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PubMed1992

Direct gene transfer to the liver with herpes simplex virus type 1 vectors: transient production of physiologically relevant levels of circulating factor IX.

We have used gene transfer vectors derived from a replication-defective mutant of herpes simplex virus type 1 (HSV-1) expressing the hepatitis B virus surface antigen (HBsAg), Escherichia coli beta-galactosidase (beta-gal), or canine factor IX (cFIX) from the immediate early promoter of human cytomegalovirus (hCMV) to infect mouse liver by direct injection or through the portal vein. By either route, high levels of transgene expression were demonstrated by the detection of immunoreactive HBsAg or cFIX in the circulation and by histochemical detection of beta-gal activity in situ. The results were striking in that the serum level of cFIX reached 10% of the normal murine levels. Although the level of transgene expression from the hCMV promoter was transient, a significant number of persistent vectors could be rescued from the livers of recipient mice up to 2 months after inoculation. Replacement of the hCMV promoter with the HSV-1 latency-associated transcript (LAT) promoter resulted in reduced but prolonged expression of both HBsAg and cFIX. The very high level of factor IX expression suggests that clinically useful gene transfer may eventually be feasible through direct vector delivery to the liver.

Animals↗
PubMed1990

Effect of feed flow-rate, antigen concentration and antibody density on immunoaffinity purification of coagulation factor IX.

A simple physical model of immunoaffinity chromatography (IAC) demonstrates that immobilized monoclonal antibody (MAb) capacity in IAC purification will be a function of many parameters, including feed flow-rate and antigen concentration, and MAb density (mg MAb immobilized/ml resin). We studied IAC of factor IX, and examined the effect of parameter variation on MAb capacity. MAb capacity (1) was not affected by feed flow-rate or antigen concentration, and (2) decreased as MAb density increased. (1) Suggested that diffusion of factor IX into the resin bead was not limiting. Characteristic diffusion, convection and reaction times were calculated and used in dimensional analysis to compare their relative magnitudes. If MAb was assumed to be localized to the outer 10% of the bead volume, this analysis concluded that diffusion was not limiting, consistent with the suggestions of our experimental data. (2) Suggests that high MAb densities make MAb less accessible.

Antibodies, Monoclonal↗
PubMedDate not supplied

Standardization of assays of factor VIII and factor IX.

The development of international standards over the last 15-20 years has led to improved interlaboratory agreement on assays of factor VIII and factor IX. In the most recent international collaborative study, the coefficient of variation for one-stage assays (26 laboratories) was 5.6%. However, in quality assurance surveys, carried out in the UK and USA, agreement between laboratories is much less good, with coefficients of variation ranging from 30% to over 50%. Improvements in agreement between clinical laboratories could be obtained by increasing the amount of testing on each sample, especially the number of dilutions, and reducing the number of reagent systems used. A large number of laboratories now use immunodepleted plasmas instead of congenitally deficient plasmas as substrates for one-stage assays. These plasmas may give satisfactory assays, but many of them have not been thoroughly evaluated in comparison with congenitally deficient plasma. In assessment of potency of very high purity (VHP) factor VIII concentrates, some immunodepleted plasmas were found to give lower potencies than hemophilic plasma. This is partly due to the fact that VHP concentrates contain little or no von Willebrand factor (vWF), and most immunodepleted plasmas are also deficient in vWF. In recent collaborative studies, assays of VHP factor VIII concentrate were much more variable, both within and between laboratories, than assays of intermediate purity concentrates. Standardization of these new products will require careful attention to methodological detail.

Factor IX↗
PubMed1995

The high frequency of the -6G-->A factor IX promoter mutation is the result both of a founder effect and recurrent mutation at a CpG dinucleotide.

We report a new Liverpool family with a mild haemophilia B Leyden phenotype caused by a -6G-->A mutation in a CpG dinucleotide in the promoter of the clotting factor IX gene. This mutation had previously been identified in three other U.K. pedigrees and six others worldwide. To investigate whether these mutations were of independent origin, the haplotype was determined for eight polymorphic loci, within or immediately adjacent to the factor IX gene, for nine of the 10 existing patients. Six probands had identical haplotypes, including all four U.K. probands, suggesting that they arose from a common founder. The other three probands differed in haplotype from the common haplotype, and from each other, suggesting that they were independent mutations at this CpG dinucleotide.

Child↗
PubMed2003

Effect of manufacturing process parameters on virus inactivation by solvent-detergent treatment in a high-purity factor IX concentrate.

BACKGROUND AND OBJECTIVES: Treatment with solvent-detergent is widely used for ensuring the virus safety of plasma products. Laboratory studies have shown this procedure to be effective for inactivating enveloped viruses under manufacturing conditions. In the present study, the effect of different manufacturing process parameters on virus inactivation by treatment with polysorbate 80 and tri-n-butyl phosphate were investigated for a high-purity factor IX concentrate in order to evaluate the robustness of this step. MATERIALS AND METHODS: Samples of factor IX intermediate were obtained, virus was added and the kinetics of virus inactivation followed during incubation. The effect of altering the conditions on virus inactivation was tested. RESULTS: Solvent-detergent treatment was confirmed to effectively inactivate, i.e. by > or = 5 log, a wide range of representative enveloped viruses under standard conditions. Virus inactivation was consistently effective in a number of different manufacturing batches. Of the parameters tested, only solvent-detergent concentration and temperature significantly effected virus inactivation. CONCLUSIONS: The robustness of the solvent-detergent step for virus inactivation has been confirmed. Using the data generated, appropriate limits can be set for this manufacturing process step.

Detergents↗
PubMed2001

Risk and prevention of anti-factor IX formation in AAV-mediated gene transfer in the context of a large deletion of F9.

The safety of several gene therapy approaches for treatment of the severe, X-linked bleeding disorder hemophilia is currently being evaluated in early phase clinical trials. One strategy seeks to correct deficiency of functional coagulation factor IX (hemophilia B) by intramuscular (IM) administration of an adeno-associated viral (AAV) vector. A potentially serious complication of any treatment for hemophilia is formation of inhibitory antibodies against the coagulation factor protein, a risk that increases in the setting of null mutations in the factor IX gene (F9). Here, we describe hemophilia B mice with a large F9 deletion that form inhibitors within 1 to 2 months after IM administration of an AAV vector expressing mouse F9 or after repeated intravenous infusion of mouse F9 concentrate. In both cases, inhibitors are primarily IgG1 immunoglobulins representing a Th2-driven humoral immune response. We further demonstrate that anti-mouse F9 antibody formation in the gene-based approach can be reduced by transient immune modulation at the time of vector administration. Moreover, this maneuver resulted in complete absence of anti-mouse F9 and sustained expression of functional mouse F9 in some hemophilia B mice, particularly in those animals treated with the immunosuppressive drug cyclophosphamide. These data have direct relevance for design of clinical trials and strategies aimed at avoiding immune responses against a secreted transgene product.

Animals↗
PubMed1990

Manic depressive illness not linked to factor IX region in an independent series of pedigrees.

We studied seven informative kindreds segregating for manic depressive illness (MDI), consistent with X-chromosome transmission of the trait (families do not show affective disease in both a father and a son), using markers mapped to the region of Xq27-Xq28. The lod scores were consistently below -2 in the region extending from about 10 cM centromeric from the Factor IX locus (F9) to the colorblindness region. This study does not replicate previous reports of linkage of MDI to Factor IX (Xq27) and colorblindness region (Xq28) chromosomal markers in other kindreds.

Bipolar Disorder↗
PubMed2000

Absence of correlation between X chromosome inactivation pattern and plasma concentration of factor VIII and factor IX in carriers of haemophilia A and B.

Haemophilia A and B are X-linked disorders which are due to a reduced activity of coagulation factor VIII or IX, respectively. Female carriers have a wide range of plasma concentration of factor VIII or factor IX, and may in rare cases have an affected phenotype. In order to investigate if this variation is related to X chromosome inactivation, we determined the X inactivation pattern in 31 haemophilia A and 15 haemophilia B carriers, using a PCR in the androgen receptor locus in blood DNA. Seven of the haemophilia A carriers and none of the haemophilia B carriers had a skewed pattern (> or =80:20). One of the skewed haemophilia A carriers had a low plasma concentration of factor VIII (0.15 U/ml), but the remaining 6 carriers did not differ in factor VIII concentration from that of carriers with a random X inactivation pattern. One carrier with a high factor VIII concentration (2.0 U/ml) did not have a skewed pattern. Similarly, for the haemophilia B carriers, there was no tendency to a more skewed X inactivation pattern in the carriers with low or high factor IX concentrations. In addition, we analysed a female with haemophilia B who was heterozygous for the mutation R180W in the factor IX gene. She had a random X chromosome inactivation pattern. We conclude that the wide range in plasma concentration of factor VIII and factor IX in haemophilia A and B carriers cannot in general be explained by the X chromosome inactivation pattern in peripheral blood cells.

Adolescent↗
PubMed1985

More precise localization of the human factor IX gene by in situ hybridization.

The location of the human antihemophilic Factor IX has been more specifically assigned from the region Xq27----qter to Xq26----q27 by quantitative in situ hybridization. The present study utilized a complex hybridization probe and prephotographed G-banded human chromosomes to improve analytical sensitivity and accuracy.

Chromosome Banding↗
PubMed2005

Structural determinants of factor IX(a) binding in nitrophorin 2, a lipocalin inhibitor of the intrinsic coagulation pathway.

Nitrophorin 2 (NP2) is a salivary lipocalin from Rhodnius prolixus that binds with coagulation factors IX (fIX) and IXa (fIXa). Binding of NP2 with fIXa results in potent inhibition of the intrinsic factor Xase complex. A panel of site-directed surface mutants of NP2 was generated to locate determinants of high affinity fIX(a) binding. The locations of the mutations were based on comparisons with the related, but less potent, inhibitor nitrophorin 3 (NP3). Three point mutants (K21A, K92A, and V94A) were found that clearly reduced the inhibitory potency as measured by the activity of a reconstituted factor Xase system. Binding of NP2 with fIXa and fIX as measured by surface plasmon resonance and isothermal titration calorimetry was reduced in a similar manner. Of the three mutants, two (K92A and V94A) were located on the loop connecting beta-strands E and F of the lipocalin beta-barrel. The largest changes were seen with the K92A mutation, which lies at the apex of the loop, with a smaller effect being seen with mutation of Val(94). Combination of four E-F loop mutations (K92A, A93K, V94A, E97A) in a single mutant reduced the inhibitory potency and binding to levels similar to those seen with NP3 without affecting heme or histamine binding.

Amino Acid Sequence↗
PubMed1995

Gene therapy for hemophilia B: host immunosuppression prolongs the therapeutic effect of adenovirus-mediated factor IX expression.

Hemophilia B is caused by a deficiency of blood clotting factor IX (FIX). Previous studies have shown that the delivery of a recombinant adenoviral vector expressing canine FIX (cFIX) resulted in a complete correction of hemophilia B in FIX-deficient dogs, but that cFIX expression decreased to only about 1-2% of normal levels 3 weeks after treatment. In the present study, therapeutic levels of cFIX expression capable of producing a partial correction of hemophilia B were maintained for at least 6 months after the coadministration of the cFIX-expressing adenovirus and the immunosuppressive agent cyclosporin A (CsA). These findings support a recent report (Yang et al., 1994) that host T-cell-mediated immunity against virally transduced cells is a major contributing factor to the transient nature of adenovirus-mediated gene expression in immunocompetent animals. Although a second administration of the cFIX-expressing adenovirus 6 months after the first infusion had only a minimal effect on plasma FIX levels in a dog that had been continuously treated with CsA, the prolonged expression of the transgene indicates that immunosuppression may be applicable in attaining long-term treatment of clinically relevant disorders.

Adenoviridae↗
PubMed1996

Factor VIIa-tissue factor interactions: an evaluation using factor VII-factor IX chimeras.

Using molecular biological techniques, a series of factor VII-factor IX chimeric molecules were made for both kinetic and competition binding assays to tissue factor in order to understand the interaction between factor VIIa and tissue factor. The results of the binding studies indicated that the major interaction between factor VIIa and tissue factor. The results of the binding studies indicated that the major interaction between factor VIIa and tissue factor involved both the first epidermal growth factor-like (EGF1) and catalytic domains of factor VII. In addition, both the gamma-carboxyglutamic acid-containing (Gla) and EGF2 domains also play a minor role in binding to tissue factor. The Gla and EGF-2 domain might either impart structure to the rest of the molecule or contribute a relatively small amount of energy to direct binding.

Binding, Competitive↗
PubMed1999

Identification of twenty-one new mutations in the factor IX gene by SSCP analysis.

In this study we have analyzed the factor IX gene from 84 hemophilia B patients of Spanish origin. It included single-strand conformation polymorphism (SSCP) analysis of all functional regions of the gene and further sequencing of all fragments showing abnormal migration. In 76 patients (90.4%), it was possible to identify molecular alterations leading to the appearance of the disease. Twenty-one new mutations were identified, including 13 missense mutations, two nonsense mutations, three splice-site mutations, one frameshift deletion, one frameshift insertion, and one non-frameshift deletion. The approach appears to be very suitable for molecular diagnosis of hemophilia B.

DNA Mutational Analysis↗
PubMed1991

The varying frequencies of five DNA polymorphisms of X-linked coagulant factor IX in eight ethnic groups.

Five RFLPS of X-linked coagulation factor IX were evaluated in more than 500 normal persons (723-804 X chromosomes) of both sexes who belonged to eight ethnic groups: Anglo-Americans, Basques, Swedes, African-Americans, East Africans, East Indians, Chinese, and Malays. The polymorphisms, 5' to 3', were BamHI, XmnI, TaqI, MnlI, and HhaI. A PCR procedure was developed for three previously described RFLPs-XmnI, TaqI, and MnlI; a PCRP procedure was developed for BamHI, and a PCRP which had been described by others was used for HhaI. Europeans were the most polymorphic, African-Americans and East Africans were intermediate, and Orientals were the least polymorphic. Extragenic 3' HhaI was highly polymorphic in most groups, and extragenic 5' BamHI was polymorphic only in persons with African ancestry. Two major haplotypes predominated among 247 men, and the expected and observed heterozygosities were concordant among women. Allelic association was very strong between the three intragenic PCRPs; it was present but weak between 5' extragenic BamHI and XmnI. No association was found between 3' extragenic HhaI and MnlI.

Asian People↗
PubMed2002

A new adenoviral helper-dependent vector results in long-term therapeutic levels of human coagulation factor IX at low doses in vivo.

We have developed a new helper-dependent (HD) adenoviral vector FTC that contains 3 cis-acting sequences as stuffer DNA: a human fragment of alphoid repeat DNA, matrix-attachment regions (MARs), and the hepatocyte control region enhancer. To determine the most robust human coagulation factor IX (hFIX) expression cassette in an adenovirus, we first tested different hFIX expression sequences with or without flanking MARs in first-generation adenoviral vectors. After intravenous infusion of the vector, serum levels of up to 100 000 ng/mL hFIX (normal level, 5000 ng/mL) were obtained at nontoxic doses. In order to make a direct comparison, a first-generation and a gene-deleted vector with identical hFIX expression cassettes were constructed. Both first-generation and HD adenovirus-treated animals demonstrated a threshold effect in a dose-response study. With the administration of 2 x 10(9) transducing particles of either vector, supraphysiological serum levels of hFIX were obtained, with the highest expression (41 000 ng/mL) occurring during the first 2 months after injection. The serum factor IX concentrations, while remaining in the therapeutic range, slowly declined by 95% over a period of 1 year. At this dose, interleukin-6 and tumor necrosis factor-alpha serum concentrations were elevated in animals that received the first-generation but not the HD vector. This study compares the properties of a gene-deleted and first-generation adenovirus with equivalent expression cassettes and suggests that the cis-DNA elements contained in the vector and expression cassette have important effects on gene expression in vivo.

Adenoviruses, Human↗
PubMed1996

Recombinant human factor IX: replacement therapy, prophylaxis, and pharmacokinetics in canine hemophilia B.

Recombinant human factor IX (rFIX) has been expressed in transduced cultured cell systems since 1985. Because there has been limited in vivo testing of rFIX in hemophilia B subjects, this study was undertaken using the severe hemophilia B canines of the Chapel Hill strain. Three groups of hemophilic dogs received either 50, 100, or 200 IU/kg of rFIX. As a control, a fourth group of hemophilic dogs received 50 IU/kg of a high purity, plasma-derived human FIX (pdFIX). The coagulant and hemostatic effects of rFIX and pdFIX were similar with all comparative dosing regimens. Based on activity data, the elimination half-life of rFIX was 18.9 +/- 2.3 hours and pdFIX was 17.9 +/- 2.1 hours. A prophylactic regimen administering rFIX daily resulted in a continuous therapeutic level of plasma FIX and was accompanied by a two-fold increase in recovery levels by day 5, compared to that observed with administration of a single bolus. The mechanisms of the high to complete recovery of FIX with the prophylactic regimen could depend not only on the degree of saturation of the vascular endothelial binding sites but also on the altered dynamics of the balance of FIX distribution between the intravascular and extravascular compartments. The pharmacokinetic (PK) parameters for rFIX and pdFIX were similar. However, the relative PK values for V1 and V5s of both products on day 5 differed greatly from day 1 and may reflect the changing equilibrium of FIX between compartments with elevated levels of plasma FIX. Neutralizing antihuman FIX antibodies resulting from human FIX antigen being administered to FIX deficient dogs were observed beginning at 14 days. The antigenicity of rFIX and pdFIX appeared to be comparable. Despite the very different procedures used for production of rFIX and pdFIX products, in vivo testing in hemophilia B dogs showed the functional behavior of these products is similar; they are highly effective for replacement therapy and for prophylaxis.

Animals↗
PubMed2004

Hybrid retroviral vector with MCK enhancers inserted in LTR for stable and specific expression of human factor IX in skeletal muscle.

BACKGROUND: Retroviral vectors have been widely used to introduce foreign into various target cells in vitro, thus showing relatively high systemic delivery efficiency of various transgene products. The authors investigated the stability and efficiency of skeletal muscle-specific hybrid retroviral vectors in expression of human factor IX (FIX) in vitro and iv vivo. METHODS: FIX cDNA in LIXSN vector was replaced with a FIX minigene containing splicing donor and splicing acceptor sequence of first intron of human FIX gene. Two copies of muscle creatine kinase enhancer (MCK, Me2) were inserted in forward or reverse orientation at NheI site of 3' long terminal repeat (LTR), resulting in two hybrid vectors, which were designated as LMe2IXm2SN(F) and LMe2IXm2SN(R), respectively. The vectors were tested in vitro and in vivo for stability and muscle-specificity of factor IX expression with SCID mice. RESULTS: Muscle cells carrying vector with Me2 expressed significantly higher levels of FIX (up to 1800 ng/106.24 h) than those without Me2, thus suggesting that Me2 could specifically increase expression level of FIX in muscle cells. Myoblasts transduced with LMe2IXm2SN(R) produced much less FIX in vivo in SCID mice than LMe2IXm2SN(F). One or two copies of Me2 sequence were deleted in myoblasts transduced with LMe2IXm2SN(R) without changing the orientation of Me2. CONCLUSIONS: LTR inserted with MCK enhancers can specifically increase human FIX expression in skeletal muscle cells in vitro and in vivo, and MCK enhancer should be positioned in the same orientation as that of LTR promoter.

Animals↗