Background Hemophilia B is an X-linked bleeding disorder that results in a qualitative or quantitative factor IX (FIX) deficiency. A subset of hemophilia B patients treated with FIX replacement therapy develop antibodies against FIX. The development of FIX inhibitors can present as allergic or anaphylactic reactions in some patients. It is currently unknown why a subset of hemophilia B patients develop inhibitors. CD4+ T cells recognizing FIX-derived peptides on major histocompatibility complex (MHC) class II on antigen-presenting cells are crucial for the development of class-switched, high-affinity anti-FIX antibodies. Objectives In this study, we explored MHC class II presentation of FIX-derived peptides by antigen presenting cells. Methods We incubated purified FIX with monocyte derived dendritic cells of healthy, human leukocyte antigen-typed donors. MHCII peptide complexes were immunopurified, followed by peptide elution and mass spectrometry-based analysis of eluted peptides. Results We analyzed 12 healthy donors; of them, 8 presented FIX-derived peptides. The majority of FIX peptides were derived from the serine protease domain of FIX. With the exception of 1 donor, all of them presented at least one peptide containing the same core sequence, “NVIRIIPHHN” (residues 295-306 of FIX). Conclusions Taken together, we identified a promiscuously presented peptide derived from the protease domain of FIX. Based on its frequent presentation in multiple donors, we speculate that this peptide may be recognized by FIX-specific CD4+ T cells that develop in hemophilia B patients with inhibitors. Peptides identified in our study can be used for inhibitor risk stratification and development of novel therapies to induce tolerance.

In contrast to major innovations in treating severe hemophilia, treatment of severe von Willebrand disease (VWD) remains limited to intravenous infusion of von Willebrand factor (VWF) concentrates. To date, no gene therapy-based approaches for treatment of VWD have been developed, largely due to the disease’s heterogeneous mutational landscape and the challenge of specifically targeting VWF production in endothelial cells. In this study we developed a novel gene therapy strategy for patients suffering from VWD caused by heterozygous dominant-negative VWF variants. Our strategy permanently inactivates VWF variants by selectively disrupting the pathogenic allele’s open reading frame (ORF) via introduction of indels by Cas9. To circumvent the challenge of designing variant-specific strategies, we targeted the common SNP rs1800378 in VWF. We used endothelial colony forming cells (ECFCs) from VWD2A and VWD2B patients with heterozygous p.C1190R and p.R1306W variants, respectively, to demonstrate ex vivo proof-of-principle. Using NGS analysis we show efficient and allele-selective knock-out of VWF, while maintaining VWF expression of the non-targeted allele. Variant mapping mass spectrometry that discriminates between wild type and variant VWF proteoforms confirmed selective reduction of variant allele expression, which was accompanied by reversal of cellular disease phenotypes in ECFCs. This study shows the feasibility of a novel gene editing strategy for VWD that, by virtue of its targeting of a common SNP, can be broadly applicable and can be used to design treatments for VWD without being constrained by the disease-causing variant, pathogenic mechanism, or VWD subtype.

Bleeding crises are common complications in hemato-oncological patients undergoing chemotherapy and stem cell transplantation. Platelet transfusions are frequently administered to treat and prevent bleeding events at low platelet counts. However, the association between thrombocytopenia and bleeding events remains unclear, arguing for an improved understanding of pathophysiological changes underpinning bleeding events. Therefore, this study aims to identify plasma protein levels associated with bleeding events in a hemato-oncological patient population using unbiased mass spectrometry-based plasma profiling. Plasma samples and clinical records from 176 hemato-oncological patients included in the Pathogen Reduction Evaluation and Predictive Analytical Rating Score study were analyzed. Of 600 identified proteins, abundance levels of 55 proteins were found to be associated with bleeding severity at the time of sampling. Among these, a large cluster of platelet proteins (n = 48) as well as coagulation factor VII and vitamin K-dependent protein C were observed. Interestingly, 9 of these 55 proteins were also associated with the time to major bleeding event (World Health Organization grade ≥2). Platelet protein levels correlated with platelet counts but not with white cell counts or platelet transfusions. Taken together, our results suggest that lower abundance of platelet and coagulation proteins in plasma are associated with increased bleeding grade and time to major bleeding events in hemato-oncological patients. This highlights the potential for improved hemostatic monitoring of hemato-oncological patients at risk of bleeding, beyond platelet count alone.

Background: Antiphospholipid syndrome (APS) is an autoimmune disease characterized by the persistent presence of antiphospholipid antibodies (aPL), mainly targeted against β2 glycoprotein 1 (β2GP1). The autoimmune response to β2GP1 is aimed at several B cell and T cell epitopes. Molecular mimicry of these epitopes by gut commensal proteins, so-called mimotopes, causing cross-immunization might contribute to the formation of aPL. Objective: To study the potential role of gut microbiome cross-immunization in APS by examining cross-reactivity of aPL with gut commensal mimotope containing proteins. Methods: Fecal microbial metagenome of APS patients was determined using shotgun sequencing. An in-house developed in silico pipeline was used to identify gut commensal proteins that show sequence homology with known β2GP1 B and T cell epitopes in the metagenomic data. An enzyme-linked immunosorbent assay was used to test the identified microbial proteins for IgG cross-reactivity, with plasma of 21 APS patients and 17 control participants. Results: The in silico pipeline resulted in the identification of six gut commensals with a B cell and T cell β2GP1 epitope homologue. Of these, YjjG family noncanonical pyrimidine nucleotidase, one of the candidate-β2GP1 B cell mimicking proteins, showed significantly increased IgG reactivity in APS patients compared to control participants, as well as higher binding of a specific anti-β2GP1 monoclonal antibody than a negative control. Conclusion: Our study shows reactivity of IgG antibodies to YjjG family noncanonical pyrimidine nucleotidase from Roseburia amylophila in APS patients. Insights into the origins of antibody formation may yield new therapeutic targets for improvement of APS treatment.

Background:Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is a rare and potentially fatal autoimmune disease caused by a severe ADAMTS13 functional deficiency, mediated by autoantibodies targeting ADAMTS13. Despite high survival rates achieved with current treatments, between 20% and 50% of patients experience relapses. Ensuring longer-lasting recovery by restoring immune tolerance toward ADAMTS13 remains a significant unmet need. Objectives:To address this, we aimed to exploit the tolerogenic nature of the red blood cell (RBC) clearance pathway by using RBCs as antigen carriers. This approach allows for persistent exposure to ADAMTS13-derived peptides, promoting antigen-specific attenuation of autoreactive CD4+ T cells and induction of regulatory T cells. Methods:Specifically, we designed a fusion peptide comprising the trans-activator of transcription cell-penetrating peptide and an immunodominant ADAMTS13-derived T cell epitope (FINVAPHAR core amino acid sequence) to enable RBC surface loading. Results:We found that the fusion peptide binds to RBCs in a concentration-dependent manner. To assess whether our approach supports antigen presentation via human leukocyte antigen (HLA) class II molecules, macrophages were incubated with peptide-loaded RBCs, followed by analysis of HLA-DR-bound peptides via mass spectrometry. FINVAPHAR-containing peptides of varying lengths were mainly presented on macrophages from donors carrying the iTTP-associated HLA risk allele DRB1∗11, confirming functional antigen presentation and supporting the biological relevance of our model. Hemoglobin-derived peptides were also abundantly presented, supporting erythrophagocytosis as the entry route of our trans-activator of transcription-FINVAPHAR fusion peptide. Conclusion:Based on our results, we propose RBC-mediated delivery of ADAMTS13-derived peptides as a potential strategy for promoting tolerance in patients with iTTP.

Thrombotic thrombocytopenic purpura (TTP) is a thrombotic microangiopathy that is caused by a deficiency of ADAMTS13. The majority of patients with TTP have an immune mediated form of the disease (iTTP), for which the loss of activity results from the development of autoantibodies directed towards ADAMTS13. Pre-emptive treatment of iTTP with rituximab when ADAMTS13 activity drops to ~20% (i.e. an 'ADAMTS13 relapse') can effectively prevent clinical relapse. Nevertheless, patients still undergo prolonged periods of low ADAMTS13 activity. We employed unbiased mass spectrometry based plasma profiling to study alterations in protein levels in a cohort of patients with iTTP treated with elective rituximab. Protein levels in samples obtained during an ADAMTS13 relapse, after recovery of ADAMTS13 activity; during follow-up in remission; and during a subsequent ADAMTS13 relapse were measured. When comparing the plasma profile of all patients during ADAMTS13 relapse and remission, signatures associated with upregulation of hemolysis and platelet activation were observed. These findings show that biological pathways linked to the onset of acute TTP are upregulated in iTTP patients with ADAMTS13 deficiency before any clinical or routine biochemical changes are seen.

Abstract Understanding how specific VWF variants disrupt endothelial processing and function is central to elucidating von Willebrand disease (VWD) pathophysiology. However, current in vitro systems lack either the endothelial specificity or the genetic flexibility required for systematic variant characterization. Here, we present a genetically defined VWF-knockout cord-blood–derived endothelial colony-forming cell (VWF-KO cbECFC) model that enables controlled reintroduction of VWF variants in a physiologically relevant endothelial context. Using a patient with type 3 VWD carrying the homozygous pathogenic variant p.M771V and a second homozygous variant of uncertain significance p.R2663P as a reference, we demonstrate that expression of p.M771V in VWF-KO cbECFCs reproduces the patient’s intracellular processing defect and loss of high-molecular-weight multimers, whereas p.R2663P behaves as a benign allele. These findings establish the model’s ability to accurately distinguish pathogenic from non-pathogenic variants. Comparative analyses with HEK293 cells show that VWF-KO cbECFCs provide superior subcellular resolution, reliably forming authentic Weibel–Palade bodies (WPBs) and faithfully revealing ER retention phenotypes that remain ambiguous in non-endothelial systems. The proliferative capacity of cbECFCs further enables scalable and reproducible experimentation, overcoming major limitations associated with patient-derived ECFCs. Looking ahead, the VWF-KO cbECFC platform offers broad potential for VWF and VWD research. Its endothelial identity and genetic flexibility make it suitable for investigating VWF biosynthesis and trafficking, secretion dynamics, WPB biology, angiogenic processes, and shear-dependent VWF function. This system therefore provides a versatile foundation for mechanistic studies, systematic variant assessment, and future translational applications.

In Immune-mediated Thrombotic Thrombocytopenic Purpura (iTTP), patients develop antibodies against ADAMTS13. The majority of patients exhibit inhibitory anti-spacer antibodies. Non-inhibitory antibodies binding to the carboxy-terminal CUB domains have been suggested to enhance the clearance of ADAMTS13 in iTTP. Furthermore, anti-CUB antibodies induce an open conformation, which has been shown to be an important biomarker for disease severity and relapse risk. We explored whether introduction of N-glycans in the CUB domains of ADAMST13 can reduce the binding of pathogenic anti-CUB autoantibodies. The binding of a panel of iTTP patient derived anti-CUB monoclonal antibodies to newly designed N-glycan modified ADAMTS13 CUB domain variants was assessed by ELISA. Additionally, a subset of these variants was screened against plasma samples of iTTP patients which primarily contain antibodies directed towards the carboxy-terminal domains of ADAMTS13. Introduction of N-glycans at amino acid positions of 1251, 1255 and 1368 in the CUB1/2 domains of ADAMTS13 can effectively reduce binding of 6 out of 7 iTTP patient-derived anti-CUB antibodies. Reduced binding to CUB N-glycan variants was observed in 8 out of 9 patient samples. Binding was decreased from 81% to 47% for NGLY3+CUB-NGLY and 60% to 28% for 5ALA+CUB-NGLY variants. Collectively our findings show that the introduction of N-glycans within the CUB-domain of ADAMTS13 is able to prevent the binding of anti-CUB antibodies in patients with iTTP. Based on these findings, we propose that CUB-NGLY modified ADAMTS13 variants can be used for improved treatment of patients with iTTP.

Treatment options for the bleeding disorder von Willebrand disease type 2B (VWD2B) are insufficient and fail to address the negative effects of circulating mutant von Willebrand factor (VWF). The dominant-negative nature of VWD2B makes functionally defective VWF an interesting therapeutic target. Previous in vitro studies have demonstrated the feasibility of allele-selective silencing of mutant VWF using small interfering RNAs (siRNAs) targeting common single nucleotide polymorphisms (SNPs) in the human VWF gene, an approach that can be applied irrespective of the disease-causing VWF mutation. This study aims to extend this concept to a heterozygous VWD2B mouse model (c.3946G>A; p.Val1316Met) here using mouse strain-specific genetic differences as proxy for human SNPs. Homozygous VWD2B C57BL/6J (2B-B6) mice were crossed with homozygous wild-type 129S1/SvImJ (129S) mice to create heterozygous 2B-B6.129S F1 offspring. These 2B-B6.129S mice were intravenously injected with endothelial-specific lipid nanoparticles loaded with the alleleselective siVwf.B6 or control and 96 hours later, lung Vwf messenger RNA, plasma VWF levels, and phenotypic characteristics were evaluated. Treatment with siVwf.B6 reduced total VWF levels by 50%, with an expected selective reduction in mutant VWF protein. This coincided with normalization of multimeric structure, improved VWF collagen binding/ VWF antigen ratio, and normalized bleeding times in two-thirds of heterozygous 2B-B6.129S mice. Being a novel approach in the field of hemostasis, we proved, for VWD, in mice, the concept of selectively inhibiting a mutant dominant-negative allele with siRNAs targeting a single nucleotide variation rather than the disease-causing mutation. For dominantnegative VWD, this offers potential for a customized therapeutic strategy.

IntroductionThe development of neutralizing antibodies (inhibitors) against coagulation factor VIII (FVIII) remains the most serious complication in the treatment of hemophilia A. While immune tolerance induction (ITI) is the standard strategy to eliminate these antibodies, it fails in approximately 30% of patients with severe hemophilia A, underscoring the need for innovative approaches to promote FVIII-specific tolerance.MethodsTo address this challenge, we generated fusion proteins composed of A2, A3-C1-C2 (light chain, LCh), and C2 domains of FVIII linked to Annexin A5 (AnxA5), a protein that binds phosphatidylserine (PS), a hallmark of apoptotic cells.ResultsELISA confirmed high-affinity binding of all fusion proteins to immobilized PS. To model PS exposure in vitro, red blood cells (RBCs) were treated with phorbol 12-myristate 13-acetate (PMA), leading to the release of PS-exposing microvesicles. Flow cytometry showed that FVIII-AnxA5 fusion proteins selectively bound to PS-exposing microvesicles but not to intact RBCs. Using mass spectrometry-based immunopeptidomics, we demonstrated that macrophages pulsed with FVIII-AnxA5 fusion proteins efficiently processed and presented FVIII-derived peptides on HLA-DR molecules.ConclusionsThese findings suggest that FVIII-AnxA5 fusion proteins can engage apoptotic cell clearance pathways to facilitate antigen presentation in a potentially tolerogenic context. This strategy may offer a novel means of inducing immune tolerance to FVIII in hemophilia A.

In recent years gene therapy has emerged as a powerful technology for treatment of a large variety of inherited disorders. With the FDA approval of in vivo gene therapy of hemophilia A and B using AAV-mediated transgene delivery to hepatocytes, the path towards a new treatment era seemed paved. Also, CRISPR-Cas based approaches have reached the clinic, as in the ex vivo treatment of hematopoietic stem cells for sickle cell disease and thalassemia patients. The question arises whether these innovative strategies will also be suitable for patients with von Willebrand Disease (VWD). Whilst in and ex vivo delivery to endothelial cells (ECs) has been demonstrated, and CRISPR-Cas9 gene editing has been successful in ECs, there are currently no gene therapy options available for VWD. The wide variety of pathogenic VWF mutations makes development of broadly applicable, cost-effective gene therapies challenging. While delivery of von Willebrand factor (VWF) as a therapeutic transgene would be optimal, the size of VWF challenges efficient delivery. Therefore, treatment of VWD requires targeted, personalized gene therapy; for instance by using the newest CRISPR-Cas technologies which can be tailored to facilitate alteration and restoration of various pathogenic VWD variants. This review describes the inherited bleeding disorder VWD and potential gene therapy approaches for management of the disease. Thereby we are exploring different CRISPR-Cas technologies and recent developments in the field. Moreover, we will discuss the ongoing advances of in vivo delivery systems, all with the scope on ECs.

Background:Immune tolerance induction (ITI) is the only treatment to eradicate inhibitors in people with severe hemophilia A (SHA). Successful ITI restores factor VIII (FVIII) tolerance. ITI is demanding and successful in approximately 70% of people. Objectives:Identifying predictors of ITI outcome is essential to guide clinical decision making. We aimed to identify genetic predictors of ITI success in people with SHA and inhibitors who underwent ITI. Methods:This observational multicenter study included people with SHA who underwent ITI, between 2015 and 2023. Clinical and patient data, including FVIII gene (F8) mutation type, and DNA samples were collected. Successful ITI was defined by a negative inhibitor titer and an adequate response to FVIII concentrates. By employing a global screening array, the associations between ITI success and F8 genotype and 216 candidate predictors, including single nucleotide polymorphisms and human leukocyte antigen variants, CA dinucleotide short tandem repeat polymorphisms in the IL10 promoter region, and FCGR2/3 gene locus variations, were analyzed. Results:Of 204 participants, 147 (72.1%) achieved ITI success. The majority (52.0%) of participants had F8 intron 22 inversion. None of the candidate single nucleotide polymorphisms/human leukocyte antigen variants, IL10 CA dinucleotide short tandem repeats, or FCGR2/3 gene locus variations were associated with ITI success. F8 large deletions were negatively associated with ITI success (odds ratio, 0.15; 95% CI, 0.04-0.51; P = .002). Conclusion:Our study of 204 people with SHA identified F8 large deletions as a predictor of ITI failure. Pooling cohorts may allow the identification of additional genetic predictors of ITI success in the future.

Background Gene therapy for hemophilia has recently been implemented as standard clinical care, requiring organizational and multistakeholder preparedness and clear guidelines. In addition to pharmaceutical summaries of product characteristics (SMPCs), various (inter)national guidance documents have been published. However, no guidance document or SMPC covers the entire gene therapy care pathway. Objectives This study provides a complete and comprehensive overview of current guidance documents and SMPCs to develop a comprehensive care pathway for hemophilia gene therapy delivery. Methods Published gene therapy guidance documents and collected SMPCs were complemented by a selective search in online databases, including PubMed and scientific societies’ websites. Reference lists were checked for additional relevant articles. Results Four SMPCs and 11 (inter)national guidance documents and recommendations were collected. The documents were focused on either the intervention or the care pathway, and none were comprehensive covering all aspects of hemophilia gene therapy delivery. Considerable differences were found between the 2 approved gene therapy products and between the SMPCs issued by the 2 regulatory authorities, the Food and Drug Administration and the European Medicines Agency. (Inter)national guidance documents provided additional information and recommendations not covered in SMPCs. Conclusion Based on SMPCs and (inter)national guidance documents and recommendations a care pathway has been developed and visualized in a Metro Map. This provides a clear and comprehensive overview of all activities, contact moments, and responsibilities within the longitudinal gene therapy treatment process. This comprehensive care pathway may help navigate gene therapy implementation, providing guidance to clinicians, patients, and caregivers.

Factor VII (FVII) deficiency is a rare bleeding disorder with a prevalence of approximately 1:300,000-500,000 individuals. We explored whether adeno-associated virus (AAV)-mediated gene therapy can achieve durable and functional expression of human FVII (hFVII) in vivo. Wild-type hFVII (hFVIIwt) and a naturally occurring splice variant designated hFVII(-22) (GenBank: NM_019616.4) were expressed under the control of the Apolipoprotein E-derived hepatic locus control region and the α1-anti-trypsin promoter. Expression cassettes were packaged in either recombinant AAV5 (rAAV5) or recombinant AAV8 (rAAV8). For both hFVIIwt and hFVII(-22), 5- to 20-fold higher plasma levels of hFVII could be obtained when rAAV8 was used as a vector as opposed to rAAV5. Interestingly, hFVII levels obtained by employing rAAV8 expressing the hFVII(-22) cDNA variant were approximately 10 times higher than those obtained using rAAV8 expressing hFVIIwt. Based on these results, we generated an rAAV8-based gene therapy vector encoding hFVII(-22) and evaluated long-term expression in vivo. Employing a vector dose of 0.8 × 1012 genome copies (gc)/kg, we observed 48 weeks of functional hFVII expression which peaked at 16 IU/mL and stabilized at 7 IU/mL. These results support the pre-clinical development of AAV8-mediated delivery employing the splice-variant hFVII(-22) for patients suffering from FVII deficiency.

ABSTRACT:Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is a rare life-threatening thrombotic disorder, which results from the development of autoantibodies targeting ADAMTS13. Most patients (>90%) with iTTP display antibodies against a shared epitope in the spacer domain of ADAMTS13. Nevertheless, a smaller population of patients (20%-40%) also has antibodies directed toward the CUB (complement C1r/C1s, Uegf, Bmp1) domains of ADAMTS13. Here, we explored whether anti-CUB antibodies have a shared epitope located on CUB domains of ADAMTS13 that overlaps with the spacer-CUB domain interface. Hydrogen-deuterium exchange mass spectrometry revealed that a panel of patient-derived human monoclonal anti-CUB domain antibodies specifically targeted peptides 1248-1253 and 1359-1377 in the CUB1 and CUB2 domains, respectively. A parallel alanine screen showed that residues W1250, K1252, and R1367 are crucially involved in binding of anti-CUB domain antibodies. A triple alanine variant containing W1250A/K1252A/R1367A ameliorated the binding of all patient-derived monoclonal antibodies. This triple-alanine variant also showed greatly reduced binding of anti-CUB antibodies upon analysis of plasma samples of a panel of 27 patients with iTTP. Functional analysis of the anti-CUB antibodies showed that all antibodies were able to induce an open conformation but did not inhibit activity toward either peptide substrates or von Willebrand factor multimers under flow conditions. Collectively, our findings show that anti-CUB antibodies target residues W1250/K1252 and R1367. Binding of pathogenic antibodies disrupt the spacer-CUB domain interface, thereby inducing an open conformation in ADAMTS13.

[This corrects the article DOI: 10.3389/fgeed.2025.1620438.].