Emicizumab is a recombinant, humanized, bispecific, monoclonal antibody that bridges activated factor IX and X to restore the function of deficient VIII. Treatment bleeding in patients with hemophilia inhibitors involves use bypassing agents (BPA).These molecules are also used for management breakthrough bleeds on prophylaxis emicizumab, increased concerns about risks combining two procoagulant drugs. Using thrombin generation assay, we tailored dosage prothrombin complex concentrate (APCC)...

Inhibition of specific transcriptional regulatory proteins is a new approach to control gene expression. Transcriptional activity DNA-binding can be inhibited by the use double-stranded (ds) oligodeoxynucleotides that compete for binding their target sequences in promoters and enhancers. As model, we used phosphodiester dumbbell oligonucleotides containing site liver-enriched transcription factor HNF-1 (Hepatocyte Nuclear Factor 1). Binding affinity was same as ds oligonucleotides,...

The biosynthesis of coagulation factor VIII (FVIII) is hampered by successive controls that limit its production. To improve this production, a truncated intron I sequence IX (TFIXI1) was inserted in FVIII cDNA place introns 1, 12 and 13 also as combination between 1 12, 13. locations were targeted because region previously shown to contain transcriptional silencer. expression CHO HepG2 cells revealed important variations the properties minigenes depending on TFIXI1 insertion sites. In...

We have developed a gene therapy project for haemophilia B which aims to express factor IX (FIX) in haematopoietic lineage. Haematopoietic stem cells and subsequent megakaryocyte-derived represent the target of this approach. Our speculation is that platelets can deliver coagulation at site injury, subsequently correct haemostasis defect. In order direct FIX expression from megakaryocytic lineage, we designed cassette where cDNA was placed under control tissue-specific glycoprotein IIb...

Abstract Gene therapy using recombinant adeno-associated virus (AAV) has induced sustained long-term coagulation human factor IX (hFIX) levels in hemophilia B (HB) patients. However, asymptomatic transient liver toxicity was observed at high vector doses, highlighting the need to improve potency of these vectors. We report generation an AAV transgene cassette containing hyperfunctional hFIX-E456H variant showing improved binding platelets, with a comparison wild-type hFIX (hFIX-WT) and...

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 the propositus. This leads to synthesis truncated TM which lost last three amino-acids transmembrane domain and cytoplasmic tail.We investigated mechanism responsible for shedding endothelial cells with mutation.Complementary DNA wild type (TM-WT) incorporated into pcDNA3.1...

Prophylaxis is currently considered the optimal care for severe haemophilia. For patients and their families one of major difficulties with prophylaxis need frequent venipunctures. The half-life standard factor IX (FIX) concentrates approximately 18 hours, which requires 2 or 3 intravenous infusions per week to achieve bleeding prevention in haemophilia B. Prolonging FIX can therefore reduce frequency infusions. Recently, extended recombinant (rFIX) have been developed. We designed a new...

Summary Three in-frame potential methionine codons have been identified in human factor IX gene and are clustered at amino acids -46, -41 -39. In view of initiating a therapy approach, production has evaluated after modifications these first three translation start sites. To characterize the most efficient initiation context, five cDNA expression vectors directed by CMV promoter-enhancer were generated. These contained different starting site combinations including one, two or ATG. A...

Summary Factor VIII (FVIII) processing within mammalian cells is demonstrated to be much less efficient than proteins of similar size. The deletion the B-domain from FVIII improves level production, due partly increase in mRNA synthesis. We aimed characterise cellular fate and intracellular molecule devoid B-domain. A deleted factor (BDD-FVIII) possessing a furin consensus cleavage site connecting segment between heavy light chain, was produced CHO cell line. In such cells, retained as two...

Introduction Post‐translational modifications of the CHO‐cell‐derived‐recombinant human factor IX (FIX) currently used for treatment hemophilia B (HB) are different from plasma derived FIX. Our previous studies described a rFIX (HIX) having better profile post‐translational than produced by CHO cells. The aim study consisted to verify improved effect HIX on in vivo recovery. Materials and methods has been bioreactor then purified supernatants. In vitro activation activity were evaluated...

Abstract Introduction The variety of treatment for haemophilia B (HB) has recently improved with the emergence both AAV‐based gene therapy and bioengineered human factor IX (hFIX) molecules prolonged half‐life due to fusion either albumin (Alb) or immunoglobulin Fc fragment (Fc). Aim Adeno‐associated viral vectors (AAV) mediating expression hFIX‐Alb hFIX‐Fc proteins was investigated HB explore if their extended translates higher plasma levels FIX. Methods Single‐stranded cross‐packaged...

Abstract Introduction and aim Severe haemophilia B (HB) is characterized by spontaneous bleeding episodes, mostly into joints. Recurrent bleeds lead to progressive joint destruction called haemophilic arthropathy. The current concept of prophylaxis aims at maintaining the FIX level >3–5 IU/dL, which effective reducing incidence Extended half‐life molecules make it easier achieve these target trough levels compared standard concentrates. We previously reported that fusion a recombinant...

Summary Haemophilia A is an attractive target for gene therapy. We designed a haemophilia therapy strategy involving the genetic modification of haematopoietic stem cells to achieve tissue‐specific expression factor VIII (FVIII) transgene in megakaryocytic lineage. Platelets would then serve as vehicles store expressed FVIII and deliver coagulation at site vascular injury. local correction haemostasis defect could, therefore, be expected following platelet activation secretion. In this...

Patients with severe haemophilia have impaired haemostatic response, delayed clot formation and fibrin clots that are vulnerable to fibrinolysis. Emicizumab is a bispecific antibody mimics activity of activated factor VIII (FVIII) increases capacity the level moderate-to-mild haemophilia, thereby used for prophylaxis. Regardless impressive clinical performance emicizumab, breakthrough bleeds may still occur. We aimed study, in FVIII knockout mice (FVIII-KO), whether haemostasis improved...

Hemophilia B (HB) is a bleeding disorder characterized by coagulation factor (F) IX (FIX) deficiency. The current standard-of-care for severe HB prophylaxis with long-term repetitive intravenous (i.v.) infusions of recombinant FIX (rFIX) standard half-life or extended half-life. Unmet needs remain regarding the development non-invasive administration routes factors. aim this study was to evaluate effectiveness intranasal delivery (IND) rFIX and fused Fc fragment (rFIX-Fc) in mice.Drops...