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PubMed1998

Sustained expression of high levels of human factor IX from human cells implanted within an immunoisolation device into athymic rodents.

Immunoisolation of allogeneic cells within a membrane-bound device is a unique approach for gene therapy. We employed an immunoisolation device that protects allograft, but not xenograft, cells from destruction, to implant a human fibroblast line (MSU 1.2) in athymic rodents. Cells, transduced with the MFG-human factor IX retroviral vector, and expressing 0.9 microg/10(6) cells/day in vitro, were implanted in rats (four 40-microl devices, each containing 2 x 10(7) cells, two subcutaneously, two in epididymal fat) and in mice (two 20-microl devices, each containing 2 x 10(6) cells, subcutaneously). Plasma factor IX levels increased for 50 days, reaching maxima of 203 ng/ml (rat) and 597 ng/ml (mouse), and both continued at greater than 100 ng/ml for more than 140 days. A clone derived from the transduced cells, making 5 microg of factor IX/10(6) cells/day, was implanted within a device (one 20-microl device containing 2.5 x 10(6) cells), or without a device (1 x 10(7) cells implanted freely), either subcutaneously or in epididymal fat. The freely implanted cells expressed transiently, reaching more than 100 ng/ml in each site by day 4, but dropped to zero by day 20 (subcutaneous) or day 90 (epididymal fat). In devices, levels gradually increased to 100 ng/ml (subcutaneous) or 300 ng/ml (epididymal fat), remaining high for more than 100 days. These results show long-term, high-level expression of a human protein: (1) when cells are implanted within a cell transplantation device, but not when the cells are freely implanted, and (2) from a transgene driven by a viral promoter. An alloprotective device will enable the use of cloned cell lines that can be subjected to stringent quality control assessment that is impossible to achieve with autologous approaches.

Animals↗
PubMed1996

The factor IX gene as a model for analysis of human germline mutations: an update.

The variation generated by germline mutation is essential for evolution, but individuals pay a steep price in the form of Mendelian disease and genetic predisposition to complex disease. Indeed, the health of a species is determined ultimately by the rate of germline mutation. Analysis of the factor IX gene in patients with hemophilia B has provided insights into the human germline mutational process. Herein, seven topics will be reviewed with emphasis on recent advances: (i) proposed mechanisms of deletions, inversions, and insertions; (ii) discordant sex ratios of mutation and associated age effects; (iii) somatic mosaicism; (iv) founder effects; (v) mutation rates; (vi) the factor IX gene as a germline mutagen test; and (vii) cancer as a possible mechanism for maintaining a constant rate of germline mutation.

Age Factors↗
PubMed1993

Recurrent mutations in the factor IX gene: founder effect or repeat de novo events. Investigation of the German haemophilia B population and review of de novo mutations.

The investigation of 114 unrelated patients, representing about half the sample of the German haemophilia B population, enabled us to delineate the causative mutation in 103 (90.4%) haemophilic factor IX genes. Of these 103 cases 84 (81.6%) turned out to be unique molecular events, the remainder being repeats. Haplotype analysis revealed that the great majority, if not all, of these recurrent observations occurred independently. This conclusion is supported by our finding that three de novo mutations could be demonstrated at two sites of frequent mutation. A further 20 de novo events could be established in an unselected sample of 37 families with sporadic haemophilia B and 37 families with a history of the disease. Altogether, the germ line of origin could be determined in 21 of these 23 cases, thereby indicating a ratio of male to female mutation rates close to 2. On the basis of the data available, it is becoming clear that rearrangements in the factor IX gene (35.4% of de novo cases) are responsible for haemophilia B at a higher frequency than has been observed today (12.3%). More than two-thirds of the de novo cases cause the severe form of the disease, thereby reflecting the deficit of these haemophilic genes in the actual gene pool because of excess mortality in the past. In addition 40% (12/30) of the de novo single-base mutations were transitions at CpG dinucleotides. Compared with the expected at-random frequency, this observation indicates an 83-fold enhancement of mutation at CpG.

DNA Mutational Analysis↗
PubMed1992

Missense mutations and the magnitude of functional deficit: the example of factor IX.

Some missense changes are compatible with normal protein function while others compromise essential aspects of protein maturation, specific activity, or stability. For those missense changes that alter function in the intact organism, how likely is it for the mutated protein to retain appreciable residual activity? By genetic analysis of patients with hemophilia B of known severity, this question can be addressed for missense mutations that reduce factor IX activity by fourfold or more below the average. We estimate that missense changes cause only 59% of moderate and severe disease, but these mutations are almost always (95%) of independent origin. In contrast, missense mutations are found in virtually all (97%) families with mild disease, but only a minority of these (41%) are of independent origin. From the aggregate data, we estimate that most (71%) of the independent deleterious missense mutations cause at least a 20-fold decrease in factor IX activity.

Factor IX↗
PubMed1993

Identification and structural analysis of the tetrasaccharide NeuAc alpha(2-->6)Gal beta(1-->4)GlcNAc beta(1-->3)Fuc alpha 1-->O-linked to serine 61 of human factor IX.

O-Linked fucose has been found attached to Thr/Ser residues within the sequence Cys-X-X-Gly-Gly-Thr/Ser-Cys in the N-terminal EGF domains of several coagulation/fibrinolytic proteins. Carbohydrate composition and mass spectrometric analyses of tryptic and thermolytic peptides containing the corresponding site (Ser-61) in the first EGF domain of human factor IX indicated the presence of a tetrasaccharide containing one residue each of sialic acid, galactose, N-acetylglucosamine, and fucose. The Ser-61 tetrasaccharide was not susceptible to alpha-fucosidase digestion. Fragments generated during mass spectrometric analysis indicated that fucose was the attachment sugar residue. The involvement of fucose in the carbohydrate-peptide linkage was confirmed by two-dimensional 1H NMR spectroscopic analysis of the glycopeptide containing factor IX residues 57-65. The complete structure of the tetrasaccharide was obtained by methylation analysis and two-dimensional 1H TOCSY and ROESY experiments as NeuAc alpha(2-->6)Gal beta(1-->4)GlcNAc beta(1-->3)Fuc alpha 1-->O-Ser61.

Amino Acid Sequence↗
PubMed2003

Anticoagulant activity of M-LAO, L-amino acid oxidase purified from Agkistrodon halys blomhoffii, through selective inhibition of factor IX.

One of haemorrhagic toxins present in snake venoms is L-amino acid oxidase (LAO), which catalyzes the oxidative deamination of L-amino acids with the generation of hydrogen peroxide. Although it is widely accepted that LAO alters platelet function, the effects of LAO on human blood coagulation remain largely unknown. The present study demonstrated, for the first time, that M-LAO, LAO purified from the venom of Agkistrodon halys blomhoffii (Japanese mamushi), possesses an anticoagulant activity. Thrombelastography (TEG) showed that M-LAO significantly delayed the onset and the progress of the coagulation process. In addition, the enzyme prolonged the activated partial thromboplastin time (aPTT) dose-dependently, but had little effect on the prothrombin time (PT), suggesting that its principal activity was mediated in the intrinsic coagulation pathway. Furthermore, M-LAO reduced factor IX procoagulant activity in a dose-dependent manner and did not affect other coagulation factors. These results indicate that M-LAO has an anticoagulant activity that impairs the intrinsic clotting by inhibiting factor IX.

Agkistrodon↗
PubMed2000

Comparing the antithrombotic efficacy of a humanized anti-factor IX(a) monoclonal antibody (SB 249417) to the low molecular weight heparin enoxaparin in a rat model of arterial thrombosis.

A humanized inhibitory anti-factor IX(a) antibody (SB 249417) has been compared to enoxaparin (Lovenox) in a rat model of arterial thrombosis. Pretreatment of rats with either SB 249417 (3.0 mg/kg, i. v.) or enoxaparin (30.0 mg/kg, i.v. or s.c.) resulted in comparable and significant reductions in thrombus formation. However, the efficacious dose of enoxaparin resulted in >30-fold increase in the aPTT over baseline, while the efficacious dose of SB 249417 prolonged the aPTT by only approximately 3-fold. Additionally, pretreatment with SB 249417 resulted in sustained blood flow and arterial patency throughout the experiment in >80% of rats treated. In contrast, <30% of rats pretreated with enoxaparin remained patent throughout the experiment. The data in this report indicate that the selective inhibition of factor IX(a) with the monoclonal antibody SB 249417 produces a superior antithrombotic profile to that of the low molecular weight heparin enoxaparin.

Animals↗
PubMed1997

Persistent expression of human clotting factor IX from mouse liver after intravenous injection of adeno-associated virus vectors.

We previously found that gene transduction by adeno-associated virus (AAV) vectors in cell culture can be stimulated over 100-fold by treatment of the target cells with agents that affect DNA metabolism, such as irradiation or topoisomerase inhibitors. Here we show that previous gamma-irradiation increased the transduction rate in mouse liver by up to 900-fold, and the topoisomerase inhibitor etoposide increased transduction by about 20-fold. Similar rates of hepatic transduction were obtained by direct injection of the liver or by systemic delivery via tail vein injection. Hepatocytes were much more efficiently transduced than other cells after systemic delivery, and up to 3% of all hepatocytes could be transduced after one vector injection. The presence of wild-type AAV, which contaminates many AAV vector preparations, was required to observe a full response to gamma-irradiation. Injection of mice with AAV vectors encoding human clotting factor IX after gamma-irradiation resulted in synthesis of low levels of human clotting factor IX for the 5-month period of observation. These studies show the potential of targeted gene transduction of the liver by AAV vectors for treatment of various hematological or metabolic diseases.

Animals↗
PubMed2003

Binding of the factor IX gamma-carboxyglutamic acid domain to the vitamin K-dependent gamma-glutamyl carboxylase active site induces an allosteric effect that may ensure processive carboxylation and regulate the release of carboxylated product.

Propeptides of the vitamin K-dependent proteins bind to an exosite on gamma-glutamyl carboxylase; while they are bound, multiple glutamic acids in the gamma-carboxyglutamic acid (Gla) domain are carboxylated. The role of the propeptides has been studied extensively; however, the role of the Gla domain in substrate binding is less well understood. We used kinetic and fluorescence techniques to investigate the interactions of the carboxylase with a substrate containing the propeptide and Gla domain of factor IX (FIXproGla41). In addition, we characterized the effect of the Gla domain and carboxylation on propeptide and substrate binding. For the propeptide of factor IX (proFIX18), FIXproGla41, and carboxylated FIXproGla41, the Kd values were 50, 2.5, and 19.7 nM and the koff values were 273 x 10(-5), 9 x 10(-5), and 37 x 10(-5) s(-1), respectively. The koff of proFIX18 is reduced 3-fold by FLEEL and 9-fold by the Gla domain (residues 1-46) of FIX. The pre-steady state rate constants for carboxylation of FIXproGla41 was 0.02 s(-1) in enzyme excess and 0.016 s(-1) in substrate excess. The steady state rate in substrate excess is 4.5 x 10(-4) s(-1). These results demonstrate the following. 1) The pre-steady state carboxylation rate constant of FIXproGla41 is significantly slower than that of FLEEL. 2) The Gla domain plays an allosteric role in substrate-enzyme interactions. 3) Carboxylation reduces the allosteric effect. 4) The similarity between the steady state carboxylation rate constant and product dissociation rate constant suggests that product release is rate-limiting. 5) The increased dissociation rate after carboxylation contributes to the release of product.

1-Carboxyglutamic Acid↗
PubMed2004

Two novel mutations in EGF-like domains of human factor IX dramatically impair intracellular processing and secretion.

We investigated the mechanisms responsible for severe factor IX (FIX) deficiency in two cross-reacting material (CRM)-negative hemophilia B patients with a mutation in the first and second epidermal growth factor (EGF) domains of FIX (C71Y and C109Y, respectively). We have determined the kinetics of mutant FIX biosynthesis and secretion in comparison with wild-type FIX (FIXwt). In transfected cells, FIXwt was retrieved as two intracellular molecular forms, rapidly secreted into the culture medium. One appeared to be correctly N-glycosylated, and corresponded to a form trafficking between the endoplasmic reticulum (ER) and Golgi apparatus. The other corresponded to the mature form, ready to be secreted, exhibiting correct N-glycosylation and sialylation. In contrast, the two mutants, FIXC71Y and FIXC109Y, were not secreted from the cells and did not accumulate intracellularly. Relative to FIXwt, they were retained longer in the ER and were only N-glycosylated. In addition, the intracellular concentration of the FIX mutants increased when ALLN, an inhibitor of cysteine proteases and of the proteasome degradation pathway, was added to the culture medium. Both the FIX mutants and FIXwt were associated in the ER with the 78-kDa glucose-regulated protein (GRP78/BiP) and calreticulin (CRT), though the amount of CRT associated with the two mutants was twice as strong as with FIXwt. These results strongly suggest that chaperone and lectin molecules act in concert to ensure both proper folding of FIXwt and the retention of mutant molecules.

Biological Transport↗
PubMed1991

Protein engineering of the propeptide of human factor IX.

Vitamin-K-dependent plasma proteins contain a highly conserved propeptide sequence located between the classical hydrophobic leader sequence and the N-terminus of the mature protein. This acts as a recognition sequence for the vitamin-K-dependent carboxylase which catalyses the conversion of specific glutamate residues to gamma-carboxyglutamate (Gla) residues in the adjacent Gla domain. Protein engineering of the 18 residue propeptide from human factor IX has highlighted the importance of residues -16Phe and -10Ala with respect to carboxylase recognition. In addition, studies of haemophilia B patients have shown that C-terminal propeptide residues -4Arg and -1Arg are required for proteolysis of the propeptide from the mature protein. To extend these previous studies we have introduced two novel mutations into the propeptide of human factor IX at positions -17(Val----Asp) and -6(Leu----AsP), and studied the effect of these changes on gamma-carboxylation and proteolytic processing. Both mutations reduce the expression of a calcium-dependent epitope in the Gla domain; however, only -6Leu----Asp shows reduced binding to barium sulphate. In addition, this latter mutation prevents proteolytic processing of the propeptide. These data support the current hypothesis that the propeptide contains two recognition elements: one for carboxylase recognition located towards the N-terminus, and one for propeptidase recognition located near the C-terminus.

Amino Acid Sequence↗
PubMed1983

Detection of factor IX inhibitors by immunoradiometric assay.

An immunoradiometric assay (IRMA) for the determination of factor IX inhibitors in haemophilia B was developed. The assay was based on competitive binding between radiolabelled anti-IX and inhibitors in the test material of immobilized IX:C. 5 haemophiliacs with known inhibitors were investigated with the new method and with a conventional neutralization test. An inhibitor titre as low as 5 X 10(-4) U/ml could be measured by IRMA, showing it to be about 500 times as sensitive as the conventional neutralization assay used, and in 2 cases the inhibitors could only be detected by IRMA. The new method is thus useful for the investigation of patients with low inhibitor titres, or when inhibitors with divergent properties are suspected.

Antibodies↗
PubMed2007

In vivo recovery of factor VIII and factor IX: intra- and interindividual variance in a clinical setting.

In vivo recovery (IVR) is traditionally used as a parameter to characterize the pharmacokinetic properties of coagulation factors. It has also been suggested that dosing of factor VIII (FVIII) and factor IX (FIX) can be adjusted according to the need of the individual patient, based on an individually determined IVR value. This approach, however, requires that the individual IVR value is more reliably representative for the patient than the mean value in the population, i.e. that there is less variance within than between the individuals. The aim of this investigation was to compare intra- and interindividual variance in IVR (as U dL1 per U kg1) for FVIII and plasma-derived FIX in a cohort of non-bleeding patients with haemophilia. The data were collected retrospectively from six clinical studies, yielding 297 IVR determinations in 50 patients with haemophilia A and 93 determinations in 13 patients with haemophilia B. For FVIII, the mean variance within patients exceeded the between-patient variance. Thus, an individually determined IVR value is apparently no more informative than an average, or population, value for the dosing of FVIII. There was no apparent relationship between IVR and age of the patient (1.5-67 years). For FIX, the mean variance within patients was lower than the between-patient variance, and there was a significant positive relationship between IVR and age (13-69 years). From these data, it seems probable that using an individual IVR confers little advantage in comparison to using an age-specific population mean value. Dose tailoring of coagulation factor treatment has been applied successfully after determination of the entire single-dose curve of FVIII:C or FIX:C in the patient and calculation of the relevant pharmacokinetic parameters. However, the findings presented here do not support the assumption that dosing of FVIII or FIX can be individualized on the basis of a clinically determined IVR value.

Adolescent↗
PubMed1986

Factor IX concentrate therapy and thrombosis: relation to changes in plasma antithrombin III.

Commercial factor IX (fIX) concentrate therapy has been associated with thrombogenic complications, the cause of which is uncertain. We have previously reported that infusion of these fIX concentrates led to a decrease in antithrombin III (ATIII) functional activity, but not antigen, from pre-infusion levels. The patient plasmas also showed a cathodal shift in ATIII antigen by crossed immunoelectrophoresis (CIEP). We chose to characterize these ATIII changes more extensively in additional patients, by functional assays, radial immunodiffusion (RID), and CIEP. The effects of commercial fIX concentrate therapy on plasma ATIII levels appear to be dose-related, with most pronounced effects at 100 U/Kg; the effects are also cumulative, with a persistence of ATIII changes 24 hours after infusion in patients on daily therapy. We also studied pre- and post-infusion ATIII levels in patients who received 100 U/Kg of a more purified fIX concentrate (American Red Cross), which contains little or no activated clotting factors and has been shown to be non-thrombogenic in animal models. These patients showed no change post-infusion in ATIII levels by clotting, amidolytic, or RID assays, nor any significant change by CIEP. These data suggest that the ATIII changes that occur predictably after commercial fIX concentrate therapy are caused by a contaminating protein or proteins other than fIX and that the ATIII changes observed are related to the thrombotic complications of these commercial concentrates.

Antigens↗
PubMed1975

The preparation of the factors IX, II, and X concentrate by means of DEAE cellulos and its therapeutic effectiveness.

Starting from a plasma used for producing cryoprecipitate a concentrate was gained by means of adsorption on DEAE cellulose, the factor IX activity of which was six to thirteen times higher than that of the original plasma. Even the factors X and II are enriched to the same extent approximately. In patients with haemophilia B the concentrate proved to be very efficient and compatible during surgical operations.

Blood Coagulation Factors↗
PubMed2003

Reduced bleeding events with subcutaneous administration of recombinant human factor IX in immune-tolerant hemophilia B dogs.

Intravenous administration of recombinant human factor IX (rhFIX) acutely corrects the coagulopathy in hemophilia B dogs. To date, 20 of 20 dogs developed inhibitory antibodies to the xenoprotein, making it impossible to determine if new human FIX products, formulations, or methods of chronic administration can reduce bleeding frequency. Our goal was to determine whether hemophilia B dogs rendered tolerant to rhFIX would have reduced bleeding episodes while on sustained prophylactic rhFIX administered subcutaneously. Reproducible methods were developed for inducing tolerance to rhFIX in this strain of hemophilia B dogs, resulting in a significant reduction in the development of inhibitors relative to historical controls (5 of 12 versus 20 or 20, P <.001). The 7 of 12 tolerized hemophilia B dogs exhibited shortened whole blood clotting times (WBCTs), sustained detectable FIX antigen, undetectable Bethesda inhibitors, transient or no detectable antihuman FIX antibody titers by enzyme-linked immunosorbent assay (ELISA), and normal clearance of infused rhFIX. Tolerized hemophilia B dogs had 69% reduction in bleeding frequency in year 1 compared with nontolerized hemophilia B dogs (P =.0007). If proven safe in human clinical trials, subcutaneous rhFIX may provide an alternate approach to prophylactic therapy in selected patients with hemophilia B.

Animals↗