Gene therapy for haemophilia A and B, from basic principles to clinical implementation: An illustrated review.

INTRODUCTION: With recent approval of the first two gene therapies for haemophilia A and B, educational materials about AAV-based gene therapy are needed by the haemophilia community for a better understanding of this novel therapeutic approach and helping healthcare providers and patients making personalized choices amongst an increasing array of therapeutic options. AIM: To provide a comprehensive summary of the whole process of AAV-based gene therapy from basic principles to clinical implementation through an illustrated review. METHODS: The authors, with expertise in and knowledge about gene therapy for haemophilia A and B, reviewed relevant articles from PubMed database and translated them into illustrations. RESULTS: The review is divided into eight illustrated sections providing an overview of gene therapy for haemophilia A and B from haemophilia basics and current treatment landscape, principles of the AAV-based liver-directed gene therapy, through exploring the efficacy and safety results of published phase III clinical trials, current and future challenges, to implementation in clinical practice, including the hub and spoke models and the patient journey. CONCLUSION: This illustrated review educates healthcare professionals on AAV-based gene therapy for haemophilia A and B enabling them to further educate their peers and their patients.

Novel manifestations of immune dysregulation and granule defects in gray platelet syndrome.

Gray platelet syndrome (GPS) is a rare recessive disorder caused by biallelic variants in NBEAL2 and characterized by bleeding symptoms, the absence of platelet α-granules, splenomegaly, and bone marrow (BM) fibrosis. Due to the rarity of GPS, it has been difficult to fully understand the pathogenic processes that lead to these clinical sequelae. To discern the spectrum of pathologic features, we performed a detailed clinical genotypic and phenotypic study of 47 patients with GPS and identified 32 new etiologic variants in NBEAL2. The GPS patient cohort exhibited known phenotypes, including macrothrombocytopenia, BM fibrosis, megakaryocyte emperipolesis of neutrophils, splenomegaly, and elevated serum vitamin B12 levels. Novel clinical phenotypes were also observed, including reduced leukocyte counts and increased presence of autoimmune disease and positive autoantibodies. There were widespread differences in the transcriptome and proteome of GPS platelets, neutrophils, monocytes, and CD4 lymphocytes. Proteins less abundant in these cells were enriched for constituents of granules, supporting a role for Nbeal2 in the function of these organelles across a wide range of blood cells. Proteomic analysis of GPS plasma showed increased levels of proteins associated with inflammation and immune response. One-quarter of plasma proteins increased in GPS are known to be synthesized outside of hematopoietic cells, predominantly in the liver. In summary, our data show that, in addition to the well-described platelet defects in GPS, there are immune defects. The abnormal immune cells may be the drivers of systemic abnormalities such as autoimmune disease.

Report of surgeries, their outcome and the thrombin generation assay in patients with Factor XI deficiency: A retrospective single-centre study.

BACKGROUND: In patients with FXI deficiency, the risk of surgery-related bleeding is poorly correlated with plasma FXI activity (FXI:C); the latter can therefore not be used as a reliable predictor of bleeding in surgeries. OBJECTIVES: The aim of this retrospective study was to determine whether thrombin generation assay (TGA) could be used to evaluate the risk of surgery-related bleeding in FXI-deficient patients. TGA parameters were compared to FXI:C values, haemostatic treatments and surgical outcomes. PATIENTS: All patients followed at the haemophilia treatment care centre (Lyon, France) with a FXI:C < 50IU/dL, and for whom a baseline TGA was performed between January 2014 and December 2019, were included. RESULTS: Among the 175 surgeries reported herein in 49 patients, FXI concentrates were used for 11 (6%) surgeries and fresh frozen plasma was used for five (3%) surgeries; these surgeries were performed in patients with two or three impaired TGA parameters. No haemostatic treatment was prescribed for 119 (68%) surgeries. A surgery-related bleeding occurred in 12 patients during 21 (12%) surgeries. Thrombin generation was significantly reduced or delayed in patients who reported surgery related-bleeding. Among the 34 (68%) surgeries performed without haemostatic treatment in patients with three impaired TGA parameters, a surgery-related bleeding was reported in 44% of cases (15 surgeries out of 34). CONCLUSION: The present study confirmed that TGA is an interesting laboratory test in FXI deficiency, for determining the bleeding risk and guiding the haemostatic management of surgeries, while taking into account the surgical bleeding risk and the history of bleeding.

Immune tolerance induction in the era of emicizumab - still the first choice for patients with haemophilia A and inhibitors?

INTRODUCTION: The development of inhibitory antibodies is a severe complication of clotting factor replacement therapy in patients with severe haemophilia A (HA). Current World Federation of Hemophilia (WFH) guidelines for haemophilia care indicate that eradication of inhibitors is best achieved through immune tolerance induction (ITI) therapy. AIM: The European Collaborative Haemophilia Network conducted a survey to determine whether ITI is still used in the routine management of patients with HA, and whether the availability of emicizumab prophylaxis has influenced treatment decisions. METHODS: The survey was conducted in late 2020/early 2021 in 18 centres representing 17 countries in the Europe/Middle East region treating a total of 4955 patients, and included sections specific to patient and centre demographics, treatment protocols (both ITI and prophylactic), inhibitor development and initiation of ITI, treatment success, and the incidence of adverse events. RESULTS: While our results indicate that ITI can still be considered a mainstay of treatment for patients with HA with inhibitors, less than daily dosing of ITI in combination with emicizumab prophylaxis is becoming commonplace across the spectrum of disease severity, with initiation being guided by bleeding patterns. The most frequently cited reasons for not initiating emicizumab prophylaxis were availability or reimbursement issues. CONCLUSION: ITI remains a mainstay for haemophilia treatment of patients with HA with inhibitors, but emicizumab has become a preferred first-line approach to protect against bleeds and represents an alternative to burdensome ITI in certain patient groups.

FcRn as a Transporter for Nasal Delivery of Biologics: A Systematic Review.

FcRn plays a major role in regulating immune homeostasis, but it is also able to transport biologics across cellular barriers. The question of whether FcRn could be an efficient transporter of biologics across the nasal epithelial barrier is of particular interest, as it would allow a less invasive strategy for the administration of biologics in comparison to subcutaneous, intramuscular or intravenous administrations, which are often used in clinical practice. A focused systematic review was conducted following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. It was registered on the international prospective register of systematic reviews PROSPERO, which helped in identifying articles that met the inclusion criteria. Clinical and preclinical studies involving FcRn and the nasal delivery of biologics were screened, and the risk of bias was assessed across studies using the Oral Health Assessment Tool (OHAT). Among the 12 studies finally included in this systematic review (out of the 758 studies screened), 11 demonstrated efficient transcytosis of biologics through the nasal epithelium. Only three studies evaluated the potential toxicity of biologics' intranasal delivery, and they all showed that it was safe. This systematic review confirmed that FcRn is expressed in the nasal airway and the olfactory epithelium, and that FcRn may play a role in IgG and/or IgG-derived molecule-transcytosis across the airway epithelium. However, additional research is needed to better characterize the pharmacokinetic and pharmacodynamic properties of biologics after their intranasal delivery.

Potential limits of AAV-based gene therapy with the use of new transgenes expressing factor IX fusion proteins.

INTRODUCTION: The variety of treatment for haemophilia B (HB) has recently improved with the emergence of both AAV-based gene therapy and bioengineered human factor IX (hFIX) molecules with prolonged half-life due to fusion to either albumin (Alb) or immunoglobulin Fc fragment (Fc). AIM: Adeno-associated viral vectors (AAV) mediating expression of hFIX-Alb and hFIX-Fc fusion proteins was investigated for gene therapy of HB to explore if their extended half-life translates to higher plasma levels of FIX. METHODS: Single-stranded cross-packaged AAV2/8 vectors expressing hFIX-Alb, hFIX-Fc and hFIX were evaluated in vitro, and in mice. RESULTS: Both hFIX-Alb and hFIX-Fc fusion proteins were synthesized and expressed as single chains of expected size following AAV-mediated gene transfer in vitro and in vivo. The procoagulant properties of these hFIX-fusion proteins were comparable to wild-type hFIX. However, their expression levels were threefold lower than wild-type hFIX in vivo most likely due to inefficient secretion. CONCLUSION: This, the first, evaluation of hFIX-fusion proteins in the context of AAV gene transfer suggests that the hFIX-fusion proteins are secreted inefficiently from the liver, thus preventing their optimal use in gene therapy approaches.

Differential diagnosis of neonatal alloimmune thrombocytopenia: Type 2B von Willebrand disease.

At birth, severe thrombocytopenia without context of infection should mainly suggest neonatal alloimmune thrombocytopenia (NAIT), especially in case of a platelet count below 20 GL-1. We report two cases of severe neonatal thrombocytopenia, first suspected as being NAIT. Both had a platelet count below 20 GL-1 with platelet clumps. The absence of alloantibodies and failure of platelet transfusion and intravenous immunoglobulins to improve the platelet count led to question the diagnosis and to evoke inherited bleeding disorders. Measurements of Von Willebrand factor (VWF) levels showed a marked reduction of VWF:RCo and a normal VWF:Ag, suggesting a type 2B Von Willebrand disease (VWD2B). Ristocetin-induced platelet aggregation could not be performed because of the very low platelet count. In the first case, after sequencing VWF exon 28, a heterozygous p.Leu1460Pro mutation was found consistent with VWD2B. In the second case, the genetic analysis of VWF exon 28 identified a homozygous mutation: p.Pro1337Leu confirming type VWD2B and also the p.Arg854Gln homozygous mutation in exon 20 confirming type 2N (ratio FVIII/VWF:Ag <0.5). The two cases underline that, even if NAIT remains the most common diagnosis in severe neonatal thrombocytopenia, it should be challenged in the absence of documented incompatibility, chronic evolution, or treatment failure. Diagnosis of VWD2B should be considered in early thrombocytopenia, even without familial history. In the cases presented, genotyping confirmed the subtype of VWD and helped to guide the therapeutic management of bleeding episodes.

A 360-degree perspective on adeno-associated virus (AAV)-based gene therapy for haemophilia: Insights from the physician, the nurse and the patient.

BACKGROUND: Adeno-associated virus (AAV)-based gene therapy for haemophilia has advanced substantially in the last 13 years; recently, three products have received approvals from regulatory authorities. Although the impact on quality of life seems promising, some limitations remain, such as the presence of pre-existing anti-AAV neutralising antibodies and the occurrence of hepatotoxicity. This review follows the CSL Behring-sponsored symposium at the 27th Congress of the European Hematology Association (EHA) 2022 that examined the haemophilia gene therapy process from a 360-degree multidisciplinary perspective. Here, the faculty (haematologist, nurse and haemophilia patient) summarised their own viewpoints from the symposium, with the aim of highlighting the key considerations required to engage with gene therapy effectively, for both patients and providers, as well as the importance of multidisciplinary collaboration, including with industry. RESULTS: When considering these new therapies, patients face a complex decision-making process, which includes whether gene therapy is right for them at their current stage of life. The authors agreed that collaboration and tailored education across the multidisciplinary team (including patients and their carers/families), starting early in the process and continuing throughout the long-term follow-up period, is key for the success of gene therapy. Additionally, patient expectations, which may surround eligibility, follow-up requirements and treatment outcomes, should be continually explored. During these ongoing discussions, transparent communication of the unknown factors, such as anticipated clotting factor levels, long-term factor expression and safety, and psychological changes, is critical. To ensure efficiency and comprehensiveness, clearly­defined protocols should outline the whole process, which should include the recording and management of long-term effects. CONCLUSION: In order to engage effectively, both patients and providers should be familiar with these key considerations prior to their involvement with the haemophilia gene therapy process. The future after the approval of haemophilia gene therapies remains to be seen and real-world evidence is eagerly awaited.

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.

Septic arthritis caused by noncapsulated Haemophilus influenzae.

Since the introduction of type b Haemophilus influenzae vaccination, noncapsulated H. influenzae has become responsible for most cases of invasive H. influenzae diseases. In our two cases of septic arthritis, we isolated strains with ß-lactamase-positive amoxicillin-clavulanate resistance (BLPACR). Thus, the increasing prevalence of BLPACR should be taken into account when empirical therapy is chosen for septic arthritis.

In vivo intranasal delivery of coagulation factor IX: a proof-of-concept study.

BACKGROUND: Hemophilia B (HB) is a bleeding disorder characterized by coagulation factor (F) IX (FIX) deficiency. The current standard-of-care for severe HB is prophylaxis with long-term repetitive intravenous (i.v.) infusions of recombinant FIX (rFIX) with standard half-life or extended half-life. Unmet needs remain regarding the development of non-invasive administration routes for coagulation factors. The aim of this study was to evaluate the effectiveness of intranasal delivery (IND) of rFIX and rFIX fused to Fc fragment (rFIX-Fc) in mice. METHODS: Drops of rFIX and rFIX-Fc were deposited in the nostrils of wild-type, FcRn knock-out, FcRn humanized, and FIX knock-out mice. rFIX mucosal uptake was evaluated by measuring plasma FIX antigen and FIX activity (FIX:C) levels, and by performing histologic analysis of the nasal mucosa following IND. RESULTS: After IND, both rFIX and rFIX-Fc were equally delivered to the blood compartment, irrespective of the mouse strain studied, mostly through a passive mechanism of transportation across the mucosal barrier, independent of FcRn receptor. Both plasma FIX antigen and FIX:C activity levels increased following IND in FIX knock-out mice. CONCLUSION: This proof-of-concept study describes evidence supporting the nasal route as an alternative to FIX i.v. infusion for the treatment of HB.

Stable and durable factor IX levels in patients with hemophilia B over 3 years after etranacogene dezaparvovec gene therapy.

Etranacogene dezaparvovec (AMT-061) is a recombinant adeno-associated virus serotype 5 (AAV5) vector containing a codon-optimized Padua variant human factor IX (FIX) transgene with a liver-specific promoter. Here, we report 3-year outcomes from a phase 2b, open-label, single-dose, single-arm, multicenter trial conducted among adults with severe or moderately severe hemophilia B (FIX ≤2%). All participants (n = 3) received a single intravenous dose (2 × 1013 gene copies per kg) and will be followed up for 5 years. The primary end point of FIX activity ≥5% at 6 weeks was met. Secondary end points included bleed frequency, FIX concentrate use, joint health, and adverse events (AEs). All participants required routine FIX prophylaxis and had neutralizing antibodies to AAV5 before etranacogene dezaparvovec treatment. After administration, FIX activity rose to a mean of 40.8% in year 1 and was sustained in year 3 at 36.9%. All participants discontinued FIX prophylaxis. Bleeding was completely eliminated in 2 out of 3 participants. One participant required on-demand FIX replacement therapy per protocol because of elective surgical procedures, for 2 reported bleeding episodes, and twice for a single self-administered infusion because of an unreported reason. One participant experienced 2 mild, self-limiting AEs shortly after dosing. During the 3-year study period, there were no clinically significant elevations in liver enzymes, no requirement for steroids, no FIX inhibitor development, and no late-emergent safety events in any participant. Etranacogene dezaparvovec was safe and effective in adults with hemophilia B over 3 years after administration. This trial was registered at www.clinicaltrials.gov as #NCT03489291.

Recombinant porcine factor VIII corrects thrombin generation in vitro in plasma from patients with congenital hemophilia A and inhibitors.

Background: Neutralizing factor VIII (FVIII) antibodies are a major complication in hemophilia A. Antihemophilic factor VIII (recombinant), porcine sequence (rpFVIII; susoctocog alfa; Baxalta US Inc., a Takeda company) has low cross-reactivity to anti-human FVIII antibodies and can provide functional FVIII activity in the presence of FVIII inhibitors. Objectives: Evaluate in vitro thrombin generation and clot formation responses to rpFVIII in blood from patients with congenital hemophilia A. Methods: In this multicenter study, blood was obtained for in vitro analyses that included human and porcine FVIII inhibitors, low <5 Bethesda units (BU)/ml or high ≥5 BU/ml titer (Nijmegen-modified Bethesda assay); thrombin generation assay (TGA), clot viscoelasticity (thromboelastography), fibrin clot structure analysis (scanning electron microscopy), and epitope mapping. Results: Blood samples were from 20 patients with congenital hemophilia A (FVIII activity <1%, mean [range] inhibitor titers: anti-human FVIII, 14 [1-427] BU/ml [n = 13 high, n = 6 low, n = 1 data unavailable]); anti-porcine FVIII, 12 (0-886) BU/ml (n = 11 high, n = 8 low, n = 1 data unavailable). Porcine inhibitor titer and TGA response measured by endogenous thrombin potential showed an inverse correlation (2.7-10.8 U/ml rpFVIII Spearman correlation coefficient: -0.594 to -0.773; p < 0.01). Clot structures in low anti-porcine inhibitor titer plasmas were similar to those in noninhibitor plasma. Conclusions: Recombinant porcine factor VIII demonstrated a dose-dependent correction of thrombin generation and clot formation in vitro, dependent on the anti-porcine FVIII inhibitor titer. Procoagulant responses to rpFVIII occurred in plasma containing FVIII inhibitors.

Clinical Evidence and Safety Profile of Emicizumab for the Management of Children with Hemophilia A.

Emicizumab is a bispecific, humanized, monoclonal antibody mimicking the factor (F) VIII cofactor activity in mediating the generation of FXa by FIXa in patients with hemophilia A (HA). This subcutaneous non-factor agent has been recently extensively approved for the prophylaxis of patients of HA patients with and without FVIII-inhibitors of all ages, although few data are currently available in children. In Phase 3 clinical trials and case series, emicizumab prophylaxis significantly reduced bleeding rates compared to previous treatment in HA adolescents and children with or without FVIII-inhibitors and was generally well tolerated. In addition, subcutaneous administration of emicizumab provided beneficial effects on health-related quality of life, and lessened the burden of the disease in HA patients as well as in their caregivers. However, additional prospective studies are required to evaluate the long-term safety of emicizumab prophylaxis in very young patients, including previously untreated patients. The aim of this paper was to review the limited data available on the use of emicizumab prophylaxis in children and to highlight the need for further studies to address remaining concerns.

[Discrepancies in FVII:C levels depending on the thromboplastin: about a case]. / Discordances du taux de facteur VII:C selon la thromboplastine : à propos d'un cas.

Factor VII deficiency is the most common of the rare coagulation deficiencies. A hemorrhagic syndrome may occur in patients with FVII deficiency below 20%, although no correlation exist between the plasma FVII activity level (FVII:C) and the bleeding risk. Therefore, the management of surgery in patients with FVII deficiency remains challenging. Laboratory monitoring of FVII:C level may be helpful but should be interpreted with caution, because the dosage of FVII:C level may vary depending on the origin of the thromboplastin used. Herein, we report the case of the management of a woman who had been fortuitously diagnosed during pregnancy with FVII deficiency due to FVII variant Padua, which have induced discrepant results between two different laboratories.

A Prolonged Treatment Response in Acquired Von Willebrand Syndrome.

Acquired von Willebrand syndrome (AVWS) is a rare bleeding disorder. We report herein a case of AVWS due to a monoclonal gammopathy of undetermined significance, in which a transient but prolonged response to a treatment by intravenous immunoglobulin (IVIG) was observed. The diagnosis was fortuitously made in a preoperative setting for neurosurgery, after biological exploration of an isolated prolonged activated partial thromboplastin time. AVWS was confirmed by an accelerated clearance of an infused plasma-derived von Willebrand factor (VWF) concentrate. High doses of IVIG were used to perform the neurosurgery. Fifty-four days after IVIG, the patient was still responding to treatment with normal levels of factor VIII and VWF.