Quand la création de nouvelles molécules recombinantes de facteur IX de la coagulation rencontre la thérapie génique : pourrait-on davantage améliorer le niveau plasmatique de facteur IX chez les patients hémophiles B ?

Introduction: Haemophilia B (HB) is an inherited bleeding disorder due to coagulation factor IX (FIX) deficiency. Adeno-associated virus (AAV)-based gene therapy for HB has shown promising results but can cause liver toxicity after administration of high dose of AAV vectors.The design of new transgene expressing modified FIX that would allow injecting fewer doses of AAV is a real challenge. Materials & Methods: Therapeutic transgene expressing human FIX with prolonged half-life due to fusion to mature albumin (hFIX-Alb) or expressing FIX with improved specific activity, hFIX-E410H, were designed and injected to murine animal model. A novel recombinant FIX molecule exhibiting enhanced half-life through fusion to the FXIIIB subunit via activated factor X-cleavable linker was design, produced and characterised. Results: The hFIX-Alb transgene did not increase the plasma FIX clotting activity compared to the transgene expressing wild-type hFIX. Experiments were undertaken to understand the mecanisms responsible for lower expression. The hFIX-E410H transgene, which showed improved specific activity in vitro and in vivo in HB mice, allowed injecting a 2.5-fold lower dose of AAV. The hFIX-LXa-FXIIIB molecule was functional, corrected the generation capacity in HB mice, and exhibited a 3.9-fold and 2.2-fold enhanced half-life in mice and in rats, respectively, compared to wild-type FIX. Conclusion: We have developed and characterised new transgenes expressing modified FIX, and a novel FIX molecule with prolonged half-life, which could become interesting perspectives for the treatment of HBIntroduction : L’hémophilie B (HB) est une maladie hémorragique héréditaire caractérisée par un déficit en facteur IX (FIX) de la coagulation. La thérapie génique de l’HB par injection de virus adéno-associés (AAV) montre des résultats prometteurs, mais entraine une toxicité hépatique à forte dose. La création de nouveau transgène de FIX permettant d’injecter de moindres doses d’AAV est un réel enjeu. Matériel et Méthodes : Des transgènes thérapeutiques exprimant une protéine humaine de FIX à demi-vie prolongée par fusion à l’albumine (hFIX-Alb) ou exprimant un FIX une activité spécifique augmentée, le hFIX-E410H, ont été créés et injectés à des modèles murins. Une nouvelle molécule recombinante de hFIX à demi-vie prolongée par fusion à la sous-unité B du FXIII via un linker clivable par le facteur X activé (hFIX-LXa-FXIIIB) a été crée, produite et caractérisée. Résultats : Le transgène hFIX-Alb n’accumulait pas le niveau plasmatique du FIX par rapport au FIX sauvage. Des expériences ont été entreprises pour comprendre les mécanismes responsables du défaut d’expression. Le transgène hFIX-E410H, montrant une activité spécifique augmentée in vitro et in vivo chez les souris HB, permettait de diminuer les doses d’AAV d’environ 2,5 fois. La molécule hFIX-LXa-FXIIIB était fonctionnelle, corrigeait la génération de thrombine chez les souris HB, et présentait une demi-vie augmentée 3,9 fois chez la souris et 2,3 fois chez le rat. Conclusion : Nous avons développé et caractérisé de nouveaux transgènes de FIX modifiés et une nouvelle molécule de FIX à demi-vie prolongée, qui pourraient constituer de nouvelles perspectives thérapeutiques de l’H

Le facteur VIII porcin recombinant chez les patients atteints d'hémophilie A avec inhibiteurs

L'hémophilie A est une maladie hémorragique héréditaire rare caractérisée par un déficit en facteur VIII (FVIII) de la coagulation, pouvant se compliquer par le développement d'inhibiteurs anti-FVIII. Les inhibiteurs du FVIII sont souvent moins réactifs vis-à-vis du FVIII porcin (FVIIIp) que du FVIII humain (FVIIIh), supposant qu'un concentré du FVIHp pourrait devenir une option thérapeutique pour les patients ayant développé des inhibiteurs. L'objectif était de déterminer l'efficacité hémostatique in vitro de doses croissantes d'OBI-1, un concentré de facteur VIII (FVIII) recombinant d'origine porcine,avec délétion du domaine B, à l'aide de tests globaux de l'hémostase, en se basant sur les titres des inhibiteurs anti-FVIIIhumain (anti-FVIIIh) et anti-FVIII porcin (anti-FVIIIp) et sur les épitopes ciblés par ces inhibiteurs. OBI-1 était ajouté in vitro dans les plasmas de patients hémophiles A sévères avec inhibiteurs à raison de 0, 2,7, 5,4 et 10,8 U mL- 1 correspondant aux doses 0, 100,200 et 400 U kg-1 , respectivement. Les inhibiteurs anti-FVIIIh et anti-FVIIIp étaient considérés comme faibles, modérés et forts après titrage par la méthode Nijmegen à 5-10 et> 10 UB mL-\ respectivement. Des tests globaux de l'hémostase tels que la thromboélastographie (TEG), le test de génération de thrombine (TGT) suivi de l'analyse par microscopie électronique du caillot, étaient réalisés pour l'ensemble des plasmas. L'épitope mapping était réalisé par ELISA directe. Nous avons utilisés des analyses statistiques de variance (ANOV A), de corrélation de Spearman et de t-test. Une valeur de p <0,05 considérée comme significative. Vingt-et-un patients atteints d'hémophilie A sévères avec inhibiteurs ont été inclus dans l'étude. Respectivement, des titres faibles, modérés et forts étaient retrouvés pour 6, 2 et 13, et 10, 0 et 11 des inhibiteurs anti-FVIIlh et anti-FVIIIp. Parmi 16 patients n'ayant pas reçu de traitement antérieur par FVIIIp, la réactivité croisée était de 62%. Trois paramètres de TGT, l'ETP, le pic de thrombine et le temps jusqu'au pic, montraient une amélioration de la capacité de génération de thrombine dépendante de la dose (p<O,OOO 1, p=0,0006 et p=0,0001, respectivement) et du titre des inhibiteurs anti-FVIIIp (r=-0,67; p=0,0009). Le même effet était retrouvé pour le raccourcissement du temps de coagulation exploré par TEG (p<O,OOO 1 ). Les domaines C2 et A2 représentaient 40 et 35% des épitopes, respectivement. Aucune relation n'apparaissait entre les épitopes et la restauration de la capacité hémostatique des plasmas contenant des inhibiteurs. 081-1 a prouvé son efficacité à restaurer une capacité hémostatique satisfaisante dans les plasmas, dépendante de la dose et du titre des inhibiteurs anti-FVIIIp. Des études cliniques actuellement en cours d'investigation devraient permettre de confirmer ces résultats in vivoLYON1-BU Santé (693882101) / SudocSudocFranceF

Why patients with THBD c.1611C>A (p.Cys537X) nonsense mutation have high levels of soluble thrombomodulin?

Recently our group has described a new autosomal dominant bleeding disorder characterized by very high plasma levels of soluble thrombomodulin (TM). The THBD c.1611C>A (p.Cys537X) mutation in heterozygous state was found in the propositus. This mutation leads to the synthesis of a truncated TM which has lost the last three amino-acids of the transmembrane domain and the cytoplasmic tail.We investigated the mechanism responsible for TM shedding in endothelial cells with THBD c.1611C>A mutation.Complementary DNA of TM wild type (TM-WT) was incorporated into a pcDNA3.1 vector for transient transfection in COS-1 cells. Mutagenesis was performed to create the c.1611C<A (TM1-536) mutant and 4 other TM mutants (TM1-515, TM1-525, TM1-533 and TM1-537) with a transmembrane domain having different lengths. The effect of shear stress, metalloprotease inhibitor, certain proteases and reducing agents were tested on TM shedding.Western blot and immunofluorescent analysis showed that TM1-536 was produced and a certain amount of TM1-536 was anchored on the cell membrane. A significantly higher levels of soluble TM was observed in the TM1-536 cell medium in comparison with TM-WT (56.3 +/- 5.2 vs 8.8 +/- 1.6 ng/mL, respectively, p = 0.001). The shedding of TM1-536 was 75% decreased in cells cultured in the presence of a metalloprotease inhibitor. No difference was observed between TM1-536 and TM-WT shedding after cell exposure to cathepsin G, elastase, several reducing agents and high shear stress (5000 s-1). Significantly higher levels of soluble TM were observed in the cell media of TM1-533, TM1-525, TM1-515 in comparison with TM-WT (p < 0.05).The mechanism responsible for TM shedding is complex and is not completely understood: higher sensitivity of the TM1-536 to the proteolysis by metalloproteases and a defect of synthesis due to the decreased size of the transmembrane domain might explain the high levels of soluble TM in plasma of the carriers

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

International audienceFcRn 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

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 knockout , FcRn humanized, and FIX knockout 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 knockout 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

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