Long-term follow-up of liver-directed, adeno-associated vector-mediated gene therapy in the canine model of hemophilia A.

Questions remain concerning the long-term efficacy, safety, and site(s) of transgene expression following adeno-associated vector (AAV) therapy. We report a long-term follow-up of 8 (male = 4, hemizygous, and female = 4, homozygous) dogs with severe hemophilia A treated with a single portal vein infusion of a B-domain-deleted (BDD)-canine FVIII (cFVIII) AAV vector (median dose = 1.25 × 1013 vg/kg, AAV2 = 4, AAV6 = 3, and AAV8 = 1). After a median follow-up of 10.8 years (8.2-12.0 years), persistent FVIII:C (median one-stage = 12.7%, chromogenic = 7.2%) was seen in all responding dogs (n = 6), with improvement in annualized bleed rates (pre = 3.9 vs post = 0.3 event per year; P = .003). Anti-AAV capsid neutralizing antibodies (nAbs) toward the dosed capsid were detected throughout the study, with limited cross-reactivity to other capsids. nAb titers for all capsid serotypes declined with time, although they remained at levels precluding redosing with the same capsid. AAV-BDD-cFVIII DNA was detected in the liver of all dogs (median = 0.15 vg per diploid genome), with lower levels in the spleen in 4 dogs (median = 0.005 vg per diploid genome). Consistent with the liver-specific promoter, BDD-cFVIII mRNA was only detected in the liver. Postmortem examination demonstrated no evidence of chronic liver disease or liver malignancy. Persistent FVIII expression and an improved bleeding phenotype was seen for more than a decade after vector delivery. This is the longest follow-up reported in a preclinical model supporting long-term efficacy and safety of AAV-mediated gene therapy.

The Jamaican Haemophilia Registry: Describing the burden of disease.

INTRODUCTION: Jamaica has an estimated 200 persons with haemophilia (PWH), who face significant constraints in access to specialized haemophilia care, including access to clotting factor concentrates. AIM: The aim of this paper is to establish the current burden of disease in PWH in Jamaica. METHODS: PWH were enrolled through the University Hospital of the West Indies, Jamaica. The impact of haemophilia was assessed using a comprehensive battery of heath outcome measures that included the following: laboratory, clinical information and validated outcome measures of joint structure and function, activity, and health-related quality of life (HRQoL) to provide a health profile of the Jamaican haemophilia population. RESULTS: In all, 45 PWH were registered (mean age: 29, range: 0.17-69 years), including 13 children (<18 years of age) and 32 adults. In this sample, 41 had haemophilia A (30 severe) and 4 had haemophilia B (3 severe); 10 patients with haemophilia A were inhibitor positive. The results indicate that adults with haemophilia in Jamaica have significant joint damage: mean Haemophilia Joint Health Score (HJHS) = 42.1 (SD = 17.3); moderate activity levels - mean Haemophilia Activities List (HAL) score = 64.8 (SD = 17.8); and low HRQoL scores - mean Haemo-QoL-A score = 62.3 (SD = 19.4). Results for children are also reported but should be interpreted with caution due to the small sample size. CONCLUSIONS: There is a very high burden of disease in PWH in Jamaica. The health profiles reported in this paper are an essential first step in advocating for a multidisciplinary Comprehensive Care Program for assessment and care of PWH in Jamaica.

Alternative therapies for the management of inhibitors.

The development of inhibitors to factor VIII (FVIII) or factor IX (FIX) remains a major treatment complication encountered in the treatment of haemophilia. Not all patients with even the same severity and genotype develop inhibitors suggesting an underlying mechanism of tolerance against FVIII- or FIX-related immunity. One mechanism may be central tolerance observed in patients in whom the FVIII mutation enables some production of the protein. The other is a peripheral tolerance mechanism which may be evident in patients with null mutation. Recently, recombinant porcine FVIII (rpFVIII, Obixur, OBI-1, BAX801) has been developed for the haemostatic treatment of both congenital haemophilia with inhibitor (CHAWI) and acquired haemophilia A (AHA). In 28 subjects with AHA with life-/limb-threatening bleeding, rpFVIII reduced or stopped bleeding in all patients within 24 h. The cross-reactivity of anti-human FVIII antibodies to rpFVIII remains around 30-50%. Recently, new therapeutics based on the quite novel concepts have been developed and clinical studies are ongoing. These are humanized asymmetric antibody mimicking FVIIIa function by maintaining a suitable interaction between FIXa and FX (Emicizumab, ACE910), and small interfering RNAs (siRNA, ALN-AT3) suppress liver production of AT through post-transcriptional gene silencing and a humanized anti-TFPI monoclonal antibody (Concizumab). Their main advantages are longer half-life, subcutaneous applicability and efficacy irrespective of the presence of inhibitors which will make it easier to initiate more effective treatment especially early childhood.

Porcine recombinant factor VIII (Obizur; OBI-1; BAX801): product characteristics and preclinical profile.

INTRODUCTION: Acquired haemophilia A (AHA) is a rare, often severe, auto-immune bleeding disorder caused by the development of inhibitory antibodies (inhibitors) to factor VIII (FVIII). Bypassing agents, recombinant activated FVII or activated prothrombin complex concentrate, are currently recommended as first-line treatments to control bleeding events in patients with AHA. AIM: A plasma-derived porcine FVIII (Hyate:C, Ipsen, UK) was used as a first-line treatment for AHA but was discontinued in 2004 due to viral safety concerns. A recombinant pFVIII (rpFVIII), Obizur (OBI-1; BAX801), which is expected to have a similar efficacy profile to Hyate:C but with a superior safety profile was developed and recently approved by the US Food and Drug Administration for the treatment of AHA. METHODS: Obizur manufacturing begins with the expression of B domain deleted rpFVIII by genetically modified baby hamster kidney-derived cells. The final purified and lyophilized drug product has a negligible risk of viral contamination and contains no animal-derived plasma proteins. Obizur was evaluated for immunogenicity, tolerability, pharmacokinetics and bleeding times in preclinical models including in haemophiliac dogs, cynomolgus monkeys and FVIII-knockout mice. RESULTS: Preclinical animal studies show that the efficacy and immunogenicity of Obizur are similar to that of Hyate:C and that Obizur has a more favourable safety profile. CONCLUSIONS: Obizur is a highly purified recombinant porcine FVIII drug product that has been demonstrated to have a favourable safety and efficacy profile when compared with Hyate:C and can be a valuable treatment option for control of bleeding in AHA patients.

Changes in von Willebrand factor level and von Willebrand activity with age in type 1 von Willebrand disease.

In a normal population, VWF plasma levels (VWF:Ag) and VWF activity (VWF:RCo) increase by approximately 0.17 and 0.15 IU mL(-1) per decade, but the influence of age is unknown in patients with type 1 von Willebrand disease (VWD). In a retrospective cohort study, the medical records of 31 type 1 VWD patients over the age of 30, who had been followed for ≥5 years, were reviewed for baseline clinical data and previously performed VWF:Ag, VWF:RCo and factor VIII levels ( FVIII: C). VWF multimer analysis was normal in 28/31 cases performed. Mean age at diagnosis was 33 (range 16-60 years), and duration of follow-up ranged from 5 to 26 years (mean 11 years). Patients had 2-10 time points of VWD testing (mean of 5.2). The mean VWF:Ag, VWF:RCo and FVIII: C at time of diagnosis were 0.44 IU mL(-1) 0.34 IU mL(-1) and 0.75 IU mL(-1) . At last follow-up, the mean VWF:Ag, VWF:RCo and FVIII: C were significantly increased to 0.71 IU L(-1) , 0.56 IU mL(-1) and 0.90 IU mL(-1) (P ≤ 0.001, <0.001, and 0.0081 respectively). Here 18/31 patients had VWF:Ag, VWF:RCo and FVIII: C levels that increased into the normal range. The rate of change in VWF:Ag, VWF:RCo and FVIII was 0.30 IU mL(-1) (0.21-0.39, CI 95%, P < 0.0001), 0.20 IU mL(-1) per decade (0.13-0.27, CI 95%, P = 0.0001) and 0.20 IU mL(-1) (0.11-0.29, CI 95%, P = 0.0011). Patients with type 1 VWD experience age-related increases to VWF:Ag and VWF:RCo which can result in normalization of VWF levels. Further studies are required to determine if the bleeding phenotype resolves with the increases in VWF:Ag and VWF:RCo levels.

Inhibitors - genetic and environmental factors.

It is known that a large number of both genetic and environmental factors contribute to the risk of inhibitor development, but underlying pathogenetic mechanisms are still under investigation. The clinical research on inhibitors towards factor VIII (FVIII) is challenged by the fact that this is an infrequent event occurring in a rare disease. Therefore, it is widely accepted that complementary studies involving animal models can provide important insights into the pathogenesis and treatment of this complication. In this respect, mouse models have been studied for clues to FVIII immunogenicity, natural history of immunity and for different approaches to primary and secondary tolerance induction. In the clinical setting, the type of FVIII product used and the occurrence of product switching are considered important factors which may have an influence on inhibitor development. The evaluation of data currently available in the literature does not prove unequivocally that a difference in the immunogenicity exists between particular FVIII products (e.g. recombinant vs. plasma-derived, full length vs. B-domainless). In addition, national products switches have occurred and, in this context, switching was not associated with an enhanced inhibitor risk. In contrast with severe haemophilia A, patients with moderate and mild haemophilia A receive FVIII treatment infrequently for bleeds or surgery. In this condition the inhibitor risk is low but remains present lifelong, requiring continuous vigilance, particularly after intensive FVIII exposure.

Current status of haemophilia gene therapy.

After many reports of successful gene therapy studies in small and large animal models of haemophilia, we have, at last, seen the first signs of success in human patients. These very encouraging results have been achieved with the use of adeno-associated viral (AAV) vectors in patients with severe haemophilia B. Following on from these initial promising studies, there are now three ongoing trials of AAV-mediated gene transfer in haemophilia B all aiming to express the factor IX gene from the liver. Nevertheless, as discussed in the first section of this article, there are still a number of significant hurdles to overcome if haemophilia B gene therapy is to become more widely available. The second section of this article deals with the challenges relating to factor VIII gene transfer. While the recent results in haemophilia B are extremely encouraging, there is, as yet, no similar data for factor VIII gene therapy. It is widely accepted that this therapeutic target will be significantly more problematic for a variety of reasons including accommodating the larger factor VIII cDNA, achieving adequate levels of transgene expression and preventing the far more frequent complication of antifactor VIII immunity. In the final section of the article, the alternative approach of lentiviral vector-mediated gene transfer is discussed. While AAV-mediated approaches to transgene delivery have led the way in clinical haemophilia gene therapy, there are still a number of potential advantages of using an alternative delivery vehicle including the fact that ex vivo host cell transduction will avoid the likelihood of immune responses to the vector. Overall, these are exciting times for haemophilia gene therapy with the likelihood of further clinical successes in the near future.

First analysis of 10-year trends in national factor concentrates usage in haemophilia: data from CHARMS, the Canadian Hemophilia Assessment and Resource Management System.

The Canadian Hemophilia Assessment and Resource Management System (CHARMS) tracks factor concentrates (FC) from the sole suppliers, Canadian Blood Services (CBS) and Hema-Quebec (HQ), to hospitals and to patients' homes. Patients FC infusion data are entered into CHARMS at Canadian Hemophilia Treatment Centres (HTCs) then exported to the national database (CentrePoint). From 2000 to 2009, 2260 registered haemophilia A or B patients received FVIII (1,009,097,765 IU) and FIX (272,406,859 IU). Over 91% of FVIII and over 84% of FIX was infused at home. Utilization of FVIII progressively increased; this was accounted for by an increase in the number of patients treated (r = 0.97; P < 0.001), there being a linear relationship between the increase in utilization and the increase in number of patients treated (P < 0.001). There was also a correlation with the annual amount used per patient (r = 0.95; P < 0.001). Utilization of FIX did not increase over time. The highest proportional utilization of both FVIII and FIX was for prophylaxis, and this proportion progressively increased being, in year 10 (2009), 77% and 66% for FVIII and FIX respectively. The proportion used for bleeding remained steady; in year 10 that proportion was 14% for FVIII and 26% for FIX, the use per patient for bleeding decreasing. The HTC-based CHARMS tracking system is essential, in Canada, for analysing indications for infusion, for predicting utilization and planning for future needs.

Incidence and risk factors for inhibitor development in previously untreated severe haemophilia A patients born between 2005 and 2010.

The objective of this study was to evaluate the inhibitor development (ID) in previously untreated patients (PUPs) with severe haemophilia A (FVIII ≤ 0.01 IU mL(-1) ). All Canadian Haemophilia Treatment Centres completed a questionnaire on patients born between September 2005 and August 2010 and followed for up to 7 years. Eligible patients had at least 20 exposure days (ED) or had developed an inhibitor. The odds ratio (OR) and 95% confidence intervals (95% CI) for risk factors to develop an inhibitor were estimated using unconditional logistic regression. A total of 99 haemophilia A PUPs were studied. Thirty-four (34%) developed an inhibitor (24/34 of high titre). Inhibitors developed in 25/63 (40%) patients with a high-risk mutation. ID was most frequent in Aboriginals (86%). Dose intensity (IU kg(-1)  day(-1) X number of ED) at first exposure to factor VIII (FVIII) was associated with a crude OR increase of 1.10 (95% CI: 0.99-1.23) with each increase of 100 dose-intensity units. Haemarthrosis and intracranial bleeding as the indication for first exposure to FVIII concentrate were associated with a crude OR for ID of 7.63 (95% CI: 2.14-27.17) and 5.08 (95% CI: 1.11-23.31) respectively. ID according to FVIII concentrate used was: Advate (®) 18/50 (36%), Kogenate FS(®) or Helixate FS(®) 15/36 (42%), Wilate(®) 0/11 and Xyntha(®) 1/2. In multivariate analysis, Aboriginal ethnicity (OR = 11.69; 95% CI: 1.11-122.86) and haemarthrosis (OR = 4.49; 95% CI: 1.08-18.61) were statistically significant. The cumulative incidence of ID in severe haemophilia A PUPs was 34% and varied according to ethnicity, type of bleeding at first ED, type of FVIII product and dose intensity at first exposure.

Switching clotting factor concentrates: considerations in estimating the risk of immunogenicity.

The development of neutralizing antibodies to factor VIII (FVIII) is the most serious complication of therapy for haemophilia A. There is now excellent documentation that a large number of both genetic and environmental factors contribute to the risk of FVIII inhibitor incidence. One of the environmental factors that has been proposed as an influence on this complication is the occurrence of FVIII product switching. There are only a small number of clinical studies that have addressed this question, and thus, the amount of objective information available to assess this association is limited. In this review, in addition to summarizing past evidence pertinent to this subject, we present the results of a complementary strategy, a Delphi analysis, to add to the considerations of product switching and FVIII immunogenicity. With the imminent arrival in the clinic of several new FVIII products, the haemophilia community must be prepared to collect prospectively controlled data to better address this important management issue.

The molecular characterization of von Willebrand disease: good in parts.

Since the cloning of the gene that encodes von Willebrand factor (VWF), 27 years ago, significant progress has been made in our understanding of the molecular basis of the most common inherited bleeding disorder, von Willebrand disease (VWD). The molecular pathology of this condition represents a range of genetic mechanisms, some of which are now very well characterized, and others that are still under investigation. In general, our knowledge of the molecular basis of type 2 and 3 VWD is now well advanced, and in some instances this information is being used to enhance clinical management. In contrast, our understanding of the molecular pathogenesis of the most common form of VWD, type 1 disease, is still at an early stage, with preliminary evidence that this phenotype involves a complex interplay between environmental factors and the influence of genetic variability both within and outside of the VWF locus.

Quantification of perioperative changes in von Willebrand factor and factor VIII during elective orthopaedic surgery in normal individuals.

von Willebrand's disease (VWD) patients undergoing major surgery are prophylactically treated to promote haemostasis. There is variability in perioperative clinical practice; however, most guidelines suggest replacing the deficient factor to a level of 1.0 IU mL(-1) (or 100%). A review of the literature reveals a paucity of well constructed descriptive data quantifying the changes in coagulation that occur in response to surgical stress. The aim of this study was to quantify the changes in haemostatic variables occurring in response to elective orthopaedic surgery in normal individuals. Eligible subjects >18 years of age undergoing total hip or knee replacement were recruited. Blood samples were drawn at five time points: baseline, preoperatively, 30 min after surgical incision, 30 min postoperatively, postoperative day (POD) 1. Analyses included t-tests and repeated measures anova. Overall 30 patients, 21 women and 9 men, with a mean age of 65 were included in the final analysis. All von Willebrand factor (VWF) variables were seen to significantly decrease intraoperatively and increase postoperatively. VWF multimers showed a statistically significant decrease in high molecular weight multimers intraoperatively and an increase postoperatively. On subgroup analysis, age, gender and anaesthesia type were significantly correlated with changes in VWF parameters. Data presented in the current study establish a physiological baseline for VWF parameters in the normal population and demonstrate mean VWF/factor VIII levels greater than 1.0 IU mL(-1) intraoperatively. As such, current management in VWD patients does not appear to mimic the normal physiological response to surgery.

Bleeding disorders, menorrhagia and iron deficiency: impacts on health-related quality of life.

von Willebrand disease (VWD) is a bleeding disorder that occurs in up to 1% of the general population. The great majority of females with VWD experience menorrhagia. The morbidity burden in females with VWD may relate to iron deficiency resulting from menorrhagia. To explore relationships between bleeding disorders, menorrhagia, iron deficiency and the outcomes of health-related quality of life (HRQL) and educational attainment. All subjects with VWD, and females with other bleeding disorders, in the Canadian national registry who were more than 12 years of age were eligible for survey. Survey measures included the HEALTH UTILITIES INDEX(®); abridged Clinical History Assessment Tool; socio-demographic questions and serum ferritin. Statistical analyses included testing differences among groups of means using analysis of variance and of proportions using chi-squared test. Significant size differences in mean HRQL scores were detected between VWD females and both females with other bleeding disorders [diff = (-0.08); P = 0.017] and VWD males [diff = (-0.07); P = 0.039]. Mean HRQL scores differed between females with and without menorrhagia (P < 0.001). Mean HRQL scores were not significantly different between females with and without iron deficiency. Educational attainment was not associated with disease group, menorrhagia status or iron status. Females with VWD have a greater morbidity burden than females in the general population, females with other bleeding disorders and males with VWD. Menorrhagia is associated with low HRQL scores in females with bleeding disorders, including VWD. Further investigation should assess how menorrhagia impacts HRQL in females with bleeding disorders.

Third Åland islands conference on von Willebrand disease, 26-28 September 2012: meeting report.

The first meeting of international specialists in the field of von Willebrand disease (VWD) was held in the Åland islands in 1998 where Erik von Willebrand had first observed a bleeding disorder in some members of a family from Föglö and a summary of the meeting was published in 1999. The second meeting was held in 2010 and a report of the meeting was published in 2012. Topics covered included progress in understanding of VWD over the last 50 years; multimers; classification of VWD; pharmacokinetics and laboratory assays; genetics; treating the paediatric patient; prophylaxis; geriatrics; gene therapy and treatment guidelines. This third meeting held over 3 days covered the structure and function of von Willebrand factor (VWF); type 1 VWD, the most common form of the disease; a lifespan of pharmacokinetics in VWD; detecting inhibitors in VWD patients; and special challenges in understanding and treating the female VWD patient.

Synergy between transcription factors DBP and C/EBP compensates for a haemophilia B Leyden factor IX mutation.

Haemophilia B Leyden is characterized by low childhood levels of factor IX which gradually increase after puberty, eventually resulting in a return to health. The disease is the result of single nucleotide substitutions within a 40 bp region encompassing the major transcriptional start site. We have characterized transcription factor binding sites within the factor IX promoter. Five sites were identified and a Leyden mutation at nucleotide -5 was shown to interfere with the binding of proteins to one of three newly identified sites. The correlation between the post-pubertal recovery of these mutants and the induction of the transcription factor DBP led us to the discovery of a synergistic interaction between DBP and C/EBP responsible for the recovery of normal transcriptional activity of the -5 mutant promoter and may play a role in the resolution of other Leyden mutants.

Low prevalence of inhibitor antibodies in the Canadian haemophilia population.

Annual reporting of inhibitors to factors (FVIII) and IX (FIX) to the Canadian Haemophilia Registry has suggested a lower prevalence than that published in the literature. We performed a prospective study to determine the prevalence of patients with inhibitors directed against either FVIII or FIX. Patients with inhibitors were classified as: (i) inhibitor test positive; (ii) inhibitor test negative but on immune tolerance induction (ITI); (iii) inhibitor test negative but bypass treatment recommended; or (iv) inhibitor resolved. One year later, the cohort was re-classified. The prevalence of inhibitors on 1 May, 2007 was 3.3% for haemophilia A, 0.6% for haemophilia B and 8.9% and 2.1% for severe haemophilia A and B. One year later 17 individuals gained and 11 individuals lost inhibitor status (10 of these with ITI). This study suggests that the prevalence of inhibitors in our population is lower than that was previously published. We hypothesize that this is primarily due to the increased use of ITI, but other factors may be the unselected nature of the cohort and the restriction of the study to one date thereby conforming as close as practical to the definition of prevalence rather than incidence. The classification system used in this study was easy for clinics to apply and was important in defining the population with inhibitors.

von Willebrand's disease: a report from a meeting in the Åland islands.

von Willebrand's disease (VWD) is probably the most common bleeding disorder, with some studies indicating that up to 1% of the population may have the condition. Over recent years interest in VWD has fallen compared to that of haemophilia, partly the result of focus on blood-borne diseases such as HIV and hepatitis. Now the time has come to revisit VWD, and in view of this some 60 international physicians with clinical and scientific interest in VWD met over 4 days in 2010 in the Åland islands to discuss state-of-the-art issues in the disease. The Åland islands are where Erik von Willebrand had first observed a bleeding disorder in a number of members of a family from Föglö, and 2010 was also the 140th anniversary of his birth. This report summarizes the main papers presented at the symposium; topics ranged from genetics and biochemistry through to classification of VWD, pharmacokinetics and laboratory assays used in the diagnosis of the disease, inhibitors, treatment guidelines in different age groups including the elderly who often have comorbid conditions that present challenges, and prophylaxis. Other topics included managing surgeries in patients with VWD and the role of FVIII in VWF replacement, a controversial subject.

International comparative field study of N8 evaluating factor VIII assay performance.

Discrepancies between the one-stage clotting assay and the chromogenic method, and also among different variations of each method, have been a significant challenge for one B-domain deleted FVIII product. N8 is a B-domain truncated FVIII product developed by Novo Nordisk. The comparison of N8 and Advate(®) was performed in an international, multicentre, randomized and blinded field study of simulated postinfusion samples. Overall, Advate(®) and N8 performed similarly in the one-stage assay. In the one-stage clotting assay, the measured mean FVIII levels of Advate(®) vs. N8 were 0.046/0.047, 0.24/0.24, 0.58/0.60 and 0.82/0.83 IU mL(-1) for the target values of 0.03, 0.2, 0.6 and 0.9 IU mL(-1) , respectively. In the chromogenic assays, the concentration estimates showed a tendency towards higher N8 values as compared with Advate(®) ; the measured FVIII levels of Advate(®) vs. N8 were 0.030/0.032, 0.22/0.24, 0.65/0.74 and 0.98/1.08 IU mL(-1) for the target values of 0.03, 0.2, 0.6 and 0.9 IU mL(-1) , respectively. In the one-stage assays, the measured values were above 150% of target at the lowest concentration, decreasing to around 90% of target at the highest concentration. In contrast, the chromogenic assays were close to target at the lowest concentration and consistently above target at the three highest concentrations. Therefore, the ratio of chromogenic/one-stage potencies was concentration dependent, ranging from 0.66 to 1.30. The SSC plasma standard was similar in both. Assay variability was similar for both compounds. The results show that N8 can be reliably measured in plasma without the need for a separate N8 standard.

Immunoglobulin isotypes and functional anti-FVIII antibodies in response to FVIII treatment in Balb/c and C57BL/6 haemophilia A mice.

Previous studies have demonstrated that genetic factors play an important role in determining the likelihood of formation of anti-factor VIII (FVIII) antibodies in haemophilia A patients. We were interested in characterizing the spectrum of FVIII antibody formation and the primary and secondary immune responses after FVIII administration in two different exon 16-disrupted haemophilia A mouse strains, Balb/c and C57BL/6. Balb/c and C57BL/6 E16 haemophilia A mice were used in all experiments. Total FVIII antibodies and FVIII inhibitors were measured using ELISA and Bethesda assays respectively. T- and B-cell cytokines were quantified using ELISA and flow cytometry. FVIII antibodies, but not functional inhibitors were detectable 1 week after the first FVIII treatment in both strains. These antibodies mainly belonged to the IgM and IgA isotypes. After the fourth FVIII treatment, neutralizing anti-FVIII antibodies were detected in both mouse strains: Balb/c (mean inhibitory titer 58 BU) and C57BL/6 (mean inhibitory titer 82 BU). IgG1 levels were similar in both strains but the IgG2A and IgG2B subclasses were higher in C57BL/6 mice. The results of intracellular cytokine staining of T cells indicated that the FVIII-treated C57BL/6 mice produced more IL10 and Th1 cytokines than the FVIII-treated Balb/c mice. These studies show that C57BL/6 mice develop a stronger immune response towards FVIII than Balb/c mice. We propose that the enhanced Th1 and IL10 cytokine micro-environment induced in C57BL/6 mice is responsible for this difference. Therefore, genetic strain-dependent differences must be considered when evaluating immunological outcomes in mouse models of haemophilia A.