The complex, confusing and poorly understood immune responses to AAV-mediated gene transfer in haemophilia-Is more or less immunosuppression required?

Attempts to achieve a functional cure or amelioration of the severe X linked bleeding disorders haemophilia A (factor VIII deficiency) and haemophilia B (factor IX deficiency) using AAV-based vectors have been frustrated by immune responses that limit efficacy and durability. The immune responses include adaptive and innate pathways as well as cytokine mediated inflammation, especially of the target organ cells-hepatocytes. Immune suppression has only been partly effective in clinical trials at ameliorating the immune response and the lack of good animal models has delayed progress in identifying mechanisms and developing more effective approaches to controlling these effects of AAV gene transfer. Here we discuss the arguments for and against more potent immunosuppression to improve factor expression after AAV-mediated gene therapy.

Hemophilia B and gene therapy: a new chapter with etranacogene dezaparvovec.

ABSTRACT: The US Food and Drug Administration (FDA)'s authorization of etranacogene dezaparvovec (Hemgenix) is a significant milestone, constituting not only the first FDA approval of a gene therapy for hemophilia but also the first approval of a liver-targeted adeno-associated virus vector gene therapy. This review summarizes the nonclinical studies and clinical development that supported regulatory clearance. Similar to other gene therapies for single gene disorders, both the short-term safety and the phenotypic improvement were unequivocal, justifying the modest-sized safety and efficacy database, which included 57 participants across the phase 2b (3 participants) and phase 3 (54 participants) studies. The most common adverse reactions included liver enzyme elevation, headache, flu-like symptoms, infusion-related reactions, creatine kinase elevation, malaise, and fatigue; these were mostly transient. One participant had hepatocellular carcinoma on a study-mandated liver ultrasound conducted 1 year after vector infusion; molecular analysis of the resected tumor showed no evidence of vector-related insertional mutagenesis as the etiology. A remarkable 96% of participants in the phase 3 trial were able to stop factor IX (FIX) prophylaxis, with the study demonstrating noninferiority to FIX prophylaxis in terms of the primary end point, annualized bleeding rate. Key secondary end points such as the annualized infusion rate, which declined by 97%, and the plasma FIX activity level at 18 months after infusion, with least squares mean increase of 34.3 percentage points compared with baseline, were both clinically and statistically significant. The FDA's landmark approval of Hemgenix as a pioneering treatment for hemophilia stands on the shoulders of >20 years of gene therapy clinical research and heralds a promising future for genomic medicines.

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.

AAV mediated gene therapy for haemophilia B: From the early attempts to modern trials.

Early gene therapy clinical trials for the treatment of Haemophilia B have been instrumental to our global understanding of gene therapy and have significantly contributed to the rapid expansion of the field. The use of adeno-associated viruses (AAVs) as vectors for gene transfer has successfully led to therapeutic expression of coagulation factor IX (FIX) in severe haemophilia B patients. Expression of FIX has remained stable following a single administration of vector for up to 8 years at levels that are clinically relevant to reduce the incidence of spontaneous bleeds and have permitted a significant change in the disease management with reduction or elimination of the need for coagulation factor concentrates. These trials have also shed light on several concerns around AAV-mediated gene transfer such as the high prevalence of pre-existing immunity against the vector capsid as well as the elevation of liver transaminases that is associated with a loss of FIX transgene expression in some patients. However, this field is advancing very rapidly with the development of increasingly more efficient strategies to overcome some of these obstacles and importantly raise the possibility of a functional cure, which has been long sought after. This review overviews the evolution of gene therapy for haemophilia B over the last two decades.

Pleiotropic effects of different exonic nucleotide changes at the same position contribute to hemophilia B phenotypic variation.

BACKGROUND: The disease-causing effects of genetic variations often depend on their location within a gene. Exonic changes generally lead to alterations in protein production, secretion, activity, or clearance. However, owing to the overlap between proteins and splicing codes, missense variants can also affect messenger RNA splicing, thus adding a layer of complexity and influencing disease phenotypes. OBJECTIVES: To extensively characterize a panel of 13 exonic variants in the F9 gene occurring at 6 different factor IX positions and associated with varying severities of hemophilia B (HB). METHODS: Computational predictions, splicing analysis, and recombinant factor IX assays were exploited to characterize F9 variants. RESULTS: We demonstrated that 5 (38%) of 13 selected F9 exonic variants have pleiotropic effects. Although bioinformatic approaches accurately classified effects, extensive experimental assays were required to elucidate and deepen the molecular mechanisms underlying the pleiotropic effects. Importantly, their characterization was instrumental in developing tailored RNA therapeutics based on engineered U7 small nuclear RNA to mask cryptic splice sites and compensatory U1 small nuclear RNA to enhance exon definition. CONCLUSION: Overall, albeit a multitool bioinformatic approach suggested the molecular effects of multiple HB variants, the deep investigation of molecular mechanisms revealed insights into the HB phenotype-genotype relationship, enabling accurate classification of HB variants. Importantly, knowledge of molecular mechanisms allowed the development of tailored RNA therapeutics, which can also be translated to other genetic diseases.

Efficacy and Safety of Adeno-Associated Virus-Based Clinical Gene Therapy for Hemophilia: A Systematic Review and Meta-Analysis.

Clinical trials of adeno-associated virus (AAV)-based gene therapy have made remarkable progress in recent years. We aimed to perform a systematic review and meta-analysis of the literature to assess the efficacy and safety of AAV-based gene therapy for hemophilia. We systematically searched the Web of Science, Embase, PubMed, and the Cochrane Database of Systematic Reviews databases, for clinical trials involving patients diagnosed with hemophilia and treated with AAV-mediated gene therapy. Data on the annualized bleeding rate (ABR), annualized infusion rate (AIR), the incidence of treatment-related adverse events (TRAEs), severe adverse events (SAEs), and alanine aminotransferase (ALT) elevation were extracted as our outcomes. A total of 12 articles from 11 clinical trials were selected from 868 articles for meta-analysis. Pooled analyses showed that AAV-based gene therapy in hemophilia patients reduced the number of bleeding events and the number of factor infusion events by an approximate average of 7 per year and 103 per year, respectively. Eighty percent, 18%, and 63% of hemophilia patients had elevated TRAE, SAE, and ALT levels, respectively. Moreover, subgroup analysis found a significant reduction in ABR and AIR 2-3 years after the therapy. Additional findings that were not pooled including coagulation factor activity are presented in the accompanying tables. Our analysis supported the efficacy and safety of AAV-mediated gene therapy for hemophilia, providing evidence for its application as a therapeutic option for widespread clinical use in hemophilia patients in the future.

Indirect treatment comparisons of the gene therapy etranacogene dezaparvovec versus extended half-life factor IX therapies for severe or moderately severe haemophilia B.

INTRODUCTION: Etranacogene dezaparvovec gene therapy for haemophilia B demonstrated superior efficacy at 24 months in reducing bleeds versus a ≥6-month lead-in period of prophylaxis with FIX products in the phase 3 trial, HOPE-B. In the absence of head-to-head comparisons of etranacogene dezaparvovec versus FIX products, indirect treatment comparisons (ITC) can be used. AIM: To compare the efficacy of etranacogene dezaparvovec versus rIX-FP, rFIXFc and N9-GP using ITC, and support HOPE-B results. METHODS: Data were leveraged from Phase 3 pivotal trials: HOPE-B, PROLONG-9FP, B-LONG and Paradigm 2. Annualised bleeding rates (ABR), spontaneous (AsBR) and joint (AjBR) bleeding rates, percentage of patients with no bleeds, and FIX consumption were assessed using inverse probability of treatment weighting and matching adjusted indirect comparisons. RESULTS: Etranacogene dezaparvovec demonstrated statistically significantly lower bleeding rates versus all comparators. Rate ratios for ABR, AsBR and AjBR versus rIX-FP were 0.19 (p < .0001), 0.08 (p < .0001) and 0.09 (p < .0001), respectively. Rate ratios for ABR, AsBR and AjBR versus rFIXFc were 0.14 (p < .0001), 0.13 (p = .0083) and 0.15 (p = .0111), respectively. Rate ratios for ABR and AsBR, versus N9-GP were 0.24 (p = .0231) and 0.13 (p = .0071), respectively. Etranacogene dezaparvovec demonstrated significantly higher percentage of patients with no bleeds versus rIX-FP and rFIXFc; odds ratios: 17.60 (p < .0001) and 5.65 (p = .0037), respectively. Etranacogene dezaparvovec resulted in significantly lower FIX consumption than all comparators. CONCLUSIONS: ITC suggests that etranacogene dezaparvovec offers patients with haemophilia B (≤2% of normal FIX expression) a single dose treatment that can significantly reduce bleeding rates and eliminate routine infusions associated with FIX therapies.

Perspectives and perception of haemophilia gene therapy by French patients.

INTRODUCTION AND AIM: A national survey was initiated by representatives of French patients with haemophilia (AFH) and the French reference centre for haemophilia, in order to appreciate the awareness and knowledge of these patients regarding haemophilia gene therapy (HGT) and understand better their position about this innovative treatment that will soon become available. RESULTS: Of 143 answers received, 137 could be analysed, representing about 3.5% of patients with severe or moderate haemophilia over 16year-old. They were 80.3% with haemophilia A and 19.7 % with haemophilia B, with a severe form of the disease for 80.3 % of them. Curiosity for HGT was formulated by 64.2% of the participants, 33.6 % being interested by this approach as soon as it will be available and 38.7 % preferring to wait until more patients have been treated. Only 3.6 % of the participants would never consider receiving HGT. The level of awareness and knowledge was estimated to be limited by 39.5 % of the patients. More than 60 % of them declared having never or almost never discussed HGT with the team of their haemophilia centre. Before deciding to get HGT, 54.4 % of the participants considered that it will be very important to compare it with their current treatment and 53.7 % would like to be better informed by their care providers. CONCLUSIONS: These results highlight the need for training and education for patients, but also for professionals at haemophilia centres, about HGT and the shared decision-making process. Objective, unbiased and transparent information must be available for patients about this very promising therapy which nonetheless carries more uncertainty and unknowns compared to other haemophilia treatments.

Clinical Implications of Discrepancy between One-Stage Clotting and Chromogenic Factor IX Activity in Hemophilia B.

BACKGROUND: Discrepancy in factor IX activity (FIX:C) between one-stage assay (OSA) and chromogenic substrate assay (CSA) in patients with hemophilia B (PwHB) introduces challenges for clinical management. AIM: To study the differences in FIX:C using OSA and CSA in moderate and mild hemophilia B (HB), their impact on classification of severity, and correlation with genotype. METHODS: Single-center study including 21 genotyped and clinically characterized PwHB. FIX:C by OSA was measured using ActinFSL (Siemens) and CSA by Biophen (Hyphen). In addition, in vitro experiments with wild-type FIX were performed. Reproducibility of CSA was assessed between three European coagulation laboratories. RESULTS: FIX:C by CSA was consistently lower than by OSA, with 10/17 PwHB having a more severe hemophilia type by CSA. OSA displayed a more accurate description of the clinical bleeding severity, compared with CSA. A twofold difference between OSA:CSA FIX:C was present in 12/17 PwHB; all patients had genetic missense variants in the FIX serine protease domain. Discrepancy was also observed with diluted normal plasma, most significant for values below 0.10 IU/mL. Assessment of samples with low FIX:C showed excellent reproducibility of the CSA results between the laboratories. CONCLUSION: FIX:C was consistently higher by OSA compared with the CSA. Assessing FIX:C by CSA alone would have led to diagnosis of a more severe hemophilia type in a significant proportion of patients. Our study suggests using both OSA and CSA FIX:C together with genotyping to classify HB severity and provide essential information for clinical management.

Knowledge and attitudes toward gene therapy of a cohort of Italian patients with hemophilia.

BACKGROUND: Gene therapy (GT) has recently become a new therapeutic option for hemophilia A and B. However, patient levels of knowledge and attitudes toward it are poorly understood. A general lack of knowledge and education has been highlighted in previous studies. To date, no studies focused on patient attitudes toward GT, priorities, concerns, and information needs, nor how these factors might influence their willingness to accept it. OBJECTIVES: To evaluate knowledge and attitudes toward GT of an Italian cohort of patients with hemophilia. METHODS: A questionnaire was administered to patients with hemophilia A and B to evaluate: (1) clinical data; (2) GT knowledge; (3) willingness to accept GT, perceived benefits and concerns, and information needs. RESULTS: Eighty-five patients participated in the study; 64 with severe hemophilia A and 4 with severe hemophilia B. Participants appeared to know only general information on GT, but little about its detailed functioning. The avoidance of frequent infusions and the reduction of bleeding episodes seem to be the most relevant expected benefits. The possibility of failing or losing effectiveness of GT over time was the main concern. Regarding willingness to undergo GT, 54.4% of respondents gave a negative response, mainly due to fear that treatment will lose effectiveness over time, fear of side effects, and lack of GT knowledge. Greater knowledge increased the acceptability of this disruptive therapy among patients with severe hemophilia. CONCLUSION: Overall, Italian patients with hemophilia showed poor knowledge of GT. However, it seems that greater knowledge was associated with a greater willingness to have GT.

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.

[Advances in AAV-CRISPR/Cas9-Mediated Hemophilia A Gene Therapy --Review].

Hemophilia A(HA) is an X-linked recessive bleeding disorder caused by mutations in coagulation factor VIII. Nowadays, exogenous coagulation factor replacement therapy is the main treatment. With the continuous development of gene therapy, new research directions have been provided for the treatment of hemophilia A. CRISPR-Cas9 technology was applied to select suitable target sites, and mediate the targeted knock-in and efficient expression of exogenous B-domain-deleted FⅧ variant gene through corresponding vectors for the treatment of hemophilia A.CRISPR-Cas9 technology is an emerging gene editing tool with great efficiency, safety and effectiveness, and has been widely used in hemophilia gene therapy research. This paper reviews the vector selection, construction of therapeutic genes, gene editing technology and selection of expression target sites for hemophilia A gene therapy at this stage.

Etranacogene dezaparvovec-drlb gene therapy for patients with hemophilia B (congenital factor IX deficiency).

INTRODUCTION: Congenital hemophilia B (HB) is an X-linked bleeding disorder resulting in Factor IX (FIX) deficiency and bleeding of variable severity. There is no cure for HB. Typical management consists of prophylactic intravenous (IV) recombinant or plasma-derived FIX infusions. Etranacogene dezaparvovec-drlb (Hemgenix, AMT-061) is an adeno-associated virus serotype 5 (AAV5) vector containing a codon-optimized Padua variant of the human F9 gene with a liver-specific promoter. Etranacogene dezaparvovec-drlb received FDA approval on 22 November 2022 for the treatment of HB in adult patients who use FIX prophylaxis therapy, have current or historical life-threatening hemorrhage, or have experienced repeated, serious spontaneous bleeding episodes. AREAS COVERED: This drug profile discusses the safety and efficacy of etranacogene dezaparvovec-drlb in patients with HB. EXPERT OPINION: Etranacogene dezaparvovec-drlb therapy results in stable and sustained expression of near-normal to normal FIX levels in patients with HB regardless of neutralizing antibodies to AAV5 up to a titer of 678. Its use has led to significant reduction in bleeding and FIX prophylaxis. Etranacogene dezaparvovec-drlb was well tolerated; however, 17% of patients required corticosteroid therapy for alanine aminotransferase (ALT) elevation. Etranacogene dezaparvovec-drlb therapy marks the beginning of an exciting era in HB treatment and opens questions regarding treatment longevity and long-term safety.

Etranacogene dezaparvovec for the treatment of adult patients with severe and moderately severe hemophilia B.

INTRODUCTION: Etranacogene dezaparvovec is the first gene therapy approved for treatment of adults with severe and moderately severe hemophilia B. AREAS COVERED: This review describes the results of the clinical trial program of AMT-060 and etranacogene dezaparvovec, outlining the pharmacokinetic, clinical efficacy and safety data. With the entry of etranacogene dezaparvovec into the market, this review summarizes the treatment landscape in hemophilia B and discusses the current unknowns in the field. EXPERT OPINION: Gene therapy appears to be a feasible option for adults with severe and moderately severe hemophilia B. Etranacogene dezaparvovec enables most patients to reach stable factor IX (FIX) levels after a single intravenous infusion, eliminating the need for regular prophylaxis; thus, drastically reducing treatment burden and avoiding variable bleeding risk owing to fluctuating FIX activity levels. Efficacy of etranacogene dezaparvovec has been demonstrated even in the presence of preexisting neutralizing antibodies (up to a titer of 1:678), with a relative low risk of transaminitis and its associated potential loss of transgene expression. However, long-term data are required to ascertain the durability of FIX levels achieved and safety. The cost-effectiveness and adoption of innovative payment models for reimbursement are key in choosing gene therapy over existing treatments.

Treatment of congenital coagulopathies, from biologic to biotechnological drugs: The relevance of gene editing (CRISPR/Cas).

Congenital coagulopathies have, throughout the history of medicine, been a focus of scientific study and of great interest as they constitute an alteration of one of the most important and conserved pathways of evolution. The first therapeutic strategies developed to address them were aimed at restoring the blood components lost during hemorrhage by administering whole blood or plasma. Later on, the use of cryoprecipitates was a significant breakthrough as it made it possible to decrease the volumes of blood infused. In the 1970' and 80', clotting factor concentrates became the treatment and, from the 1990's to the present day, recombinant factors -with increasingly longer half-lives- have taken over as the treatment of choice for certain coagulopathies in a seamless yet momentous transition from biological to biotechnological drugs. The beginning of this century, however, saw the emergence of new advanced (gene and cell) treatments, which are currently transforming the therapeutic landscape. The possibility to use cells and viruses as well as specific or bispecific antibodies as medicines is likely to spark a revolution in the world of pharmacology where therapies will be individualized and have long-term effects. Specifically, attention is nowadays focused on the development of gene editing strategies, chiefly those based on CRISPR/Cas technology. Rare coagulopathies such as hemophilia A and B, or even ultra-rare ones such as factor V deficiency, could be among those deriving the greatest benefit from these new developments.

Hemophilia Gene Therapy: The End of the Beginning?

Extensive preclinical research over the past 30 years has culminated in the recent regulatory approval of several gene therapy products for hemophilia. Based on the efficacy and safety data in a recently conducted phase III clinical trial, Roctavian® (valoctocogene roxaparvovec), an adeno-associated viral (AAV5) vector expressing a B domain deleted factor VIII (FVIII) complementary DNA, was approved by the European Commission and Food and Drug Administration (FDA) for the treatment of patients with severe hemophilia A. In addition, Hemgenix® (etranacogene dezaparvovec) was also recently approved by the European Medicines Agency and the FDA for the treatment of patients with severe hemophilia B. This product is based on an AAV5 vector expressing a hyper-active factor IX (FIX) transgene (FIX-Padua) transgene. All AAV-based phase III clinical trials to date show a significant increase in FVIII or FIX levels in the majority of treated patients, consistent with a substantial decrease in bleeding episodes and a concomitant reduction in factor usage obviating the need for factor prophylaxis in most patients. However, significant interpatient variability remains that is not fully understood. Moreover, most patients encountered short-term asymptomatic liver inflammation that was treated by immune suppression with corticosteroids or other immune suppressants. In all phase III trials to date, FIX expression has appeared relatively more stable than FVIII, though individual patients also had prolonged FVIII expression. Whether lifelong expression of clotting factors can be realized after gene therapy requires longer follow-up studies. Further preclinical development of next-generation gene editing technologies offers new prospects for the development of a sustained cure for hemophilia, not only in adults, but ultimately in children with hemophilia too.

THE JEREMIAH METZGER LECTURE: TURNING GENES INTO MEDICINES: HIGHLIGHTS AND HURDLES IN THE DEVELOPMENT OF GENE THERAPY FOR GENETIC DISEASE.

The journey from in vitro transfer of genes into mammalian cells to approved gene therapy products has spanned decades. This manuscript summarizes hurdles encountered and obstacles overcome in the development of successful adeno-associated viral (AAV) vectors for hemophilia B and for an inherited retinal dystrophy caused by mutations in the RPE65 gene. In the case of hemophilia B, careful analysis of the first unsuccessful attempts led to the realization that the human immune response to AAV vectors was preventing durable expression; elucidation of the response to the recombinant virion led to strategies that enabled successful long-lasting gene transfer. For RPE65 deficiency, a key to success was development and validation of a novel clinical endpoint for a disease that previously lacked a pharmacologic treatment.

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.