Complete short-term correction of canine hemophilia A by in vivo gene therapy

Hemophilia A is a severe bleeding disorder caused by a deficiency in clotting factor VIII (FVIII). A canine model that closely mimics the human disease was used to determine if an adenoviral vector expressing a human FVIII cDNA could be used to correct the hemophilia A phenotype. Within 48 hours after peripheral vein administration of the vector to FVIII-deficient dogs, the hemophilic phenotype was corrected, based on determination of the activated clotting time, the activated partial thromboplastin time, and the cuticle bleeding time. Direct measurement of human FVIII in the dog plasma showed FVIII expression at amounts well above the human therapeutic level. FVIII expression in treated dogs was short-term, lasting 1 to 2 weeks, due to the development of a human FVIII-specific inhibitor antibody response. These data provide the first demonstration of in vivo gene therapy of hemophilia A.     

Efficient adenoviral vector transduction and expression of functional human factor VIII in cultured primary human hepatocytes

Hemophilia A is a severe bleeding disorder caused by a deficiency in blood coagulation factor VIII (FVIII). Adenoviral vectors containing a potent human FVIII expression cassette encoding a truncated FVIII cDNA were developed that mediated sustained FVIII expression in normal and haemophiliac mice and complete phenotypic correction of the bleeding disorder in haemophiliac mice and dogs (Connelly and Kaleko, Haemophilia, 1998; 4: 380-8). Here, we evaluated two E1/E2a/E3-deleted adenoviral vectors encoding human FVIII, one containing the full-length cDNA and the second containing a truncated cDNA lacking the B-domain. Viral vectors encoding the human full-length FVIII cDNA have not been described previously. Hepatocyte transduction was efficient and dose dependent, ranging from 50% to 100%. High levels of functional FVIII were secreted from transduced cells at amounts up to 6000 mU-1 10(6)cells-1 60 h. B-domain deleted FVIII was expressed at levels at least 8-fold higher than the full-length FVIII protein, whereas FVIII RNA levels were similar with both vectors. These data provide the first demonstration of FVIII adenoviral vector function in primary human cells and verify the potential clinical utility of adenoviral vectors for the treatment of haemophilia A.     

Sustained expression of Epstein–Barr virus episomal vector mediated factor VIII in vivo following muscle electroporation

Haemophilia A treatment is an attractive candidate for gene therapy. The aim of haemophilia gene therapy is to obtain long-term therapeutic level of factor VIII (FVIII). We investigated Epstein-Barr virus (EBV)-based episomal vector combined with in vivo electroporation of naked DNA as a safe, efficient and simple method for correcting FVIII deficiency. A combinant FVIII expression EBV-based episomal vector pcDNA3-FVIII-EBVR was constructed and expressed in COS-7 cells. Then the naked plasmid DNA was injected into the quadriceps of mice following the electric pulse stimulation. Our data showed that pcDNA3-FVIII-EBVR expression in transfected COS-7 can maintain stably for at least 60 days and the hFVIII:Ag in plasma in two pcDNA3-FVIII-EBVR groups mice was higher than that in pcDNA-FVIII groups no matter with or without electric pulse stimulation. With the stimulating of electric pulse, the FVIII expression in plasma of recipient mice was increased two- to fourfolds and can be lasted for at least 90 days. No severe muscle damage was detected. So this novel strategy that FVIII expression mediated by EBV episomal vector following muscle electroporation is efficient, safe, simple and economic and may be applicable to clinical usage.     

Hepatic gene therapy for haemophilia B

Summary. Early retroviral-mediated factor IX gene transfer into deficient dogs showed that constitutive expression of low levels of factor IX which has led to persistent improvement of clinically relevant parameters such as the WBCT and PTT. Conversely, in vivo adenoviral mediated delivery of the factor IX cDNA into hepatocytes of haemophilia B dogs has resulted in greater than wild-type plasma concentrations of clotting factor with complete, albeit transient normalization of haemostasis for a short time. An immune response directed against the vector transduced cells presented a big obstacle to clinical application. However, the future of gene therapy for factor IX deficiency appears bright with the development of fully adenoviral-gene deleted vectors, rAAV and lentiviral vectors which seem to offer safety, therapeutic levels of factor IX and relatively long-term persistence. We must proceed with cautious optimism as these vector systems undergo further scrutiny.     

Inhibitor development in haemophilia A: the role of von Willebrand factor/factor VIII concentrates

Summary.  The presence of inhibitors that neutralize the function of factor VIII (FVIII) decreases the haemostatic efficacy of replacement clotting factor concentrate and increases morbidity among patients with haemophilia A. Certain genetic and environmental variables have been linked to a higher incidence of inhibitors. Conversely, the presence of von Willebrand factor (VWF) in some plasma-derived FVIII products may provide some measure of protection against inhibitor development, although the evidence is not conclusive. Clinical trials are needed to resolve this issue and determine the appropriate role of VWF-containing FVIII concentrates in the treatment of haemophilia A patients.     

Acquired haemophilia: dynamic whole blood coagulation utilized to guide haemostatic therapy

Acquired haemophilia is a rare bleeding disorder caused by autoimmune antibodies interacting with factor VIII (FVIII) or factor IX. Anticipating a high degree of heterogeneity amongst cases, we recently initiated systematic recording of whole blood (WB) coagulation dynamic profiles using our recently developed thrombelastographic method employing very small amounts of tissue factor for activation. Six newly diagnosed patients with acquired haemophilia A in our University Hospital were investigated with the purpose to characterize the WB clotting phenotypes in each patient, as well as inspecting the ex vivo and in vivo response to supplementation with various haemostatic agents. Our results show a striking heterogeneity in patients WB clotting profiles, each patient having a particular pattern and an individual type of response to bypassing agents. Profiles in some of patients resembled severe haemophilia A, even if there was a measurable residual FVIII:C activity while others were more similar moderate-to-mild haemophilia. In one case the profile was very close to normal. Each patient seemed to respond individually to bypassing agents. WB clotting profiles assisted us in selecting an optimal treatment modality in each case and whenever possible, we compared the clinical effects of the treatment selected with the appearance of the WB clotting pattern. In one patient, the ex vivo response to FVIII looked promising, and a approximately 200 IU kg-1 per 24 h high-dose programme nearly normalized the clotting profile in 2-week time. Our preliminary small series of data should be concluded with caution. However, it seems that WB clotting profile studies at baseline, with ex vivo addition of haemostasis promoting agents, and during treatment may hold the potential to predict the success of treatment.     

Factor VIII and IX deficiencies related to acquired inhibitors in a patient with chronic hepatitis C virus infection receiving treatment with pegylated interferon plus ribavirin

The development of clotting factor inhibitor autoantibodies is rarely observed, but can result in a potentially life-threatening haemorrhagic disorder. These acquired inhibitors are most frequently against factor VIII (FVIII), whilst the detection of inhibitors against other clotting factors is rarer. Inhibitors against FVIII and FIX are mostly observed in patients with classical hereditary haemophilia after receiving factor replacement therapy. We report a rare case of acquired FVIII and factor IX (FIX) inhibitors in a single, non-haemophilic patient with chronic hepatitis C virus (HCV) infection who was receiving antiviral treatment with pegylated interferon plus ribavirin. The FVIII and FIX activities were <1% and high titres of inhibitors autoantibodies were found in his serum samples. After achieving a sustained virological response, combined immunosuppression with oral corticosteroids (prednisone) and azathioprine was introduced, eradicating the inhibitory autoantibodies. The development of these inhibitors in association with antiviral therapy for chronic hepatitis C is poorly understood, and particular attention must be given to HCV-infected patients with worsening coagulopathy, particularly if coexistent with treatment related thrombocytopenia.     

Factor VIII and IX deficiencies related to acquired inhibitors in a patient with chronic hepatitis C virus infection receiving treatment with pegylated interferon plus ribavirin

Abstract

The development of clotting factor inhibitor autoantibodies is rarely observed, but can result in a potentially life‐threatening haemorrhagic disorder. These acquired inhibitors are most frequently against factor VIII (FVIII), whilst the detection of inhibitors against other clotting factors is rarer. Inhibitors against FVIII and FIX are mostly observed in patients with classical hereditary haemophilia after receiving factor replacement therapy. We report a rare case of acquired FVIII and factor IX (FIX) inhibitors in a single, non‐haemophilic patient with chronic hepatitis C virus (HCV) infection who was receiving antiviral treatment with pegylated interferon plus ribavirin. The FVIII and FIX activities were <1% and high titres of inhibitors autoantibodies were found in his serum samples. After achieving a sustained virological response, combined immunosuppression with oral corticosteroids (prednisone) and azathioprine was introduced, erradicating the inhibitory autoantibodies. The development of these inhibitors in association with antiviral therapy for chronic hepatitis C is poorly understood, and particular attention must be given to HCV‐infected patients with worsening coagulopathy, particularly if coexistent with treatment related thrombocytopenia.     

Prospective observational cohort studies for studying rare diseases: the European PedNet Haemophilia Registry

Haemophilia is a rare disease. To improve knowledge, prospective studies of large numbers of subjects are needed. To establish a large well‐documented birth cohort of patients with haemophilia enabling studies on early presentation, side effects and outcome of treatment. Twenty‐one haemophilia treatment centres have been collecting data on all children with haemophilia with FVIII/IX levels up to 25% born from 2000 onwards. Another eight centres collected data on severe haemophilia A only. At baseline, details on delivery and diagnosis, gene mutation, family history of haemophilia and inhibitors are collected. For the first 75 exposure days, date, reason, dose and product are recorded for each infusion. Clinically relevant inhibitors are defined as follows: at least two positive inhibitor titres and a FVIII/IX recovery <66% of expected. For inhibitor patients, results of all inhibitor‐ and recovery tests are collected. For continued treatment, data on bleeding, surgery, prophylaxis and clotting factor consumption are collected annually. Data are downloaded for analysis annually. In May 2013, a total of 1094 patients were included: 701 with severe, 146 with moderate and 247 with mild haemophilia. Gene defect data were available for 87.6% of patients with severe haemophilia A. The first analysis, performed in May 2011, lead to two landmark publications. The outcome of this large collaborative research confirms its value for the improvement of haemophilia care. High‐quality prospective observational cohorts form an ideal source to study natural history and treatment in rare diseases such as haemophilia.     

Prolonged activity of a recombinant factor VIII-Fc fusion protein in hemophilia A mice and dogs

Despite proven benefits, prophylactic treatment for hemophilia A is hampered by the short half-life of factor VIII. A recombinant factor VIII-Fc fusion protein (rFVIIIFc) was constructed to determine the potential for reduced frequency of dosing. rFVIIIFc has an ～ 2-fold longer half-life than rFVIII in hemophilia A (HemA) mice and dogs. The extension of rFVIIIFc half-life requires interaction of Fc with the neonatal Fc receptor (FcRn). In FcRn knockout mice, the extension of rFVIIIFc half-life is abrogated, and is restored in human FcRn transgenic mice. The Fc fusion has no impact on FVIII-specific activity. rFVIIIFc has comparable acute efficacy as rFVIII in treating tail clip injury in HemA mice, and fully corrects whole blood clotting time (WBCT) in HemA dogs immediately after dosing. Furthermore, consistent with prolonged half-life, rFVIIIFc shows 2-fold longer prophylactic efficacy in protecting HemA mice from tail vein transection bleeding induced 24-48 hours after dosing. In HemA dogs, rFVIIIFc also sustains partial correction of WBCT 1.5- to 2-fold longer than rFVIII. rFVIIIFc was well tolerated in both species. Thus, the rescue of FVIII by Fc fusion to provide prolonged protection presents a novel pathway for FVIII catabolism, and warrants further investigation.     

Immunological aspects of inhibitor development

Summary.  The development of inhibitor antibodies is perhaps the most serious complication of coagulation factor replacement therapy. A complex interaction of several variables leads to inhibitor formation in congenital haemophilia, while acquired haemophilia represents a failure of the immune tolerance mechanisms that regulate a normal immune response to factor VIII (FVIII). The immune response to FVIII is dependent upon the interaction of different CD4+ T-cell subsets (Th1, Th2 and Th3) specific for FVIII. Failure to activate regulatory CD4+ cells likely plays a crucial role in the development of FVIII inhibitors. Although the basic mechanisms of the immune response to FVIII in the setting of factor replacement therapy are being elucidated, a clear understanding of the relevance of these mechanisms in the context of successful immune tolerance therapy and ultimately gene therapy, awaits further study.     

Prophylaxis in adults with haemophilia

Summary.  Recurrent haemarthroses in patients with severe haemophilia A often result in irreversible joint damage. Treatment using routine infusions of factor VIII (FVIII) concentrate, a therapy known as prophylaxis, is currently recommended for the prevention of haemarthroses and arthropathy in persons with severe haemophilia A. However, until recently, the body of evidence supporting prophylaxis in comparison with FVIII infusions given only at the time of haemarthroses was mostly retrospective and anecdotal. Recently, two prospective randomized clinical trials have been conducted to compare prophylaxis with on-demand FVIII treatment for the prevention of arthropathy and haemarthroses in young children with haemophilia A. A third prospective, non-randomized trial evaluated a strategy of escalating the dose frequency of prophylaxis. Data from these studies will provide objective evidence for the prevention of haemarthroses and arthropathy in children with severe haemophilia A.     

Comparing prophylaxis with episodic treatment in haemophilia A: implications for clinical practice

Summary. Recurrent haemarthroses in patients with severe haemophilia A often result in irreversible joint damage. Treatment using routine infusions of factor VIII (FVIII) concentrate, a therapy known as prophylaxis, is currently recommended for the prevention of haemarthroses and arthropathy in persons with severe haemophilia A. However, until recently, the body of evidence supporting prophylaxis in comparison with FVIII infusions given only at the time of haemarthroses was mostly retrospective and anecdotal. Recently, two prospective randomized clinical trials have been conducted to compare prophylaxis with on‐demand FVIII treatment for the prevention of arthropathy and haemarthroses in young children with haemophilia A. A third prospective, non‐randomized trial evaluated a strategy of escalating the dose frequency of prophylaxis. Data from these studies will provide objective evidence for the prevention of haemarthroses and arthropathy in children with severe haemophilia A.     

Inhibitor incidence after intensive FVIII replacement for surgery in mild and moderate haemophilia A: a prospective national study in the Netherlands

Inhibitor development is currently the most severe complication in mild/moderate haemophilia A patients, causing increased bleeding tendency, hospitalization and mortality. It has been suggested that receiving high doses of factor VIII (FVIII) concentrates for surgical procedures is an important risk factor for inhibitor development in these patients. The current multicentre study aimed to determine prospectively the incidence of inhibitor development after intensive FVIII replacement therapy for surgical procedures in patients with mild/moderate haemophilia A. All consecutive patients with mild/moderate haemophilia A were included when they required at least 10 000 iu of FVIII concentrates (or 250 iu/kg) for 5 or more days for a surgical procedure. Potential clinical risk factors for inhibitor development and results of inhibitor tests were collected. Forty-six patients with a median age of 54 years (interquartile range, 40-59 years) were included in the study. F8 genotyping revealed 20 different missense mutations. Patients received either recombinant (65%) or plasma-derived FVIII concentrates (35%) by intermittent bolus injections (41%) or continuous infusion (57%). Two patients developed a low titre inhibitor post-operatively. The incidence of inhibitor development following intensive treatment for surgery in this unselected prospective cohort of mild/moderate haemophilia A patients was 4% (95% confidence interval, 0·5-14·8).     

Clinical advances in gene therapy updates on clinical trials of gene therapy in haemophilia

Gene therapy is rapidly becoming a new therapeutic strategy for haemophilia A and B treatment. In the 1990s, studies in animal models showed that adeno‐associated vectors (AAV) exhibited an efficient expression of factor IX (FIX). In the first clinical trial in patients with haemophilia B, therapeutic levels of FIX were documented but the expression remained only for few weeks. Subsequently, improvements in vector design, such as the use of different AAV serotypes, the development of the self‐complementary vector, the engineering of the transgene with codon optimization and liver‐specific expression cassette resulted in circulating FIX level between 2% and 5% for long‐lasting period. Recently, a natural gain of function FIX variant (Padua) inserted in the F9 cDNA improved the expression of FIX achieving a level of more than 30% resulting in cessation of infusions and in a greatly reduction of bleeding events. Encouraging clinical progresses have been also obtained from trials of gene therapy for haemophilia A. Transgene expression persisted for three years with circulating FVIII activity levels of 52.3% in patients treated with AAV vector containing a codon‐optimized F8 cDNA. A complication, reported in both clinical trials for haemophilia A and B, was the elevation of liver enzymes, which resolved with steroid treatment in a large group of patients. However, to date, the pathophysiological mechanism for the liver toxicity remains still unclear. Clinical trials with adeno‐associated vectors have documented a significant success for haemophilia gene therapy demonstrating potential to transform haemophilia treatment offering hope for a long‐term expression.     

Laboratory testing for factor VIII and IX inhibitors in haemophilia: A review

Inhibitors are antibodies directed against haemophilia treatment products which interfere with their function. Factor VIII (FVIII) inhibitors in haemophilia A and factor IX (FIX) inhibitors in haemophilia B are significant clinically when they require a change in a patient's treatment regimen. Their persistence may increase morbidity and mortality. Multiple laboratory tests are now available for detecting and understanding inhibitors in haemophilia. Inhibitors are traditionally measured by their interference in clotting or chromogenic factor assays. They may also be detected using immunologic assays, such as enzyme‐linked immunosorbent assay or fluorescence immunoassay. Anti‐FVIII or anti‐FIX antibodies of IgG₄ subclass best correlate with the presence of functional inhibitors. Improvements in inhibitor measurement have been recently introduced. Preanalytical heat treatment of patient specimens allows testing of patients without delaying treatment. Use of chromogenic and immunologic assays may aid in identification of false‐positive results, which are frequent among low‐titre inhibitors. Validated reagent substitutions can be used to reduce assay cost. New methods for defining assay positivity and reporting low‐titre inhibitors have been suggested. Challenges remain in the areas of quality control, assay standardization, monitoring of patients undergoing immune tolerance induction therapy and testing in the presence of modified and novel treatment products.     

Engineered factor IX variants bypass FVIII and correct hemophilia A phenotype in mice

The complex of the serine protease factor IX (FIX) and its cofactor, factor VIII (FVIII), is crucial for propagation of the intrinsic coagulation cascade. Absence of either factor leads to hemophilia, a disabling disorder marked by excessive hemorrhage after minor trauma. FVIII is the more commonly affected protein, either by X-chromosomal gene mutations or in autoimmune-mediated acquired hemophilia. Whereas substitution of FVIII is the mainstay of hemophilia A therapy, treatment of patients with inhibitory Abs remains challenging. In the present study, we report the development of FIX variants that can propagate the intrinsic coagulation cascade in the absence of FVIII. FIX variants were expressed in FVIII-knockout (FVIII-KO) mice using a nonviral gene-transfer system. Expression of the variants shortened clotting times, reduced blood loss after tail-clip assay, and reinstalled clot formation, as tested by in vivo imaging of laser-induced vessel injury. In addition, we confirmed the therapeutic efficacy of FIX variants in mice with inhibitory Abs against FVIII. Further, mice tolerant to wild-type human FIX did not develop immune responses against the protein variants. Our results therefore indicate the feasibility of using variants of FIX to bypass FVIII as a novel treatment approach in hemophilia with and without neutralizing FVIII Abs.     

PEGylated therapeutic proteins for haemophilia treatment: a review for haemophilia caregivers

PEGylation is the technology involving the covalent attachment of polyethylene glycol (PEG) to a protein‐, peptide‐ or small‐molecule drug to improve their pharmacokinetic, pharmacodynamic and immunological profiles, and thus, enhance the therapeutic effect. Today, PEGylation of proteins is a well‐established technology and is being used in the treatment of a variety of clinical disorders. Several PEGylated coagulation proteins for haemophilia A and B are under development with the goal of prolonging the circulation half‐life of factor VIII (FVIII) or factor IX. The prolongation of half‐life, resulting in less frequent injections can provide significant benefits in improving the quality of life of subjects with haemophilia and improvement in adherence to treatment. A review of published literature on PEGylated therapeutic products currently approved for human use and a discussion of a PEGylated recombinant FVIII molecule (BAY 94–9027, Bayer HealthCare, Berkeley, CA, USA) currently being investigated in the pivotal clinical trial prior to registration is provided. Available safety information of PEGylated proteins containing high molecular weight PEG does not indicate any safety concerns to date, following long‐term (chronic) use in animal models or patients. Chronic use of currently available PEGylated products has been shown to be safe, paving the way for chronic use of PEGylated coagulation products in persons with haemophilia.     

Clinical utility and impact of the use of the chromogenic vs one-stage factor activity assays in haemophilia A and B

Treatment of haemophilia A/B patients comprises factor VIII (FVIII) or factor IX (FIX) concentrate replacement therapy, respectively. FVIII and FIX activity levels can be measured in clinical laboratories using one-stage activated partial thromboplastin time (aPTT)-based clotting or two-stage chromogenic factor activity assays. We discuss strengths and limitations of these assays, providing examples of clinical scenarios to highlight some of the challenges associated with their current use for diagnostic and monitoring purposes. Substantial inter-laboratory variability has been reported for one-stage assays when measuring the activity of factor replacement products due to the wide range of currently available aPTT reagents, calibration standards, factor-deficient plasmas, assay conditions and instruments. Chromogenic activity assays may avoid some limitations associated with one-stage assays, but their regulatory status, perceived higher cost, and lack of laboratory expertise may influence their use. Haemophilia management guidelines recommend the differential application of one or both assays for initial diagnosis and disease severity characterisation, post-infusion monitoring and replacement factor potency labelling. Efficient communication between clinical and laboratory staff is crucial to ensure application of the most appropriate assay to each clinical situation, correct interpretation of assay results and, ultimately, accurate diagnosis and optimal and safe treatment of haemophilia A or B patients.