Haemophilia A and B are X-linked bleeding disorders caused by an inherited deficiency of factor VIII or factor IX, respectively. Of the approximately 1 per 5000–10 000 male births affected by haemophilia, 80% are deficient in factor VIII and 20% are deficient in factor IX. The National Foundation of Haemophilia estimates the worldwide incidence to be near 400 000 people, most of whom do not have ready access to medical care. The clinical phenotype mainly correlates with the level of endogenous factor, with the most severe phenotype presenting with factor levels below 1% of normal. As a disorder of secondary haemostasis, haemophilia is characterized by spontaneous and provoked joint, muscle, gastrointestinal and CNS bleeding, leading to major morbidity and even mortality if left untreated or under-treated. This article aims to review the current treatment options for patients with congenital haemophilia A and B, including clotting factor repletion, inhibitor reduction and adjuvant treatment. Because the treatment of haemophilia is advancing rapidly, the future direction of haemophilia treatment is reviewed.
1. Timeline of Early Haemophilia Treatment
Prior to the 1940s, treatment of haemophilia was limited to supportive care and transfusion of whole blood or fresh plasma. Neither product contains adequate levels of factor VIII or factor IX and, therefore, affected individuals still suffered from major bleeding and long-term morbidity. During this time, the average lifespan of a male with haemophilia was 27 years. With the outbreak of World War II, transfusion medicine improved and by 1960 the average life expectancy had increased to 39.7 years. In 1964, Dr Judith Graham Pool described cryoprecipitate, rich in factor VIII and fibrinogen, further expanding treatment options. The 1970s saw radical improvement in haemophilia care with the development and availability of plasma-derived clotting factors.[4,5] These commercial freeze-dried concentrations of factor VIII and factor IX, derived from pools of up to 20 000 donors, made home therapy possible. For the first time, people with haemophilia were able to travel, work and attend school with regularity. Haemophilia treatment centres saw to the holistic care of patients and by 1980 the average lifespan of those with haemophilia was 60 years. Hepatitis B and C were known to be in the factor supply but were thought to be an acceptable risk in light of such a drastic improvement in quality of life.[6-8] However, in 1982, the first report of patients with haemophilia succumbing to AIDS was received and only retrospectively was it determined that plasma-derived factor concentrate was the vector of this still unidentified infectious moiety.[9-11] HIV was isolated in early 1984 and by early 1985 heating of factor was standard practice to kill the virus.
2. Factor Repletion
2.1 Factor VIII Products
As mentioned in section 1, satisfactory management of haemophilia only became possible with the development of plasma-derived clotting factor concentrates in the late 1960s. The subsequent evolution of coagulation-factor replacement therapy focused on maximizing viral safety through the widespread implementation of donor selection and screening, chromatographic purification and viral inactivation. Even today, multiple plasma-derived factor VIII products are used due to their efficacy, safety and lower cost
The cloning and sequencing of factor VIII in 1984 led to the development of recombinant factor VIII (rFVIII).[12,13] Human rFVIII can only be produced using mammalian cell culture systems due to the complex glycosylation and other post-translational modifications required for its full cofactor activity. Scale-up of production and purification processes led to the commercial production of full-sequence-length rFVIII products. The first clinical trial establishing efficacy of an rFVIII product was published in 1990 and the first rFVIII was marketed for clinical use in 1992. These first-generation recombinant factor products required bovine or human serum to stabilize the final product. Though simultaneous advances in plasma screening and viral inactivation technology made serum safer, strides were made to remove all plasma from the manufacturing process to ensure the safety of the infused product. As a result, plasma is required during the manufacturing process of second-generation products but is removed in the final product. Third-generation recombinant products are serum-free during the manufacturing process and final product, substituting synthesized or genetically engineered stabilizing molecules. The purification process removes impurities derived from the medium and cultured cells, and concentrates the rFVIII molecule through various chromatographic steps. As a general rule, 1 unit of factor VIII per kilogram bodyweight will raise the factor activity in the recipient's plasma by 1.5–2.0 IU/dL (1.5–2.0%). First-, second- and third-generation rFVIII products available in the US are summarized in table II.
Although viral transmission has never been recorded with any rFVIII product, a theoretical risk of transmitting a human-derived infectious agent still remains in the first-generation products. In addition, emerging non-viral pathogens such as the prion responsible for variant Creutzfeldt-Jakob (vCJD) must be considered, and reducing the risks of pathogen transmission continues to be a high priority for the haemophilia community.[16,17] rFVIII products have excellent haemostatic efficacy in both previously untreated and treated haemophilia A patients. Since the manufacture of rFVIII is not limited by plasma availability, the improved supply of factor VIII has contributed to increased application of prophylactic administration and subsequent improvement of functional outcomes.
2.2 Factor IX Products
Prior to the introduction of recombinant factor IX (rFIX) concentrate, fresh frozen plasma or plasma derivatives (prothrombin complex concentrates [PCCs]) were used as the source of factor IX for patients with haemophilia B. PCCs (table III) are enriched in prothrombin and factors VII, IX and X, and also contain trace amounts of factors VIII, VIIa and IXa. The anticoagulant vitamin K-dependent factors protein C and protein S are also present at variable concentrations.
PCCs have been associated with thrombotic events, including venous thromboembolism and disseminated intravascular coagulation (DIC), as well as microvascular thrombosis and myocardial infarction.[20-23] The use of PCCs in haemophilia B fell after the introduction of high-purity plasma-derived factor IX (pdFIX) and subsequently rFIX (nonacog alfa) [table III] in the 1990s. As opposed to PCCs, infusion of high-purity factor IX products did not lead to significant activation of the coagulation system, confirming that a component other than factor IX is responsible for the thrombogenicity of PCCs in haemophilia B.
The human factor IX gene was cloned in 1982, which led to the expression of human rFIX in Chinese hamster ovary cells.[25,26] rFIX is structurally and functionally similar to pdFIX, although minor differences in the post-translational sulfation and phosphorylation of rFIX have been associated with approximately 30% lower in vivo recovery, especially in children <15 years of age. International clinical trials have demonstrated the efficacy and safety of rFIX for the treatment of haemorrhages as well as in the prophylactic and surgical settings in previously treated and untreated patients with haemophilia B. Generally, 1 unit of factor IX per kilogram bodyweight will raise the factor activity in the recipient's plasma by 0.7–1.4 IU/dL (0.7–1.4%).
2.3 Replacement Strategies
The treatment goal in haemophilia is to replace the missing coagulation factor from exogenous sources. Episodic, or ‘on demand’, replacement is the conventional treatment approach in which the missing factor is replaced as soon as possible after the onset of bleeding symptoms. More recently, prophylactic administration of clotting factor concentrate, administered to prevent bleeding, has been recommended as the standard of care by the Medical and Scientific Advisory Council of the National Hemophilia Foundation, the World Federation of Hemophilia and the WHO.
In primary prophylaxis, the concentrate is given from an early age to prevent expected complications, whereas secondary prophylaxis is begun after such events occur to prevent recurrence. The rationale for and benefits of primary prophylaxis delivered three times a week in haemophilia A have been validated in a prospective randomized controlled trial in the US. A dose-escalated prophylaxis regimen for haemophilia (increasing the frequency from once weekly until breakthrough bleeding episodes are controlled) has been investigated. This once-a-week regimen resulted in fewer bleeding episodes and the development of fewer target joints than historical controls.
There is growing evidence that prophylactic dosing of factor should start before joint damage to promote joint integrity. Several studies show that children with haemophilia exhibited better musculoskeletal outcomes when started on prophylaxis early (mean age of 3 years).[31-34] In one study, the only significant predictor for development of haemophilia arthropathy was the age of patients when prophylaxis was started. Using the Pettersson score, a scoring system that increases based on radiological evidence of haemophilic joint disease, Fischer and Van Den Berg described an 8% score increase each year in which prophylaxis was postponed after the first occurrence of haemarthrosis. These studies showed that irreversible joint damage may follow after a few joint bleeding episodes and that even early prophylaxis may not abrogate the process completely. However, establishing venous access in young children is commonly a challenge. Not surprisingly, central venous catheters are often required in this population. The benefit of easy access provided by such catheters must be balanced by their risks, in particular catheter-related infections.
Patients who have pre-existing joint disease and who experience frequent acute haemarthroses may be treated with periodic use of factor concentrates for a short or long period of time to curtail bleeding recurrence. This approach is known as secondary prophylaxis and is commonly used to minimize bleeding frequency and lessen the progression of joint disease. Even though secondary prophylaxis cannot reverse the changes of chronic arthropathy, it may be beneficial by reducing frequency of bleeding, hospital admissions and lost days from school or work, and by decreasing damage progression. The use of secondary prophylaxis versus on-demand therapy has been the subject of various studies in children and adults who have severe haemophilia.[35-37] In summary, the results indicate that the patients treated with secondary prophylaxis had a decreased number of joint bleeding episodes at the expense of higher clotting factor concentrate consumption.
3. Treatment of Patients with Inhibitors
The most serious treatment-related complication in haemophilia today is the formation of an antibody against factor VIII or factor IX. In both haemophilia A and B, these antibodies neutralize the activity of the clotting factors. In addition, factor IX inhibitors can be associated with anaphylaxis or severe allergic reactions to infused factor IX. The incidence of this adverse immune reaction is 15–50% in haemophilia A and 1–3% in severely affected patients with haemophilia B. These inhibitors are IgG antibodies, predominantly subtype 4. Inhibitors mostly appear following a median of 8–12 exposure days. The strongest determinant of the risk of inhibitor development is the type of mutation in the factor VIII or factor IX gene.[39,40] Blacks and Latinos form inhibitors more frequently.[41,42] Other patient risk factors may be associated with human leukocyte antigen types and polymorphisms in genes that code for cytokines. Multiple studies have suggested that rFVIII has an increased risk of inhibitor development.[43,44] Following a comprehensive analysis of the literature, the European Haemophilia Therapy Standardisation Board was unable to conclude that immunogenicity varies with the type of product and that, currently, treating physicians should not choose one factor product over another based on concerns about inhibitor risk alone.
Most centres consider ≥0.6 Bethesda units (BU) positive for having an inhibitor. If the inhibitor titre is low (generally, <5 BU), bleeding can typically resolve with normal to increased quantities of exogenous factor. However, for high titres (≥5 BU), successful management depends on two components: control of acute bleeding episodes and the reduction of the inhibitor titre.
3.1 Inhibitors and Acute Bleeding
As transmission of blood-borne pathogens has decreased, the development of inhibitory antibodies to transfused clotting factor has become the most serious treatment complication. The need for therapies to control bleeding in haemophilia patients affected by high-titre inhibitors to factor VIII or factor IX, i.e. to ‘bypass’ the factor VIII/IX complex in coagulation, led to a number of early clinical trials exploring the efficacy and safety of PCCs.[47-49] In the 1970s, the first activated PCCs were developed. These products were manipulated ex vivo to increase the content of activated clotting factors, especially factor VIIa. At present, only one product, FEIBA (factor VIII inhibitor bypass activity) anti-inhibitor coagulant complex, is available for this indication. The vial potency labelling is in arbitrary units of factor VIII inhibitor bypassing units, where 1 unit of FEIBA shortens the activated partial thromboplastin time of high-titre factor VIII reference plasma to 50% of the blank value. The drug's mechanism of action is now believed to be dependent on its content of prothrombin and FXa. Empirically, FEIBA is administered at doses of 50–75 U/kg every 8–12 hours with a recommended maximum daily dose of 200 U/kg. Three prospective randomized clinical trials on the early treatment of acute haemarthrosis established the efficacy and safety of FEIBA.[47,48,51] The response rate, judged subjectively by joint pain resolution, was only 50–60% at 6 hours after the first infusion (with significantly higher rates of response for the drug compared with a non-activated PCC) compared with a placebo response rate of 25%. These response rates at 6 hours were significantly lower than would be expected when using factor VIII to treat acute haemarthrosis in haemophilia A uncomplicated by an inhibitor. With repeated dosing over longer periods, however, the efficacy rate for FEIBA in the management of acute bleeding events was substantially higher, generally more than 85%.[52,53]
Recombinant factor VIIa (rFVIIa; eptacog alfa [activated]) is almost structurally identical to native factor VIIa. This agent is widely licensed for the management of bleeding in haemophilia A or B complicated by inhibitory antibodies (at doses of 90–120 μg/kg), for inherited factor VII deficiency (at a dose of 15–30 μg/kg), and in Europe for bleeding in Glanzmann thrombasthenia with refractoriness to platelet transfusions. In haemophilia, high-dose eptacog alfa is speculated to act by producing a ‘thrombin burst’ on the surface of activated platelets by proteolytic activation of factors IX and X (and ultimately prothrombin) in the absence of tissue factor. Although some data have suggested increased efficacy with even higher doses of eptacog alfa (usually 270 μg/kg) in haemophilia-related bleeding, supportive data from prospective randomized clinical trials have thus far only shown equivalence. Like FEIBA, the haemostatic efficacy rate for eptacog alfa varies depending on when it is assessed after administration; indeed, a recent multinational randomized crossover clinical trial demonstrated equivalence of an 85 U/kg dose of FEIBA and two 105 μg/kg doses of eptacog alfa. Response to both was judged to be effective in about 80% of cases at 6 hours. Regardless of the indication, administration of eptacog alfa invariably results in shortening of the prothrombin time, although this does not correlate with haemostatic efficacy. As with FEIBA, a validated method for monitoring eptacog alfa is an area of active investigation.
3.2 Inhibitor Reduction
3.2.1 Immune Tolerance Therapy
Long-term management of patients with haemophilia with factor VIII inhibitors is aimed at eliminating the inhibitors. The primary method attempted is immune tolerance induction via the administration of repetitive doses of factor VIII with or without immunosuppressive therapy. Many responders have an initial rise in the antibody titre caused by the anamnestic response, followed by a progressive reduction to a low or undetectable titre. Immune tolerance usually must be maintained by continued exposure to infused clotting factor.
How desensitization works is not completely understood. In one study, desensitization did not change the concentration of antibodies or their ability to inhibit the procoagulant function of factor VIII. The associated reduction in the Bethesda assay to undetectable levels appeared to involve the production of anti-idiotypic antibodies that neutralized the inhibitory capacity of the factor VIII antibodies. In another report, tolerant patients had circulating antibodies against factor VIII that, compared with the original inhibitor, differed in specificity, lacked coagulation inhibitory activity and did not enhance the rate of elimination of factor VIII.
A variety of different regimens have been used. In one report, 12 patients were treated with factor VIII at 50 U/kg/day without adjunctive immunosuppressive therapy. The inhibitor became undetectable in nine patients at 1–10 months; these patients required smaller and less frequent infusions. The three nonresponders had the highest inhibitor levels. In a multicentre survey of 158 patients from an international registry, 68% had complete tolerance, 7% partial tolerance and 25% did not respond. Even though most patients were high responders, success was most often observed in those receiving higher doses of factor VIII (≥100 U/kg/day) and those with low levels of inhibitor (<10 BU) at the start of therapy. Once achieved, tolerance was long-lasting, as only 1 of 107 responders relapsed. Data on 188 courses of immune tolerance therapy (ITT) were obtained via questionnaire by the North American Immune Tolerance Registry. Three factors have been found to be predictive of successful tolerance in patients with haemophilia A: (i) historical peak inhibitor titre prior to initiation of ITT; (ii) titre when ITT is initiated; and (iii) peak titre during ITT. As an example, ITT was successful in 83% of patients with haemophilia A when the pre-ITT titre was <10 BU compared with 40% when the titre was ≥10 BU. The Italian Immune Tolerance Induction registry (the PROFIT [PROgnostic Factors in Immune Tolerance] study) found that certain factor VIII mutations are associated with a high risk of factor VIII inhibitor formation. These high-risk mutations include large deletions, inversions, nonsense and splice site mutations. Small insertions/deletions and nonsense mutations are associated with a low risk of factor VIII inhibitor formation. Although ITT was not performed in a uniform manner or at a uniform time after inhibitor development, the low-risk mutation group showed a significantly higher ITT success rate than those with high-risk mutations (81% vs 41%); and mutation risk class (odds ratio 6.2), inhibitor titre <5 BU at the start of ITT (odds ratio 11.8) and peak titre <100 BU during ITT (odds ratio 11.4) were predictors of successful ITT following multivariate analysis.
The first published report of partially successful suppression of factor VIII inhibitors in two haemophilia A patients was published in 1965 using mercaptopurine. In these early days, immunosuppression was used in conjunction with very high-dose factor repletion as intervention for acute bleeding episodes in haemophilia patients with inhibitors. Often, outcomes were still poor. Since bypassing agents as described in section 3.1 can control acute bleeding episodes, clinicians currently reserve the use of immunosuppression for long-term inhibitor suppression. The literature abounds with observational cases and studies reporting successful inhibitor eradication using agents such as corticosteroids, cyclophosphamide, azathiaprine, mercaptopurine, vincristine, ciclosporin, tacrolimus, mycophenolate mofetil and rituximab. These have been used as a single agent or in combination with each other, ITI, lower doses of clotting factor and/or immunoadsorption. Although these reports give us hope, no randomized controlled trial has definitively identified which regimen, if any, best suppresses inhibitory antibody formation.
Intravenous immunoglobulin (IVIg) was first described in 1983 as an adjuvant immunomodulating therapy in haemophilia patients with inhibitors and continues to be used today. When used as a single agent, outcomes ranged from no change in inhibitor titres to complete eradication for 8 months following repeated doses of IVIg. When used with immunoadsorption, a second objective of IVIg is to replete immunoglobulins nonspecifically adsorbed to the column to minimize risk of infections.
3.2.3 Immunoadsorption and Plasmapheresis
Immunoadsorption and plasmapheresis are extracorporeal strategies for removing pathological antibodies rapidly, albeit transiently, with minimal adverse effects. Plasmapheresis replaces the patient's plasma with donor plasma, thereby reducing the inhibitor titre. Several case reports are published where plasmapheresis successfully reduced the inhibitor titre sufficiently enough to allow for control of active bleeding or successful completion of an invasive procedure.[69-74] In these reports, successful outcomes were seen in patients with haemophilia A and B, low- and high-titre inhibitors, and in patients as young as 18 months old. Outcomes included suppression of the inhibitor for 40 months when plasmapheresis was used in conjunction with high-dose PCC and corticosteroids. In another case, the inhibitor was suppressed for at least 10 months with plasmapheresis and 6 weeks of cyclophosphamide and corticosteroids. Plasmapheresis is rapid and relatively inexpensive, but requires specialized personnel and equipment and a large-bore intravascular catheter. In addition, plasmapheresis exposes the patient to plasma, which can elicit an anamnestic response and increase the risk of plasma-associated infections. Adverse effects included vasovagal episodes, tachycardia, tachypnoea and blood loss requiring a blood transfusion.
Extracorporeal adsorption of the Fc segment of antibodies to protein A has also been shown to be an efficient strategy in reducing the inhibitor titre. Two case series have been published on immunoadsorption in patients with haemophilia inhibitors. Thirteen patients in the US prospectively underwent immunoadsorption with concurrent cyclophosphamide administration. Eight of 11 patients with haemophilia were considered ‘responders’ with ≤15 BU/mL for up to 12 days following immunoadsorption. A risk factor for not responding included a high pre-immunoadsorption inhibitor titre. In a Swedish series, ten patients underwent one to four sessions of immunoadsorption and nine cases obtained sufficiently low titres to achieve haemostasis with clotting factor concentrates for 5–9 days. As in the US series, the patient with the highest titre could not achieve haemostatic levels of coagulation factor following two separate attempts at immunoadsorption. As with plasmapheresis, immunoadsorption requires specialized personnel, equipment and a large-calibre venous catheter. Acute adverse effects were mild, and included paraesthesias, headache, nausea/vomiting, hypotension, hypertension, fever, chills, tachycardia, bradycardia, dizziness, thrombocytopenia and pain. Since protein A binds immunoglobulins nonspecifically, plasma levels of IgM, IgA and IgG were significantly reduced following immunoadsorption.[
4. Adjuvant Therapy
Desmopressin has been used to control or prevent bleeding in mild haemophilia A, some cases of moderate haemophilia A and some types of von Willebrand's disease since 1977. Its mechanism of action appears multifactorial, including an increase in plasma levels of factor VIII and von Willebrand's factor, stimulation of platelet adhesion and an increased expression of tissue factor.[77,78] Desmopressin is not effective for the treatment of patients who have haemophilia B. For mild to moderate haemophilia A, indications for desmopressin are determined by the type of bleeding episode, baseline symptoms and desired level of factor VIII activity. However, some patients have a low biological response to desmopressin, precluding its use as prophylaxis or treatment of acute bleeding. Thus, a test dose, also known as a desmopressin challenge, is often performed under controlled conditions where blood pressure and heart rate can be monitored. A blood sample is obtained before the test dose and approximately 1 and 4 hours afterwards to monitor factor VIII and sodium levels. Following desmopressin administration, plasma factor VIII levels typically increase 2- to 6-fold and return to baseline within 6–14 hours.[7,79-81] This rise in factor VIII levels explains why patients who have severe haemophilia A are not candidates for this type of therapy to control bleeding. The intranasal route of desmopressin administration is convenient for outpatient treatment and commonly used before dental procedures and for oral or nasal mucosal bleeding. The intravenous route also is available and typically used in the inpatient setting. One advantage of intravenous administration is that peak factor VIII levels tend to be higher and achieved faster. Patients should be advised to limit water intake during desmopressin treatment and to avoid using more than three consecutive daily doses to reduce the risk of hyponatraemia. Other adverse effects are generally mild and include facial flushing, headache, nausea, and increased heart rate or blood pressure.
The antifibrinolytic agents ϵ-aminocaproic acid and tranexamic acid, both lysine derivatives, are also useful adjuvant therapies for patients who have mild to severe haemophilia. They exert their effect by inhibiting the proteolytic activity of plasmin and, therefore, inhibiting fibrinolysis. The use of antifibrinolytic agents is indicated in the presence of mucosal bleeding, primarily oral, nasal and menstrual blood loss. Its use is contraindicated in the presence of DIC or thromboembolic disease, and for bleeding in the upper urinary tract due to increased risk for intrarenal or ureteral thrombosis.
Topical agents can be applied directly to tissue to stop bleeding. Fibrin sealants are prepared by mixing fibrinogen and thrombin concentrates, with or without factor XIII or aprotinin, derived from human or bovine plasma. They have been used in Europe for decades but were only approved in the US in 1998 due to concerns regarding a high rate of antibody formation (up to 20%) against thrombin and factor V or the potential risk for transmitting blood-borne pathogens, such as hepatitis or vCJD.
Other haemostatic topical agents are on the market, but their use has not been extensively reported in haemophilia patients. FloSeal Matrix™ (Baxter Healthcare, Fremont, CA, USA) is a topical haemostatic agent using bovine-derived gelatin and human-derived thrombin. THROMBIN-JMI® (King Pharmaceuticals, Inc., Bristol, TN, USA) is bovine-derived thrombin. Like fibrin sealants, there is a risk of the formation of an antibody against endogenous thrombin. BioGlue® (CryoLife, Kennesaw, GA, USA) is composed of purified bovine serum albumin and glutaraldehyde. The glutaraldehyde molecules covalently bond albumin molecules to each other, creating a mechanical seal independent of the body's clotting mechanism. CoSeal® (Baxter Healthcare, Hayward, CA, USA) is a biocompatible polyethylene glycol (PEG) polymer that rapidly cross-links with proteins in exposed tissues to immediately adhere and form a sealant. Lastly, Quikclot® (Z-Medica Corporation, Wallingford, CT, USA) is an over-the-counter product made of zeolite, an inert, porous, natural material that absorbs water from blood, thereby concentrating clotting factors.
5. Future Directions
5.1 Factor Repletion
In recent years, research efforts have been focused on improving haemophilia management by prolonging the half-life of factor concentrates utilizing different technologies. Some of the more promising approaches being explored include sustained delivery, chemical modification and genetic mutation/fusion. The sustained-delivery approach utilizes methods that release the protein more slowly into the circulation over time, thereby further increasing the bioavailability and stability of the protein. Studies showing prolongation in the mean number of days without bleeding episodes in patients receiving pegylated liposomal factor VIII (PEGLip-FVIII) compared with standard factor VIII have been published.[84-86] Despite these promising results, the phase II trial evaluating the efficacy of one formulation of PEGLip-FVIII was unfortunately discontinued early due to futility.
A second approach is to chemically modify factor VIII in order to block factor VIII receptor-mediated clearance and/or reduce renal clearance. This strategy involves conjugating the protein with a hydrophilic polymer, such as PEG, via certain functional groups on the protein. PEG creates a molecular ‘shield’ around the protein and prevents the approach of antibodies and proteolytic enzymes. Pegylation can effectively modify the immunological, pharmacokinetic and pharmacodynamic properties of proteins. Although this is an established technique with proof-of-principle for complex proteins, several factors remain unknown, such as the clearance mechanisms of large PEG-protein conjugates over an extended time period and the impact on immunogenicity. Pegylation technology continues to advance and become more versatile, but is no longer the only available option as other biological molecules are also being utilized. Polysialylation, which involves the addition of sialic acid residues to the protein, is a suitable alternative to pegylation.
The three-dimensional crystal structure and domain organization of human factor VIII have been described.[89,90] This knowledge may be helpful not only to better understand the role of factor VIII in blood coagulation but also to predict some of the structural outcomes when protein modifications are made to factor VIII. Genetic engineering modifies the protein to reduce its metabolism. This can be achieved either by directly altering the amino acids or by fusing the protein of interest with another protein that has a longer biological half-life.
Factor VIII is activated by proteolytic cleavage, which generates an unstable heterotrimer that is susceptible to rapid dissociation of the A2 subunit. The activity of factor VIII can be extended by preventing either spontaneous dissociation or activated protein C (APC)-mediated proteolysis of the A2 subunit. A modified factor VIII molecule, in which the A2 was covalently cross-linked to the A3 domain by disulfide bonds, has been described. The resultant molecule prevented spontaneous A2 dissociation while maintaining good functional activity. The molecule appeared to be more stable and cofactor activity was extended. In another approach, a genetically modified factor VIIIa molecule was resistant to proteolytic inactivation and subunit dissociation. In this molecule, the APC cleavage sites at residues 336 and 562 on the A1 and A2 subunits, respectively, were genetically modified so that the A2 subunit was covalently attached to the light chain. The resultant molecule displayed higher in vitro- and in vivo-specific activity than that of wild-type factor VIII.
Interfering with factor VIII clearance is another method that could potentially result in prolonged factor VIII activity. There are at least three interactive sites in the A2, A3 and C-terminal domains of the factor VIII protein. However, mutating these binding regions to remove the clearance sites may also interfere with other critical functional regions of factor VIII that are essential for interacting with phospholipids, factor IXa and von Willebrand's factor.
A technique that is growing in interest and showing considerable promise is the generation of factor VIII fusion proteins. This strategy uses recombinant DNA technology to fuse a carrier protein, which has a longer half-life, to the C- or N-terminus of the factor VIII molecule. Albumin and the Fc fragment of IgG are the best candidate fusion partners: they are present in the circulation at high concentrations, have long half-lives (>20 days) and have the same mode of recycling through the Fcγ receptor.[93,94] Through fusion with these proteins, factor VIII is rescued from degradation, recycled back into the plasma and released into the circulation by the process of repeated endocytosis.[
5.2 Genetic Therapies
5.2.1 Gene Therapy
Gene therapy is a highly active field of research in haemophilia. The overreaching goal of gene therapy is to replace the dysfunctional gene with an exogenous functional gene to cure the disease phenotype. Six clinical trials opened in the 1990s to study the role of gene therapy in the treatment of patients with haemophilia. Three of these trials were in patients with factor VIII deficiency and three were in patients with factor IX deficiency. As these studies have been thoroughly reviewed,[96-98] details are not presented here.
Haemophilia is an attractive disease process for gene therapy for multiple reasons. Currently available therapies are episodic with peaks and troughs in plasma factor levels allowing for break-through bleeding episodes. Treatment can be expensive and inconvenient. Furthermore, haemophilia is relatively prevalent and its molecular pathway is well studied. Haemophilia A and B are each caused by mutations in a single gene. Both factor VIII and factor IX have a wide therapeutic window and even slight increases in either factor level might reduce morbidity. Even though endogenous factor VIII and factor IX are secreted by endothelial cells and hepatocytes, the transgene may be expressed in other types of cells without altering the efficacy of the protein product. Lastly, patients can be readily monitored for plasma factor levels and correction of the bleeding phenotype.
On the other hand, gene therapy for haemophilia presents challenges. Factor VIII is a large molecule, making its insertion into gene delivery systems tricky. While results from murine, canine and non-human primate gene therapy models have been impressive in the past, none predicted the short-term efficacy and adverse effects seen in the human clinical trials performed to date. In addition, the formation of neutralizing antibodies against exogenous factor VIII or factor IX, whether supplied by infused factor or a transgene, is still a major challenge in gene therapy for haemophilia. The death of one patient with ornithine transcarbamylase deficiency following a serious inflammatory reaction to an adenoviral vector and the diagnosis of leukaemia in several other patients who were successfully treated with gene therapy for severe combined immunodeficiency (SCID) temporarily stalled further clinical trials.
Using in vitro and animal models, researchers continue to investigate an ideal combination of gene delivery systems (viral and non-viral), promoters, enhancers, methods of administration of the vector to increase long-term expression of the transgene product, and various methods of reducing the risk of inhibitor formation following gene therapy. As research in gene therapy continues, clinical trials in humans have cautiously restarted. At the time of publication, two phase I/II gene therapy trials in haemophilia B are registered with ClinicalTrials.gov.[101,102] The recent publication of a successful gene therapy clinical trial in adenosine deaminase-deficient SCID patients provides hope that gene therapy in haemophilia is a tangible possibility.[
5.2.2 Premature Termination Codon Suppression
Nonsense mutations in both factor VIII and factor IX typically cause a severe bleeding phenotype.[104,105] Currently, the Haemophilia A Mutation, Structure, Test and Resource Site (HAMSTeRS) has 131 nonsense mutations registered out of 1209 total unique mutations (11%). These nonsense mutations, also known as premature termination codons (PTCs), are caused by base pair substitutions, insertions or deletions causing the translation of truncated proteins, which undergo nonsense-mediated decay and can lead to a disease phenotype. A novel area of research has flourished in past years to identify and develop interventions to promote ribosome read-through of PTCs in order to translate and express full-length functional proteins. First described in 1964, this strategy utilized the aminoglycoside streptomycin to correct the phenotype of mutant strains of Escherichia coli. Not until 30 years had passed was this strategy used to restore the cystic fibrosis protein following PTC suppression.[108,109]
Ataluren (PTC124®; PTC Therapeutics, South Plainfield, NJ, USA) is an oral investigational drug that promotes dose-dependent read-through of nonsense codons. Early-phase clinical trials to investigate the safety of ataluren in healthy volunteers and its efficacy in the treatment of cystic fibrosis have been published.[110,111] No major adverse event occurred during either trial. At the time of publication, a phase II trial studying the effects of two 28-day treatment cycles of ataluren in patients with haemophilia A and B is recruiting patients. Ataluren, if proven to be efficacious for nonsense mutations in haemophilia, would offer a convenient alternative to intravenous infusions of factor and would avoid the risks associated with gene therapy. If PTC suppression could be initiated prior to the induction of central tolerance, ataluren might decrease the risk of inhibitor formation that complicates factor replacement and gene therapy regimens. However, as only a fraction of patients with haemophilia carry nonsense mutations and PTC suppressors may only be efficacious for a fraction of these patients, ataluren and other PTC suppressors hold hope only for a small percentage of the haemophilia population.
The history of haemophilia is one of tragedy and triumph, but the collaborative work of researchers, clinicians, patients and patient advocates has made numerous treatment options available to patients with congenital haemophilia today. With continued innovation and cooperation, the future is bound to hold still more options so that patients with haemophilia can live longer, better lives.
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