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PubMed1990

Characterization of recombinant human factor IX expressed in transgenic mice and in derived trans-immortalized hepatic cell lines.

Transgenic mice were generated in which 5 kb of the 5' flanking promoter region of the human Factor IX (FIX) gene fused to various FIX constructs (gene, minigene and cDNA) were stably integrated in the germ line. Several transgenic mouse lines expressed high circulating levels of active and correctly processed recombinant human FIX. The presence of at least one FIX intron had a positive effect on the expression. The FIX transgenes were expressed in a tissue-specific manner in the liver of transgenic mice. By crossing transgenic mice synthesizing FIX with others prone to develop hepatoma, progeny which co-express the transgenes in hepatocytes were obtained. Hepatoma-derived cell lines were shown to have a differentiated phenotype and secrete active human FIX for many generations.

Amino Acid Sequence↗
PubMed1993

Germline mutations in the factor IX gene: a comparison of the pattern in Caucasians and non-Caucasians.

DNA samples of patients from around the world have been sequenced to precisely define the mutations in the factor IX gene resulting in hemophilia B. This study compares the patterns of independent mutation between 127 Caucasian and 44 non-Caucasian patients with hemophilia B. Caucasians and non-Caucasians are found to have similar patterns of transitions and transversions (both at CpG and at non-CpG sites) as well as insertions, deletions, microdeletions, and complex changes (chi 2 = 2.71, p = 0.922). An analysis of subgroups of transitions and transversions shows similar patterns among Caucasians and non-Caucasians (chi 2 = 2.98, p = 0.83). If the subset of non-Caucasian samples (24) in which the mutation is known to have occurred outside of the United States, Canada, and Europe (UCE) is compared to the Caucasian subset, the above conclusions are unchanged. The invariant nature of this pattern of mutation is most simply compatible with a predominance of endogenous processes or common mutagen exposure rather than mutagen exposure specifically associated with non-Caucasian status or non-Western lifestyle.

DNA Mutational Analysis↗
PubMed1984

The contribution of Ca2+ and phospholipids to the activation of human blood-coagulation Factor X by activated Factor IX.

The role of the cofactors Ca2+ and phospholipid in the activation of human Factor X by Factor IXa was investigated. By use of a sensitive spectrophotometric Factor Xa assay, it was demonstrated that human Factor IXa can activate Factor X in the absence of cofactors. The presence of Ca2+ as the only cofactor resulted in a 7-fold stimulation of the Factor Xa formation. Kinetic analysis of the Ca2+-stimulated reaction showed that the apparent Km of Factor X was 4.6 microM, whereas the apparent Vmax. for Factor Xa formation was 0.0088 mol of Xa/min per mol of IXa. The presence of phospholipid as the only cofactor had no effect on the rate of Factor Xa formation. However, a several-hundred-fold stimulation was observed when Ca2+ and phospholipid were present in combination. The activation of Factor X in the presence of Ca2+ and phospholipid was found to be kinetically heterogeneous, involving both phospholipid-bound and free reactants. Quantitative data concerning the phospholipid binding of Factors IXa and X were used to study the relation between the rate of Factor Xa formation and the binding of enzyme and substrate to the phospholipid membrane. The results support the hypothesis that phospholipid-bound Factor X is the substrate in the phospholipid-stimulated reaction; however, phospholipid-bound and free Factor IXa seem to be equally efficient in catalysing the activation of phospholipid-bound Factor X.

Calcium↗
PubMed1994

Properties of recombinant chimeric human protein C and activated protein C containing the gamma-carboxyglutamic acid and trailing helical stack domains of protein C replaced by those of human coagulation factor IX.

The properties of a recombinant (r) chimeric human protein C (PC) containing replacement of its gamma-carboxyglutamic acid (Gla) and helical stack (HS) domains by those of human coagulation factor IX (fIX) have been examined. Titration with Ca2+ of the divalent cation-induced intrinsic fluorescence quenching of this chimera (r-GDIX/PC) allowed determination of the [Ca2+], of 1.8 mM, required to produce this alteration in 50% of the protein molecules. These values were 0.41 and 0.61 mM for wtr-PC and fIX, respectively. The chimera did not react with a Ca(2+)-dependent, Gla domain-directed conformational monoclonal antibody (MAb) to r-PC but did interact with a similar MAb (H5B7) to fIX. The [Ca2+] required to induced H5B7 binding to 50% of the r-GDIX/PC molecules was 6.6 mM, while this same value for fIX was a nearly identical 7.2 mM. The [Ca2+] needed for binding of 50% of r-GDIX/PC to acidic phospholipid (PL) vesicles was 0.58 mM, while that for wtr-PC and fIX were 1.2 and 0.55 mM, respectively. The [protein] required for 50% binding of r-GDIX/PC to PL at 20 mM Ca2+ was 0.29 microM. These same values for r-PC and fIX were 0.38 and 1.8 microM, respectively. The Ca(2+)-mediated inhibition of the thrombin-catalyzed activation of r-GDIX/PC was characterized by a Ki of 118 microM, a value similar to that of 125 microM obtained for this same inhibition of wtr-PC activation. The thrombin-catalyzed activation of both r-GDIX/PC and wtr-PC was stimulated by soluble r-thrombomodulin. Similar to the case of wtr-PC, Ca2+ initially enhanced and, at higher concentrations, inhibited the activation of r-GDIX/PC. The Km and kcat values for this latter activation at optimal [Ca2+] (100 microM) were 4.1 microM and 2.5 s-1, respectively. These same kinetic constants for activation of wtr-PC were 4.3 microM and 2.9 s-1, respectively. These results show that many of the features needed for functional integrity of the Ca2+-bound Gla/HS domains of PC are also present in those same modules of fIX, a finding that points to a generalized functional role for the Ca2+-induced conformation of the structural unit consisting of the Gla and HS domains. The data also suggest that the Ca2+-bound form of the Gla/HS region is an independently folded unit in PC and perhaps in fIX.(ABSTRACT TRUNCATED AT 400 WORDS)

1-Carboxyglutamic Acid↗
PubMed2000

Single nucleotide polymorphisms of the factor IX gene for linkage analysis in the southern Chinese population.

Carrier detection and prenatal testing for haemophilia B in Oriental populations have been hampered by the lack of informative markers within the factor IX (FIX) gene. We detected a T/C nucleotide variation at nucleotide 32770 in the poly-A region of the FIX gene in the mother of a haemophilia B child. Analysis of 139 unrelated alleles revealed a heterozygosity rate of 0.193, thus offering an additional marker for linkage analysis. Together with two other polymorphic sites (5' MseI and 3' HhaI) found in Chinese and Thai populations, these polymorphisms were useful in 66% of the families studied.

China↗
PubMed1979

Transmission of non-A non-B hepatitis to chimpanzees by factor-IX concentrates after fatal complications in patients with chronic liver disease.

6 cases of non-A non-B hepatitis which followed administration of four different batches of concentrates of coagulation factor IX from commercial and non-commercial sources are described. Of 17 patients who received the concentrate on account of chronic liver disease, 4 developed hepatitis, and in 3 of these the illness proved fatal. The incubation periods ranged from 42 to 103 days (mean 65 days). 3 chimpanzees were inoculated with concentrate from the same batch used on the above patients, a further commercial batch upon which no adverse reactions had been reported, and plasma from a known non-A non-B carrier. All developed hepatitis after 10 weeks' incubation. Liver biopsy when serum-aminotransferase was at its highest level showed features consistent with acute hepatitis. As in the patients, viral markers for hepatitis A and B, cytomegalovirus, and Epstein-Barr virus were unchanged.

Acute Disease↗
PubMed1996

Implantation of autologous skin fibroblast genetically modified to secrete clotting factor IX partially corrects the hemorrhagic tendencies in two hemophilia B patients.

OBJECTIVE: To examine the safety and effects of gene therapy for hemophilia B by implantation of autologous fibroblasts genetically modified to secrete clotting factor IX (hFIX). PATIENTS AND METHODS: Two hemophilia B patients LD and LW were selected from one family to accept gene transfer study. The hFIX protein of both patients were about 100 ng/ml plasma and hFIX activity was about 2%. The autologous skin fibroblasts of the two patients were genetically modified by retrovirus-mediated gene transfer with XL-IX and N2CMVIX vector (HBSF-IX). Human hFIX protein was measured by ELISA, hFIX activity was measured by one-stage clotting assay and barium citrate sorbent method. hFIX inhibitor was assayed by Bethesda methods. Human hFIX cDNA was detected by PCR. HBSF-IX cells were mixed with collagen for injection after safety assessments. RESULTS: The HBSF-IX cells from the two patients secreted hFIX at high levels in vitro. After implantation of autologous HBSF-IX cells, no treatment-related side effects were observed. Plasma hFIX protein in both patients increased over 2 folds after several injections of HBSF-IX cells and persisted for more than 420 days. Blood clotting activity increased significantly in both patients, hemorrhagic tendencies have been partially corrected after treatment. Further elevation of hFIX can be achieved by repeating the same treatment 420 days later in Patient LD. CONCLUSIONS: Implantation of autologous fibroblast genetically modified to secrete human hFIX offers a simple, safe and effective approach to gene therapy of hemophilia B.

Adolescent↗
PubMed1992

Determination of total protein in highly purified factor IX concentrates.

Protein determination by the methods of Kjeldahl, Biuret, Bradford and UV absorbance at 280 nm have been studied in regard to accuracy, precision and simplicity. A reference preparation of a highly purified factor IX concentrate, Nanotiv, reconstituted to 1/5 of ordinary volume was used in the study in order to make a comparison between the different procedures. The Kjeldahl method resulted in a protein concentration of 3.7 mg/ml, whereas the Biuret, Bradford (BSA) and UV absorbance at 280 nm resulted in protein concentrations of 3.6, 2.5 and 2.8 mg/ml, respectively. The corresponding values for specific activity were 136, 140, 200 and 179 IU/mg, respectively. These results demonstrate a great variation in the response obtained by different methods for determination of total protein.

Biuret Reaction↗
PubMed1993

Variations among Japanese of the factor IX gene (F9) detected by PCR-denaturing gradient gel electrophoresis.

In the course of feasibility studies to examine the efficiencies and practicalities of various techniques for screening for genetic variations, the human coagulation factor IX (F9) genes of 63 Japanese families were examined by PCR-denaturing gradient gel electrophoresis (PCR-DGGE). Four target sequences with lengths of 983-2,891 bp from the F9 genes of 126 unrelated individuals from Hiroshima and their 100 children were amplified by PCR, digested with restriction enzymes to approximately 500-bp fragments, and examined by DGGE--a total of 6,724 bp being examined per individual. GC-rich sequences (GC-clamps) of 40 bp were attached to both ends of the target sequences, as far as was feasible. Eleven types of new nucleotide substitutions were detected in the population, none of which produced RFLPs or caused hemophilia B. By examining two target sequences in a single lane, approximately 8,000 bp in a diploid individual could be examined. This approach is very effective for the detection of variations in DNA and is applicable to large-scale population studies.

Base Sequence↗
PubMed2004

Therapeutic levels of factor IX expression using a muscle-specific promoter and adeno-associated virus serotype 1 vector.

Extensive studies in animal models of the X-linked bleeding disorder hemophilia B (deficiency in functional coagulation factor IX, F.IX) have shown that muscle-directed adeno-associated (AAV)-mediated F.IX gene transfer can be used to treat this disease. However, large vector doses of AAV-2 vector are required for therapeutic levels of expression, and the number of vector doses that can be injected per intramuscular site is limited. Several studies have shown that some of these limitations can be overcome by use of AAV serotype 1 vector. Here, we demonstrate levels of F.IX transgene expression from a synthetic muscle-specific promoter (C5-12) that were higher than from the cytomegalovirus (CMV) immediate-early enhancer-promoter in cultured muscle cells in vitro and approximately 50% of CMV-driven expression in vivo in murine skeletal muscle after AAV-1 gene transfer. These data show for the first time that a tissue-specific promoter can be used to achieve therapeutic levels of muscle-derived F.IX expression in the context of viral gene transfer. However, use of a muscle-specific promoter did not prevent antibody formation in response to a murine F.IX transgene product in mice with F.IX gene deletion, indicating that the risk of humoral immune responses remains in the context of an immunologically unfavorable mutation.

Animals↗
PubMed1999

Persistent expression of canine factor IX in hemophilia B canines.

We previously demonstrated that direct intramuscular injection of recombinant adeno-associated virus (rAAV) carrying the human FIX (hFIX) cDNA can safely be administered to hemophilic B canines and express human factor IX protein; however, the functional activity of the hFIX protein could not be assessed due to anti-human FIX antibody (inhibitor) formation. To test the therapeutic efficacy of rAAV in hemophilic dogs, rAAV type 2 (rAAV2) carrying canine FIX (cFIX) cDNA was injected into the skeletal muscle of two dogs at doses of 1012-13particles. Circulating cFIX protein levels were maintained for 1 year at levels of 1-2% of normal. Hemostatic correction (WBCT and APTT) paralleled plasma FIX antigen levels. Both dogs still required plasma infusion for spontaneous and traumatic bleeding events. Inhibitors to cFIX protein were not detected in either animal by Bethesda assay. Neutralizing antibodies directed against AAV-2 capsid were pronounced and persistent. Vector DNA and mRNA transcripts were detected only at the injected skeletal muscle tissue. Analysis of both high and low molecular weight DNA identified both replicative episomal and integrated AAV species. These results demonstrate that persistent secretion of the FIX transgene protein, necessary for successful gene therapy of hemophilia B, can be achieved using the parvovirus-based rAAV vector

Animals↗
PubMed2000

Evidence for gene transfer and expression of factor IX in haemophilia B patients treated with an AAV vector.

Pre-clinical studies in mice and haemophilic dogs have shown that introduction of an adeno-associated viral (AAV) vector encoding blood coagulation factor IX (FIX) into skeletal muscle results in sustained expression of F.IX at levels sufficient to correct the haemophilic phenotype. On the basis of these data and additional pre-clinical studies demonstrating an absence of vector-related toxicity, we initiated a clinical study of intramuscular injection of an AAV vector expressing human F.IX in adults with severe haemophilia B. The study has a dose-escalation design, and all patients have now been enrolled in the initial dose cohort (2 x 10(11) vg/kg). Assessment in the first three patients of safety and gene transfer and expression show no evidence of germline transmission of vector sequences or formation of inhibitory antibodies against F.IX. We found that the vector sequences are present in muscle by PCR and Southern-blot analyses of muscle biopsies and we demonstrated expression of F.IX by immunohistochemistry. We observed modest changes in clinical endpoints including circulating levels of F.IX and frequency of FIX protein infusion. The evidence of gene expression at low doses of vector suggests that dose calculations based on animal data may have overestimated the amount of vector required to achieve therapeutic levels in humans, and that the approach offers the possibility of converting severe haemophilia B to a milder form of the disease.

Adult↗
PubMed1978

Spontaneously acquired factor IX inhibitor in a nonhemophiliac child.

A 2 1/2-year-old, previously healthy child developed progressive swelling of the left leg and a hematoma of the anterior chest wall associated with a falling hemoglobin concentration, as a result of a spontaneously acquired Factor IX inhibitor. Successful management of her condition required a four-volume exchange transfusion and immunosuppressive therapy consisting of cyclophosphamide for four days and prednisone for one month. A brief review of the literature regarding the occurrence and nature of acquired coagulation factor inhibitors and the role of immunosuppressive therapy is also presented.

Blood Coagulation↗
PubMed1989

Aspartyl beta-hydroxylase: in vitro hydroxylation of a synthetic peptide based on the structure of the first growth factor-like domain of human factor IX.

beta-Hydroxylation of aspartic acid is a post-translational modification that occurs in several vitamin K-dependent coagulation proteins. By use of a synthetic substrate comprised of the first epidermal growth factor-like domain in human factor IX and either mouse L-cell extracts or rat liver microsomes as the source of enzyme, in vitro aspartyl beta-hydroxylation was accomplished. Aspartyl beta-hydroxylase appears to require the same cofactors as known alpha-ketoglutarate-dependent dioxygenases. The hydroxylation reaction proceeds with the same stereospecificity and occurs only at the aspartate corresponding to the position seen in vivo. Further purification and characterization of this enzymatic activity should now be possible.

Decarboxylation↗
PubMed2004

Factor IX variants improve gene therapy efficacy for hemophilia B.

Intramuscular injection of adeno-associated viral (AAV) vector to skeletal muscle of humans with hemophilia B is safe, but higher doses are required to achieve therapeutic factor IX (F.IX) levels. The efficacy of this approach is hampered by the retention of F.IX in muscle extracellular spaces and by the limiting capacity of muscle to synthesize fully active F.IX at high expression rates. To overcome these limitations, we constructed AAV vectors encoding F.IX variants for muscle- or liver-directed expression in hemophilia B mice. Circulating F.IX levels following intramuscular injection of AAV-F.IX-K5A/V10K, a variant with low-affinity to extracellular matrix, were 2-5 fold higher compared with wild-type (WT) F.IX, while the protein-specific activities remained similar. Expression of F.IX-R338A generated a protein with 2- or 6-fold higher specific activity than F.IX-WT following vector delivery to skeletal muscle or liver, respectively. F.IX-WT and variant forms provide effective hemostasis in vivo upon challenge by tail-clipping assay. Importantly, intramuscular injection of AAV-F.IX variants did not trigger antibody formation to F.IX in mice tolerant to F.IX-WT. These studies demonstrate that F.IX variants provide a promising strategy to improve the efficacy for a variety of gene-based therapies for hemophilia B.

Adenoviridae↗
PubMed2001

Factors influencing the development of an anti-factor IX (FIX) immune response following administration of adeno-associated virus-FIX.

The present study sought to determine the impact of the route of administration of an adeno-associated virus (AAV) vector encoding human factor IX (hFIX) on the induction of an immune response against the vector and its xenogenic transgene product, hFIX. Increasing doses of AAV-hFIX were administered by different routes to C57Bl/6 mice, which typically demonstrate significant immune tolerance to hFIX. The route of delivery had a profound impact on serum hFIX levels as well as the induction of an anti-hFIX humoral immune response. At all dose levels tested, delivery of AAV-hFIX by an intramuscular (IM) route induced an antibody response against the human FIX protein and no hFIX was detected in the serum of animals even at doses of 2 x 10(11) DNA viral particles (vp) of AAV-hFIX. This was in stark contrast to the mice that received AAV-hFIX by intraportal vein (IPV) administration. No anti-hFIX inhibitors were observed in any of these mice and therapeutic levels of hFIX were detected in the serum of all mice that received doses of 2 x 10(10) vp AAV-hFIX and higher. When pre-existing neutralizing immunity to AAV was established in mice, AAV-hFIX administration by either the IM or IPV routes did not result in detectable serum hFIX. Although hFIX expression was not observed in mice with pre-existing neutralizing immunity to AAV, an anti-hFIX response was induced in all of the animals that received AAV-hFIX by the IM route. This was not observed in the preimmune mice that received AAV-hFIX by IPV administration. These results suggest that the threshold of inducing an immune response against a secreted transgene product, in this case the xenoprotein hFIX, is lower when the vector is administered by the IM route even in animals with pre-existing immunity to AAV. (Blood. 2001;97:3733-3737)

Adenoviridae↗
PubMed1995

Transfer of specific endothelial cell-binding properties from the procoagulant protein human factor IX into the anticoagulant protein human protein C.

A series of recombinant (r) chimeric mutants of human coagulation protein C (PC) and activated protein C (APC) containing replacements of homologous PC domains by those of human coagulation factor IX (fIX) were generated, with the intention of determining whether the specific bovine aortic endothelial cell (BAEC) receptor-binding characteristics of fIX could be incorporated into the chimeric r-PC while maintaining the essential properties of PC and APC. Using a competitive BAEC displacement assay with [125I]fIX, we found that a chimeric r-PC (r[delta PC1-46/delta fIX1-47]PC), consisting of the entire gamma-carboxyglutamic domain ([GDIX], residues 1-38) and helical stack ([HSIX], residues 38-47) of fIX as replacements for these same domains of PC, provided an IC50 for fIX-related BAEC binding of 13 nM, as compared to 10 nM for that of unlabeled fIX. This showed that all of the BAEC tight binding determinants for fIX existed within the [GDIX/HSIX]. Additionally, this chimera reacted to the same extent as fIX with the Ca(2+)-dependent, [GDIX]-specific monoclonal antibody H5B7 and lost its reactivity to a similar antibody specific for the [GDPC], JTC1. A synthetic peptide containing residues 1-47 of fIX also competed effectively (IC50 = 16 nM) with intact fIX for BAEC binding. Displacement of [125I]fIX from BAEC did not occur with a chimera containing the [HSIX] alone or with another mutant protein possessing a replacement of the two epidermal growth factor (EGF) homology regions of r-PC (residues 47-137) with those same domains of fIX.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1990

In vitro gamma-carboxylation of a 59-residue recombinant peptide including the propeptide and the gamma-carboxyglutamic acid domain of coagulation factor IX. Effect of mutations near the propeptide cleavage site.

We report the expression in Escherichia coli of a fusion protein that contains the propeptide sequence and gamma-carboxyglutamic acid domain (residues -18 to 41) of human factor IX (FIXGla). CNBr was used to release FIXGla from the fusion protein. The 59-amino acid peptide is an efficient substrate for in vitro gamma-carboxylation. Its Km,app (0.55 microM) is several thousand-fold lower than that of the commonly used substrate FLEEL and about 5 times lower than proPT28 or proFIX28, (Hubbard, B. R., Jacobs, M., Ulrich, M. M. W., Walsh, C., Furie, B., and Furie, B. C. (1989) J. Biol. Chem. 264, 14145-14150). In addition, FIXGla is the first peptide substrate that is carboxylated in vitro to more than one gamma-carboxyglutamic acid/molecule (6-11 gamma-carboxyglutamic acids/molecule). We created peptides with mutations identical to FIXSan Dimas or FIXCambridge as well as a peptide with both mutations in the propeptide sequence and examined the effect of the mutations on in vitro carboxylation. Enzyme kinetic studies revealed no significant difference in Vmax/Km values between normal and mutant substrates. Maximum carbon dioxide incorporation was achieved with the double mutant. From these data we conclude the following. 1) FIXGla and its mutants are excellent substrates for studying the mechanism of gamma-carboxylase. 2) Although arginines at positions -4 and -1 are highly conserved in the propeptide sequence of all the vitamin K-dependent proteins, neither is critical for gamma-carboxylation.

1-Carboxyglutamic Acid↗