Factor 4 Research Articles

Valoctocogene Roxaparvovec and Etranacogene Dezaparavovec: Novel Gene Therapies for Hemophilia A and B.

To review efficacy and safety data of valoctocogene roxaparvovec (Roctavian) and etranacogene dezaparavovec (Hemgenix), novel gene therapies for the treatment of the life-threatening bleeding disorders hemophilia A and B, respectively. A PubMed/Google Scholar search from inception through August 11, 2023 was conducted using the following keywords: gene therapy, hemophilia A, hemophilia B, etranacogene dezaparavovec, valoctocogene roxaparvovec, and bleeding. Data, including phase 1 to 3 clinical trials (non-comparator), were obtained from primary literature and package inserts. These reports evaluated clinical pharmacology, efficacy, safety, adverse events, warnings, and precautions. Valoctocogene phase 3 study in males (n = 134): 87% had factor VIII (FVIII) levels that at least met criteria for mild hemophilia. Etranacogene phase 3 study in males (n = 54): within 3 weeks of infusion, mean factor IX (FIX) levels had reached 26.8 IU/dL. Both therapies provided clinically and statistically significant decreases in bleeding events and prophylactic factor infusions. Most common adverse event was elevations in liver function tests that were treated with glucocorticoids. The endogenous production of clotting factors mimics physiological production while decreasing morbidity and mortality related to bleeding events similar to the effects of existing replacement strategies. Gene therapy was also shown to increase patient quality of life. Valoctocogene and etranacogene provide another treatment for selected patients with hemophilia. Treatment for the patient with hemophilia (gene therapy vs replacement strategy) must be personalized as new clinical data are published being cognizant of drug affordability.

Random X-Chromosome Inactivation in Adult Hemophilia B Carriers Does Not Attenuate Bleeding Risk

Introduction: The X-linked bleeding disorder Hemophilia B (HB) results from deficiency in coagulation Factor IX (FIX). In contrast to Hemophilia A, genotype-phenotype correlations in HB males are complex, as some F9 variants in males are associated with low plasma FIX activity (FIX:C) but milder than expected bleeding phenotypes. At least 75% of FIX is extravascular and not adequately measured from plasma. Hemostasis is likely influenced by this extravascular FIX which is bound reversibly to type IV collagen in the subendothelial basement membrane. We predict that genotype-phenotype correlations in female carriers of F9 mutations may have additional complexity as the mutant FIX can competitively interfere with wild type (WT) FIX for extravascular binding sites. F9 expression in female HB carriers is influenced by X-chromosome inactivation (XCI), a process that silences one X in XX females. XCI results in cells that randomly inactivate either the X carrying the mutant or wild type F9, although skewed XCI (>80:20) is found in ~25% of the general population. XCI skewing in HB carriers can lead to preferential expression of the X carrying the F9 mutation, which in turn influences plasma and extravascular levels of FIX and bleeding tendency. How frequently XCI is skewed in HB carriers, and the role of XCI in modulating FIX levels and bleeding phenotypes, remains poorly understood. We sought to determine whether XCI skewing may further differentiate bleeding risk in HB carriers. Methods: Subjects were enrolled in a prospective, observational cross-sectional study with informed consent. Both the Condensed MCMDM-1 bleeding assessment tool (BAT) and Pictorial Blood-loss Assessment Chart (PBAC) were completed at the time of the visit. F9 mutation analysis confirmed carrier status. FIX:C and FIX antigen ELISA assays were performed on all subjects. XCI skewing was assessed using established assays at the Androgen Receptor (AR) and Fragile X Mental Retardation (FMR1) loci. Results: A total of 15 subjects (11 adults) were confirmed F9 mutation carriers; six missense mutations and one frameshift mutation were identified. Abnormal bleeding is frequent in adult HB carriers, with 5/11 (45%) having BAT scores >4. Heavy menstrual bleeding (HMB) is particularly frequent, with all adult HB carriers reporting PBAC > 100, and 8/11 with BAT HMB subcategory score >1. FIX assessment by activity (mean 56% ± 28) and antigen (mean 0.55 IU/mL ± 0.2) assays are highly correlated (r 2= 0.83, p < 0.0001). Notably, only two adult HB carriers with significant bleeding have FIX levels < 50%, while two HB carriers with FIX levels < 50% have BAT scores < 4, indicating that neither FIX:C nor FIX antigen levels adequately predict bleeding risk in all HB carriers. Indeed, for all 11 adult HB carriers, no significant differences in either FIX:C or FIX antigen were observed between those with BAT scores ≥ 4 and < 4. We asked if XCI explained FIX levels or bleeding phenotypes. Nearly half (5/11) of the adult carriers have moderately skewed XCI (> 70:30); only one is extremely XCI skewed (> 80:20), and of the individuals with skewed XCI, 3/5 have BAT scores ≥ 4. Although random XCI is proposed to attenuate HB carrier bleeding phenotype, 2/6 HB carriers with random XCI also have BAT scores ≥ 4. XCI weakly correlated with FIX antigen (n = 15; r 2= 0.27, p < 0.05) but not FIX:C. When assessing this relationship for only the individuals with F9 c.1025C>T (n = 6), there was significant correlation with FIX:C (R 2= 0.73, p < 0.05) but not with FIX antigen or bleeding phenotype. The lack of correlation between XCI and FIX antigen in individuals with an identical F9 mutation argues for additional influences on intravascular, and likely extravascular, FIX levels and will require further investigation. Conclusions: Bleeding tendency in HB carriers is not rare and does not correlate with plasma FIX:C or FIX antigen. Random XCI is not fully protective against abnormal bleeding. Future studies are needed to better understand bleeding phenotypes in HB carriers that consider XCI, F9 mutation type and influences on circulating levels of WT and mutant FIX.

Long-Term Bleeding Protection, Sustained FIX Activity, Reduction of FIX Consumption and Safety of Hemophilia B Gene Therapy: Results from the HOPE-B Trial 3 Years after Administration of a Single Dose of Etranacogene Dezaparvovec in Adult Patients with Severe or Moderately Severe Hemophilia B

Introduction: Etranacogene dezaparvovec (formerly AMT-061) is the first approved gene therapy for hemophilia B in the US and Europe. It is an adeno-associated virus serotype 5 (AAV5) vector containing a codon-optimized, highly active factor IX (FIX) Padua R338L transgene under the control of the liver-specific promoter LP-1. The pivotal phase 3 HOPE-B clinical trial (NCT03569891) of etranacogene dezaparvovec demonstrated superiority of bleeding protection compared to standard of care FIX prophylaxis up to 24 months post-treatment; long-term follow-up from Year 2 post-administration onward is currently ongoing. Aim: To report long-term efficacy and safety data of etranacogene dezaparvovec from the HOPE-B trial over a period of 3 years post-treatment. Methods: In this pivotal phase 3 open-label, single-arm trial, adult male participants with severe or moderately severe hemophilia B (FIX ≤2%), with or without preexisting AAV5 neutralizing antibodies (NAbs), were infused with a single dose (2×10 13 gc/kg) of etranacogene dezaparvovec, following a ≥6-month lead-in period of receiving their usual FIX prophylaxis. Efficacy (bleeding rates, aPTT-based FIX activity levels, FIX consumption) and safety data (adverse events [AEs]) during Years 1, 2, and 3 post-treatment with etranacogene dezaparvovec are reported. Results: Of 54 participants who received etranacogene dezaparvovec, 52 completed 36 months of follow-up. Mean annualized bleeding rate (ABR) for all bleeds during Months 7-36 post-treatment was significantly reduced by 64% (mean ABR 1.52) compared with the ≥6-month lead-in period (mean ABR 4.17; P=0.0004). Total number of bleeds (all types) were 136 during the ≥6-month lead-in period and decreased to 55 during Year 1, 48 during Year 2, and 37 during Year 3 post-treatment. Median [range] bleeds per participant decreased from 2.0 [0-10] during the lead-in period and remained stable to 0.0 [0-4] during Year 1, 0.0 [0-10] during Year 2, and 0.0 [0-8] during Year 3. Superior bleeding protection was in line with the level of transgene-derived endogenous FIX expression. The mean±SD (median; range) endogenous FIX activity level (ie. in the absence of exogenous FIX exposure) of participants was 41.5 IU/dL ±21.7 (39.9; 5.9-113, n=50) at Year 1, 36.7 IU/dL ±19.0 (33.9; 4.7-99.2, n=50) at Year 2, and sustained at 38.6 IU/dL ±17.8 (36.0; 4.8-80.3, n=48) at Year 3 post-treatment. Pharmacodynamic profile was not significantly different in participants with AA5 NAb undetected or titer ≤1:678. At 3 years post-treatment, 51 (94%) remained free of continuous FIX prophylaxis. One participant who lacked efficacy (highest AAV5 NAbs titer of 1:3212) and 1 who received a 10% partial dose of treatment did not discontinue prophylaxis; 1 participant eventually had his FIX levels declined to 2-5% range; his bleeding phenotype returned, and he resumed prophylaxis per protocol at month 30 post-treatment. During Year 2 and Year 3 post-treatment, 37 (70%) and 39 (75%) participants received no FIX infusion, respectively. Overall mean annualized FIX consumption decreased by 96% over 3 years post-treatment compared to the ≥6-month lead-in period (-246,763 IU/kg/participant, including those receiving FIX prophylaxis post-treatment; P<0.0001). During the 3 years post-dose, all participants experienced at least 1 treatment-emergent AE (TEAE); of 709 events, 541 (76%) were mild, 137 (19%) were moderate, and 31 (4%) were severe. There were no serious AEs related to treatment [a serious AE of hepatocellular carcinoma (HCC) and a death were reported previously before Year 2 and determined to be unrelated to treatment]. A total of 38/54 (70%) participants experienced 96 treatment-related TEAEs, of which 95% occurred before 6 months post-treatment. The most common AE was an increase in alanine transaminase (ALT), for which 9 (16.7%) participants received supportive care with reactive corticosteroids for a mean duration of 81.4 days (SD: 28.6; range: 51-130 days). No new deaths, no new HCC, and no late treatment-related ALT elevations or thromboembolic events were reported. Conclusion: Long-term follow-up during the HOPE-B trial has shown that a single-dose of etranacogene dezaparvovec resulted in long-term endogenous FIX Padua expression and superior bleeding protection compared to FIX prophylaxis in participants without or with AAV NAb titer ≤1:678, with a favorable safety profile over 3 years post-administration.

Stable Therapeutic Transgenic FIX Levels for More Than 10 Years in Subjects with Severe Hemophilia B Who Received scAAV2/8-LP1-Hfixco Adeno-Associated Virus Gene Therapy

Background: Previously, we reported data from a first-in-human, dose-escalation, Phase 1 gene therapy trial demonstrating a dose-dependent increase in transgenic factor IX (FIX) expression in 10 adults with severe hemophilia B (FIX activity ≤1%). This was achieved following a single intravenous infusion of a self-complementary adeno-associated virus (scAAV) vector containing the wild-type FIX gene (codon-optimized), under the control of a synthetic liver-specific promoter and pseudotyped AAV8 capsid (scAAV2/8-LP1-hFIXco; ClinicalTrials.gov:NCT00979238). We now report durable efficacy and long-term safety over at least 10 years of follow-up in these participants. Methods: FIX activity, annualized bleeding rate (ABR), use of FIX concentrates, safety (specifically transaminitis and oncogenesis), and FIX expression kinetics were evaluated in the 10 participants following a bolus infusion of scAAV2/8-LP1-hFIXco at a dose of 2x10 11vg/kg (N=2), 6x10 11vg/kg (N=2), or 2x10 12vg/kg (N=6), between 2010 and 2012. Results: As of December 31, 2022, the median follow-up was 10.7 years (range 4-12 years). A total of 11 treatment-related adverse events occurred in 10 participants, including transient elevation of liver transaminases within 3 months of vector infusion in 4 of 6 patients treated with the high vector dose, without any recurrence. Two new serious adverse events were reported: (1) non-mucinous lung adenocarcinoma in situ, identified incidentally following a bullectomy for spontaneous pneumothorax 5 years after gene therapy in a 48-year-old participant considered not related to gene therapy following molecular studies, and (2) adenocarcinoma of the prostate in a 72-year-old participant 12 years after gene therapy. Tissue evaluation of the latter case is ongoing. No FIX inhibitors, thrombosis, persistent transaminitis or deaths were observed. Transgenic FIX expression has remained stable, with mean (±SD) FIX activity (one-stage) for the three dose cohorts of 1.7±0.9, 2.3±0.9 and 4.9±2.2 IU/dL, respectively (Figure 1). The mean and median ABR over the 10-year period following gene therapy of all 10 participants were 1.95 and 1.6 compared to 16.5 and 14, respectively, before gene therapy. This represents an 8.5-fold reduction in bleeding events (Wilcoxon signed rank test p=0.002). In the 6 high-dose participants, the ABR was 21-fold lower (mean=1.16, median 1, paired t-test, p<0.008). Mean and median FIX concentrate usage before gene therapy in the 10 participants were 2869 and 2526 IU/kg dropping to 945 and 274 IU/kg, respectively, over a 10-year period after gene therapy (p=0.0003). All participants developed a persistent, high-titer polyclonal anti-AAV8 capsid-specific antibody response after administration of scAAV2/8-LP1-hFIXco. Conclusion: Expression of transgenic FIX has remained stable over a period of 10 years following systemic administration of scAAV2/8-LP1-hFIXco resulting in sustained clinical benefit, with substantial reduction in ABR and FIX concentrate use.

Characterizing a Cohort of Patients with Hemophilia B Treated with Fidanacogene Elaparvovec from the Phase 3 Benegene-2 Study Who Returned to Factor IX Prophylaxis

Background: Fidanacogene elaparvovec (PF-06838435, formerly SPK-9001) is an adeno-associated virus (AAV)-based gene therapy designed to deliver a high-activity human factor IX (FIX) variant transgene, FIX-R338L, resulting in endogenous FIX production in people with hemophilia B. To date, 45 participants with moderately severe to severe (FIX:C ≤2%) hemophilia B have received fidanacogene elaparvovec as part of the ongoing phase 3 study, BENEGENE-2 (NCT03861273). Of these 45 participants, 6 returned to prophylaxis (RTP) of FIX after initially responding to treatment. We describe the characteristics of the RTP participants. Methods: Participants with baseline FIX activity ≤2% received a single dose of 5e11 vg/kg fidanacogene elaparvovec (AAVrh74 variant) as part of the phase 3 study (N=45). Participants suspended prophylaxis following vector infusion (1 participant continued for 2 weeks post infusion), which could be resumed per the investigator's discretion and the protocol provided guidance for when to consider resuming prophylaxis: ≥2 consecutive central laboratory FIX activity levels ≤2% at least 2 weeks apart and/or ≥2 spontaneous joints bleeds within 4 weeks and/or ≥3 spontaneous bleeds overall (joint and non-joint). Results: Prior to fidanacogene elaparvovec infusion, all 45 participants had completed at least 6 months of prophylaxis as part of the lead-in study (BENEGENE-1, NCT03587116). The mean age (range) of all 45 study participants was 33.2 y (18-62) and the 6 RTP participants had a mean age (range) of 28.3 y (18-47), of whom 4 were <30 y old. The region, race, and weight of the 6 RTP participants were representative of the entire study population (Table 1). All RTP participants initially responded to gene therapy and achieved peak FIX activity levels >5%, determined by one-stage actin-FSL (7-22.1%) and one-stage Synthasil (18.3-45.5%) across Days 36-97. Time to RTP from fidanacogene elaparvovec dose ranged from Days 155 to 623. The reasons reported for RTP were low FIX activity in 5 participants, of whom 1 had a prior history of intracerebral hemorrhage, and increased bleeds in 1 participant. Five participants recorded ≥1 bleeding event prior to resumption of prophylaxis. All RTP participants were treated with ≥1 course of corticosteroids for presumed cellular immune response. In all cases, maximum alanine aminotransferase was 1-2x upper limit of normal. Two RTP participants had an ELISPOT drawn within ±1 day of starting corticosteroids; both were negative for capsid peptides (Table 2). In comparison, 4 participants who took corticosteroids but did not resume prophylaxis were positive for capsid. All 6 RTP participants had a decline in FIX activity from peak levels in the absence of inhibitors, but displayed variable decline prior to and during corticosteroid treatment, or after completion of corticosteroid wean, with and without some elevation of liver enzyme at the time of the decline. Conclusion: The 6 RTP participants who received fidanacogene elaparvovec in the phase 3 study (BENEGENE-2) initially responded to therapy before a heterogenous decline in FIX activity. The limited number of participants and lack of consistent patterns and demographic features make identifying predictors of potential RTP challenging. Although all RTP participants were treated with corticosteroids during this study, not all participants treated with corticosteroids RTP of FIX. Predictors of loss of response have not been identified and further work is ongoing to potentially identify factors associated with increased risk of RTP.

Health-Related Quality of Life in Adults with Hemophilia B after Receiving Gene Therapy with Fidanacogene Elaparvovec

Introduction : The burden of the management and clinical sequelae of hemophilia B (HB) negatively impacts health-related quality of life (HRQoL), including chronic pain and mental health. Fidanacogene elaparvovec (PF-06838435, formerly SPK-9001) is an adeno-associated virus-based gene therapy vector transferring the high activity variant of human factor IX (FIX), FIX-R338L, aimed at enabling endogenous FIX expression in individuals with hemophilia B. We present descriptive data on the impact on HRQoL for participants in the fidanacogene elaparvovec phase 1/2a study. Methods : Fifteen participants with moderately severe to severe hemophilia B (FIX activity ≤2%) received a single infusion of fidanacogene elaparvovec 5e11 vg/kg as part of a phase 1/2a study (NCT02484092). The study was 52 weeks in duration, after which participants were eligible to enroll in a long-term follow-up (LTFU) study for up to 5 years (NCT03307980). All 15 participants completed the phase 1/2a study and 14 subsequently enrolled in the LTFU study (1 participant declined participation). Participants in both studies completed patient-reported outcome (PRO) HRQoL assessments, including the Haemophilia Quality of Life Questionnaire for Adults (Haem-A-QoL), the visual analog scale of the EQ-5D (EQ-VAS), and the Change in Level of Activity questionnaire, every 6 months through Week 156 post infusion and then once annually during the LTFU study. The Haem-A-QoL questionnaire consists of 46 items pertaining to 10 domains to assess HRQoL, and domains scores and the Total Score ranging from 0-100 based on the individual items (high scores indicate high impairment in HRQoL). In the EQ-VAS, participants rate their current health state for endpoints of 100 (best imaginable health) to 0 (worst imaginable health). In the Change in Level of Activity questionnaire, participants assess whether they have been doing more/fewer/the same amount or intensity of physical activities in the past month. The data cutoff date was August 2, 2022. Results : For 14 participants (aged 20 to 63 years), the mean (SD) age at entry to the LTFU study was 42 (13.7) years, body mass index was 26.4 (4.4) kg/m 2, and 85.7% were White. Of the 14 participants, 13 had HRQoL data available through Week 156; beyond this time point, there were 10 or fewer participants with both post- and pre-vector infusion (baseline) values. For the Haem-A-QoL assessment, the mean (SD) Total Score at Week 156 (n=12) decreased by 15.2 (10.3) from pre-vector infusion, indicating an improvement in HRQoL ( Fig. 1). A 7-point reduction in the total score has been shown to be clinically meaningful in previous studies (Von Mackensen S et al, Haemophilia 2020;26:1019-30; Wyrwich KW et al, Haemophilia 2015;21:578-84). Of the 12 participants with data at Week 156, 8 had Total Score decreases of 7 or more at each visit through Week 156. Similarly, participant scores for each individual domain decreased over time (implying an improvement in HRQoL) from pre-vector infusion in all instances except for the Dealing with Hemophilia domain ( Fig. 1). Mean EQ-VAS scores were above mean baseline score and were consistently greater than baseline by 7 to 8 points during Year 2 onward, suggesting an improvement in overall health status. At Week 156, the EQ-VAS score increased from baseline by mean (SD) 7.2 (6.2): baseline 83.7 (7.3), n=13; Week 156 91.5 (5.4), n=12. An EQ-VAS score change of 7 points has been shown to be clinically meaningful (Pickard AS et al. Health Qual Life Outcomes 2007;21:5:70). Responses in the Change in Level of Activity questionnaire showed that a greater proportion of participants at each visit (n=12-14) reported doing the same or more intensive physical activities up to Week 156 than reported at baseline ( Fig. 2). No participant reported a reduction in the amount or intensity of physical activities at Week 156 (n=13) ( Fig. 2). Conclusion : HRQoL improvements after gene therapy are an indicator that fidanacogene elaparvovec can reduce the burden associated with hemophilia. PRO assessments will be a part of BENEGENE-2, an ongoing pivotal phase 3 study to demonstrate the efficacy of fidanacogene elaparvovec and will provide additional insights via the analyses of secondary endpoints into HRQoL benefits for participants with HB following gene therapy with fidanacogene elaparvovec.

AAV-Mediated Expression of a Single-Chain FVIII Mimetic Antibody Efficiently Corrects Bleeding in Haemophilia a Mice

Background: The recent approval of gene therapy for haemophilia A (HA) using adeno-associated viruses (AAVs) marks an important and long-awaited landmark as it is associated with markedly reduced bleeding events whilst overcoming the demands of regular life-long administration of factor concentrates or antibodies with FVIII mimetic activity. However, the benefits of AAV gene therapy in severely affected HA patients are likely to be short-lived as clinical data from most of the advanced trials show a gradual decline in transgenic Factor VIII (FVIII) levels over time. The reasons for this decline in expression remain unexplained but may be due in part to the packaging of an oversized FVIII expression cassette into AAV vectors. This hypothesis is supported by the fact that such decline in transgene expression has not been observed in other gene therapy approaches in clinic where the transgene expression cassette is within the packaging limits of AAV vectors. Therefore, we explored an alternative therapy approach for HA that utilises an expression cassette encoding a novel FVIII mimetic bispecific antibody (Bi8) that remains within the packaging constrains of AAV vectors, thus potentially improving the prospects of long-term control of the bleeding diathesis in severely affected HA subjects. . Methods: Bi8, is a small 54kDa single-chain bispecific antibody designed as a tandem of single-chain fragment variables (scFvs) targeting Factors IXa (FIXa) and X (FX). FVIII mimetic activity of recombinant Bi8 protein was evaluated in vitro using (1) the catalytic conversion of purified FX into FXa in a chromogenic assay, and (2) factor XIa (FXIa)-triggered thrombin generation assay (TGA) and activated partial thromboplastin time (aPTT) using spiked human HA plasma. Bi8 sequences were cloned in an AAV expression cassette downstream of a strong liver-specific promoter, pseudotyped with AAV serotype 8 capsid and evaluated in vitro as well as in vivo, including in FVIIIKO mice to assess potency in a tail vein transection assay, kinetics of Bi8 expression and the occurrence of anti-drug antibodies (ADAs). Results: In preliminary experiments, we demonstrated that the single-chain format used for Bi8 retained haemostatic functions. When tested in a chromogenic assay, recombinant Bi8 protein showed kinetics of FXa formation identical to that of emicizumab. Furthermore, Bi8 spiked in human HA plasma at 350nM corrected the clotting time (17.2 sec) in an aPTT assay to levels observed with emicizumab (22.7 sec). TGA with recombinant Bi8 and emicizumab showed comparable efficacy with peaks of thrombin generation at 354.2 and 358.4nM respectively. Next, human hepatocyte HuH7 cells were transduced with AAV8 encoding Bi8 (AAV8_Bi8) vectors in vitro, leading to the expression of a fully functional bispecific antibody with an activity/antigen ratio of 0.99 when compared to the recombinant Bi8 protein. Three doses of AAV8_Bi8 vector were then evaluated in 6-8 weeks old, male FVIIIKO mice, with a single intravenous infusion of 4e 11; 4e 12 and 1.2e 13 vector genome (vg) per kilo. Expression of the bispecific antibody was detectable in plasma for all 3 doses and peaked 2 weeks post-infusion with respective concentrations of 4.5; 47.4 and 178.8nM (Panel A). Transgenic expression was then stably maintained for 12 weeks and ADAs were not detected in any of the treated animals. When challenged in a bleeding assay, haemostasis was secured in all cohorts of mice infused with AAV-Bi8 vectors, including at the lowest dose of 4e 11 vg/kg where the average total blood loss was reduced to 401.2μL (median 406.2μL) compared to 760.4μL (median 773.1μL) in non-treated FVIIIKO mice (Panel B). Conclusion: This study establishes the feasibility of using AAV vectors to deliver an expression cassette encoding Bi8, a novel FVIII mimetic antibody formatted as a single-chain tandem-scFv, leading to circulating concentrations of bispecific antibody at levels that can secure haemostasis and reduce blood loss in a murine model of HA. This non FVIII-based expression cassette meets the packaging capacity of AAVs and therefore provides an alternative approach for AAV-mediated gene therapy for HA that may be better suited for stable long-term expression than FVIII transgenes.

Epidemiological profile of hemophilia A in Karbala-Iraq.

Hemophilia is an X-linked congenital bleeding disorder caused by a deficiency of coagulation factor VIII (FVIII) (in hemophilia A) or factor IX (FIX) (in hemophilia B) and is one of the most important hereditary conditions in Iraq. The current study tried to provide a glimpse into the epidemiological and clinical status, as well as complications and treatment used for patients with hemophilia A in Karbala, Iraq. This retrospective research was carried out by reviewing the medical records of 90 male patients diagnosed with hemophilia A registered at the Hereditary Blood Disease Center in Karbala Teaching Hospital for children in Karbala, Iraq. The data was collected from May 10, 2023, until June 15, 2023, and included age, severity, type of bleeding, therapeutic approach, chronic viral infections including hepatitis B, hepatitis C, and human immunodeficiency virus (HIV). The median age of the patients was 15 years (18.86±14.42). In this study, one-third of patients' presentations started in the first year of life. The most common type was severe hemophilia A (factor VIII activity < 1%). Consequently, more than half of the patients were treated with factor VIII concentrate. Four patients had hepatitis C, while HIV was confirmed in one patient. The epidemiological or clinical parameters of hemophilia A patients in Karbala seem similar to those in other cities in Iraq. By considering this preliminary data, further comprehensive studies for patients with hemophilia and associated complications in other provinces or cities in Iraq are highly suggested to provide a clear perspective about the prevalence and epidemiological burdens.

Protein S antibody as an adjunct therapy for hemophilia B

AbstractHemophilia B (HB) is caused by an inherited deficiency of plasma coagulation factor IX (FIX). Approximately 60% of pediatric patients with HB possess a severe form of FIX deficiency (<1% FIX activity). Treatment typically requires replacement therapy through the administration of FIX. However, exogenous FIX has a limited functional half-life, and the natural anticoagulant protein S (PS) inhibits activated FIX (FIXa). PS ultimately limits thrombin formation, which limits plasma coagulation. This regulation of FIXa activity by PS led us to test whether inhibiting PS would extend the functional half-life of FIX and thereby prolong FIX-based HB therapy. We assayed clotting times and thrombin generation to measure the efficacy of a PS antibody for increasing FIX activity in commercially obtained plasma and plasma from pediatric patients with HB. We included 11 pediatric patients who lacked additional comorbidities and coagulopathies. In vivo, we assessed thrombus formation in HB mice in the presence of the FIXa ± PS antibody. We found an accelerated rate of clotting in the presence of PS antibody. Similarly, the peak thrombin formed was significantly greater in the presence of the PS antibody, even in plasma from patients with severe HB. Furthermore, HB mice injected with PS antibody and FIX had a 4.5-fold higher accumulation of fibrin at the thrombus induction site compared with mice injected with FIX alone. Our findings imply that a PS antibody would be a valuable adjunct to increase the effectiveness of FIX replacement therapy in pediatric patients who have mild, moderate, and severe HB.

Molecular Dynamics Simulation Study of the Selective Inhibition of Coagulation Factor IXa over Factor Xa.

Thromboembolic disorders, arising from abnormal coagulation, pose a significant risk to human life in the modern world. The FDA has recently approved several anticoagulant drugs targeting factor Xa (FXa) to manage these disorders. However, these drugs have potential side effects, leading to bleeding complications in patients. To mitigate these risks, coagulation factor IXa (FIXa) has emerged as a promising target due to its selective regulation of the intrinsic pathway. Due to the high structural and functional similarities of these coagulation factors and their inhibitor binding modes, designing a selective inhibitor specifically targeting FIXa remains a challenging task. The dynamic behavior of protein-ligand interactions and their impact on selectivity were analyzed using molecular dynamics simulation, considering the availability of potent and selective compounds for both coagulation factors and the co-crystal structures of protein-ligand complexes. Throughout the simulations, we examined ligand movements in the binding site, as well as the contact frequencies and interaction fingerprints, to gain insights into selectivity. Interaction fingerprint (IFP) analysis clearly highlights the crucial role of strong H-bond formation between the ligand and D189 and A190 in the S1 subsite for FIXa selectivity, consistent with our previous study. This dynamic analysis also reveals additional FIXa-specific interactions. Additionally, the absence of polar interactions contributes to the selectivity for FXa, as observed from the dynamic profile of interactions. A contact frequency analysis of the protein-ligand complexes provides further confirmation of the selectivity criteria for FIXa and FXa, as well as criteria for binding and activity. Moreover, a ligand movement analysis reveals key interaction dynamics that highlight the tighter binding of selective ligands to the proteins compared to non-selective and inactive ligands.

Perioperative management of a patient with haemophilia B and PSVT undergoing radiofrequency ablation: A case report.

Haemophilia B is a rare inherited bleeding disorder in which patients have impaired coagulation. This study describes a patient with Haemophilia B and paroxysmal supraventricular tachycardia (PSVT) who underwent radio frequency catheter ablation (RFCA). The perioperative replacement therapy with coagulation factor IX (FIX) was agreed upon after an interdisciplinary consultation involving a team of specialists in haematology, cardiovascular medicine and cardiothoracic surgery. There were no obvious bleeding points or complications during the perioperative period following the treatment, nor recurrence of PSVT within a three-year follow-up period. In summary, RFCA can be performed safely in patients with haemophilia B on the premise of developing an individualized perioperative exogenous coagulation factor supplementation regimen based upon an adequate preoperative evaluation and clinical monitoring and management by an interdisciplinary team.

Computational design of N-linked glycans for high throughput epitope profiling.

Efficient identification of epitopes is crucial for drug discovery and design as it enables the selection of optimal epitopes, expansion of lead antibody diversity, and verification of binding interface. Although high-resolution low throughput methods like x-ray crystallography can determine epitopes or protein-protein interactions accurately, they are time-consuming and can only be applied to a limited number of complexes. To overcome these limitations, we have developed a rapid computational method that incorporates N-linked glycans to mask epitopes or protein interaction surfaces, thereby providing a mapping of these regions. Using human coagulation factor IXa (fIXa) as a model system, we computationally screened 158 positions and expressed 98 variants to test experimentally for epitope mapping. We were able to delineate epitopes rapidly and reliably through the insertion of N-linked glycans that efficiently disrupted binding in a site-selective manner. To validate the efficacy of our method, we conducted ELISA experiments and high-throughput yeast surface display assays. Furthermore, x-ray crystallography was employed to verify the results, thereby recapitulating through the method of N-linked glycans a coarse-grained mapping of the epitope.

Targeted delivery of miR125a-5p and human Factor VIII attenuates molecular mediators of hemophilic arthropathy

Hemophilic arthropathy (HA) due to repeated bleeding into the joint cavity is a major cause of morbidity in patients with hemophilia. The molecular mechanisms contributing to this condition are not well characterized. MicroRNAs (miRs) are known to modulate the phenotype of multiple joint diseases such as osteoarthritis (OA) and rheumatoid arthritis (RA). Since miR125a is known to modulate disease progression in OA and RA, we performed a targeted screen of miR125a-5p and its target genes in a murine model of chronic HA. A digital PCR analysis demonstrated significant downregulation of miR125a-5p (2-fold vs control joint). Further molecular evaluation revealed elevated expression of the immunological markers STAT1 (7.6-fold vs control joint) and TRAF6 (10.6 fold vs control joint), which are direct targets of miR125a-5p. We then studied the impact of targeted overexpression of miR125a-5p using an Adeno-associated virus (AAV) vector in modulating the molecular mediators of HA. AAV5-miR125a vectors were administered intra-articularly either alone or in combination with a low dose of AAV8-based human factor 8 (F8) gene in a murine model of HA. We observed significantly increased expression of miR125a-5p in AAV5-miR125a administered mice (~12 fold vs injured joint) or in combination with AAV8-F8 vectors (~44 fold vs injured joint). The activity assay revealed ~17 %–20 % FVIII levels in mice that received low dose liver-directed F8 gene therapy. Further immunohistochemical analysis, demonstrated a decrease in inflammatory markers (STAT1 and TRAF6) and cartilage-degrading matrix metalloproteinases (MMPs) 3, 9, 13 in the joints of treated animals. These data highlight the crucial role of miR125a-5p in the development of HA.

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

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. 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. 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. 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.

Hemophilia Gene Therapy: The End of the Beginning?

Extensive preclinical research over the past 30 years has culminated in the recent regulatory approval of several gene therapy products for hemophilia. Based on the efficacy and safety data in a recently conducted phase III clinical trial, Roctavian® (valoctocogene roxaparvovec), an adeno-associated viral (AAV5) vector expressing a B domain deleted factor VIII (FVIII) complementary DNA, was approved by the European Commission and Food and Drug Administration (FDA) for the treatment of patients with severe hemophilia A. In addition, Hemgenix® (etranacogene dezaparvovec) was also recently approved by the European Medicines Agency and the FDA for the treatment of patients with severe hemophilia B. This product is based on an AAV5 vector expressing a hyper-active factor IX (FIX) transgene (FIX-Padua) transgene. All AAV-based phase III clinical trials to date show a significant increase in FVIII or FIX levels in the majority of treated patients, consistent with a substantial decrease in bleeding episodes and a concomitant reduction in factor usage obviating the need for factor prophylaxis in most patients. However, significant interpatient variability remains that is not fully understood. Moreover, most patients encountered short-term asymptomatic liver inflammation that was treated by immune suppression with corticosteroids or other immune suppressants. In all phase III trials to date, FIX expression has appeared relatively more stable than FVIII, though individual patients also had prolonged FVIII expression. Whether lifelong expression of clotting factors can be realized after gene therapy requires longer follow-up studies. Further preclinical development of next-generation gene editing technologies offers new prospects for the development of a sustained cure for hemophilia, not only in adults, but ultimately in children with hemophilia too.

HTRS2023.O6B.2 Allosteric regulation of coagulation factor IXa

HTRS2023.O6B.2 Allosteric regulation of coagulation factor IXa

Mathematical modelling of gene delivery in patients with haemophilia B

Haemophilia B is a bleeding disorder resulting from a deficiency of coagulation factor IX (FIX). Although gene therapy is a potentially curative treatment option, optimising the dosing of therapeutic genes for patients remains a challenge. Detailed simulation of gene delivery systems is required for an improved understanding of the system. Hence, the purpose of this paper is to develop a modelling framework to predict the physiological response of a subject affected by type B haemophilia to a dose of the vector. To address this, an integrated pharmacokinetic/pharmacodynamic (PK/PD) modelling platform was developed based on in vivo clinical data for three patients with severe haemophilia B, whose functional plasma levels of FIX are less than 1% of the normal value. The plasma FIX activity was considered as the pharmacological effect, while the level of serum alanine aminotransferase (ALT) demonstrated the hepatocellular toxicity. Both an individual-based modelling approach and a population modelling approach were used to estimate the physiological parameters of the developed PK/PD models. The models were then validated using data from the clinical study before being used in a simulation-based modelling approach to provide dosing recommendations. The results obtained from the study demonstrate a good prediction of the pharmacokinetics and pharmacodynamics of the vector. Model-based simulations were subsequently performed to guide initial dose selection to provide clinicians with better tools to simplify the decision-making process for designing more effective treatment plans.

Efficacy of rFIXFc versus N9-GP Prophylaxis in Patients with Hemophilia B: Matching-Adjusted Indirect Comparison of B-LONG and PARADIGM 2 Trials.

For patients with hemophilia B, extended half-life factor IX (FIX) products are available for prophylaxis and for treating bleeds. Different methods are used to extend the half-lives of recombinant FIX Fc fusion protein (rFIXFc) and nonacog beta pegol (N9-GP). This affects their biodistribution and plasma FIX levels, although differences do not always correlate with clinical outcomes. A matching-adjusted indirect comparison (MAIC) of prophylaxis with rFIXFc and N9-GP was performed, based on licensed dosing in the European Union. Combined rFIXFc data from the weekly and individualized interval prophylaxis arms of the B-LONG clinical trial, and N9-GP data from the 40 IU/kg once-weekly prophylaxis arm of PARADIGM 2 were used in a MAIC. Individual patient data for rFIXFc (n=87) were matched to aggregated data for N9-GP (n=29). Estimated annualized bleeding rates (ABRs) for rFIXFc were recalculated using a Poisson regression model with adjustment for over-dispersion, and compared with ABRs reported for N9-GP, using incidence rate ratios (IRRs) with 95% confidence interval (CI). There was no evidence of significant differences in estimated ABRs between prophylaxis with rFIXFc and N9-GP. Analysis of pooled rFIXFc weekly and interval-adjusted dosing compared with N9-GP 40 IU/kg once weekly produced estimated ABRs of 2.59 versus 2.51 (IRR 1.03; 95% CI 0.56-1.89), as well as 1.34 versus 1.22 (IRR 1.10; 95% CI 0.42-2.91) and 1.13 versus 1.29 (IRR 0.88; 95% CI 0.47-1.63) for overall, spontaneous, and traumatic bleeding events, respectively. The study did not reveal any significant differences in the efficacy of rFIXFc and N9-GP prophylaxis. Given differences in trough levels (rFIXFc dosing was targeted to achieve a trough 1-3 IU/dL above baseline versus a reported estimated N9-GP mean trough of 27.3 IU/dL), interpreting plasma FIX levels as potential surrogate efficacy markers requires consideration of compound-specific pharmacokinetic profiles.

Plasma levels of coagulation factors VIII and IX and risk of venous thromboembolism: Systematic review and meta-analysis

IntroductionThe associations of plasma factor VIII (FVIII) and factor IX (FIX) levels with risk of venous thromboembolism (VTE) are not well defined. We performed a systematic review and meta-analysis of these associations. MethodsRandom effects inverse-variance weighted meta-analysis was used to estimate pooled odds ratios for comparisons across equal quartiles of the distributions and 90 % thresholds (higher versus lower), and for testing linear trends. ResultsAmong 15 studies (5327 cases) the pooled odds ratio of VTE for the fourth versus first quarter was 3.92 (95 % confidence interval 1.61, 5.29) for FVIII level; and among 7 studies (3498 cases) 1.57 (1.32, 1.87) for FIX level. Comparing factor levels above, versus below, the 90th percentile, the estimated pooled odds ratios were 3.00 (2.10, 4.30) for FVIII; 1.77 (1.22, 2.56) for FIX; and 4.56 (2.73, 7.63) for both FVIII and FIX considered jointly. ConclusionsWe confirm increases in risk of VTE across population distributions of FVIII and FIX levels. Levels above the 90th percentile have almost twice the risk for FIX level compared to levels below; three-fold risk for FVIII level; and almost five-fold risk for both FVIII and FIX levels elevated.

Model-Informed Support of Dose Selection for Prophylactic Treatment with Dalcinonacog Alfa in Adult and Paediatric HemophiliaB Patients.

Dalcinonacog alfa (DalcA), a novel subcutaneously administered recombinant human factorIX (FIX) variant is being developed for adult and paediatric patients with hemophiliaB (HB). DalcA has been shown to raise FIX to clinically meaningful levels in adults with HB. This work aimed to support dosing regimen selection in adults and perform first-in-paediatric dose extrapolations using a model-based pharmacokinetic (PK) approach. A population PK model was built using adult data from two clinical trials (NCT03186677, NCT03995784). With allometry in the model, clinical trial simulations were performed to study alternative dosing regimens in adults and children. Steady-state trough levels and the time-to-reach target were derived to inform dose selection. Almost 90% of the adults were predicted to achieve desirable FIX levels, i.e. 10% FIX activity, following daily 100IU/kg dosing, with 90% of the subjects reaching target within 1.6-7.1days. No every-other-day regimen met the target. A dose of 125IU/kg resulted in adequate FIX levels down to 6years, whereas a 150IU/kg dose was needed below 6 down to 2years of age. For subjects down to 6years that did not reach target with 125IU/kg, a dose escalation to 150IU/kg was appropriate. The children below 6 to 2years were shown to need a dose escalation to 200IU/kg if 150IU/kg given daily was insufficient. This study supported the adult dose selection for DalcA in the presence of sparse data and enabled first-in-paediatric dose selection to achieve FIX levels that reduce risk of spontaneous bleeds.