Hemophilia B and gene therapy: a new chapter with etranacogene dezaparvovec.

ABSTRACT: The US Food and Drug Administration (FDA)'s authorization of etranacogene dezaparvovec (Hemgenix) is a significant milestone, constituting not only the first FDA approval of a gene therapy for hemophilia but also the first approval of a liver-targeted adeno-associated virus vector gene therapy. This review summarizes the nonclinical studies and clinical development that supported regulatory clearance. Similar to other gene therapies for single gene disorders, both the short-term safety and the phenotypic improvement were unequivocal, justifying the modest-sized safety and efficacy database, which included 57 participants across the phase 2b (3 participants) and phase 3 (54 participants) studies. The most common adverse reactions included liver enzyme elevation, headache, flu-like symptoms, infusion-related reactions, creatine kinase elevation, malaise, and fatigue; these were mostly transient. One participant had hepatocellular carcinoma on a study-mandated liver ultrasound conducted 1 year after vector infusion; molecular analysis of the resected tumor showed no evidence of vector-related insertional mutagenesis as the etiology. A remarkable 96% of participants in the phase 3 trial were able to stop factor IX (FIX) prophylaxis, with the study demonstrating noninferiority to FIX prophylaxis in terms of the primary end point, annualized bleeding rate. Key secondary end points such as the annualized infusion rate, which declined by 97%, and the plasma FIX activity level at 18 months after infusion, with least squares mean increase of 34.3 percentage points compared with baseline, were both clinically and statistically significant. The FDA's landmark approval of Hemgenix as a pioneering treatment for hemophilia stands on the shoulders of >20 years of gene therapy clinical research and heralds a promising future for genomic medicines.

Use of Andexanet Alfa for Factor Xa Inhibitor Reversal in US Verified Trauma Centers: A National Survey.

Direct oral factor Xa inhibitors are replacing vitamin K-dependent antagonists as anticoagulation treatment in many clinical scenarios. Trauma centers are noting an increase in patients presenting on these medications. The 2018 Food and Drug Administration approval of andexanet alfa provides an alternative anticoagulation reversal. Barriers may limit utilization of new medications including a lack of grade 1A evidence supporting the use of prothrombin complex concentrate (PCC) versus andexanet alfa and cost. To evaluate barriers of andexanet alfa utilization by trauma surgeons, a 15-question survey was conducted. There was a 9% completion rate (n = 89). The results revealed 23.5% would choose andexanet alfa as first-line treatment in children, and 25.8% as first-line treatment in adults. The majority of respondents, 64.7% and 67.4%, would use PCC preferentially in children and adults, respectively. Respondents indicated that cost burden was an overriding factor (76.3%); 42.4% cited lack of high-level efficacy data of andexanet alfa for reversal of factor Xa inhibitors. Additional double-blinded multi-institutional randomized controlled trials comparing 4F-PCC and andexanet alfa for factor Xa inhibitor reversal are needed to support efficacy especially with the increased cost associated.

Utilization and safety of off-label prothrombin complex concentrate (four-factor prothrombin complex concentrate) in a surgical population.

The aim of this study is to evaluate and describe the utilization and safety of 4F-PCC in a nonanticoagulated, surgical patient population at an academic, tertiary care center. This retrospective, single-center chart review evaluated nonanticoagulated adult patients at least 18 years of age who had at least one dose of 4F-PCC administered between 1 January 2017 and 30 September 2022 for a surgical or peri-procedural indication. Hemostatic efficacy following 4F-PCC administration was the primary outcome, assessed by subsequent blood product administration and hemoglobin and hematocrit reduction. Secondary outcomes included an assessment of thrombotic events within 30 days post-4F-PCC administration, in-hospital mortality, and the length of hospital stay. A total of 71 patients met the inclusion criteria, with 61 patients receiving 4F-PCC for cardiac surgery and 10 patients for other intraoperative or peri-procedural indications. The mean total 4F-PCC dose was 25.0 U/kg. For the primary outcome of hemostatic efficacy, 81% of patients had excellent hemostasis; however, blood product administration was reported in 95.8% of patients post-4F-PCC. Thromboembolic events occurred in 10 (14.1%) patients and 21.1% of patients expired prior to discharge in the total cohort. Off-label 4F-PCC use in nonanticoagulated patients is reported despite a lack of robust guidance for use. Following 4F-PCC administration, hemostatic efficacy based on hemoglobin and hematocrit changes was observed; however, blood product use was frequent, and 4F-PCC administration was not without risks, including thromboembolic complications such deep vein thrombosis (DVT), pulmonary embolism, and stroke. Further studies are needed to validate the off-label administration of 4F-PCC in nonanticoagulated patients.

Etranacogene dezaparvovec gene therapy for haemophilia B (HOPE-B): 24-month post-hoc efficacy and safety data from a single-arm, multicentre, phase 3 trial.

BACKGROUND: Etranacogene dezaparvovec, the first gene therapy approved for haemophilia B treatment, was shown to be superior to treatment with continuous prophylactic factor IX in terms of bleeding protection 18 months after gene therapy in a phase 3 trial. We report post-hoc 24-month efficacy and safety data from this trial to evaluate the longer-term effects of etranacogene dezaparvovec in individuals with haemophilia B. METHODS: The phase 3 HOPE-B trial enrolled males aged 18 years or older with inherited haemophilia B, classified as severe (plasma factor IX activity level <1%) or moderately severe (plasma factor IX activity level ≥1% and ≤2%), with a severe bleeding phenotype and who were on stable continuous factor IX prophylaxis. Participants were treated with a single infusion of etranacogene dezaparvovec (2 × 1013 genome copies per kg of bodyweight). The primary endpoint, reported previously, was non-inferiority of the annualised bleeding rate (ABR) during the 52 weeks following stable factor IX expression (defined as months 7-18 after treatment) versus an at least 6-month lead-in period in which participants received their usual continuous factor IX prophylaxis, and is updated here up to month 24. Additional, post-hoc efficacy analyses, including adjusted ABR, factor IX activity, participants within factor IX ranges, and factor IX use, and safety analyses were performed at 24 months after gene therapy. Data were analysed in the full analysis set, which comprised the 54 patients who received at least a partial dose of gene therapy. The trial is ongoing and is registered with ClinicalTrials.gov, number NCT03569891. FINDINGS: The study began on June 27, 2018, and participants were treated between January, 2019, and March, 2020; the date of data cutoff was April 21, 2022. 54 adult males (40 White, two Asian, one Black or African American, 11 other or missing) received a single intravenous infusion of etranacogene dezaparvovec and were followed for a median of 26·51 months (IQR 24·54-27·99), after a lead-in period of 7·13 months (6·51-7·82). In the updated analysis comparing months 7-24 after gene therapy to the lead-in period, mean adjusted ABR significantly reduced from 4·18 to 1·51 (p=0·0002) for all bleeds and from 3·65 to 0·99 (p=0·0001) for factor IX-treated bleeds. During each 6-month period after gene therapy, at least 67% of participants experienced no bleeding (36 of 54 during months 0-6 and stable thereafter), compared with 14 (26%) of 54 during the lead-in period. 24 months after gene therapy, 1 (2%) participant had one-stage factor IX activity less than 5%, whereas 18 (33%) had factor IX activity more than 40% (non-haemophilia range), with mean factor IX activity stable and sustained at 36·7% (SD 19·0%). 52 (96%) of 54 participants expressed endogenous factor IX, remaining free of factor IX prophylaxis at month 24. No new safety concerns were identified and no treatment-related serious adverse events or treatment-related deaths occurred. The most common treatment-related adverse events were an increase in alanine aminotransferase (nine [17%] of 54 patients), headache (eight [15%]), influenza-like illness (seven [13%]), and an increase in aspartate aminotransferase (five [9%]). INTERPRETATION: By providing durable disease correction throughout the 24 months after gene therapy, etranacogene dezaparvovec provides a safe and effective therapeutic option for patients with severe or moderately severe haemophilia B. FUNDING: uniQure and CSL Behring.

Recombinant factor IX Fc for the treatment of hemophilia B.

Current hemophilia B treatment guidelines recommend routine prophylaxis with factor IX (FIX) replacement products, tailored to maintain plasma activity at levels that will prevent bleeds. However, plasma FIX activity may not be the primary determinant or best indicator of hemostatic efficacy due to its extravascular distribution. FIX replacement therapy has evolved to include extended half-life (EHL) products that provide effective bleed protection when administered at intervals of 7 days or longer. rFIXFc is a recombinant fusion protein with an extended circulation time. rFIXFc has a biodistribution profile consistent with distribution into extravascular space, where it may support hemostasis at sites of vessel injury independent of circulating plasma activity levels. The safety and efficacy of rFIXFc prophylaxis is well established in adults, adolescents and children including previously untreated patients with hemophilia B, with substantial evidence from clinical trials and real-world clinical practice. This review describes the pharmacokinetic characteristics of rFIXFc, summarizes available safety and efficacy data, and evaluates the use of rFIXFc in special populations. Current hemophilia B treatment challenges, including target FIX plasma levels, perioperative use, and management of patients with comorbidities, are discussed together with the potential role of EHL products in the future treatment landscape of hemophilia B.

Multiple venous thrombosis caused by F9 gene duplication and treated with catheter-directed thrombolysis, AngioJet-assisted pharmaco-mechanical thromboectomy and manual aspiration thromboectomy.

Inferior vena cava thrombosis (IVCT) is rare. Thrombophilia is one of the important risk factors. It is also uncommon for gene mutations in F9 gene to cause thrombosis but not hemorrhage. A 35-year-old male patient was admitted to our department with left lower limb swelling without an obvious cause for 1 day. Through contrast-enhanced computed tomography and color Doppler ultrasound, he was found to have lower extremity deep vein thrombosis, IVCT and pulmonary embolism. Through whole-exome sequencing analysis, he was found to carry a 925.7 kb duplication (chrX:137939698-138865419, hg19) encompassing ATP11C , SRD5A1P1 , MCF2 , FGF13 and F9 genes. This duplication of F9 gene was not detected in his parents. Other thrombophilic genes defects were not found. The factor IX activities of this patient, his father and mother were 194, 70 and 148, respectively. He was treated with catheter-directed thrombolysis, AngioJet-assisted pharmaco-mechanical thromboectomy and manual aspiration thromboectomy. Complete recanalization of left femoral, iliac veins and inferior vena cava was achieved. F9 gene duplication is a rare mutation, which can induce multiple venous thrombosis through increasing the activity level of factor IX in plasma. IVCT is a serious type of venous thrombosis. Personalized intervention treatment plans should be developed based on the different clinical characteristics of each case to achieve a higher benefit-risk ratio.

AAV mediated gene therapy for haemophilia B: From the early attempts to modern trials.

Early gene therapy clinical trials for the treatment of Haemophilia B have been instrumental to our global understanding of gene therapy and have significantly contributed to the rapid expansion of the field. The use of adeno-associated viruses (AAVs) as vectors for gene transfer has successfully led to therapeutic expression of coagulation factor IX (FIX) in severe haemophilia B patients. Expression of FIX has remained stable following a single administration of vector for up to 8 years at levels that are clinically relevant to reduce the incidence of spontaneous bleeds and have permitted a significant change in the disease management with reduction or elimination of the need for coagulation factor concentrates. These trials have also shed light on several concerns around AAV-mediated gene transfer such as the high prevalence of pre-existing immunity against the vector capsid as well as the elevation of liver transaminases that is associated with a loss of FIX transgene expression in some patients. However, this field is advancing very rapidly with the development of increasingly more efficient strategies to overcome some of these obstacles and importantly raise the possibility of a functional cure, which has been long sought after. This review overviews the evolution of gene therapy for haemophilia B over the last two decades.

Prothrombin Complex Concentrate vs Conservative Management in ICH Associated With Direct Oral Anticoagulants.

Importance: Intracerebral hemorrhage (ICH) associated with direct oral anticoagulant (DOAC) use carries extremely high morbidity and mortality. The clinical effectiveness of hemostatic therapy is unclear. Objective: To compare the clinical and radiological outcomes of DOAC-associated ICH treated with prothrombin complex concentrate (PCC) vs conservative management. Design, Setting, and Participants: In this population-based, propensity score-weighted retrospective cohort study, patients who developed DOAC-associated ICH from January 1, 2016, to December 31, 2021, in Hong Kong were identified. The outcomes of patients who received 25 to 50 IU/kg PCC with those who received no hemostatic agents were compared. Data were analyzed from May 1, 2022, to June 30, 2023. Main Outcomes and Measures: The primary outcome was modified Rankin scale of 0 to 3 or returning to baseline functional status at 3 months. Secondary outcomes were mortality at 90 days, in-hospital mortality, and hematoma expansion. Weighted logistic regression was performed to evaluate the association of PCC with study outcomes. In unweighted logistic regression models, factors associated with good neurological outcome and hematoma expansion in DOAC-associated ICH were identified. Results: A total of 232 patients with DOAC-associated ICH, with a mean (SD) age of 77.2 (9.3) years and 101 (44%) female patients, were included. Among these, 116 (50%) received conservative treatment and 102 (44%) received PCC. Overall, 74 patients (31%) patients had good neurological recovery and 92 (39%) died within 90 days. Median (IQR) baseline hematoma volume was 21.7 mL (3.6-66.1 mL). Compared with conservative management, PCC was not associated with improved neurological recovery (adjusted odds ratio [aOR], 0.62; 95% CI, 0.33-1.16; P = .14), mortality at 90 days (aOR, 1.03; 95% CI, 0.70-1.53; P = .88), in-hospital mortality (aOR, 1.11; 95% CI, 0.69-1.79; P = .66), or reduced hematoma expansion (aOR, 0.94; 95% CI, 0.38-2.31; P = .90). Higher baseline hematoma volume, lower Glasgow coma scale, and intraventricular hemorrhage were associated with lower odds of good neurological outcome but not hematoma expansion. Conclusions and Relevance: In this cohort study, Chinese patients with DOAC-associated ICH had large baseline hematoma volumes and high rates of mortality and functional disability. PCC treatment was not associated with improved functional outcome, hematoma expansion, or mortality. Further studies on novel hemostatic agents as well as neurosurgical and adjunctive medical therapies are needed to identify the best management algorithm for DOAC-associated ICH.

The use of andexanet alfa vs. 4-factor prothrombin complex concentrates in the setting of life-threatening intracranial hemorrhage.

OBJECTIVE: Andexanet alfa is a targeted reversal agent for life threatening hemorrhage associated with direct acting oral anticoagulants (DOACs), but there is uncertainty regarding the benefit when compared to 4-factor prothrombin complex concentrate (4F-PCC) for this indication. We investigated the clinical outcomes and cost associated with reversal of DOACs in the setting of life-threatening intracranial hemorrhage (ICH). METHODS: A retrospective evaluation was conducted to evaluate patients with ICH in the setting of anticoagulation with DOAC from 9/1/2013 to 4/30/2020. Patients were included in the study if they received reversal with either andexanet alfa or 4F-PCC. RESULTS: Eighty-nine patients were included in the study. There was no statistically significant difference in 30-day mortality between patients who received andexanet alfa or 4F-PCC (52% vs. 35%, P  = 0.14). Radiographic stability of bleed was identified in 57% of patients receiving andexanet alfa vs. 58% of patients receiving 4F-PCC ( P  = 0.93). Median length of stay was not different between the andexanet alfa and 4F-PCC populations (7 days [IQR 6 - 12] vs. 6 days [IQR 3-12], P  = 0.66). Median cost of reversal agent was higher in patients receiving andexanet alfa compared to 4F-PCC ($15 000 [IQR 15 000-$27 000] vs. $11 650 [IQR $8567-$14 149]). CONCLUSION: Among patients with life-threatening intracranial hemorrhage in the setting of DOAC therapy, no clinical differences were observed with respect to selection of reversal agent. Prothrombin complex concentrates remain a viable alternative to reversal of DOAC therapy though multicenter, randomized, prospective studies are needed to further evaluate the role of 4F-PCC in the reversal of DOAC therapy.

Trends in the Use of Recombinant Activated Factor VII and Prothrombin Complex Concentrate in Heart Transplant Patients in Virginia.

OBJECTIVES: To explore trends in intraoperative procoagulant factor concentrate use in patients undergoing heart transplantation (HTx) in Virginia. Secondarily, to evaluate their association with postoperative thrombosis. DESIGN: Patients who underwent HTx were identified using a statewide database. Trends in off-label recombinant activated factor VII (rFVIIa) use and on-label and off-label prothrombin complex concentrate (PCC) use were tested using the Mantel-Haenszel test. Multivariate logistic regression was used to test for an association between procoagulant factor concentrate administration and thrombosis. SETTING: Virginia hospitals performing HTx. PARTICIPANTS: Adults undergoing HTx between 2012 and 2022. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Among 899 patients who required HTx, 100 (11.1%) received off-label rFVIIa, 69 (7.7%) received on-label PCC, and 80 (8.9%) received off-label PCC. There was a downward trend in the use of rFVIIa over the 10-year period (p = 0.04). There was no trend in on-label PCC use (p = 0.12); however, there was an increase in off-label PCC use (p < 0.001). Patients who received rFVIIa were transfused more and had longer cardiopulmonary bypass time (p < 0.001). Receipt of rFVIIa was associated with increased thrombotic risk (odds ratio [OR] 1.92; 95% CI 1.12-3.29; p = 0.02), whereas on-label and off-label PCC use had no association with thrombosis (OR 0.98, 95% CI 0.49-1.96, p = 0.96 for on-label use; and OR 0.61, 95% CI 0.29-1.30, p = 0.20 for off-label use). CONCLUSIONS: Use of rFVIIa in HTx decreased over the past decade, whereas off-label PCC use increased. Receipt of rFVIIa was associated with thrombosis; however, patients who received rFVIIa were more severely ill, and risk adjustment may have been incomplete.

Pleiotropic effects of different exonic nucleotide changes at the same position contribute to hemophilia B phenotypic variation.

BACKGROUND: The disease-causing effects of genetic variations often depend on their location within a gene. Exonic changes generally lead to alterations in protein production, secretion, activity, or clearance. However, owing to the overlap between proteins and splicing codes, missense variants can also affect messenger RNA splicing, thus adding a layer of complexity and influencing disease phenotypes. OBJECTIVES: To extensively characterize a panel of 13 exonic variants in the F9 gene occurring at 6 different factor IX positions and associated with varying severities of hemophilia B (HB). METHODS: Computational predictions, splicing analysis, and recombinant factor IX assays were exploited to characterize F9 variants. RESULTS: We demonstrated that 5 (38%) of 13 selected F9 exonic variants have pleiotropic effects. Although bioinformatic approaches accurately classified effects, extensive experimental assays were required to elucidate and deepen the molecular mechanisms underlying the pleiotropic effects. Importantly, their characterization was instrumental in developing tailored RNA therapeutics based on engineered U7 small nuclear RNA to mask cryptic splice sites and compensatory U1 small nuclear RNA to enhance exon definition. CONCLUSION: Overall, albeit a multitool bioinformatic approach suggested the molecular effects of multiple HB variants, the deep investigation of molecular mechanisms revealed insights into the HB phenotype-genotype relationship, enabling accurate classification of HB variants. Importantly, knowledge of molecular mechanisms allowed the development of tailored RNA therapeutics, which can also be translated to other genetic diseases.

Pattern of use and clinical outcomes with rIX-FP in pediatric/adolescent patients with haemophilia B in Italy: Results from IDEAL real-world study.

OBJECTIVES: To evaluate pattern of use and clinical outcomes in pediatric/adolescent patients enrolled in the IDEAL study. METHODS: This post-hoc analysis of IDEAL retrospective-prospective observational study focused on patients <18 years, 100% on prophylaxis during the entire observation period. RESULTS: Thirteen subjects (median age 10.0 years; 61.5% ≤ 11 years) were analyzed. The infusion frequency changed from 2/week in 84.6% (N = 11) of patients with previous rFIX, to less than 1/weekly in 76.9% (N = 9) with rIX-FP and the annualized number of infusions reduced of 57% (p = .002), from a mean ± SD of 95.1 ± 22.77 to 40.4 ± 6.79, respectively. Annualized mean consumption decreased of about 56% (p = .001), from 3748.4 ± 1155.40 IU/kg with previous rFIX, to 1656.8 ± 456.63 IU/kg of rIX-FP. Mean FIX trough level changed from 3.0% ± 1.98% to 10.92% ± 3.6%. Low mean Annualized Bleeding Rate was maintained across all prophylaxis regimens (0.8 ± 1.69 vs. 0.3 ± 0.89) and zero bleeding patients moved from 69.2% (N = 9) with previous rFIX to 84.6% (N = 11) with rIX-FP (p = .63). Two adverse events, none related to rIX-FP, occurred in two patients. No inhibitors development was reported. CONCLUSIONS: The results in this pediatric/adolescent subgroup support rIX-FP prophylaxis may reduce infusion frequency, while providing high FIX trough levels, stable annualized bleeding rate and a good safety profile.

Indirect treatment comparisons of the gene therapy etranacogene dezaparvovec versus extended half-life factor IX therapies for severe or moderately severe haemophilia B.

INTRODUCTION: Etranacogene dezaparvovec gene therapy for haemophilia B demonstrated superior efficacy at 24 months in reducing bleeds versus a ≥6-month lead-in period of prophylaxis with FIX products in the phase 3 trial, HOPE-B. In the absence of head-to-head comparisons of etranacogene dezaparvovec versus FIX products, indirect treatment comparisons (ITC) can be used. AIM: To compare the efficacy of etranacogene dezaparvovec versus rIX-FP, rFIXFc and N9-GP using ITC, and support HOPE-B results. METHODS: Data were leveraged from Phase 3 pivotal trials: HOPE-B, PROLONG-9FP, B-LONG and Paradigm 2. Annualised bleeding rates (ABR), spontaneous (AsBR) and joint (AjBR) bleeding rates, percentage of patients with no bleeds, and FIX consumption were assessed using inverse probability of treatment weighting and matching adjusted indirect comparisons. RESULTS: Etranacogene dezaparvovec demonstrated statistically significantly lower bleeding rates versus all comparators. Rate ratios for ABR, AsBR and AjBR versus rIX-FP were 0.19 (p < .0001), 0.08 (p < .0001) and 0.09 (p < .0001), respectively. Rate ratios for ABR, AsBR and AjBR versus rFIXFc were 0.14 (p < .0001), 0.13 (p = .0083) and 0.15 (p = .0111), respectively. Rate ratios for ABR and AsBR, versus N9-GP were 0.24 (p = .0231) and 0.13 (p = .0071), respectively. Etranacogene dezaparvovec demonstrated significantly higher percentage of patients with no bleeds versus rIX-FP and rFIXFc; odds ratios: 17.60 (p < .0001) and 5.65 (p = .0037), respectively. Etranacogene dezaparvovec resulted in significantly lower FIX consumption than all comparators. CONCLUSIONS: ITC suggests that etranacogene dezaparvovec offers patients with haemophilia B (≤2% of normal FIX expression) a single dose treatment that can significantly reduce bleeding rates and eliminate routine infusions associated with FIX therapies.

Health-related quality of life and physical activity in Nordic patients with moderate haemophilia A and B (the MoHem study).

INTRODUCTION: The impact of moderate haemophilia on health-related quality of life (HRQoL) and physical activity (PA) is not well known. In previous studies, persons with factor VIII/factor IX activity (FVIII/FIX:C) below 3 IU/dL were associated with a more severe bleeding phenotype than predicted. AIM: To explore HRQoL and PA in patients with moderate haemophilia A (MHA) and B (MHB). METHODS: A cross-sectional, multicentre study covering patients with MHA and MHB in Sweden, Finland, and Norway. HRQoL was assessed with the EuroQoL 5-Dimensions (EQ-5D) form and PA with the International Physical Activity Questionnaire among participants aged ≥15 years. RESULTS: We report on 104 patients aged 15-84 years from the MoHem study. Overall, EQ-5D utility was .85 (median) (Q1-Q3 0.73-1.0) with corresponding visual analogue scale (VAS) 80 (70-90), which were similar regardless of treatment modality, FVIII/FIX:C, and MHA or MHB. Pain and mobility were most frequently affected dimensions. Utility (r = -.54), VAS (r = -.42), and PA (r = -.32) correlated negatively with arthropathy (HJHS). Only patients aged 41-50 years displayed lower utility (p = .02) and VAS (p < .01) than the Norwegian population norm. Patients on prophylaxis aged 35-54 years reported higher PA than those treated on-demand (p = .01). CONCLUSION: Haemophilic arthropathy had negative impact on HRQoL and PA in Nordic patients with moderate haemophilia. Middle-aged patients captured lower utility and VAS than observed in the general population. Tailored prophylaxis and improved joint health may influence positively on HRQoL and PA also in moderate haemophilia.

Comparative analysis of andexanet alfa and prothrombin complex concentrate in reversing anticoagulation by rivaroxaban ex vivo.

BACKGROUND: The comparative effectiveness of the specific antidote andexanet alfa vs the nonspecific therapy four-factor prothrombin complex concentrates (4F-PCCs) as reversal agents for direct factor Xa (FXa) inhibitors in severely bleeding patients is unclear. We hypothesised that specific reversal using andexanet alfa would be more effective than a high dose of PCC (50 IU kg-1) for reversing the FXa inhibitor rivaroxaban. METHODS: The reversal potential of andexanet alfa, various 4F-PCCs, and activated PCC was investigated ex vivo in human blood anticoagulated with rivaroxaban (37.5, 75, 150, and 300 ng ml-1) using a panel of coagulation parameters, including conventional coagulation assays, thrombin generation, and a newly developed viscoelastometric device. We simulated in vivo conditions of coagulation activation and fibrin formation using flow chamber experiments of thrombogenicity potential under arterial flow conditions. RESULTS: The 4F-PCCs normalised clotting profiles only at low rivaroxaban concentrations, whereas andexanet alfa and activated PCC significantly shortened clotting time at all rivaroxaban concentrations. Only andexanet alfa restored thrombin generation to baseline. Flow chamber results showed that various 4F-PCCs concentration-dependently restored clot formation. CONCLUSIONS: In contrast to thrombin generation measurements, haemostatic reversal of rivaroxaban using high-dose 4F-PCCs exhibited similar efficacy as andexanet alfa in flow chamber experiments. The haemostatic effects of 4F-PCCs and andexanet alfa in the context of bleeding patients taking FXa inhibitors requires further study.

Humanization and functional characterization of enhanced coagulation factor IX variants identified through ancestral sequence reconstruction.

BACKGROUND: Laboratory resurrection of ancient coagulation factor (F) IX variants generated through ancestral sequence reconstruction led to the discovery of a FIX variant, designated An96, which possesses enhanced specific activity independent of and additive to that provided by human p.Arg384Lys, referred to as FIX-Padua. OBJECTIVES: The goal of the current study was to identify the amino acid substitution(s) responsible for the enhanced activity of An96 and create a humanized An96 FIX transgene for gene therapy application. METHODS: Reductionist screening approaches, including domain swapping and scanning residue substitution, were used and guided by one-stage FIX activity assays. In vitro characterization of top candidates included recombinant high-purity preparation, specific activity determination, and enzyme kinetic analysis. Final candidates were packaged into adeno-associated viral (AAV) vectors and delivered to hemophilia B mice. RESULTS: Five of 42 total amino acid substitutions in An96 appear sufficient to retain the enhanced activity of An96 in an otherwise human FIX variant. Additional substitution of the Padua variant further increased the specific activity 5-fold. This candidate, designated ET9, demonstrated 51-fold greater specific activity than hFIX. AAV2/8-ET9 treated hemophilia B mice produced plasma FIX activities equivalent to those observed previously for AAV2/8-An96-Padua, which were 10-fold higher than AAV2/8-hFIX-Padua. CONCLUSION: Starting from computationally inferred ancient FIX sequences, novel amino acid substitutions conferring activity enhancement were identified and translated into an AAV-FIX gene therapy cassette demonstrating high potency. This ancestral sequence reconstruction discovery and sequence mapping refinement approach represents a promising platform for broader protein drug and gene therapy candidate optimization.

Clinical Implications of Discrepancy between One-Stage Clotting and Chromogenic Factor IX Activity in Hemophilia B.

BACKGROUND: Discrepancy in factor IX activity (FIX:C) between one-stage assay (OSA) and chromogenic substrate assay (CSA) in patients with hemophilia B (PwHB) introduces challenges for clinical management. AIM: To study the differences in FIX:C using OSA and CSA in moderate and mild hemophilia B (HB), their impact on classification of severity, and correlation with genotype. METHODS: Single-center study including 21 genotyped and clinically characterized PwHB. FIX:C by OSA was measured using ActinFSL (Siemens) and CSA by Biophen (Hyphen). In addition, in vitro experiments with wild-type FIX were performed. Reproducibility of CSA was assessed between three European coagulation laboratories. RESULTS: FIX:C by CSA was consistently lower than by OSA, with 10/17 PwHB having a more severe hemophilia type by CSA. OSA displayed a more accurate description of the clinical bleeding severity, compared with CSA. A twofold difference between OSA:CSA FIX:C was present in 12/17 PwHB; all patients had genetic missense variants in the FIX serine protease domain. Discrepancy was also observed with diluted normal plasma, most significant for values below 0.10 IU/mL. Assessment of samples with low FIX:C showed excellent reproducibility of the CSA results between the laboratories. CONCLUSION: FIX:C was consistently higher by OSA compared with the CSA. Assessing FIX:C by CSA alone would have led to diagnosis of a more severe hemophilia type in a significant proportion of patients. Our study suggests using both OSA and CSA FIX:C together with genotyping to classify HB severity and provide essential information for clinical management.

Matching-Adjusted Indirect Comparison of Recombinant Factor IX Albumin Fusion Protein Versus Recombinant Factor IX Fc Fusion Protein for Weekly Prophylactic Treatment of Hemophilia B.

INTRODUCTION: Prophylactic treatment of hemophilia B with recombinant factor IX (rFIX) molecules with enhanced pharmacokinetics including rIX-FP (albutrepenonacog alfa; Idelvion©) and rFIXFc (eftrenonacog alfa; Alprolix©) have commonly been used in the clinic. In the absence of head-to-head comparative trials, the aim of this study was to estimate the efficacy of rIX-FP versus rFIXFc using matching-adjusted indirect comparisons (MAICs). METHODS: MAIC analyses leveraged individual patient data from the PROLONG-9FP trial and published summary-level data from the B-LONG trial for subjects who received weekly prophylaxis regimens. Individual patient data were used to assign weights and balance subjects from PROLONG-9FP with subjects from B-LONG on baseline disease severity, age, prior FIX regimen, and body mass index (BMI). Six efficacy outcomes were analyzed including annualized bleeding rate (ABR), annualized spontaneous bleeding rate (AsBR), annualized joint bleeding rate (AjBR), and the proportion of subjects without bleeding events (for total, spontaneous, and joint bleeding events). RESULTS: After adjustment for baseline disease severity, age, prior FIX regimen, and BMI, rIX-FP was associated with a statistically significant decrease in AsBR (rate ratio [RR] 0.42; 95% confidence interval [CI] 0.22, 0.82; P = 0.0107), and the proportion of patients without bleeding events (odds ratio [OR] 3.24; 95% CI 1.41, 7.45; P = 0.0057), spontaneous bleeding events (OR 3.47; 95% CI 1.56, 7.73; P = 0.0023), and joint bleeding events (OR 2.41; 95% CI 1.10, 5.26; P = 0.0274) compared with rFIXFc. Prophylactic treatment with rIX-FP was also associated with a numerically lower ABR (RR 0.75; 95% CI 0.32, 1.75; P = 0.5095) and AjBR (RR 0.82; 95% CI 0.37, 1.82; P = 0.6178). CONCLUSION: The MAICs demonstrated that weekly prophylaxis treatment of severe hemophilia B with rIX-FP resulted in favorable efficacy outcomes as compared to rFIXFc. These findings suggest rIX-FP may offer improved clinical benefits over rFIXFc.

High variability in Factor IX one-stage assay in samples spiked with nonacog beta pegol among different pairs of reagent/detection system.

INTRODUCTION: Haemophilia B (HB) is an X-linked hereditary bleeding disorder characterized by coagulation factor IX (FIX) deficiency. To improve the quality of life of patients and adherence to treatment, recombinant factor concentrates modified to extend their half-life have been developed, called extended half-life factors (EHL: extended half-life). Nonacog beta pegol (N9-GP) is a glycopegylated recombinant human FIX molecule that has a half-life of 93 h with a single dose and has shown a higher recovery percentage than other molecules. To diagnose and monitor the treatment of haemophiliac patients, FIX activity is determined with the one-stage clotting assay (OSA) and/or the chromogenic assay. The objective of this work, carried out in three centres, was to measure the recovery of N9-PG with 10 different activated partial thromboplastin time (APTT) reagents on three platforms, in samples spiked in vitro with N9-GP, at four different concentration levels. METHODS: It was measured the recovery of N9-GP with 10 different APTT reagents (polyphenol, ellagic acid, silice dioxide, colloidal silica as APTT activator on three platforms, in sample spiked in vitro with N9-GP. RESULTS: The results show heterogeneity in the activity of N9-GP measured by OSA with the different APTT reagents when the calibrations were performed with the specific calibrator of each coagulometer. A recovery percentage between 87% and 108% was obtained only with polyphenol and ellagic acid as activator in the three platforms evaluated. The other reagents studied overestimate or underestimate, with no clear profile. When a calibration curve was performed with a calibrator prepared from the N9-GP vial, all APTT reagents met the established recovery requirement. CONCLUSION: APTT reagents with polyphenol or ellagic acid as activator would be the only ones appropriate when using the commercially available OSA with specific calibrator to monitor patients treated with N9-GP.