Heterogeneity of the factor IX locus in nine hemophilia B inhibitor patients.

DNA from nine hemophilia B patients who produce anti-factor IX inhibitors (antibodies), including two brothers, was analyzed by the Southern blotting method and hybridization with factor IX cDNA, intragenomic, and 3'-flanking probes. Two inhibitor patients were shown to have total deletions of the factor IX gene. Two other inhibitor patients, the brothers, were shown to have a presumably identical complex rearrangement of the factor IX gene involving two separate deletions. The first deletion is of approximately 5.0 kb and removes exon e. The second deletion is between 9 and 29 kb and removes exons g and h but leaves exon f intact. An abnormal Taq I fragment at one end of the deletion junctions acted as a marker for the inheritance of hemophilia B in the patients' family. Five other inhibitor patients have a structurally intact factor IX gene as detected by this method. Our studies indicate that whereas large structural factor IX gene defects predispose hemophilia B patients to developing an anti-factor IX inhibitor, the development of an inhibitor can be associated with other defects of the factor IX gene.     

Partial factor IX protein in a pedigree with hemophilia B due to a partial gene deletion.

A partial gene product was identified in a pedigree with hemophilia B due to a partial deletion of the Factor IX gene (Chen, S.-H.,S. Yoshitake, P.F. Chance, G.L. Bray, A.R. Thompson, C.R. Scott, and K. Kurachi, 1985, J. Clin. Invest., 76:2161-2164). Levels of this mutant protein in plasma of affected family members studied ranged from 24 to 36 ng/ml (0.6-0.9 U/dl or percent of normal) by a solid-phase immunoassay which is sensitive and specific for the calcium-dependent conformation of human Factor IX. No Factor IX antigen could be detected in patients' plasmas by a non-calcium-requiring monoclonal anti-Factor IX antibody (less than 2 ng/ml). The unconcentrated urine from the five affected family members and four obligate heterozygotes the five affected family members and four obligate heterozygotes tested contained calcium-dependent Factor IX antigen levels ranging from 64 to 160 ng/ml (1.6-4.0 U/dl) and from 10 to 68 ng/ml (0.25-1.7 U/dl), respectively. Of nine normal volunteers screened, three had detectable calcium-dependent antigen in unconcentrated first morning-voided urines with 9.6-16.8 ng/ml (0.24-0.42 U/dl), while the remaining six had detectable urinary antigen only after a 10-fold concentration. Abnormal and normal urinary Factor IX antigen species were concentrated, immunoaffinity purified, electrophoresed, immunoblotted, and distinguished by autoradiography after incubation with 125I-polyclonal calcium-requiring anti-Factor IX. After reducing purified or concentrated samples, a single abnormal 36,000-mol-wt band was identified in the urines from the four affected family members and four obligate heterozygotes tested. Electrophoresis of the reduced urinary Factor IX antigen from the one normal subject tested showed a broad 15,000-20,000-mol-wt band. This normal band was smaller than the species in patients' urines, and was seen as a minor component in the samples from the heterozygotes. No abnormal antigen could be detected in urine from the two other female family members tested. Thus, abnormal urinary Factor IX antigen represents a marker for the presence of the hemophilic Factor IX gene in this family.     

Human coagulation factor IX gene therapy in murine hemophilia B by hydrodynamic delivery and site-specific genomic integration

OBJECTIVE: To investigate whether the plasmid bearing attB and human coagulation factor IX (hFIX) coding sequence could insert into hemophilia B mice genome and persistently express hFIX with co-injected integrase. METHODS: The plasmid attB-hFIX-pIRES2-EGFP was constructed, which bore attB site and hFIX coding sequence and was proved in vitro to express hFIX. The plasmid and CMV-int expressing integrase was co-infused rapidly in a large-volume solution through tail vein of hemophilia B mice. Mice infused with the plasmid alone served as controls. ELISA was performed to determine serum hFIX level. Correction of coagulation defect in vivo by plasmid infusion was assessed by bleeding time. Genomic integration of the plasmid was determined by nested PCR. RESULTS: The plasmid attB-hFIX-pIRES2-EGFP was successfully constructed. The hemophilia B mice produced (1533 ± 239) ng/ml hFIX at 24 hour after infusion of the hFIX encoding plasmid and the bleeding diathesis of the hemophilia B mice was significantly corrected as measured by clotting assays. However, whether or not co-injected with CMV-int, the serum hFIX level decreased to background level in 10 days after infusion. Nested-PCR results indicated that the integrase phiC31 resulted in the integration of the plasmid in the mouse liver chromosomes. CONCLUSION: Integrase phiC31 can catalyze recombination of 34 bp attB and pseudo-attP. Human FIX driven by CMV promoter can be transiently and highly expressed after infusion, but rapidly silenced in vivo.     

Serologic evidence that factor IX inhibitor in the plasma of hemophilia B patients detects factor IX on normal red cells

BACKGROUND: Patients with hemophilia B lack factor IX (F IX). These patients may become alloimmunized after the transfusion of F IX concentrates and may develop F IX inhibitors, which have been characterized as polyclonal IgG4 alloantibodies. Two cases in which F IX inhibitors caused difficulty in compatibility testing and antibody identification were encountered. It was hypothesized that, because F IX is present in normal plasma, it might be adsorbed by red cells in vivo and then be detected during antibody screening tests with serum containing F IX inhibitors. CASE REPORT: Sera from two African American half-brothers with hemophilia B were incompatible with all common and rare red cell phenotypes tested in the anti-human globulin test, but did not react with each other's red cells. The brothers' red cell antibodies were neutralized with both normal plasma and a commercially available F IX concentrate, which indicated that the red cell incompatibility was most probably caused by their F IX inhibitors. Red cells from an unrelated patient with hemophilia B and a very low titer of F IX inhibitor were tested against the half-brothers' sera and did not react. The compatible red cells from one of the half-brothers and the unrelated patient with hemophilia B adsorbed F IX from normal plasma or F IX concentrate after 37 degrees C incubation; this rendered them incompatible with the plasma containing F IX inhibitor from the other half-brother. CONCLUSION: F IX appears to be present on normal red cells and may be detected during compatibility and antibody identification procedures when serum or plasma containing F IX inhibitors is tested.     

Functions of AAV-CMV-F.IX And AAV-EF1alpha-F.IX in gene therapy for hemophilia B

There has been substantial progress in using gene therapy to treat animals with hemophilia. Adeno-associated viral (AAV) gene transfer of coagulation factor IX to skeletal muscle and liver of murine and canine models of hemophilia has resulted in sustained systemic expression and, in several studies, in complete cure of the bleeding disorder. Two AAV vectors widely used at present are AAV-CMV-F.IX and AAV-EF1alpha-F.IX. This work compares the predicted molecular functions of AAV-CMV-F.IX and AAV-EF1alpha -F.IX by sequence docking and gene ontology. It is shown that both AAV-CMV-F.IX and AAV-EF1alpha -F.IX induce coagulation factor IXa activity; however, AAV-CMV-F.IX administration also yields coagulation factor XIa activity and AAV-EF1alpha -F.IX treatment results in coagulation factor Xa activity. Therefore, AAV-CMV-F.IX might be useful for factor XI deficiency. AAV-CMV-F.IX has several additional molecular functions and processes compared with AAV-CMV-F.IX.     

An intragenic deletion of the factor IX gene in a family with hemophilia B.

A family of seven patients severely afflicted with hemophilia B has been studied for their factor IX genes through the use of factor IX cDNA and genomic DNA probes. The patients had detectable (less than 10% of normal) factor IX antigen in urine and no detectable inhibitors in sera to factor IX protein. Based on the DNA hybridization analysis, these patients showed a partial intragenic deletion in their factor IX gene. The deletion included two exons (exons V and VI) coding for the amino acid sequence from number 85 to 195 of the factor IX protein. The deleted portion of the gene contained the entire factor IX activation peptide. The length of the deletion was estimated to be 10 +/- 0.3 kilobase pairs. This specific gene has been named FIXSeattle. In this family both the deletion and a Taq 1 restriction fragment length polymorphism can be used as a useful marker for accurate detection of female carriers of the deficient factor IX gene.     

Evidence of multiyear factor IX expression by AAV-mediated gene transfer to skeletal muscle in an individual with severe hemophilia B.

In a phase I study, administration of an AAV2-FIX vector into the skeletal muscle of eight hemophilia B subjects proved safe and achieved local gene transfer and FIX expression for at least 10 months after vector injection, the last time point assessed by muscle biopsy. In hemophilia B dogs we have demonstrated FIX in both muscle biopsies and circulation >4 years following AAV2-FIX injection. Because circulating FIX levels remained less than 1% of normal in human subjects from the study, the duration of AAV2-mediated transgene expression in humans is unknown. We sought to determine if FIX gene transfer and expression persisted locally at injection sites. Muscle biopsies were obtained from one subject 3.7 years following treatment and revealed transgene FIX DNA and protein by quantitative PCR, DNA fluorescence in situ hybridization, and immunohistochemistry for FIX. These results demonstrate, for the first time, multiyear FIX expression by AAV2 vector in humans and suggest that improved muscle delivery provides effective treatment for protein deficiencies or muscle-specific diseases.     

Persistent delivery of factor IX in mice: gene therapy for hemophilia using implantable microcapsules.

Severe hemophilia B is a life-threatening, life long condition caused by absence of or defective coagulation factor IX. Gene therapy could provide an alternative treatment to repeated injection of plasma-derived concentrate or recombinant factor IX. We have previously described the use of implantable microcapsules containing recombinant myoblasts to deliver human factor IX in mice. This study reports the generation of improved myoblast-specific expression vectors. Mouse myoblast clones transfected with the various vectors secreted factor IX in vitro, at rates between 70 and 1000 ng/10(6) cells/day. The recombinant myoblast clones were then encapsulated and implanted into mice. Immunocompetent mice implanted with encapsulated myoblasts had up to 65 ng of factor IX per milliliter in their plasma for up to 14 days, after which antibodies to human factor IX became detectable, and this coincided with decreased factor IX in mouse plasma. In immunodeficient mice, however, factor IX delivery was maintained at a constant level for at least 6 weeks (end of experiment). Interestingly, the highest-secreting myoblast clone in vitro did not deliver the highest level of hFIX in vivo. This discrepancy observed between performance in vitro and in vivo may have important implications for the development of gene therapy protocols based on recombinant cells.     

The measurement of low levels of factor VIII or factor IX in hemophilia A and hemophilia B plasma by clot waveform analysis and thrombin generation assay

BACKGROUND: Precise assessment of clotting function is essential for monitoring of hemostatic treatment for hemophilias A and B. MATERIALS AND METHODS: Clot waveform analysis and thrombin generation assays were performed on factor (F) VIII- and FIX-deficient plasmas, which had been reconstituted with known amounts of recombinant FVIII (rFVIII) and affinity-purified FIX respectively. Clot waveforms were assessed qualitatively and quantitatively by measuring the parameters clotting time, maximum coagulation velocity (Min1), and maximum coagulation acceleration (Min2). The thrombin generation assay was also assessed qualitatively and measurements made of time to peak and peak height. RESULTS: Overall results obtained with both assays showed good correlation for both clotting factors confirming that the changes in clotting waveform reflected changes in thrombin generation. Both assays demonstrated a predictable dose response to the addition of FVIII or IX. However, clot waveform analysis was more sensitive than the thrombin generation assay, particularly in detecting very low levels (0-0.1 IU dL(-1)) of both factors. CONCLUSIONS: These data suggest that the application of clot waveform analysis to the routine management of the hemophiliacs could increase our understanding of the clinical significance of low levels of FVIII and FIX that cannot be measured by assays in current use. This may be particularly useful in the management of hemophiliacs with inhibitors or undergoing gene therapy.     

Three point mutations in the factor IX genes of five hemophilia B patients. Identification strategy using localization by altered epitopes in their hemophilic proteins.

In five patients with hemophilia B and detectable Factor IX antigen, altered reactivity to a specific polyclonal antibody fraction or monoclonal anti-Factor IX antibodies was noted. Amplification of selected portions of their Factor IX genes by polymerase chain reaction allowed rapid identification of a single base transition in each of the five families tested. In a patient with severe hemophilia and an altered calcium binding domain, a G to A transition in exon II changed the codon for Glu-27 to Lys (Factor IXSeattle 3). Patients from two families with mild hemophilia with decreased reactivity to a MAb that binds to a site within the sequence coded for by exon IV had a G to A transition changing the codon for Gly-60 to Ser (Factor IXDurham). Two unrelated patients with moderately severe hemophilia lacked reactivity to another murine monoclonal anti-Factor IX which binds to an epitope in the amino-terminal half of the heavy chain of Factor IXa. In these patients, exon VIII contained a G to A transition changing Arg-248 to Gln (Factor IXSeattle 4).     

Replacing the first epidermal growth factor-like domain of factor IX with that of factor VII enhances activity in vitro and in canine hemophilia B.

Using the techniques of molecular biology, we made a chimeric Factor IX by replacing the first epidermal growth factor-like domain with that of Factor VII. The resulting recombinant chimeric molecule, Factor IXVIIEGF1, had at least a twofold increase in functional activity in the one-stage clotting assay when compared to recombinant wild-type Factor IX. The increased activity was not due to contamination with activated Factor IX, nor was it due to an increased rate of activation by Factor VIIa-tissue factor or by Factor XIa. Rather, the increased activity was due to a higher affinity of Factor IXVIIEGF1 for Factor VIIIa with a Kd for Factor VIIIa about one order of magnitude lower than that of recombinant wild-type Factor IXa. In addition, results from animal studies show that this chimeric Factor IX, when infused into a dog with hemophilia B, exhibits a greater than threefold increase in clotting activity, and has a biological half-life equivalent to recombinant wild-type Factor IX.     

Treatment with a pure factor IX concentrate in a patient with moderate hemophilia B undergoing bilateral total hip replacement

Recently, a pure factor IX concentrate, licensed in Sweden as Immunine, was prepared through ion exchange and hydrophobic chromotography. Virus inactivation included two steps of steam treatment. Reconstituted Immunine could be kept at room temperature for at least 7 days without any substantial loss of activity. A patient with moderate hemophilia B undergoing bilateral total hip replacement was continuously infused with Immunine by use of a pump. There were no bleeding complications or signs of thrombosis, and the operation markedly improved the patient's range of motion and quality of life.