Mutational Profile in Romanian Patients with Hemophilia A.

Hemophilia A (HA) is an X-linked recessive bleeding disorder caused by mutations in the F8 gene, resulting in deficient or dysfunctional factor VIII (FVIII). This study aimed to characterize the mutational profile of HA in Romanian patients using next-generation sequencing (NGS) and multiplex ligation-dependent probe amplification (MLPA). A total of 107 patients were analyzed, revealing pathogenic or likely pathogenic variants in 96.3% of cases. The identified mutations included missense (30.5%), nonsense (9.1%), small deletions (6.4%), small insertions (2.1%), splice-site variants (4.3%), large deletions (1.6%), and large duplications (1.1%). Large intron inversion was previously found in 37.5% of the patients. Novel variants accounted for 21.5% of identified mutations, expanding the spectrum of F8 variants in this population. This study underscores the genetic heterogeneity of HA and provides insights into genotype-phenotype correlations, aiding in clinical management and prenatal diagnosis.

Pre-clinical evaluation of an enhanced-function factor VIII variant for durable hemophilia A gene therapy in male mice.

Durable factor VIII expression that normalizes hemostasis is an unrealized goal of hemophilia A adeno-associated virus-mediated gene therapy. Trials with initially normal factor VIII activity observed unexplained year-over-year declines in expression while others reported low-level, stable expression inadequate to restore normal hemostasis. Here we demonstrate that male mice recapitulate expression-level-dependent loss of factor VIII levels due to declines in vector copy number. We show that an enhanced function factor VIII variant (factor VIII-R336Q/R562Q), resistant to activated protein C-mediated inactivation, normalizes hemostasis at below-normal expression without evidence of prothrombotic risk in male hemophilia A mice. These data support that factor VIII-R336Q/R562Q may restore normal factor VIII function at low levels of expression to permit durability using low vector doses to minimize dose-dependent adeno-associated virus toxicities. This work informs the mechanism of factor VIII durability after gene transfer and supports that factor VIII-R336Q/R562Q may safely overcome current hemophilia A gene therapy limitations.

Deciphering the circulating microRNA signature of hemophilic arthropathy.

BACKGROUND: Haemophilic arthropathy (HArt) is a serious complication in patients with hemophilia. Early diagnosis and treatment are essential to minimise the development of HArt. The use of biomarkers may improve early diagnosis of HArt. Circulating microRNAs (miRNAs) are small, non-coding RNAsthat regulate gene expression, and are being investigated as promising biomarkers due to their role in joint and bone metabolism. AIMS: To investigate differential expression of miRNAs and their relationship to arthropathy in patients with hemophilia A. METHODS: miRNA expression was examined in a pilot study followed by a validation study (100 hemophilia A patients with [n = 83] and without HArt [n = 17], 14 controls). Differential miRNA expression was investigated using real-time quantitative PCR. RESULTS: The pilot study identified 2 miRNAs differentially expressed in patients with Hart (Pettersson score ≥ 1), after adjusting for the false discovery rate (FDR). The validation study evaluated these 2 miRNAs. The results demonstrated that two miRNAs (miR- 208a-3p and 524-3p) were significantly underexpressed in plasma of patients with HArt compared to patients without arthropathy, with FDR <0.05 (Fig. 1). In addition, 3 miRNAs (130a-3p, miR- and 506-3p) were significantly underexpressed in patients with moderate HArt (Pettersson score 4 to 7). CONCLUSIONS: In this proof of concept study we identified a signature of 5 circulating miRNAs associated with Hart with potential as diagnosis tools for HArt. These miRNAs are potential negative regulators of gene expression, suggesting their activity in HArt by interfering with osteoblastic (miR- 208a-3p) and osteoclastic (miR-506-3p) differentiation to impair bone mineralization and remodeling processes, or regulating chondrogenesis (miR-335-5p). miRNAs associated with earlier stages of HArt will be further investigated in a sub-study of the prospective clinical trial PROVE, which will investigate the effects of long-term prophylaxis with simoctocog alfa versus emicizumab in adults with hemophilia A.

International Society on Thrombosis and Haemostasis clinical practice guideline for treatment of congenital hemophilia A and B based on the Grading of Recommendations Assessment, Development, and Evaluation methodology.

BACKGROUND: Hemophilia is a rare congenital bleeding disorder that results from complete or partial deficiency of blood coagulation factor (F)VIII (hemophilia A) or FIX (hemophilia B) due to pathogenic variants in their coding genes. Hemophilia requires complex management. To date, there is no evidence-based clinical practice guideline on hemophilia treatment based on the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) approach. OBJECTIVES: This evidence-based clinical practice guideline from the International Society on Thrombosis and Haemostasis aims to provide an overview of evidence and support patients, caregivers, hematologists, pediatricians, other clinicians, researchers, and stakeholders in treatment decisions about congenital hemophilia A and B. METHODS: The International Society on Thrombosis and Haemostasis formed a multidisciplinary guideline panel of physicians and patients with global representation, balanced to minimize potential bias from conflicts of interest. The panel prioritized a set of clinical questions and outcomes according to their importance for clinicians and patients. A methodological team supported the guideline development process, including searching for evidence and performing systematic reviews. The GRADE approach was used, including GRADE Evidence to Decision frameworks. The recommendations were subject to public comment. RESULTS: The panel selected 13 questions, of which 11 addressed the treatment of hemophilia A and 2 the treatment of hemophilia B. Specifically, the panel addressed questions on prophylactic and episodic treatment with FVIII concentrates, bypassing agents, and nonfactor therapy (emicizumab) for hemophilia A (with and without inhibitors) as well as immune tolerance induction for hemophilia A. For hemophilia B, the panel addressed questions on prophylactic and episodic treatment of bleeding events with FIX concentrates. Agreement was reached for all 13 recommendations, of which 7 (54%) were based on evidence from randomized clinical trials, 3 (23%) on observational studies, and 3 (23%) on indirect comparisons. CONCLUSION: Strong recommendations were issued for prophylactic over episodic treatment for severe and moderately severe hemophilia A and B. Only conditional recommendations were issued for the remaining questions. Future research should focus on direct treatment comparisons and the treatment of hemophilia B with and without inhibitors. Future updates of this guideline will provide an updated evidence synthesis on the current questions and focus on new FVIII and FIX concentrates, novel nonfactor therapies, and gene therapy for severe and nonsevere hemophilia A and B.

Detection of hemophilia A genetic variants using third-generation long-read sequencing.

BACKGROUND: Hemophilia A (HA) is an X-linked recessive genetic disorder caused by pathogenic variations of the factor VIII -encoding gene, F8 gene. Due to the large size and diverse types of variations in the F8 gene, causative mutations in F8 cannot be simultaneously detected in one step by traditional molecular analysis, and genetic molecular diagnosis and prenatal screening of HA still face significant difficulties and challenges in clinical practice. Therefore, we aimed to develop and validate an efficient, accurate, and time-saving method for the genetic detection of HA. METHODS: A comprehensive analysis of hemophilia A (CAHEA) method based on long-range PCR and long-read sequencing (LRS) was used to detect F8 gene mutations in 14 clinical HA samples. The LRS results were compared with those of the conventional methods to evaluate the accuracy and sensitivity of the proposed approach. RESULTS: The CAHEA method successfully identified 14 F8 variants in all probands, including 3 small insertion deletions, 4 single nucleotide variants, and 7 intron 22 inversions in a "one-step" manner, of which 2 small deletions have not been reported previously. Moreover, this method provided an opportunity to analyze the mechanism of rearrangement and the pathogenicity of F8 variants. The LRS results were validated and found to be in 100% agreement with those obtained using the conventional method. CONCLUSION: Our proposed LRS-based F8 gene detection method is an accurate and reproducible genetic screening and diagnostic method with significant clinical value. It provides efficient, comprehensive, and accurate genetic screening and diagnostic services for individuals at high risk of HA as well as for premarital and prenatal populations.

Recent Advances in Gene Therapy for Hemophilia: Projecting the Perspectives.

One of the well-known X-linked genetic disorders is hemophilia, which could be hemophilia A as a result of a mutation in the F8 (factor VIII) gene or hemophilia B as a result of a mutation in the F9 (factor IX) gene, leading to insufficient levels of the proteins essential for blood coagulation cascade. In patients with severe hemophilia, factor VIII or factor IX activities in the blood plasma are considerably low, estimated to be less than 1%. This is responsible for spontaneous or post-traumatic bleeding episodes, or both, leading to disease complications and death. Current treatment of hemophilia relies on the prevention of bleeding, which consists of expensive lifelong replacement infusion therapy of blood plasma clotting factors, their recombinant versions, or therapy with recombinant monoclonal antibodies. Recently emerged gene therapy approaches may be a potential game changer that could reshape the therapeutic outcomes of hemophilia A or B using a one-off vector in vivo delivery and aim to achieve long-term endogenous expression of factor VIII or IX. This review examines both traditional approaches to the treatment of hemophilia and modern methods, primarily focusing on gene therapy, to update knowledge in this area. Recent technological advances and gene therapeutics in the pipeline are critically reviewed and summarized. We consider gene therapy to be the most promising method as it may overcome the problems associated with more traditional treatments, such as the need for constant and expensive infusions and the presence of an immune response to the antibody drugs used to treat hemophilia.

Hemophilia Gene Therapy: Another Blessing or Another Curse?

Medical therapies for hemophilia patients over the past 60 years have included several blessings and a curse. The long-sought cure with gene therapy may have finally arrived. Unfortunately, preclinical animal models are now raising concerns for genotoxicity with gene therapy. Although no cancers have been detected in humans, it may be a few decades before we know if gene therapy for hemophilia is another blessing, or another curse.

Liver health in hemophilia in the era of gene therapy.

Gene therapy for hemophilia is a groundbreaking treatment approach with promising results and potential to reduce the burden of the disease. However, uncertainties remain, particularly regarding the liver side effects of AAV gene therapy, which are more common in hemophilia A. Unlike some other diseases, such as spinal muscular atrophy, where the target cell for gene therapy is different from the one affected by side effects, hemophilia gene therapy operates within the same cellular domain-the hepatocyte. This overlap is challenging and requires a targeted strategy to mitigate the risks associated with liver injury, which often requires temporary immunosuppressive therapy. A comprehensive approach is essential to increase the efficacy of gene therapy and reduce the likelihood of hepatocyte damage. Key components of this strategy include a thorough pre-gene therapy assessment of liver health, careful post-gene therapy liver monitoring, and prompt therapeutic intervention for loss of transgene expression and liver injury. Collaboration between hematologists and hepatologists is essential to ensure a well-coordinated management plan for patients undergoing hemophilia gene therapy. This review addresses the critical aspect of hepatic comorbidities in patients with hemophilia, emphasizing the need to identify and address these issues prior to initiating gene therapy. It examines the known mechanisms of liver damage and emphasizes the importance of liver monitoring after gene therapy. In addition, the review draws insights from experiences with other AAV-based gene therapies, providing valuable lessons that can guide hemophilia centers in effectively managing liver damage associated with hemophilia gene therapy.

Characterization of zebrafish coagulation cofactors Fviii and Fv mutants and modeling hemophilia A and factor V deficiency.

The aim of this study is to characterize zebrafish coagulation cofactors fviii and fv mutant fish and assess if they phenocopy classical hemophilia A and factor V deficiency in humans. The embryos from fviii and fv zebrafish heterozygote mutants generated by ENU mutagenesis were purchased from the ZIRC repository. They were reared to adulthood and genotyped. The heterozygote male and female were crossed to get homozygote, heterozygote, and wild-type fish. Functional kinetic coagulation assays and bleeding assays were performed on normal and mutant adult fish, and venous laser injury assays were performed on the larvae. The DNA from fviii and fv mutants were sequenced to confirm if they have a premature stop codon in exon 19, and in exon 2, respectively, and in both mutants, the amino acid glutamine is replaced with a stop codon. Homozygous and heterozygous 5 days post fertilization (dpf) larvae for fviii and fv deficient mutants exhibited prolonged time to occlusion after venous laser injury compared to wild-type controls. The homozygous and heterozygous fviii adult mutants showed modest bleeding and delayed fibrin formation in the kinetic partial thromboplastin time (kPTT) assay with their plasma. fv homozygous larvae had poor survival beyond 12 dpf. However, heterozygous fv mutants exhibited heavy bleeding and prolonged fibrin formation in the kPTT and kPT assay compared with wild-type siblings. Our characterization showed fviii and fv mutants from ZIRC phenocopied to a considerable extent classical hemophilia A and factor V deficiency in humans, respectively. These models should be useful in studying and developing novel drugs that reverse the phenotype and in generating suppressor mutations to identify novel factors that compensate for these deficiencies.

AAV mediated repression of Neat1 lncRNA combined with F8 gene augmentation mitigates pathological mediators of joint disease in haemophilia.

Haemophilic arthropathy (HA), a common comorbidity in haemophilic patients leads to joint pain, deformity and reduced quality of life. We have recently demonstrated that a long non-coding RNA, Neat1 as a primary regulator of matrix metalloproteinase (MMP) 3 and MMP13 activity, and its induction in the target joint has a deteriorating effect on articular cartilage. In the present study, we administered an Adeno-associated virus (AAV) 5 vector carrying an short hairpin (sh)RNA to Neat1 via intra-articular injection alone or in conjunction with systemic administration of a capsid-modified AAV8 (K31Q) vector carrying F8 gene (F8-BDD-V3) to study its impact on HA. AAV8K31Q-F8 vector administration at low dose, led to an increase in FVIII activity (16%-28%) in treated mice. We further observed a significant knockdown of Neat1 (~40 fold vs. untreated injured joint, p = 0.005) in joint tissue of treated mice and a downregulation of chondrodegenerative enzymes, MMP3, MMP13 and the inflammatory mediator- cPLA2, in mice receiving combination therapy. These data demonstrate that AAV mediated Neat1 knockdown in combination with F8 gene augmentation can potentially impact mediators of haemophilic joint disease.

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.

Aberrant methylation and expression of TNXB promote chondrocyte apoptosis and extracullar matrix degradation in hemophilic arthropathy via AKT signaling.

Recurrent joint bleeding in hemophilia patients frequently causes hemophilic arthropathy (HA). Drastic degradation of cartilage is a major characteristic of HA, but its pathological mechanisms has not yet been clarified. In HA cartilages, we found server matrix degradation and increased expression of DNA methyltransferase proteins. We thus performed genome-wide DNA methylation analysis on human HA (N=5) and osteoarthritis (OA) (N=5) articular cartilages, and identified 1228 differentially methylated regions (DMRs) associated with HA. Functional enrichment analyses revealed the association between DMR genes (DMGs) and extracellular matrix (ECM) organization. Among these DMGs, Tenascin XB (TNXB) expression was down-regulated in human and mouse HA cartilages. The loss of Tnxb in F8-/- mouse cartilage provided a disease-promoting role in HA by augmenting cartilage degeneration and subchondral bone loss. Tnxb knockdown also promoted chondrocyte apoptosis and inhibited phosphorylation of AKT. Importantly, AKT agonist showed chondroprotective effects following Tnxb knockdown. Together, our findings indicate that exposure of cartilage to blood leads to alterations in DNA methylation, which is functionally related to ECM homeostasis, and further demonstrate a critical role of TNXB in HA cartilage degeneration by activating AKT signaling. These mechanistic insights allow development of potentially new strategies for HA cartilage protection.

DNA base editing corrects common hemophilia A mutations and restores factor VIII expression in in vitro and ex vivo models.

BACKGROUND: Replacement and nonreplacement therapies effectively control bleeding in hemophilia A (HA) but imply lifelong interventions. Authorized gene addition therapy could provide a cure but still poses questions on durability. FVIIIgene correction would definitively restore factor (F)VIII production, as shown in animal models through nuclease-mediated homologous recombination (HR). However, low efficiency and potential off-target double-strand break still limit HR translatability. OBJECTIVES: To correct common model single point mutations leading to severe HA through the recently developed double-strand break/HR-independent base editing (BE) and prime editing (PE) approaches. METHODS: Screening for efficacy of BE/PE systems in HEK293T cells transiently expressing FVIII variants and validation at DNA (sequencing) and protein (enzyme-linked immunosorbent assay; activated partial thromboplastin time) level in stable clones. Evaluation of rescue in engineered blood outgrowth endothelial cells by lentiviral-mediated delivery of BE. RESULTS: Transient assays identified the best-performing BE/PE systems for each variant, with the highest rescue of FVIII expression (up to 25% of wild-type recombinant FVIII) for the p.R2166∗ and p.R2228Q mutations. In stable clones, we demonstrated that the mutation reversion on DNA (∼24%) was consistent with the rescue of FVIII secretion and activity of 20% to 30%. The lentiviral-mediated delivery of the selected BE systems was attempted in engineered blood outgrowth endothelial cells harboring the p.R2166∗ and p.R2228Q variants, which led to an appreciable and dose-dependent rescue of secreted functional FVIII. CONCLUSION: Overall data provide the first proof-of-concept for effective BE/PE-mediated correction of HA-causing mutations, which encourage studies in mouse models to develop a personalized cure for large cohorts of patients through a single intervention.

Clinical and molecular characteristics of hemophilia A affected individuals and carriers: A 24 years experience from three centers.

Hemophilia A is a rare bleeding disorder with variable expressivity and allelic heterogeneity. Despite the advancement of prenatal diagnostics and molecular studies, the number of studies reviewing the reproductive choices of hemophilia A carriers and affected individuals remains limited. Through this retrospective review, we hope to gain a deeper understanding of hemophilia A-affected individuals' clinical and molecular characteristics, as well as the reproductive choices of the at-risk couples. A total of 122 individuals harboring likely causative F8 gene alterations from 64 apparently unrelated families attending three centers between 3/2000 and 3/2023 were included in this study. Their clinical and molecular findings as well as reproductive choices were gathered in a clinical setting and verified through the electronic medical record database of the public health system. Forty-seven affected males and 75 female heterozygous carriers were included in the analysis. Among 64 apparently unrelated families, 36 distinct pathogenic/likely pathogenic variants were identified, of which 30.6% (11/36) of variants were novel. While the majority of clinical findings and genotype-phenotype correlations appear to be in accordance with existing literature, female carriers who had no fertility intention were significantly more likely to have affected sons than those who had fertility intention (5/19 vs. 4/5; p = 0.047). Through this retrospective review, we summarized the clinical and molecular characteristics of 122 individuals harboring pathogenic/likely pathogenic F8 variants, as well as their fertility intentions and reproductive outcomes. Further studies are required to look into the considerations involved in reproductive decision-making.

Clinical immunogenicity outcomes from GENEr8-1, a phase 3 study of valoctocogene roxaparvovec, an AAV5-vectored gene therapy for hemophilia A.

Gene transfer therapies utilizing adeno-associated virus (AAV) vectors involve a complex drug design with multiple components that may impact immunogenicity. Valoctocogene roxaparvovec is an AAV serotype 5 (AAV5)-vectored gene therapy for the treatment of hemophilia A that encodes a B-domain-deleted human factor VIII (FVIII) protein controlled by a hepatocyte-selective promoter. Following previous results from the first-in-human phase 1/2 clinical trial, we assessed AAV5-capsid- and transgene-derived FVIII-specific immune responses with 2 years of follow-up data from GENEr8-1, a phase 3, single-arm, open-label study in 134 adult men with severe hemophilia A. No FVIII inhibitors were detected following administration of valoctocogene roxaparvovec. Immune responses were predominantly directed toward the AAV5 capsid, with all participants developing durable anti-AAV5 antibodies. Cellular immune responses specific for the AAV5 capsid were detected in most participants by interferon-γ enzyme-linked immunosorbent spot assay 2 weeks following dose administration and declined or reverted to negative over the first 52 weeks. These responses were weakly correlated with alanine aminotransferase elevations and showed no association with changes in FVIII activity. FVIII-specific cellular immune responses were less frequent and more sporadic compared with those specific for AAV5 and showed no association with safety or efficacy parameters.

Comprehensive genomic filtering algorithm to expose the cause of skewed X chromosome inactivation. The proof of concept in female haemophilia expression.

BACKGROUND: Exploring the expression of X linked disorders like haemophilia A (HA) in females involves understanding the balance achieved through X chromosome inactivation (XCI). Skewed XCI (SXCI) may be involved in symptomatic HA carriers. We aimed to develop an approach for dissecting the specific cause of SXCI and verify its value in HA. METHODS: A family involving three females (two symptomatic with severe/moderate HA: I.2, the mother, and II.1, the daughter; one asymptomatic: II.2) and two related affected males (I.1, the father and I.3, the maternal uncle) was studied. The genetic analysis included F8 mutational screening, multiplex ligation-dependent probe amplification, SNP microarray, whole exome sequencing (WES) and Sanger sequencing. XCI patterns were assessed in ectoderm/endoderm and mesoderm-derived tissues using AR-based and RP2-based systems. RESULTS: The comprehensive family analysis identifies I.2 female patient as a heterozygous carrier of F8:p.(Ser1414Ter) excluding copy number variations. A consistent XCI pattern of 99.5% across various tissues was observed. A comprehensive filtering algorithm for WES data was designed, developed and applied to I.2. A Gly58Arg missense variant in VMA21 was revealed as the cause for SXCI.Each step of the variant filtering system takes advantage of publicly available genomic databases, non-SXCI controls and case-specific molecular data, and aligns with established concepts in the theoretical background of SXCI. CONCLUSION: This study acts as a proof of concept for our genomic filtering algorithm's clinical utility in analysing X linked disorders. Our findings clarify the molecular aspects of SXCI and improve genetic diagnostics and counselling for families with X linked diseases like HA.