The majority of cases of human hemophilia B are the result of missense mutations in the coagulation factor IX gene and defective circulating factor IX is detectable in most patients. The available mouse factor IX knockout models of hemophilia B (FIXKO mouse) reproduce the bleeding phenotype of human hemophilia B, but because the models produce no factor IX they fail to reproduce the dominant hu...

The majority of cases of human hemophilia B are the result of missense mutations in the coagulation factor IX gene and defective circulating factor IX is detectable in most patients. The available mouse factor IX knockout models of hemophilia B (FIXKO mouse) reproduce the bleeding phenotype of human hemophilia B, but because the models produce no factor IX they fail to reproduce the dominant hu...

To improve production of functional fully gamma-carboxylated recombinant human clotting factor IX (r-hFIX), cell lines stably overexpressing r-hFIX have been engineered to also overexpress proteins of the gamma-carboxylation system. Here we demonstrate that siRNA silencing of calumenin, an inhibitor of the gamma-carboxylation system, enhances production of functional r-hFIX produced by engineer...

Hemophilic bleeding into joints causes synovial and microvascular proliferation and inflammation (hemophilic synovitis) that contribute to end-stage joint degeneration (hemophilic arthropathy), the major morbidity of hemophilia. New therapies are needed for joint deterioration that progresses despite standard intravenous (IV) clotting factor replacement. To test whether factor IX within the joi...

Background:  Human coagulation factor IX (hFIX) is a glycoprotein with two N-glycosylation sites at the activation peptide. Since the activation peptide is removed in mature hFIX, the exact role of N-glycosylation is unclear. To investigate the role of N-glycosylation in the secretion and activity of hFIX, we inhibited N-glycosylation by tunicamycin in the stable Human Embryonic Kidney (HEK)- c...

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...

Background. Hemophilia is a rare recessive X-linked disease characterized by a deficiency of coagulation factor VIII or factor IX. Its current treatment is merely palliative. Advanced therapies are likely to become the treatment of choice for the disease as they could provide a curative treatment. Methods. The present study looks into the use of a safe non-viral transfection method based on nuc...

The effect of neonatal gene transfer on antibody formation was determined using a retroviral vector (RV) expressing human factor IX (hFIX). Normal mice from different strains injected intravenously with RV as newborns achieved therapeutic levels of hFIX without antibody production and were tolerant as adults to challenge with hFIX. Neonatal hemophilia B mice that received different amounts of R...

Following fetal or neonatal gene transfer in mice and other species immune tolerance of the transgenic protein is frequently observed; however the underlying mechanisms remain largely undefined. In this study fetal and neonatal BALB/c mice received adenovirus vector to deliver human factor IX (hFIX) cDNA. The long-term tolerance of hFIX was robust in the face of immune challenge with hFIX prote...