstrated the efficacy and safety of rFVIIIFc for use in regular prophylaxis and in management of bleeds and surgery. In these studies, just under half the participants showed a zero annualized bleed rate (ABR), and the median ABR (1.6 in the pivotal study for the individualized prophylaxis arm) showed a further decrease in the extension study. On average, the patients required fewer infusions (reduced by at least a third), and the mean weekly consumption seems to be in keeping with standard recombinant factor VIII. EHL rFVIIIFc has made decreased infusion frequency a possibility. However, the interindividual variability in dose and infusion frequency highlights the need for a personalized approach based on individual patient's half-life and/or response to treatment.

Efficacy and safety of rVIII-SingleChain: results of a phase 1/3 multicenter clinical trial in severe hemophilia A.

Recombinant VIII (rVIII)-SingleChain is a novel B-domain-truncated recombinant factor VIII (rFVIII), comprised of covalently bonded factor VIII (FVIII) heavy and light chains. It was designed to have a higher binding affinity for von Willebrand factor (VWF). This phase 1/3 study investigated the efficacy and safety of rVIII-SingleChain in the treatment of bleeding episodes, routine prophylaxis, and surgical prophylaxis. Participants were ≥12 years of age, with severe hemophilia A (endogenous FVIII <1%). The participants were allocated by the investigator to receive rVIII-SingleChain in either an on-demand or prophylaxis regimen. Of the 175 patients meeting study eligibility criteria, 173 were treated with rVIII-SingleChain, prophylactically (N = 146) or on-demand (N = 27). The total cumulative exposure was 14 306 exposure days (EDs), with 120 participants reaching ≥50 EDs and 52 participants having ≥100 EDs. Hemostatic efficacy was rated by the investigator as excellent or good in 93.8% of the 835 bleeds treated and assessed. Across all prophylaxis regimens, the median annualized spontaneous bleeding rate was 0.00 (Q1, Q3: 0.0, 2.4) and the median overall annualized bleeding rate (ABR) was 1.14 (Q1, Q3: 0.0, 4.2). Surgical hemostasis was rated as excellent/good in 100% of major surgeries by the investigator. No participant developed FVIII inhibitors. In conclusion, rVIII-SingleChain is a novel rFVIII molecule showing excellent hemostatic efficacy in surgery and in the control of bleeding events, low ABR in patients on prophylaxis, and a favorable safety profile in this large clinical study. This trial was registered at www.clinicaltrials.gov as #NCT01486927.

Long-term safety and efficacy of factor IX gene therapy in hemophilia B.

BACKGROUND: In patients with severe hemophilia B, gene therapy that is mediated by a novel self-complementary adeno-associated virus serotype 8 (AAV8) vector has been shown to raise factor IX levels for periods of up to 16 months. We wanted to determine the durability of transgene expression, the vector dose-response relationship, and the level of persistent or late toxicity. METHODS: We evaluated the stability of transgene expression and long-term safety in 10 patients with severe hemophilia B: 6 patients who had been enrolled in an initial phase 1 dose-escalation trial, with 2 patients each receiving a low, intermediate, or high dose, and 4 additional patients who received the high dose (2×10(12) vector genomes per kilogram of body weight). The patients subsequently underwent extensive clinical and laboratory monitoring. RESULTS: A single intravenous infusion of vector in all 10 patients with severe hemophilia B resulted in a dose-dependent increase in circulating factor IX to a level that was 1 to 6% of the normal value over a median period of 3.2 years, with observation ongoing. In the high-dose group, a consistent increase in the factor IX level to a mean (±SD) of 5.1±1.7% was observed in all 6 patients, which resulted in a reduction of more than 90% in both bleeding episodes and the use of prophylactic factor IX concentrate. A transient increase in the mean alanine aminotransferase level to 86 IU per liter (range, 36 to 202) occurred between week 7 and week 10 in 4 of the 6 patients in the high-dose group but resolved over a median of 5 days (range, 2 to 35) after prednisolone treatment. CONCLUSIONS: In 10 patients with severe hemophilia B, the infusion of a single dose of AAV8 vector resulted in long-term therapeutic factor IX expression associated with clinical improvement. With a follow-up period of up to 3 years, no late toxic effects from the therapy were reported. (Funded by the National Heart, Lung, and Blood Institute and others; ClinicalTrials.gov number, NCT00979238.).

Unfavourable cardiovascular disease risk profiles in a cohort of Dutch and British haemophilia patients.

Cardiovascular disease (CVD) mortality is reported to be decreased in haemophilia patients, but reports on the prevalence of CVD risk factors are conflicting. A cross-sectional assessment of CVD risk profiles was performed in a large cohort of haemophilia patients. Baseline data on CVD risk factors of 709 Dutch and UK haemophilia patients aged ≥30 years were analysed and compared with the general age-matched male population. CVD risk profiles were assessed using the QRISK®2-2011 and SCORE algorithms. Although QRISK® 2 was only validated in the UK, comparison with SCORE indicated similar properties of QRISK®2 in both Dutch and UK patients (correlation 0.86). Mean age was 49.8 years. Hypertension was more common in haemophilia patients than in the general population (49% vs. 40%), while the prevalences of obesity and hypercholesterolaemia were lower (15 vs. 20% and 44 vs. 68%, respectively), and those of diabetes and smoking were similar. The predicted 10-year QRISK®2 risk was significantly higher in haemophilia patients than in the general population (8.9 vs. 6.7%), indicating more unfavourable cardiovascular disease risk profiles. This increased risk became apparent after the age of 40 years. Our results indicate an increased prevalence of hypertension and overall more unfavourable CVD risk profiles in haemophilia patients compared with the general age-matched male population.

Adenovirus-associated virus vector-mediated gene transfer in hemophilia B.

BACKGROUND: Hemophilia B, an X-linked disorder, is ideally suited for gene therapy. We investigated the use of a new gene therapy in patients with the disorder. METHODS: We infused a single dose of a serotype-8-pseudotyped, self-complementary adenovirus-associated virus (AAV) vector expressing a codon-optimized human factor IX (FIX) transgene (scAAV2/8-LP1-hFIXco) in a peripheral vein in six patients with severe hemophilia B (FIX activity, <1% of normal values). Study participants were enrolled sequentially in one of three cohorts (given a high, intermediate, or low dose of vector), with two participants in each group. Vector was administered without immunosuppressive therapy, and participants were followed for 6 to 16 months. RESULTS: AAV-mediated expression of FIX at 2 to 11% of normal levels was observed in all participants. Four of the six discontinued FIX prophylaxis and remained free of spontaneous hemorrhage; in the other two, the interval between prophylactic injections was increased. Of the two participants who received the high dose of vector, one had a transient, asymptomatic elevation of serum aminotransferase levels, which was associated with the detection of AAV8-capsid-specific T cells in the peripheral blood; the other had a slight increase in liver-enzyme levels, the cause of which was less clear. Each of these two participants received a short course of glucocorticoid therapy, which rapidly normalized aminotransferase levels and maintained FIX levels in the range of 3 to 11% of normal values. CONCLUSIONS: Peripheral-vein infusion of scAAV2/8-LP1-hFIXco resulted in FIX transgene expression at levels sufficient to improve the bleeding phenotype, with few side effects. Although immune-mediated clearance of AAV-transduced hepatocytes remains a concern, this process may be controlled with a short course of glucocorticoids without loss of transgene expression. (Funded by the Medical Research Council and others; ClinicalTrials.gov number, NCT00979238.).

Retinoid-induced apoptosis in B-cell chronic lymphocytic leukaemia cells is mediated through caspase-3 activation and is independent of p53, the retinoic acid receptor, and differentiation.

The aim of this study was to investigate the effects of all-trans retinoic acid (ATRA) on apoptosis induction, Bcl-2 family protein expression, and differentiation in B-cell chronic lymphocytic leukaemia (B-CLL) cells. ATRA induced apoptosis in all the B-CLL samples tested, and this was accompanied by a specific reduction in Bcl-2 and Mcl-1 protein expression in the apoptotic cells. In contrast, Bax, p21, and p53 expression was not altered in either the viable or apoptotic B-CLL cells, inferring that ATRA utilises a p53-independent cell death pathway. Caspase-3 activation was shown to be a prerequisite for ATRA-induced apoptosis, which was inhibited by the pan-caspase inhibitor Z-VAD-FMK and the caspase-9 inhibitor Z-LEHD-FMK. In addition, the retinoic acid receptor (RAR) antagonist AGN194310 failed to abrogate the apoptotic effects of ATRA, indicating that RAR binding was not necessary for ATRA-induced apoptosis. Furthermore, there was no evidence of ATRA-induced differentiation of the B-CLL cells in this study either in terms of altered morphology or immunophenotype. In summary these data indicate that ATRA induces apoptosis via the intrinsic apoptotic pathway, and this is independent of RAR binding, p53 activation, and cellular differentiation in B-CLL cells.