Adeno-Associated Virus Gene Therapy for Hemophilia.

In vivo gene therapy is rapidly emerging as a new therapeutic paradigm for monogenic disorders. For almost three decades, hemophilia A (HA) and hemophilia B (HB) have served as model disorders for the development of gene therapy. This effort is soon to bear fruit with completed pivotal adeno-associated viral (AAV) vector gene addition trials reporting encouraging results and regulatory approval widely anticipated in the near future for the current generation of HA and HB AAV vectors. Here we review the clinical development of AAV gene therapy for HA and HB and examine outstanding questions that have recently emerged from AAV clinical trials for hemophilia and other monogenic disorders.

Identifying hemophilia B carriers: Utility of aPTT, factor IX levels and ratios of factor IX to other Vitamin K dependent factors.

INTRODUCTION: Diagnosing hemophilia B (HB) carrier status is important to manage bleeding in carriers and to prevent bleeding in potential offspring. Without a family history of hemophilia, diagnosing HB carrier status is challenging. Genetic testing is the gold-standard, however it is reserved for individuals with a high suspicion of carrier status. AIMS: To describe the distribution of activated partial thromboplastin time (aPTT) and factor IX coagulant (FIX:C) levels in HB carriers and assess the ratio of FIX:C to other Vitamin K dependent factors (FII:C, FVII:C, FX:C) as an indicator of HB carrier status. METHODS: In this retrospective, single-centre cohort study, subjects were included if they were obligate or genetically proven HB carriers. Distributions of aPTT and FIX:C were described and the relationship between FIX:C levels in carriers and severity of familial HB was analysed. Ratios of FIX:C to FII:C, FVII:C, FX:C were calculated. RESULTS: Seventy-two female HB carriers (median age: 34 years; IQR 24-43) were included. Median aPTT and FIX:C levels were 33.0 s [IQR 30.0-37.0] and 57 IU/dL [IQR 43-74]. Fifteen carriers (21%) had mild HB (FIX:C levels of 10-40 IU/dL). FIX:C levels trended higher in carriers of mild HB versus carriers of moderate/severe HB. In six carriers, the median ratio of FIX:C to other Vitamin K dependent factors was 0.44, with 92% of ratios being ≤ 0.75. CONCLUSION: aPTT and FIX:C levels were unreliable in diagnosing HB carrier status. A low ratio of FIX:C to other Vitamin K dependent factors may be a useful marker of HB carrier status.

The current challenges faced by people with hemophilia B.

Hemophilia B (HB) is a rare, hereditary disease caused by a defect in the gene encoding factor IX (FIX) and leads to varying degrees of coagulation deficiency. The prevailing treatment for people with HB (PWHB) is FIX replacement product. The advent of recombinant coagulation products ushered in a new era of safety, efficacy, and improved availability compared with plasma-derived products. For people with severe HB, lifelong prophylaxis with a FIX replacement product is standard of care. Development of extended half-life FIX replacement products has allowed for advancements in the care of these PWHB. Nonetheless, lifelong need for periodic dosing and complex surveillance protocols pose substantive challenges in terms of access, adherence, and healthcare resource utilization. Further, some PWHB on prophylactic regimens continue to experience breakthrough bleeds and joint damage, and subpopulations of PWHB, including women, those with mild-to-moderate HB, and those with inhibitors to FIX, experience additional unique difficulties. This review summarizes the current challenges faced by PWHB, including the unique subpopulations; identifying the need for improved awareness, personalized care strategies, and new therapeutic options for severe HB, which may provide future solutions for some of the remaining unmet needs of PWHB.

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.

Genotype-phenotype analyses of Iranian patients with hemophilia B (Leyden -) and hemophilia B (Leyden +): A single-center study.

BACKGROUND: There is a high prevalence of inherited bleeding disorders in Iran, such as hemophilia A (HA) and hemophilia B (HB). This study aimed to analyze the molecular and clinical profiles of patients with HB. METHODS: A single-center study was conducted among patients with severe HB between March 20, 2000, and June 31, 2023. The polymerase chain reaction (PCR) amplification was used for all of the major regions, such as the promoter, the exons, the adjacent intronic regions, and the untranslated regions of the F9 gene. Finally, Sanger sequencing was performed on the PCR products. RESULTS: A total of 111 HB patients (17 with HB [Leyden +] and 94 with HB [Leyden -]) were enrolled in this study. Among 94 patients with HB (Leyden -), 59 (62.8 %) had missense, 21 (22.3 %) had nonsense, and 8 (8.5 %) had frameshift mutations. Moreover, the most frequent pathogenic variant in HB (Leyden +) was c.-17 A>G in this study. CONCLUSION: The results of this study confirm that HB is caused by a wide range of molecular defects in Iran. Thus, by knowing the genotypes and phenotypes, we would be able to stratify the patients which is important in terms of their management and outcome.

Inherent hepatocytic heterogeneity determines expression and retention of edited F9 alleles post-AAV/CRISPR infusion.

Infusing CRISPR/donor-loaded adeno-associated viral vectors (AAV/CRISPR) could enable in vivo hepatic gene editing to remedy hemophilia B (HB) with inherited deficiency of clotting factor IX (FIX). Yet, current regimens focus on correcting HB with simple mutations in the coding region of the F9, overlooking those carrying complicated mutations involving the regulatory region. Moreover, a possible adverse effect of treatment-related inflammation remains unaddressed. Here we report that a single DNA cutting-mediated long-range replacement restored the FIX-encoding function of a mutant F9 (mF9) carrying both regulatory and coding defects in a severe mouse HB model, wherein incorporation of a synthetic Alb enhancer/promoter-mimic (P2) ensured FIX elevation to clinically meaningful levels. Through single-cell RNA sequencing (scRNA-seq) of liver tissues, we revealed that a subclinical hepatic inflammation post-AAV/CRISPR administration regulated the vulnerability of the edited mF9-harboring host cells to cytotoxic T lymphocytes (CTLs) and the P2 activity in a hepatocytic subset-dependent manner via modulating specific sets of liver-enriched transcription factors (LETFs). Collectively, our study establishes an AAV/CRISPR-mediated gene-editing protocol applicable to complicated monogenetic disorders, underscoring the potentiality of improving therapeutic benefits through managing inflammation.

Assessment of joint bleeding and target joints in patients with severe or moderately severe hemophilia B (factor IX ≤2%) receiving prophylaxis with rIX-FP in the PROLONG-9FP clinical trial program.

OBJECTIVE: To assess the impact of prophylaxis with rIX-FP, a fusion protein linking recombinant factor IX (FIX) with human albumin, on joint outcomes. METHODS: Joint outcomes were assessed in pediatric (<12 years) and adult/adolescent (≥12 years) patients receiving rIX-FP prophylaxis every 7, 10, or 14 days; patients (>18 years) well-controlled on a 14-day regimen could switch to a 21-day regimen. Target joints were defined as ≥3 spontaneous bleeds into a single joint within a 6-month period. RESULTS: For adult/adolescent (n = 63) and pediatric (n = 27) patients, median (Q1, Q3) annualized joint bleeding rate was 0.39 (0.00, 2.31), 0.80 (0.00, 2.85), 0.20 (0.00, 2.58), and 0.00 (0.00, 1.78) when treated with 7-, 10-, 14-, or 21-day prophylaxis. 50.0%, 38.9%, 45.5%, and 63.6% of adult/adolescent patients had no joint bleeds when treated with 7-, 10-, 14-, or 21-day prophylaxis, respectively, and 40.7%, 37.5%, and 37.5% of pediatric patients had no joint bleeds when treated with 7-, 10-, or 14-day prophylaxis. Ten adult and two pediatric patients developed target joints; all resolved by the end of the study. CONCLUSION: Prophylaxis with rIX-FP produced low joint bleeding rates and provided excellent hemostatic efficacy in the treatment of joint bleeds. All target joints reported resolved with rIX-FP prophylaxis.

Cost-effectiveness analysis of recombinant factor IX Fc fusion protein compared with recombinant factor IX for the treatment of moderate-severe to severe hemophilia B in China.

OBJECTIVE: To evaluate the lifetime cost-effectiveness of recombinant factor IX Fc fusion protein (rFIXFc) and recombinant factor IX (rFIX) for the treatment of hemophilia B (HB) in China. METHODS: We developed a decision-analytic Markov model including three health states: alive, requiring surgery, and dead. This model estimated the lifetime cost and quality-adjusted life-years (QALYs) of prophylaxis in childhood, followed by on-demand treatment in adulthood for moderate-severe to severe HB patients from China's healthcare system perspective. Efficacy data derived from pivotal clinical trials, clinical guideline recommendations, and expert consultation were applied to two scenarios (full dose and low dose). One-way sensitivity analysis and probabilistic sensitivity analysis (PSA) were performed to assess the robustness of the model. OUTCOMES: Lifetime cost, QALYs, and the incremental cost-effectiveness ratio were calculated, and the results were compared with willingness-to-pay (WTP) thresholds of one to three times the gross domestic product per capita of China in 2021 ($12,551-$37,653). RESULTS: RFIXFc was associated with lower cost and more QALYs than rFIX in both scenarios, which suggested that it is a dominant strategy (more effective and cheaper) for moderate-severe to severe HB in China. In the full-dose scenario, rFIXFc saved more money and yielded more QALYs than in the low-dose scenario (low doses are the typical clinical reality in China). PSA demonstrated that rFIXFc had an over 90% probability of being cost-effective with full-dose and low-dose treatment at WTP thresholds of $12,551-$37,653. CONCLUSIONS: Compared with rFIX, rFIXFc appears to be a cost-effective option for the lifetime management of moderate-severe to severe HB patients in China.

Successful Multidisciplinary Management of Aortic Valve Repair in Severe Hemophilia B with Extended Half-Life Recombinant Factor IX Concentrate.

Successful management of surgery in severe coagulation disorders depends on adequate replacement of the deficient factors from intervention until wound healing. Extended half-life (EHL) recombinant factor IX (rFIX) has been increasingly used in hemophilia B (HB) patients. Monitoring of blood levels of EHL rFIX allows to obtain pharmacokinetic (PK) parameters in order to optimize and personalize therapeutic scheme. We describe a case of a young male with severe HB who successfully underwent aortic valve repair. This is the first reported open-heart surgery in a patient with severe HB using EHL rFIX. The success was based on accurate PK evaluation and on meticulous preoperative planning and close cooperation among surgeons, hemophilia specialists, and laboratory team despite the long distance between hemophilia center and surgical clinic.

Perioperative continuous infusions of factor VIII versus factor IX for patients with hemophilia A or B undergoing major surgery.

Continuous factor VIII (FVIII) or factor IX (FIX) infusions are commonly used for patients with hemophilia A (HA) or B (HB) undergoing surgery to secure perioperative hemostasis. To describe differences between the initial recovery and subsequent FIX and FVIII levels, and describe clinical outcomes among HB and HA patients receiving perioperative continuous infusion (CI) of recombinant FVIII and FIX concentrates. Retrospective chart review was conducted on 8 consecutive patients with HB and 7 consecutive patients with HA who underwent major surgery between 2014 and 2018 and received continuous infusions of standard half-life factor concentrate. Median initial bolus dose per kilogram was higher for HB compared to HA patients [90.8 (IQR 78.0-98.7) vs. 52.1 (IQR 48.6-55.6) IU/kg], while initial CI dose-rates were similar [4.3 (IQR 3.8-4.6) vs. 4.2 (IQR 3.8-4.4) IU/kg/h]. Median post-bolus recovery was higher for FVIII compared to FIX [1.70 (IQR 1.23-1.75) vs. 0.88 (IQR 0.75-1.00) IU/mL]. Median factor levels also were higher for FVIII on post-operative days 1 to 3. HB patients had greater mean intraoperative estimated blood loss [285.7 (range 0-1000) vs. 142.8 (range 0-400) mL] and longer median length of hospital stay [9 (IQR 8-12) vs. 5 (IQR 4-6.5) days]. Our initial evidence suggests greater in vivo yield of rFVIII compared to rFIX in the perioperative setting. We identified poorer clinical outcomes in this small cohort of perioperative HB patients indicating that they may benefit from a higher CI rate for adequate surgical hemostatic coverage.

Gene Therapy Approaches for the Treatment of Hemophilia B.

In contrast to the standard enzyme-replacement therapy, administered from once per 7-14 days to 2-3 times a week in patients with severe hemophilia B, as a result of a single injection, gene therapy can restore F9 gene expression and maintain it for a prolonged time. In clinical research, the approach of delivering a functional copy of a gene using adeno-associated viral (AAV) vectors is widely used. The scientific community is actively researching possible modifications to improve delivery efficiency and expression. In preclinical studies, the possibility of genome editing using CRISPR/Cas9 technology for the treatment of hemophilia B is also being actively studied.

Effects of 14 F9 synonymous codon variants on hemophilia B expression: Alteration of splicing along with protein expression.

There is growing evidence that synonymous codon variants (SCVs) can cause disease through the disruption of different processes of protein production. The aim of the study is to investigate whether the 14 SCVs reported in the F9 variant database were the pathogenic causes of hemophilia B. The impacts of SCVs on splicing and protein expression were detected using a combination of in silico prediction, in vitro minigene splicing assay and cell expression detection. The splicing transcripts were identified and quantified by co-amplification fluorescent PCR. The mechanism of splicing was verified by a modified pU1snRNA and pU7snRNA approach. Aberrant splicing patterns were found in eight SCVs. Five of the 8 SCVs produced almost all aberrant splicing isoforms, which were expected to truncate protein, three of them presented a partial defect on both splicing and protein secretion, the overall effects were consistent with the residual Factor IX activity of the affected cases. Neither the pre-messenger RNA (mRNA) splicing process nor the protein function was impaired in the rest six SCVs. In conclusion, our study firstly revealed the pathogenic mechanism of the 14 F9 SCVs and highlighted the importance of performing mRNA splicing analysis and protein expression studies of SCVs in inherited disorders.

In silico evaluation of limited sampling strategies for individualized dosing of extended half-life factor IX concentrates in hemophilia B patients.

PURPOSE: Hemophilia B is a bleeding disorder, caused by a factor IX (FIX) deficiency. Recently, FIX concentrates with extended half-life (EHL) have become available. Prophylactic dosing of EHL-FIX concentrates can be optimized by assessment of individual pharmacokinetic (PK) parameters. To determine these parameters, limited sampling strategies (LSSs) may be applied. The study aims to establish adequate LSSs for estimating individual PK parameters of EHL-FIX concentrates using in silico evaluation. METHODS: Monte Carlo simulations were performed to obtain FIX activity versus time profiles using published population PK models for N9-GP (Refixia), rFIXFc (Alprolix), and rIX-FP (Idelvion). Fourteen LSSs, containing three or four samples taken within 8 days after administration, were formulated. Bayesian analysis was applied to obtain estimates for clearance (CL), half-life (t1/2), time to 1% (Time1%), and calculated weekly dose (Dose1%). Bias and precision of these estimates were assessed to determine which LSS was adequate. RESULTS: For all PK parameters of N9-GP, rFIXFc and rIX-FP bias was generally acceptable (range: -5% to 5%). For N9-GP, precision of all parameters for all LSSs was acceptable (< 25%). For rFIXFc, precision was acceptable for CL and Time1%, except for t1/2 (range: 27.1% to 44.7%) and Dose1% (range: 12% to 29.4%). For rIX-FP, all LSSs showed acceptable bias and precision, except for Dose1% using LSS with the last sample taken on day 3 (LSS 6 and 10). CONCLUSION: Best performing LSSs were LSS with samples taken at days 1, 5, 7, and 8 (N9-GP and rFIXFc) and at days 1, 4, 6, and 8 (rIX-FP), respectively.

Acute coronary syndrome in patients with hemophilia: a delicate balancing act.

Therapeutic advances have resulted in increased life expectancy in patients with hemophilia. Consequently, the prevalence of coronary artery disease in this population is increasing. Little is known about the optimal management of acute coronary syndrome in these patients. Current guidelines for the management of this condition are based mainly on expert opinion and generally recommend administration of the clotting factor prior to the anticoagulant, antiplatelet, and interventional therapies. We report a case that illustrates the potential harm that may come from this approach: evolution of non-ST-segment elevation acute coronary syndrome into ST-elevation acute coronary syndrome during the administration of recombinant clotting factor. We review available literature and describe the refined informatics-based guidelines for managing acute coronary syndrome in patients with hemophilia we developed in response to the presented clinical case. We propose adopting this novel informatics-based approach, which aids in the identification and early treatment of these patients, operationalizes timely involvement of hematology experts, and gathers data for further study.

Hemophilia: A Review of Perioperative Management for Cardiac Surgery.

Hemophilia is an X-linked, recessive bleeding disorder. Improvements in factor replacement therapy and overall approach to hemophilia management have increased the lifespan and quality of life for patients with hemophilia. Thus, they are presenting for cardiac surgery related to age-related atherosclerosis, vascular disorders, and degenerative valvular disease. Although challenging, cardiac surgery can be performed safely with appropriate planning, using a multidisciplinary approach. This article provides a narrative review and framework for the perioperative management of patients with hemophilia A and B undergoing cardiac surgery.

The Molecular Basis of FIX Deficiency in Hemophilia B.

Coagulation factor IX (FIX) is a vitamin K dependent protein and its deficiency causes hemophilia B, an X-linked recessive bleeding disorder. More than 1000 mutations in the F9 gene have been identified in hemophilia B patients. Here, we systematically summarize the structural and functional characteristics of FIX and the pathogenic mechanisms of the mutations that have been identified to date. The mechanisms of FIX deficiency are diverse in these mutations. Deletions, insertions, duplications, and indels generally lead to severe hemophilia B. Those in the exon regions generate either frame shift or inframe mutations, and those in the introns usually cause aberrant splicing. Regarding point mutations, the bleeding phenotypes vary from severe to mild in hemophilia B patients. Generally speaking, point mutations in the F9 promoter region result in hemophilia B Leyden, and those in the introns cause aberrant splicing. Point mutations in the coding sequence can be missense, nonsense, or silent mutations. Nonsense mutations generate truncated FIX that usually loses function, causing severe hemophilia B. Silent mutations may lead to aberrant splicing or affect FIX translation. The mechanisms of missense mutation, however, have not been fully understood. They lead to FIX deficiency, often by affecting FIX's translation, protein folding, protein stability, posttranslational modifications, activation to FIXa, or the ability to form functional Xase complex. Understanding the molecular mechanisms of FIX deficiency will provide significant insight for patient diagnosis and treatment.

In vivo enrichment of genetically manipulated platelets for murine hemophilia B gene therapy.

Our previous studies have demonstrated that platelet-targeted factor IX (FIX) gene therapy can introduce sustained platelet-FIX expression in hemophilia B (FIXnull ) mice. In this study, we aimed to enhance platelet-FIX expression in FIXnull mice with O6 -methylguanine-DNA-methyltransferase (MGMT)-mediated in vivo drug selection of transduced cells under nonmyeloablative preconditioning. We constructed a novel lentiviral vector (2bF9/MGMT lentivirus vector), which harbors dual genes, the FIX gene driven by the αIIb promoter (2bF9) and the MGMT P140K gene under the murine stem cell virus promoter. Platelet-FIX expression in FIXnull mice was introduced by 2bF9/MGMT-mediated hematopoietic stem cell transduction and transplantation. The 2bF9/MGMT-transduced cells were effectively enriched after drug selection by O6 -benzylguanine/1,3-bis-2-chloroethyl-1-nitrosourea. There were a 2.9-fold higher FIX antigen and a 3.7-fold higher FIX activity in platelets, respectively, posttreatment compared with pretreatment. When a 6-hr tail bleeding test was used to grade the bleeding phenotype, the clotting time in treated animals was 2.6 ± 0.5 hr. In contrast, none of the FIXnull control mice were able to clot within 6 hr. Notably, none of the recipients developed anti-FIX antibodies after gene therapy. One of four recipients developed a low titer of inhibitors when challenged with rhF9 together with adjuvant. In contrast, all FIXnull controls developed inhibitors after the same challenge. Anti-FIX immunoglobulin G were barely detectable in recipients (1.08 ± 0.54 µg/ml), an 875-fold lower level than in the FIXnull controls. Our data demonstrate 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 tolerance in FIXnull mice.

Novel treatments for hemophilia through rebalancing of the coagulation cascade.

Hemophilia A and B are inherited hemorrhagic disorders that result from alterations in the coagulation cascade. Aside from spontaneous bleeding, the main complication of hemophilia is hemarthrosis. Progress over the last three decades, specifically prophylaxis using recombinant factor, has prevented hemarthrosis and lengthened patient life expectancies. However, many treatments require frequent dosing up to three times a week, and alloantibodies (inhibitors) against replacement factor continues to be an issue. These problems call for novel treatments for patients with hemophilia. Although there has been progress in extended half-life factors and mimetics of factor VIII, an alternative treatment methodology is to rebalance the activities of pro- and anticoagulant factors through inhibition of the natural anticoagulants: antithrombin, tissue factor pathway inhibitor, protein C, and protein S. This review will explore the efficacy of targeting these inhibitory pathways from preclinical development through clinical trials, and delve into concerns of thrombotic risk.

Prophylaxis Using a Mixture of Plasma-Derived Activated Factor VII and Factor X (pdFVIIa/FX) in a Patient with Hemophilia B Complicated by Inhibitors and Allergy to Factor IX Concentrates: A Case Report.

Treating patients with hemophilia and inhibitors is often problematic. The presence of inhibitors negatively impacts the effectiveness of treatment to achieve hemostasis especially in patients with hemophilia B, owing mainly to allergic reactions to factor IX (FIX) concentrates and the low success rate of immune tolerance therapy. A 9-month-old boy had intracranial hemorrhage and was diagnosed with hemophilia B. After replacement therapy, he developed inhibitors and an allergic reaction to FIX. Prophylactic therapy was initiated with recombinant activated factor VII (rFVIIa) and later switched to pdFVIIa/factor X (FX; 120 µg/kg as the FVII dose, every other day) because of a recurrence of intracranial hemorrhage. Since then, he remained well without life-threatening bleeding for more than 2 years. Our case suggests that pdFVIIa/FX may be useful for prophylactic therapy in hemophilia B complicated by inhibitors and allergic reaction to FIX concentrates.

In silico comparison of pharmacokinetic properties of three extended half-life factor IX concentrates.

PURPOSE: Pharmacokinetic (PK) differences between the extended half-life (EHL) factor IX (FIX) concentrates for hemophilia B exist, which may influence hemostatic efficacy of replacement therapy in patients. Therefore, we aimed to evaluate the PK properties of three EHL-FIX concentrates and compare them to a standard half-life (SHL) recombinant FIX (rFIX) concentrate. METHODS: Activity-time profiles of PEGylated FIX (N9-GP), FIX linked with human albumin (rIX-FP), FIX coupled to human IgG1 Fc-domain (rFIXFc), and SHL rFIX were simulated for 10,000 patients during steady-state dosing of 40 IU/kg once weekly (EHL-FIX) and biweekly (rFIX) using published concentrate specific population PK models. RESULTS: Half-lives were respectively 80, 104, and 82 h for N9-GP, rIX-FP, and rFIXFc versus 22 h for rFIX. Between the EHL concentrates, exposure was different with area under the curve (AUC) values of 78.5, 49.6, and 12.1 IU/h/mL and time above FIX target values of 0.10 IU/mL of 168, 168, and 36 h for N9-GP, rIX-FP, and rFIXFc, respectively. N9-GP produced the highest median in vivo recovery value (1.70 IU/dL per IU/kg) compared with 1.18, 1.00, and 1.05 IU/dL per IU/kg for rIX-FP, rFIXFc, and rFIX, respectively. CONCLUSIONS: When comparing EHL products, not only half-life but also exposure must be considered. In addition, variation in extravascular distribution of the FIX concentrates must be taken into account. This study provides insight into the different PK properties of these concentrates and may aid in determination of dosing regimens of EHL-FIX concentrates in real-life.