Inhibitor antibodies to factor VIII and factor IX: management

Inhibitor antibodies directed against factor VIII or factor IX present challenges to the clinician. Fortunately, several management options are available, although each has disadvantages as well as advantages. Alloantibodies against factor VIII (which develop in 25 to 50% of children with severe hemophilia A, as well as in a small percentage of children with mild or moderate hemophilia A) may be low titer and transient or may be high titer. Most patients with high-titer problematic inhibitors now try to eliminate the inhibitor by using one of several immune tolerance induction (ITI) regimens. For treatment of bleeding episodes in patients who have high-titer (> or = 5 Bethesda units) inhibitors, one can use a prothrombin complex concentrate (PCC) (preferably an activated PCC [APCC]), recombinant (r) factor VIIa, or porcine factor VIII. The choice of product is generally dependent on the type and severity of the patient's bleeding, degree of cross-reactivity of the patient's inhibitor with porcine factor VIII, physician familiarity with the product, product availability, and cost. In persons with hemophilia B, alloantibodies occur in only 1 to 3% of severely affected individuals. However, in roughly half of those who develop inhibitors, anaphylaxis or severe allergic reactions occur on infusion of any type of factor IX-containing product. This phenomenon usually develops after relatively few exposures to factor IX; thus it is recommended that the first 10 to 20 infusions of factor IX given to children with severe hemophilia B be given in a setting equipped for treatment of shock. For treatment of bleeding episodes in patients with severe allergic reactions, rF VIIa is the treatment of choice. ITI has been less successful in hemophilia B patients with inhibitors than in those with hemophilia A, and in a subgroup of patients with severe allergic reactions who were desensitized to factor IX and then tried on ITI, results were even poorer. Additionally, several developed nephrotic syndrome while on ITI. For hemophilia B patients with inhibitors who do not have allergic reactions to factor IX, bleeding episodes can be treated with PCC or APCC or with rF VIIa. Autoantibodies directed against factor VIII are rare but can occur in a variety of settings. They occur mainly in adults, and bleeding is often severe and life threatening. Although some factor VIII autoantibodies disappear spontaneously, most require immunosuppression. Corticosteroids and cyclophosphamide are generally recommended. For treatment of bleeding, therapeutic options include (human) factor VIII concentrates, porcine factor VIII, APCC, and rFVIIa. The choice of product is generally determined by the consulting hematologist's familiarity with the product, product availability and cost, as well as response to treatment.     

Current status of hemophilia patients and recombinant coagulation factor concentrates in Japan

The first recombinant factor VIII concentrate was introduced in 1987 to treat hemophilia A patients, and the product was licensed in the United States in 1992. More than 10 years have passed since the recombinant products have been used for treatment of hemophilia A. The new therapeutic options seem to be safe and effective through the gathered experiences. Recently, recombinant factor VIIa concentrate has become available to treat hemophilia patients with inhibitor and factor VII deficiency patients in Europe and also recombinant factor IX for the treatment of hemophilia B has been licensed in the United States and Europe. The usage of recombinant coagulation factors has expanded the routine therapy for hemophilia in many countries. In Japan, the consumption of recombinant factor VIII is increasing year by year, because many patients have started to think that the recombinant technology seems to be safe. Unfortunately, though, the factor VIIa and factor IX products have not been licensed yet in Japan. This article discusses the current status of patients with hemophilia and recombinant coagulation factor products in Japan.     

The use of continuous infusion of factor concentrates in the treatment of hemophilia

Bolus infusion of clotting factor concentrates remains the most common approach to the treatment or prevention of bleeding in patients with hemophilia. Although successful use of continuous infusion of such concentrates has been reported by several groups, this alternative treatment method has not achieved widespread popularity. We report here our experience in one hemophilia center with the use of continuous infusion of factor VIII and factor IX concentrates in 13 patients, 11 with hemophilia A, and 2 with hemophilia B. All patients were treated successfully for bleeding episodes (e.g., hemarthroses, intracranial, or gastrointestinal bleeding) or for surgical procedures (appendectomy, thoracotomy, etc.). Three patients with low titer factor VIII inhibitors were treated successfully with constant infusion therapy, requiring a mean dose of factor VIII concentrate 2.3 fold (8.20 u/kg/h) higher than that of the patients without inhibitors (3.63 u/kg/h) to maintain a circulating plasma level of factor VIII of 1 u/ml. The use of constant infusion of clotting factor concentrates is safe, efficacious, and more convenient than bolus therapy of factor concentrates and should be considered for hospitalized hemophilia patients requiring replacement therapy.     

Gene therapy of the hemophilias

Development of hemophilia gene therapy depends on testing gene transfer vectors in hemophilic and nonhemophilic animals. Available animal models include factor VIII or factor IX knockout mice as well as dogs with spontaneous hemophilia A or B. Large animals (particularly dogs) more closely replicate the requirements for correction of human hemophilia than do mice. Small animals are more convenient to maintain and require significantly less vector for testing than do large animals. Nonhemophilic animals (mice or nonhuman primates), whose endogenous factor VIII and factor IX complicate analysis of the human proteins, have utility for safety testing of vectors; some assays can discriminate between human coagulation factors and the endogenous coagulation factors. Most animal models suffer the limitations imposed by the immune response to human factor VIII or IX protein. Clinical trials have failed to achieve significant factor VIII expression in hemophilia A patients, while one clinical trial in hemophilia B patients showed only transient therapeutic increments of factor IX expression. Gene therapy remains an investigational method with many obstacles to overcome before it can be widely used as treatment for hemophilia.     

The Pennsylvania hemophilia program 1973-1978

In Pennsylvania, the prevalence of hemophilia is one per 10,000 males. Factor VIII deficiency is five times more frequent than Factor IX deficiency, and 34% of the patients have no relatives affected with the disease. The mean age is 23 years old, and 50% of the patients are less than 20 years old. Approximately one-third of the patients with Factor VIII deficiency and one fourth of the patients with Factor IX deficiency have levels of < 0.01 μ/ml. By clinical criteria, 55% of those with Factor VIII deficiency are severe compared to 45% of those with Factor IX deficiency. Factor VIII-deficient patients are treated an average of 18 times per year compared to ten times per year for patients with Factor IX deficiency. Hemarthroses account for 70% of the hemorrhages treated and for 40% of the concentrate usage. Home therapy patients use an average of 45,950 Factor VIII units per year at a cost of $4,170 per patient and their use accounts for 60% of the total Factor VIII usage of 1.7 million units. Less than five days per patient per year are lost from school or work because of bleeding, and patients are hospitalized for bleeding an average of only two to three days per patient per year. Adverse immediate reactions to therapy are infrequent. Five percent of patients have persistence of the hepatitis B virus, and 7.5% have inhibitors. The mortality rate is 0.04% per year, with half of the deaths being hemophilia-related.     

Immunologic status of hemophilia patients treated with cryoprecipitate or lyophilized concentrate

We evaluated 37 patients with moderate or severe hemophilia A and six patients with severe factor IX deficiency for clinical or laboratory evidence of immune abnormalities. Patients were assigned to one of four groups according to the type of clotting factor replacement. Twenty patients had received only cryoprecipitate during the two years preceding the evaluation (group I); 11 additional patients were treated predominantly with cryoprecipitate but had also received up to nine bottles of factor VIII concentrate (group II); six patients received factor VIII concentrate (group III); six patients received factor IX concentrate (group IV). There was no clinical or laboratory evidence of immunodeficiency among the 43 patients. The mean absolute number of Th cells was normal in all patient groups, but the mean absolute number of Ts cells was increased compared with controls, both in patients treated with cryoprecipitate and in patients treated with factor VIII or factor IX concentrate. There was no correlation between the Th/Ts ratio and patient age, alanine aminotransferase level, hepatitis serology, in vitro lymphocyte function, or amount of clotting factor administered. Our observations demonstrate that the volunteer or commercial origin of clotting factor replacement cannot fully explain the alterations in lymphocyte subset distribution previously described in patients with hemophilia A.     

A new therapy of hemophilia with ranitidine

20 cases of hemophilia, including ten of hemophilia A , nine of hemophilia B and one of von Willebrand's disease (VWD), were treated with ranitidine. The results revealed that the levels of both factor VIII and IX were increased and the clinical symptoms of bleeding were ameliorated. The level of factor VIII in hemophilia A ranged from 5.27 +/- 3.94% before treatment and rose to 14.68 +/- 4.70% during the treatment period (P less than 0.001). The level of the factor IX in the patients with hemophilia B increased from 4.42 +/- 3.01% before treatment to 20.33 + 9.31% under treatment (P less than 0.001). Tn the patient with VWD the level of the factor VIII rose but the level of von Willebrand factor did not change. The drug has no side effect. The results of our study suggests that ranitidine therapy is effective and safe in hemophilia.     

Decreased helper/suppressor cell ratios after treatment with factor VIII and IX concentrates and fresh frozen plasma

The immunologic status of three groups of multiply transfused asymptomatic patients was evaluated. These included five with acquired inhibitors to factor VIII treated with both factor VIII and factor IX concentrates (Group A), seven with hemophilia B treated with factor IX concentrate (Group B), and six with hemophilia B treated with fresh frozen plasma (Group C). Mean helper/suppressor T cell ratios (± SEM) for the three groups were 0.72 ± 0.09, 1.35 ± 0.18, and 1.37 ± 0.12, respectively. All three differed significantly (p < 0.01) from the control mean ratio of 2.22 ± 0.16. In addition, the mean ratio of Group A patients was significantly different (p < 0.01) from those of Groups B and C. An inverted ratio (less than 1.00) was found in all Group A patients and only one Group B patient. Increased IgG levels were found in 80, 57, and 50 percent of each group, respectively. These immunologic findings bear a striking resemblance to those of the acquired immunodeficiency syndrome (AIDS) of homosexuals, intravenous-drug abusers, Haitian immigrants, and factor VIII concentrate-treated hemophiliacs. Transmission via a blood-borne infectious agent seems likely.     

Successful treatment of high titer inhibitors in mild hemophilia A with avoidance of factor VIII and immunosuppressive therapy.

We describe two patients with mild hemophilia A (MHA) who developed high titer inhibitor (HTI) following intensive recombinant factor VIII (rFVIII) concentrate replacement for surgery and trauma. Intranasal desmopressin was instituted shortly following immunosuppressive therapy (IST) and activated prothrombin complex concentrate (APCC) in one case, and following APCC alone in the second case. Avoidance of factor VIII (FVIII) coupled with intranasal desmopressin prophylaxis three times a week resulted in undetectable inhibitor levels. Both patients have had no further bleeding episodes and have been maintained on desmopressin prophylaxis prior to activity for the past 2 to 3 years. Recombinant factor VIIa (rFVIIa) was used successfully prior to a second surgery in one patient without complication.     

Acquired hemophilia: a case report

Acquired hemophilia is a severe bleeding diathesis that affects both males and females. It is caused by suddenly appearing autoantibodies that interfere with coagulation factor VIII activity. This disorder is characterized by spontaneous and post-traumatic subcutaneous bleeds and massive mucosal hemorrhages. We report in the current article a case of acute renal failure and bleeding from the urinary tract caused by idiopathic acquired hemophilia in a 54-year-old woman. Hemostatic tests indicated prolonged activated partial thromboplastin time (APTT) to 107.8 sec (norm 26-36 sec), normal value of the prothrombin index which was 82% (norm 70-130%), increased fibrinogen concentration to 583 mg/dl (normal value 200-400 mg/dl), the bleeding time was 5 min and 20 s (norm < 10 min) and the platelet count was 366 x 10(9)/l (norm 130-400 x10(9)/l). The autoantibody against factor VIII in a titer of 121 Bethesda Units/ml (BU/ml) and decreased factor VIII activity to 2% (norm 50-150%) with normal plasma concentration of factor IX. Activated (FEIBA, Baxter) and nonactivated prothrombin complex concentrates (factor IX concentrate) have been used in the treatment of bleeding episode. Immunosuppressive treatment with the combination of oral prednisone 60 mg/24h and cyclophosphamide 150 mg/24h was administered in order to remove the factor VIII inhibitor. Reduction of the factor VIII inhibitor titer to 38 BU/ml and increase of factor VIII activity to 4% was initially achieved. This treatment has been continued for two years and led to normalization of hemostatic parameters (APTT 26 sec, factor VIII activity 108%) which means a total removal of factor VIII inhibitor.     

Comparative efficacy of nonheated and heat-treated factor IX complex concentrate in treatment of hemophiliacs with inhibitors

To determine if heat-treated factor IX complex concentrate is as effective as nonheated factor IX complex concentrate for treatment of subjects with hemophilia A and antifactor VIII antibodies (inhibitor patients), we have retrospectively reviewed consecutive home treatment records of ten inhibitor patients who had been receiving nonheat-treated factor IX complex concentrate (NHT-Konyne) and subsequently converted to heat-treated factor complex concentrate (Konyne-HT) when it was licensed in late 1984. Overall, 162 of 284 (57%) separate bleeding episodes treated with NHT-Konyne and 53 of 80 (66.3%) separate bleeding episodes treated with Konyne-HT required only one treatment course of approximately 60-75 U/kg. The distribution of bleeding sites and the absolute factor IX unitage required per treatment episode were similar for both preparations. These data suggest that the percentage of hemophilic inhibitor patients responding to factor IX complex concentrate remains at least 50%, as was reported several years ago in a controlled study, and that inhibitor bypass activity has not altered by heat treatment.     

Noncoagulation inhibitory factor VIII antibodies after induction of tolerance to factor VIII in hemophilia A patients

We recently described tolerance induction with factor VIII/IX, cyclophosphamide, and high-dose intravenous IgG in hemophilia A or B patients with coagulation inhibitory antibodies. Circulating noninhibitory antibodies complexed with factor IX have been demonstrated in tolerant hemophilia B patients. Similar findings are now described in six tolerant hemophilia A patients. Complexes between factor VIII and the 'tolerant' antibody were demonstrated by subjecting plasma to gel filtration chromatography, void fractions containing factor VIII/vWF complexes being collected and adsorbed to protein A. Using 125I-labeled F(ab')2 fragments against IgG subclass and factor VIII antigen, complexes between an IgG4 antibody and factor VIII were found to adsorb to protein A. After infusion of factor VIII to tolerant patients, all factor VIII circulated in complex with IgG4 antibody. In three of the patients, the 'tolerant' antibodies inhibited an ELISA specific for factor VIII light chain but, unlike the pretolerant antibodies, did not bind radiolabeled factor VIII heavy chain. Although after induction of tolerance the patients still have circulating IgG4 antibodies against factor VIII, the antibodies differ in specificity, lack coagulation inhibitory activity, and do not enhance the rate of elimination of factor VIII.     

Current status and future prospects for the prophylactic management of hemophilia patients with inhibitor antibodies

Inhibitor antibodies to factor VIII arise in a substantial minority of patients with hemophilia A treated with replacement therapy; factor IX inhibitors in treated hemophilia B patients are considerably less common. As replacement therapy is not feasible in most such patients, hemostasis can generally only be achieved with “inhibitor bypassing agents”, namely (activated) prothrombin complex concentrates and recombinant factor VIIa. These agents are widely used to treat active bleeding in inhibitor patients but they have been used relatively infrequently as prophylactic agents to prevent bleeding and its consequences, mainly progressive joint damage. This is in contrast to hemophilia patients without inhibitors, in whom prophylactic replacement with concentrates of factor VIII or IX has become widely accepted as the optimal strategy to prevent these adverse outcomes. This review addresses the current experience and evidence and the future prospects regarding prophylaxis in inhibitor patients.     

Effect of danazol on clotting factor levels, bleeding incidence, factor infusion requirements, and immune parameters in hemophilia

Several recent studies have reported conflicting results on the effectiveness of danazol, an attenuated androgen, in raising plasma levels of clotting factors VIII and IX in patients with hemophilia. We undertook a randomized, double-blind cross-over trial using 8 weeks' administration of danazol (D), 600 mg/d, and 8 weeks' administration of placebo (P) separated by 2 weeks of rest in 12 patients with hemophilia A and four patients with hemophilia B. Plasma factor VIII and IX levels, frequency and type of bleeding episodes, amount of factor concentrate infused, fibrinogen, fibrinolysis assays, antithrombin III, liver function, and immune parameters were followed. During the danazol phase a minimal increase was noted in the average clotting factor levels, an increase that, although statistically significant, was of hemostatically marginal magnitude. Significant increases in protein C and plasminogen levels, however, were observed during the danazol period, suggestive of danazol-mediated enhanced fibrinolysis. Clinically, bleeding frequency was significantly increased, and more clotting factor was consumed during the danazol period. Furthermore, eight episodes of hematuria and oral mucosal bleeding was reported during the danazol phase in contrast to only one episode of hematuria during the placebo phase, consistent with an enhancement of fibrinolytic activity with danazol. We conclude that danazol does not have a hemostatically significant effect on plasma levels of factor VIII and IX but may be associated with enhancement of fibrinolytic activity, resulting in increased bleeding frequency and requiring more clotting factor infusions. Therefore, danazol is not a viable alternative in the treatment of hemophilia.     

1986 update of HIV seroprevalence, seroconversion, AIDS incidence, and immunologic correlates of HIV infection in patients with hemophilia A and B

A cohort of 181 patients with hemophilia A (149) and hemophilia B (32) cared for at the Hemophilia Center of Western Pennsylvania was followed to determine human immunodeficiency virus (HIV) seroprevalence, seroconversion rate, and clinical and immunologic correlates of HIV infection. By December 1986, 82 (45%) were HIV seropositive, and of these, ten (12%) had developed AIDS, 28 (34%) had symptomatic HIV infection (CDC class III, IV), of whom 14 (17%) had AIDS-related complex (ARC), and 44 (54%) had asymptomatic HIV infection (CDC class II). The HIV seropositive group included 82% of those treated with factor VIII concentrate (97% severe, 5% moderate), 48% of those treated with factor IX concentrate (92% severe, 8% moderate), 10% of those treated with cryoprecipitate (67% severe, 33% moderate), and none of those treated with fresh frozen plasma. Based on 77 serially sampled HIV seropositive hemophiliacs (1977 to 1986), peak seroconversion occurred in 1982, with 14% (11 of 77) occurring since 1984. With increasing time from seroconversion, both T4 lymphocyte number and function (the latter measured by growth in soft agar [T colony assay]) progressively declined; T4 number declined to 135 +/- 26/mm3 (SEM), and colony count declined 1193 +/- 537 (control 3851 +/- 387) by 5 years after seroconversion. In those developing AIDS, total T4 fell below 100/mm3 (33 +/- 8/mm3) at diagnosis. In this cohort, the overall AIDS incidence is 5.5% (12% among the HIV seropositive) and in those seropositive 5 or more years, the AIDS incidence approaches 32%.     

AIDS retrovirus antibodies in hemophiliacs treated with factor VIII or factor IX concentrates, cryoprecipitate, or fresh frozen plasma: prevalence, seroconversion rate, and clinical correlations

Antibodies to the AIDS retrovirus, specifically to human T cell lymphotropic virus, type III, and AIDS-associated retrovirus, were detected with increasing prevalence in a population of 190 hemophiliacs from western Pennsylvania between 1981 and 1984: 7.7% in 1981, 20.0% in 1982, 45.5% in 1983, and 62.5% in 1984. The seropositive included approximately three fourths of those receiving factor VIII concentrate, nearly one third of those receiving factor IX concentrate, nearly one fifth of those receiving cryoprecipitate, and none of those receiving fresh frozen plasma. The seroconversion rate, determined on 43 seropositive hemophiliacs from this group who were serially sampled, was 0% in 1977, 4.7% in 1978, 4.9% in 1979, 2.6% in 1980, 10.5% in 1981, 52.9% in 1982, 87.5% in 1983, and 100% in 1984. Of 27 seropositive for three or more years (since 1982 or before), four (15%) have developed AIDS and seven (26%), diffuse lymphadenopathy (ARC); of 16 seropositive for less than three years, none has developed AIDS and three (19%) have developed ARC. The mean time from seroconversion to onset of ARC, 0.8 +/- 0.2 years (SEM), was shorter (P less than .001) than the time to onset of AIDS, 4.1 +/- 0.6 years. These findings confirm the widespread presence of AIDS retrovirus and support the association of these retroviruses with the acquired immunodeficiency syndrome and related conditions.     

Antibodies reactive with human T cell leukemia viruses in the serum of hemophiliacs receiving factor VIII concentrate

The third member of the family of T cell leukemia viruses (HTLV III) has been proposed as the primary etiologic agent of the acquired immunodeficiency syndrome (AIDS). A high risk of AIDS has been reported among patients with hemophilia, particularly those with factor VIII deficiency who receive commercial clotting factor concentrates. In a prevalence survey conducted between September 1982 and April 1984, initial serum samples from 74% of hemophiliacs who had ever been treated with commercial factor VIII concentrate, 90% of those frequently treated with factor VIII concentrate, and 50% of those treated with both factor VIII and factor IX concentrates had antibodies reactive against antigens of HTLV III, compared with none of the hemophiliacs treated only with factor IX concentrate or volunteer donor plasma or cryoprecipitate. Two of the seropositive patients have developed AIDS-related illnesses, and a third patient died of bacterial pneumonia. One initially seronegative patient developed antibodies against HTLV III during the study and is currently well. The predominant antibody specificities appear directed against p24 and p41, the presumed core and envelope antigens of HTLV III, suggesting that factor VIII concentrate may transmit the p24 and p41 antigens of HTLV III. However, the presence of infectious retroviruses in clotting factor concentrates and the effectiveness of screening and viral neutralization procedures remain to be determined.     

Orthotopic liver transplantation in a patient with combined hemophilia A and B

A 38-year-old man with severe factor IX and mild factor VIII deficiencies complicated by cirrhosis secondary to chronic non-A non-B hepatitis underwent orthotopic liver transplantation as treatment for both the cirrhosis and his congenital coagulopathy. Intraoperative hemostasis was obtained with factor VII-depleted prothrombin complex concentrate and fresh frozen plasma. Factor VIII and factor IX levels were assayed frequently in the perioperative period, and both returned to normal within 24 hr and remained normal postoperatively. Liver transplantation can be considered as definitive therapy for hemophilia A and/or B with transfusion-related liver disease.     

Factor IXa-factor VIIIa-cell surface complex does not contribute to the basal activation of the coagulation mechanism in vivo.

We have infused recombinant factor VIIa into patients with hereditary factor VII deficiency with marked reductions in plasma concentrations of factor IX activation peptide (FIXP), factor X activation peptide (FXP), and prothrombin activation fragment F1+2. These investigations show substantial elevations in these markers of coagulation activation and thereby demonstrate that the factor VII-tissue factor pathway is largely responsible for the activation of factor IX as well as factor X in the basal state (ie, the absence of thrombosis or provocative stimuli). We have administered a monoclonal antibody purified factor IX concentrate to individuals with hemophilia B. These studies show an increase in the plasma levels of FIXP that were initially greatly decreased, but no change in FXP or F1+2. We have also infused highly purified factor VIII concentrate into patients with hemophilia A. The data demonstrate no significant changes in the plasma concentrations of FXP and F1+2. The above observations indicate that factor IXa generated by the factor VII-tissue factor pathway is unable to activate factor X under basal conditions. Based upon the above findings, we outline a model of blood coagulation system function under basal conditions, and suggest a process by which the generation of factor Xa and thrombin might be accelerated during normal hemostasis and in the setting of thrombotic disorders.     

Adeno-associated virus-mediated gene transfer for hemophilia B

Hemophilia is the bleeding diathesis caused by mutations in the gene encoding factor VIII (hemophilia A) or factor IX (hemophilia B). Currently, the disease is treated by intravenous infusion of the missing purified clotting factor. The goal of gene transfer for treating hemophilia is to achieve sustained expression of factor VIII or factor IX at levels high enough to improve the symptoms of the disease. Hemophilia has proven to be an attractive model for those interested in gene transfer, and multiple gene-transfer strategies are currently being investigated for the hemophilias. The most promising preclinical studies have been with adeno-associated viral vectors (AAV); introduction of AAV vectors expressing factor IX into skeletal muscle or liver in hemophilic dogs has resulted in the long-term expression of factor IX at levels that are adequate to improve disease symptoms. Efforts to translate these findings into the clinical arena have proceeded slowly because of the lack of prior clinical experience with parenteral administration of AAV. In a staged approach, AAV-factor IX (AAV-F.IX) was first administered at doses of up to 1.8 x 10(12) vector genomes/kg (vg/kg) into the skeletal muscles of men with hemophilia B. This trial established the safety of parenteral administration and also showed that general characteristics of AAV transduction were similar in mice, dogs, and humans. In an ongoing trial, AAV-F.IX is being administered into the hepatic circulation of men with severe hemophilia B. The goal of these studies is to identify a safe dose that reliably yields circulating levels of factor IX >2% of normal levels in all subjects. This goal has already been achieved in the hemophilia B dog model; the ongoing study will determine whether a similar result can be achieved in humans with hemophilia B.