New developments in the treatment of pediatric hemophilia and bleeding disorders

Branchford, Brian R.a,b; Monahan, Paul E.c; Di Paola, Jorgea,b

doi: 10.1097/MOP.0b013e32835bf77e
HEMATOLOGY AND ONCOLOGY: Edited by Stephen Hunger

Purpose of review: Disorders of hemostasis such as hemophilia, von Willebrand disease (VWD), and other clotting protein deficiencies lead to significant morbidity in the pediatric population. Because of the limitations of current treatment options, novel therapies are being developed, many of which are reviewed here.

Recent findings: Several new observations about the nature of clotting protein physiology have been made recently, creating novel perspectives on the treatment options. This review will mostly focus on the current therapy as well as new progress in hemophilia care (particularly strategies to prolong half-life of clotting factor replacements, the management of inhibitors, gene therapy, and novel therapeutic approaches), and briefly mention some progress in VWD and fibrinogen deficiency therapies.

Summary: New therapeutic developments have the potential to dramatically decrease morbidity and improve the quality of life of children with bleeding disorders.

aDepartment of Pediatrics, Section of Hematology/Oncology/Bone Marrow Transplantation, Children's Hospital Colorado, University of Colorado – Anschutz Medical Campus

bHemophilia and Thrombosis Center, University of Colorado, Aurora, Colorado

cComprehensive Hemophilia Diagnostic and Treatment Center, University of North Carolina, Chapel Hill, North Carolina, USA

Correspondence to Jorge Di Paola, MD, Department of Pediatrics, Section of Hematology/Oncology/BMT, Children's Hospital Colorado, University of Colorado, 13123 East 16th Avenue, B115, Aurora, CO 80045, USA. Tel: +1 303 724 4000; e-mail:

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Previous advances in disorders of hemostasis (particularly plasma-based and recombinant clotting factor replacement therapy) have significantly reduced disease-related mortality and, to a lesser degree, morbidity in children. In order to reduce morbidity, even further novel therapeutic approaches are needed. This article reviews the current therapeutic approaches and recent advances (including drugs in development) for hemophilia, von Willebrand disease, and deficiencies of fibrinogen and factor XIII.

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Hemophilia A (80% of cases, 1 in 5000 live male births) and hemophilia B (20% of cases, 1 in 30 000 live male births) are disorders of secondary hemostasis related to the deficiency of clotting factors VIII and IX, respectively, and are inherited, except for rare exceptions, in an X-linked manner [1▪,2▪]. Rarely, hemophilia can be acquired by way of antibodies against the specific clotting factors. In children under 16 years of age, the incidence of acquired hemophilia A is approximately 0.045 per million per year [3▪▪]. Only a few cases of acquired hemophilia B have been reported. Disease severity is determined by the degree of factor activity: less than 1%: severe (50–70% of cases of hemophilia A and 30% of hemophilia B), 1–5%: moderate, and 6–40%: mild [1▪].

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Current therapy

Current therapy focuses on replacing deficient clotting factors to prevent or treat bleeding with clotting factor concentrates (CFCs), as seen in Table 1. Although efficient, this approach is complicated by transfusion-related infections, inhibitor development, and occasionally thrombotic events [2▪].

Plasma-derived CFCs have been available for decades, but viral safety concerns led to subsequent development of recombinant factors. Episodic or ‘on-demand’ CFC infusions comprise the conventional therapy for patients with mild or moderate disease [1▪]. More recently, however, prophylactic administration of CFC to prevent bleeding in patients with severe hemophilia (25–40 IU/kg of FVIII three times weekly or 50–100 IU/kg of FIX twice weekly) has been recommended as the standard of care by the Medical and Scientific Advisory Council (MASAC) of the National Hemophilia Foundation, the World Federation of Hemophilia, and the World Health Organization [1▪,2▪,4]. In fact, primary prophylaxis, started before age 2 or after the first joint bleed, is the evidence-based, gold standard for preserving joint health in children with severe hemophilia [5]. The frequent infusions required for effective prophylaxis are difficult in very young children, however, often requiring central venous catheters. Additionally, the high cost of CFC makes prophylaxis mostly available to hemophilia patients in industrialized countries. Adjuvant therapies such as desmopressin (a synthetic vasopressin analog) and topical agents such as fibrin sealants and others are detailed in the excellent recent review by Wong and Recht [2▪].

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Clotting factor concentrate improvement strategies

Alternative treatment strategies are being developed to extend the half-life of exogenous factor proteins, reduce the cost of factor concentrates, reduce immunogenicity of the products, and even produce alternative hemostatic agents that are resistant to inhibitors. A summary of these strategies can be found in Table 2.

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Half-life extension

Strategies to prolong exogenous factor half-life include sustained delivery mechanisms that slowly release protein into circulation (increasing bioavailability and stability), chemical modification to decrease clearance such as conjugating the protein with sialic acid residues or a hydrophilic polymer such as polyethylene glycol (PEG) or PEGylated liposomes to encapsulate the drug, formation of inactivation-resistant fusion proteins, and manufactured genetic mutations that decrease exogenous factor metabolism by preventing spontaneous dissociation, or activated protein C (APC)-mediated proteolysis of the A2 subunit of factor VIII [1▪,2▪,4,6]. The prolonged half-life exhibited by these products has the potential to decrease dosing frequency in prophylaxis regimens (which could increase compliance and quality of life) or even improve on-demand therapy options by potentially allowing bleeding treatment with a single dose.

One such strategy to prolong half-life is the PEGylation of the therapeutic clotting factor. A recombinant FVIII (rFVIII) product formulated with sucrose and bound noncovalently to PEGylated liposomes to extend hemostatic efficacy has had variable success with apparent increased efficacy, but surprisingly no difference in pharmacokinetic parameters from non-PEGylated rFVIII. No safety concerns or reports of inhibitor formation were reported in this study [7]. A glycoPEGylated FIX peptide (N9-GP) exhibits similar enzyme kinetics for factor X activation and thromboelastography parameters similar to recombinant factor IX (rFIX), with five-fold to nine-fold increase in half-life, with an excellent safety profile without inhibitor development [4,6]. Additionally, the incremental recovery (increase in plasma concentration per injected dose) of PEG-conjugated FIX was 94% higher than rFIX, suggesting that the PEG group may shield the FIX molecule from undesired interactions with the cell surface [6]. The PEGylated compounds have shown a clear advantage for half-life prolongation, but caution is urged, because of potential for hypersensitivity reactions or clearance-inducing anti-PEG antibody formation [4].

Another promising technique is the use of recombinant DNA technology to use cleaveable or noncleaveable linkers to fuse factor VIII with a carrier protein, such as albumin or IgG, with a longer half-life that can protect factor VIII from degradation [2▪,6]. A novel recombinant protein (rFIX-FP) designed using albumin fusion technology has extended half-life (up to five-fold), allowing less frequent dosing. Also, a recombinant single-chain version of factor VIII (CSL267) has improved stability and enhanced affinity for von Willebrand factor (VWF) [4,8,9]. Also, rVIII and rIX have been fused to the constant region of IgG (rFVIIIFc and rFIXFc), creating molecules that can be internalized by endothelial cells and recycled back to the cell surface, avoiding lysosomal catabolism. In the case of rFVIIIFc, it can be released back into plasma, associate with VWF and exert cofactor activity similar to native FVIII. In the case of rFIXFc, when expressed in human HEK-293H cells, it has acceptable specific procoagulant activity. Both molecules have demonstrated half-life prolongation in murine and canine hemophilia models (two-fold for rFVIIIFc and four-fold for rFIXFc), and in humans with a 1.5–1.7-fold increased half-life [4,6].

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Other strategies

The high price of manufacturing recombinant factor protein concentrate can become prohibitive. One promising cost-reduction strategy includes the production of FIX proteins in milk of transgenic sheep or pigs, as estimates based on known milk output suggest that 800 pigs could produce enough FIX to cover the world's yearly consumption [1▪,6].

Development of B-domain-deleted (BDD) factor replacement products (such as porcine/human BDD hybrid FVIII molecules) is being encouraged, as they may reduce immunogenicity and increase FVIII production, while still maintaining full procoagulant function and exhibiting higher expression levels than wildtype FVIII [1▪,10].

Engineered variants of FIX capable of direct activation of factor X may serve as therapy for hemophilia A, are insensitive to inhibitor antibodies toward factor VIII and have been shown to correct the hemophilia A phenotype in mice [6].

A unique novel approach for both hemophilia A and B is the development of interventions to promote ribosome read-through of premature termination codons (PTCs) to translate and express full-length functional proteins, thereby bypassing the nonsense mutations that comprise approximately 10–12% of factor VIII mutations and an unknown number of factor IX mutations [2▪]. Suppression therapy using aminoglycosides to promote read-through in vitro has been known since the 1960s, and new pharmacological agents with nonsense suppression activities are being developed and evaluated [11]. A nonaminoglycoside oral drug, Ataluren (PTC124), is being investigated in early phase clinical trials for its ability to promote dose-dependent read-through of nonsense codons and may decrease the risk of inhibitor formation if the PTC suppression can be initiated prior to induction of immune tolerance [2▪,11].

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Inhibitors: standard of care

The most serious complication of CFC infusion is the development of inhibitors – IgG antibodies that neutralize the activity of the clotting factors or, in the case of hemophilia B, trigger severe allergic reactions to infused factor (up to 50% of hemophilia B patients) [1▪,2▪]. Interestingly, it appears that patients exposed to plasma-derived CFCs had lower incidence of FVIII inhibitors (10.3%) compared with those treated with recombinant CFCs (28.7%) [12▪], a finding that is being explored in a prospective randomized trial. Bleeding in patients with low-titer inhibitor [<5 Bethesda Units (BU), 25–40% of all inhibitors] may resolve with increased quantities of replacement factor, while high-titer inhibitors (≥5 BU, 60–75% of all inhibitors) may prevent response to CFCs, requiring a two-pronged approach of short-term control of acute bleeding episodes with bypassing agents and inhibitor titer reduction over the long term by immune tolerance induction (ITI) [1▪,2▪]. This strategy attempts to eliminate inhibitors via administration of repetitive CFC doses with or without immunosuppressive therapy [2▪]. Proposed ITI mechanisms include clonal deletion of antibody-producing B cells, induction of suppressor T cells, and synthesis of anti-idiotype antibodies. This approach is effective in up to 70% of patients with hemophilia A, but much less so in hemophilia B [1▪]. Corticosteroids, cyclophosphamide, azathioprine, mercaptopurine, vincristine, cyclosporine, tacrolimus, mycophenolate mofetil, and rituximab have all been used in an attempt to eradicate inhibitors, though no randomized controlled trial has identified which regimen best suppresses inhibitor formation [2▪].

When bleeding in patients with inhibitors occurs in spite of ITI, a bypassing approach for short-term control is used, either with activated prothrombin complexes (aPCCs) or with recombinant activated factor VII (rVIIa), also known as NovoSeven [eptacog alfa (activated)]. A summary of these products can be found in Table 1. The only licensed aPCC in the USA is the FVIII inhibitor bypass activity (FEIBA). FEIBA exhibits high levels of clotting factors and is typically 85% effective for the management of acute bleeding events if administered at doses of 50–75 U/kg every 8–12 h with a recommended maximum daily dose of 200 U/kg [2▪]. Concern has been raised about the thrombotic complications arising from the use of FEIBA, but in a 10-year compilation of thrombotic events only 16 thrombotic adverse events occurred during the 10-year period, and most were suspected to be related to excessive dosing [13].

NovoSeven is a licensed bypass agent for bleeding management in hemophilia A or B with inhibitors (equally effective at doses of 90–120 μg/kg every 2 h or 270 μg/kg every 6 h) and likely acts by producing a thrombin burst on activated platelet surfaces by proteolytic activation of factors IX and X (and ultimately prothrombin) in the absence of tissue factor, and demonstrates higher affinity for activated platelets than wildtype FVIIa [2▪,7]. The cost of bypass therapy in patients with inhibitors is very high. NovoSeven is known by many providers for its expense. The cost of treatment for a standard bleeding episode in a 50-kg child with hemophilia and inhibitor who requires bypass treatment is approximately $23 986 (adapted from figures in a recent systematic literature review of economic analysis of the bypass agents) [14]. However, a similar course of therapy when using aPCC as initial treatment followed by NovoSeven as second-line or third-line treatment would cost approximately $26 416 or $27 500, respectively [14].

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Inhibitor bypass strategies

The short half-life of rFVIIa requires frequent dosing, so glycoPEGylated products (N7-GP) and fusion proteins (rVIIa-FP) are being developed for FVII, with prolonged half-life but somewhat lower plasma activity [4,15]. Vatreptacog alfa, a recombinant FVIIa analog with enhanced tissue factor-independent capability, is in phase 3 clinical trial and it was well tolerated in a recent randomized controlled trial with a high efficacy rate (98% of bleeds were controlled within 9 h) [16].

Another FVIIa variant with enhanced procoagulant activity and prolonged half-life was designed with γ-carboxyglutamic acid-containing domain mutations, on the basis that FVIIa's hemostatic activity is enhanced when there is increased membrane affinity, theoretically resulting in a larger number of molecules localized to the activated platelet surface. This product is currently in early stage clinical trials [17].

An alternative approach is the direct infusion of factor Xa (FXa) to bypass the intrinsic pathway and promote thrombin generation, but this protein is rapidly inactivated in plasma by tissue factor pathway inhibitor (TFPI) and antithrombin [4]. A novel engineered factor X variant, FXI16L, constitutes a functional protease that resists inactivation but retains prothrombinase activity when assembled with factors Va and prothrombin on phospholipid surfaces, and has demonstrated efficacy and long half-life in murine models [4,18▪▪]. Other novel FXa-generating molecules under development include inhibitors of TFPI [monoclonal antibodies, natural (fucoidan) or synthetic (RNA aptamers, synthetic peptides, and nonanticoagulant sulfated polysaccharides) inhibitors] and APC (synthetic aptamer inhibitors or RNA interference silencing) [1▪,4,17].

Finally, other synthetic compounds are being developed that can bind directly to FVIII inhibitors, such as the epitopes C6 and H10, mimitopes that combine into heteromultimers, resulting in bispecific molecules that block polyclonal antibody binding to FVIII [1▪]. In order to improve tolerization in hemophilia B, inactive variants of FIX are being used, such as the fusion protein of FIX and transmucosal carrier cholera toxin β-subunit expressed in tobacco chloroplasts and administered orally as frozen leaf powder to hemophilia B mice [6].

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Gene therapy

In light of its curative potential, gene therapy is an area of expanding interest and there is evidence of early clinical translational success. The goal is to deliver a functioning gene (FVIII or FIX) to the nucleus of a target cell, enabling the host to persistently produce sufficient clotting factor to prevent bleeding. Hemophilia is an ideal disease target for gene therapy, as described earlier in this article; moderate hemophiliacs (1–5% of normal factor levels) bleed much less than those severe patients with less than 1% of normal factor levels. Therefore, although ideal, a complete correction to normal clotting factor levels is not needed and gene therapy can be successful if a severe hemophiliac is ‘converted’ into a moderate or mild patient by elevating factor levels modestly.

The delivery of naked DNA is extremely inefficient. Therapeutic nucleic acid sequences, however, may be supplied via vector-mediated gene therapy, in which the gene is chemically complexed (e.g. with lipids that force interaction with host cells) or incorporated into a virus that acts as a delivery vehicle for cell entry and trafficking to the nucleus. Alternatively, the gene may be introduced ex vivo into cells, which are then inoculated into the host (cell-mediated gene therapy). Although vector strategies that result in the integration of the therapeutic FVIII or FIX transgene into the genome of host cells (e.g. retroviral or conventional lentiviral vectors) have the potential for persistent phenotypic correction, insertional mutagenesis and development of malignancy are valid concerns using integrating vectors. Insertional mutagenesis refers to the insertion-related activation of proto-oncogenes by integrated proviral vectors leading to clonal expansion and eventual development of leukemia, as previously occurred following otherwise successful gene therapy treatment in some cases of severe combined immunodeficiency, adrenoleukodystrophy, thalassemia, chronic granulomatous disease, and Wiskott–Aldrich Syndrome [19]. For this reason, approaches that result in FVIII or FIX gene transcription from extrachromosomal ‘episomal’ sequences delivered to long-lived postmitotic cells, such as skeletal muscle cells or hepatocytes [20], have received considerable attention. These include the use of the adeno-associated virus (AAV), high-capacity adenoviral vectors (HCAVs), and lentiviruses engineered to prevent integration [6,21]. Nevertheless, many viral vectors are engineered based upon wildtype viruses that are prevalent in the environment (e.g. adenovirus, AAV), and individuals may have preexisting humoral immunity to the viral structural proteins that limits initial gene transfer or triggers cell-mediated immune response to gene-corrected cells [22▪]. Humoral immune responses to the viral vector will in many cases also limit the potential for repeated administration [1▪].

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Gene therapy: human clinical application

Both AAV-based and lentiviral vectors have directed stable correction of bleeding phenotypes in hemophilia A and B in preclinical murine, canine, or nonhuman primate models. At the current time, the AAV approach is generally preferred for FIX gene delivery because of their nonpathogenic, replication-deficient nature and low probability of chromosomal integration [6]. At least five phase 1 clinical trials were completed, with more underway, using either direct in-vivo gene delivery of retroviral FVIII or AAV FIX vectors or ex-vivo genetic engineering and re-implantation of autologous cells [1▪]. In only one trial, phenotypic correction with persistent expression of greater than 1% factor activity has been demonstrated. The trial used an AAV vector engineered for factor IX expression specific to the liver, resulting in factor IX expression in each of six individuals treated [23▪▪]. As had been observed in a 2006 liver-directed AAV trial by Manno et al., a dose-dependent cytotoxic lymphocyte (CTL) response against the AAV-transduced hepatocytes was observed at the highest vector dosages used. Large and small animal data suggest that the incorporation of a gain-of-function factor IX variant (Factor IX Padua) [24] may increase the efficiency of the vector adequately so that the use of high doses of AAV vector, associated with CTL response, may be avoided [25,26]; this hypothesis is being tested in an ongoing human clinical trial.

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Gene therapy: prospects for pediatric applications

Current regulations do not permit gene therapy safety trials, including trials for hemophilia, to enroll children unless the targeted condition causes death or severe degeneration if not treated in childhood. Despite this, there are several reasons to believe that gene therapy for hemophilia may have particular advantages if delivered in childhood rather than adulthood, provided that safety data in adults can be established. The most obvious reason is that boosting endogenous factor expression from early childhood may provide all of the benefits of early continuous prophylaxis, including joint preservation and avoidance of intracranial and other life-threatening bleeding, while avoiding potential complications of repetitive protein replacement. From a practical standpoint, the production of gene vectors or cell-based therapies, highly complex biological therapeutics, at high titer is often a limiting factor, so that the small size of children is in this case advantageous. The concern of potential hepatotoxicity following liver-directed approaches excludes the majority of adults with active hepatitis from the ongoing hemophilia B gene therapy trials. The pediatric hemophilia population does not have baseline hepatic scarring or inflammation to be concerned about, although the tremendous growth of liver mass in early childhood means that FVIII or FIX transgenes that persist as episomes likely will be lost as hepatocytes divide throughout childhood. In addition, delivery at a young age, prior to natural environmental exposure to wildtype viruses upon which viral vectors are based, may avoid the effect of naturally occurring neutralizing antibodies that may abrogate gene delivery [27]. Both small and large animal models demonstrate that genes delivered in fetal or early neonatal life may have a greater potential to induce tolerance to the transgene [28]. Recent efforts have focused on using gene therapy to prevent or reverse inhibitor formation, following endogenous expression from the liver or using B-cell presentation or lymphocyte depletion techniques for tolerance induction [29–31]. As genetic and environmental factors associated with inhibitor risk are defined, it is conceivable that prophylactic tolerance induction approaches using gene therapy may be developed for particularly at-risk children.

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This section will briefly cover new treatments for other pediatric bleeding disorders.

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Von Willebrand disease

Desmopressin (1-desamino-8-D-arginine vasopressin, DDAVP), a synthetic vasopressin analog, increases endogenous VWF by secretion from its natural site of synthesis and storage, the vascular endothelial cell, following intravenous, subcutaneous, or intranasal administration (which typically increase plasma VWF : FVIII levels two-fold to four-fold above the baseline within 30 min, and in general high levels last in the plasma for 6–8 h) [32]. It is effective in von Willebrand disease (VWD) type 1, not in VWD type 3, and of variable efficacy in VWD type 2, with particular concern regarding transient thrombocytopenia in type 2B VWD (gain-of-function mutation). Limitations of this therapy include tachyphylaxis, hyponatremic seizures, or arterial thrombosis in elderly patients with atherosclerotic disease.

When desmopressin is not effective, VWF-containing factor concentrates (some with variable amounts of FVIII) are used. These products have been used for several years in children and adults without major complications. There is current emphasis on secondary long-term prophylaxis with VWF–CFCs for severe VWD patients with multiple hemarthroses, recurrent gastrointestinal bleeding, or frequent severe epistaxis [33]. Recombinant VWF has recently been entered into clinical trials and has been shown to have a higher level of high molecular weight VWF multimers than normal plasma-derived VWF [33]. However, the impact of this finding on the efficacy of the product is not clear.

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Fibrinogen deficiency

Congenital afibrinogenemia occurs in approximately 1 in 1 000 000 individuals [34], but hypofibrinogenemia can also be acquired in a variety of medical conditions (consumptive coagulopathy, hepatic failure, etc.). Until recently, the only reliable source of fibrinogen for patients with genetic or acquired (e.g. consumption in disseminated intravascular coagulation) deficiency was the plasma product cryoprecipitate. Infusions of cryoprecipitate have been complicated by volume overload as well as viral transmission, transfusion reactions, sensitization, etc. A recent, prospective, open-label, international pharmacokinetic trial of pasteurized human fibrinogen concentrate in patients with afibrinogenemia showed effective restoration of clot formation based on thromboelastography parameters, and the concentrate was well tolerated and no treatment-related adverse events were noted [35]. Multiple in-vitro experiments, animal studies, and proof-of-principle randomized, clinical studies have recently shown that hemostatic intervention with a fibrinogen concentrate may be efficient and safe in controlling perioperative bleeding [36]. Two recent trials demonstrated that patients who receive fibrinogen concentrate during major cardiothoracic surgery required significantly less allogeneic blood product support than controls [37].

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Notable advances have been made in the development of improved and novel therapeutics for hemophilia and other bleeding disorders. Indeed, it could be argued that the last decade has been characterized by an extremely productive partnership between academia and industry that has brought several investigational products to clinical trials. As a result, it is likely that we will continue to see significant improvements in the lives of children with bleeding disorders in industrialized countries. Still, the challenge remains on how to extend these discoveries to all other parts of the world.

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Conflicts of interest

There are no conflicts of interest.

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Papers of particular interest, published within the annual period of review, have been highlighted as:

▪ of special interest

▪▪ of outstanding interest

Additional references related to this topic can also be found in the Current World Literature section in this issue (pp. 146–147).

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A full-spectrum evaluation of new management strategies for hemophilia that offers a good overall review of the recent advances.

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A thorough review of new topical treatments.

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An excellent review of long-acting products.

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A good review of the different fVIII replacement options currently available and the particular strengths of each one.

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A superb research article from the Camire laboratory that demonstrates the ability of molecularly modifying coagulation factor X, conferring longer half-life and, more importantly, making it effective as a hemostatic agent in hemophilia mice.

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This study provides good background on a common form of gene transfer therapy and the potential uses for this approach in hemophilia.

23▪▪. Nathwani AC, Tuddenham EG, Rangarajan S, et al. Adenovirus-associated virus vector-mediated gene transfer in hemophilia B. N Engl J Med 2011; 365:2357–2365.

This article represents the first successful gene therapy trial for hemophilia with long-lasting, sustained coagulation factor levels and clinical efficacy.

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bioengineering; gene therapy; hemophilia; recombinant proteins; von Willebrand disease

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