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J N Lozier

Publications and source records attributed to J N Lozier.

14 recordsLinked to original sources
PubMed1999

Adenovirus-mediated expression of human coagulation factor IX in the rhesus macaque is associated with dose-limiting toxicity.

We used a first-generation adenovirus vector (AVC3FIX5) to assess whether human factor IX could be expressed and detected in the rhesus macaque, which we have shown does not make high-titer antibodies to human factor IX protein. Three animals received 1 x 10(10) to 1 x 10(11) plaque-forming units per kilogram by intravenous injection. Human factor IX was present within 24 hours of vector administration and peaked 4 days later at 4,000 ng/mL in the high-dose recipient, and lower levels were seen in the intermediate-dose recipient. No human factor IX was detected in the low-dose recipient's plasma. Serum cytokine analysis and early hypoferremia suggested a dose-dependent acute-phase response to the vector. Human factor IX was detectable in rhesus plasma for 2 to 3 weeks for the high- and intermediate-dose recipients, but disappeared concomitant with high-titer antihuman factor IX antibody development. There was substantial, dose-dependent, dose-limiting liver toxicity that was manifest as elevated serum transaminase levels, hyperbilirubinemia, hypoalbuminemia, and prolongation of clotting times. Of particular interest was prolongation of the thrombin clotting time, an indicator of decreased fibrinogen or fibrinogen dysfunction. All evidence of liver toxicity resolved except for persistent hypofibrinogenemia in the high-dose recipient, indicating possible permanent liver damage. Our data suggest a narrow therapeutic window for first-generation adenovirus-mediated gene transfer. The development of antihuman factor IX antibodies and abnormalities of fibrinogen in the rhesus macaque is of concern for application of adenovirus (or other viral) vectors to hemophilia gene therapy.

Adenoviridae↗
PubMed1999

The rhesus macaque as an animal model for hemophilia B gene therapy.

We have determined the 2905 nucleotide sequence of the rhesus macaque factor IX complementary DNA (cDNA) and found it to be greater than 95% identical to that of the human factor IX cDNA. The cDNA has a large 3' untranslated region like the human cDNA, but unlike the human cDNA has two polyadenylation sites 224 nucleotides apart that are used for transcription of the messenger RNA. The deduced amino acid sequence is greater than 97% identical to that of human factor IX, differing in only 11 of 461 amino acids in the complete precursor protein. We found a single silent polymorphism in the nucleotide sequence at the third position of the codon for asparagine at position 167 in the secreted protein (AAC/AAT). All residues subject to posttranslational modifications in the human protein are also found in the rhesus factor IX sequence. The high degree of homology between the rhesus and human factor IX proteins suggested the possibility that the human factor IX protein might be nonimmunogenic in the rhesus. We tested the immunogenicity of human factor IX in three rhesus macaques by repeated intravenous injections of monoclonal antibody-purified, plasma-derived human factor IX over the course of more than a year and assessed the recovery and half-life of the infused protein, as well as in vitro indicators of antihuman factor IX antibodies. Human factor IX recovery and half-life remained unchanged over the course of a year in the three animals studied, and aPTT mixing studies showed no evidence for neutralizing antihuman factor IX antibodies. An outbred, nonhuman primate model that permits assessment of the level and duration of factor IX expression as well as vector safety would complement the use of other (mouse and canine) hemophilia B animal models in current use for the development of gene therapy for hemophilia B.

Amino Acid Sequence↗
PubMed1997

Gut epithelial cells as targets for gene therapy of hemophilia.

Gut epithelium is an attractive target for gene therapy of hemophilia due to the large number of rapidly dividing cells that should be readily accessible to a wide range of vectors by a noninvasive route of administration. We have performed in vitro tests to determine the suitability of gut epithelial cells for gene transfer, protein synthesis, and secretion of coagulation factors VIII and IX. The results with retroviral vectors indicate that transduced epithelial cells from human, rat, or porcine small or large intestine can synthesize significant amounts of factor VIII or factor IX and that two-thirds or more of the recombinant protein is secreted in a basolateral direction (i.e., away from the lumen and toward underlying capillaries and lymphatics). Furthermore, we have demonstrated that intestinal epithelial cells are susceptible to efficient gene transfer by lipofection and adenovirus vectors. In the case of factor IX, we have produced a high-titer adenovirus vector capable of transducing gut epithelial cells resulting in synthesis of factor IX. The results of our in vitro studies indicate that gene transfer targeting gut epithelium as a new approach to hemophilia gene therapy is rational and merits in vivo studies in hemophilia animal models.

Adenoviridae↗
PubMed1996

Safety profile of porcine factor VIII and its use as hospital and home-therapy for patients with haemophilia-A and inhibitors: the results of an international survey.

A multicentre retrospective survey was conducted to re-assess the use of porcine factor VIII (HYATE:C), its side effects and the selection of patients for regular or home-therapy. 15,152,000 units of HYATE:C were used by 154 patients. The median inhibitor cross-reactivity to porcine VIIIC of 137 patients was 15%, 27% of patients lacking cross-reactivity. An absent, intermediate or brisk specific antiporcine anamnestic response was observed in 29, 40 and 31% of patients respectively. Seven patients were treated on-demand as home-therapy for a median 6.2, range 1.5-13 years, 23 further patients were treated regularly in hospital for a median of 3, range 2-7 years. This group used 8,319,000 U of porcine VIIIC for 2,000 bleeding episodes. The incidence of transfusion reactions was 0.001%, 0.64% and 2.3%, for domiciliary infusions, infusions in multiply treated in-patients, and unselected in-patient infusions, respectively. The risk of reactions was dose-related. A post-infusion fall in platelet count was common, but usually transient and clinically insignificant. This was also dose-related (r = -0.64, p = 0.002). Marked reductions in platelet count were occasionally seen, usually with intensive replacement therapy. The relative lack of side effects observed amongst patients treated at home is attributable to the low, median 33 U/kg, dose used by this group. A subgroup of inhibitor patients, identifiable by their absent or modest anamnestic response to porcine factor VIII may be treated regularly and safely with this product in small doses, over a period of years.

Animals↗
PubMed1994

Gene therapy and the hemophilias.

Gene therapy for hemophilia A and B, now in the developmental stage, holds promise of a therapeutic revolution, resulting in amelioration or cure of the hemorrhagic diatheses. The genes for factor VIII and IX have been cloned, and vectors for the transfer of their cDNA into cells have been developed. Although viral and nonviral constructs containing the hemophilia gene have been used, most often modified retroviruses or adenoviruses have been employed. Cells that have been targeted for genetic modification to produce the antihemophilic proteins include hepatocytes, muscle cells, endothelial cells, keratinocytes, and fibroblasts. The delivery system may be (1) ex vivo, with implantation of modified cultured hemophilic cells in the donor, either in tissues or in semipermeable capsules, or (2) in vivo, the vector being delivered directly to target cells, genetically modifying them in situ. Successful in vivo therapy has been demonstrated in a hemophilic animal model, with conversion to a less severe hemophilic state.

Animals↗
PubMed1994

Efficient transfection of primary cells in a canine hemophilia B model using adenovirus-polylysine-DNA complexes.

We have used molecular conjugates containing combinations of DNA, adenovirus, polylysine, and transferrin to transfect primary cells derived from canines with hemophilia B (factor IX deficiency), as well as a canine epithelial cell line. Transfection of canine hemophilia B fibroblasts with molecular conjugates resulted in efficient transfection and expression of luciferase DNA-adenovirus-polylysine (AdpL) conjugates or luciferase DNA-adenovirus-polylysine-transferrin (hTfpL/AdpL) conjugates. No expression in canine hemophilia B fibroblasts was evident after exposure to DNA alone, or DNA conjugated with polylysine and transferrin. Transfection efficiencies of 50% or more could be demonstrated in cells transfected with a beta-galactosidase reporter gene as part of an hTfpL/AdpL molecular conjugate. Transfection with canine factor IX AdpL conjugates or canine factor IX hTfpL/AdpL conjugates resulted in factor IX expression for more than 2 weeks in vitro in hemophilia B canine fibroblasts. Maximum levels of expression of over 700 ng of canine factor IX/10(6) cells/24 hr were demonstrated in fibroblasts after transfection with canine factor IX hTfpL/AdpL conjugates. Similar conjugates were used to transfect hemophilia B canine bone marrow stromal cells and Madin-Darby canine kidney cells that also expressed canine factor IX. The use of molecular conjugates to transfect primary cells may be feasible as a means of in vitro or in vivo gene therapy for hemophilia B, and can be tested in the canine hemophilia B model.

Adenoviridae↗
PubMed1993

Use of porcine factor VIII for surgical procedures in hemophilia A patients with inhibitors.

We have described the use of polyelectrolyte fractionated porcine factor VIII (HYATE:C, Porton Speywood Ltd, Wrexham, UK) to provide hemostasis in 45 patients with hemophilia A complicated by inhibitor antibodies. The cases were collected from hemophilia care providers in seven nations and represent some of the experience with porcine factor VIII generated over the past 12 years. A wide variety of procedures was performed with varying degrees of associated hemostatic challenge in a difficult treatment group. The patients were representative of the hemophilia population in general and ranged in age from 1 to 67 years. The results of our survey should encourage clinicians to consider use of porcine factor VIII to provide hemostatic coverage in hemophilia patients with inhibitors who require surgical procedures. Hemostatic coverage was satisfactory in the vast majority of episodes; there have been only a small number of easily controlled, well-tolerated adverse reactions, which were usually self-limited in nature. No life-threatening reactions to porcine factor VIII were seen during coverage for surgical procedures in our survey, although serious reactions can occur as noted above. This is remarkable, since surgical patients usually are treated with higher doses of porcine factor VIII for greater periods of time than most other hemophilic patients treated for hemarthroses or soft-tissue bleeding, for example. Thrombocytopenia in association with porcine factor VIII therapy has been a major concern since the first crude preparations were used in the 1950s. Modern series, however, note the incidence of thrombocytopenia in only a minority of patients treated with the current polyelectrolyte fractionated preparations of porcine factor VIII. In our series, thrombocytopenia with platelet counts of less than 150 x 10(9)/L occurred in 11 of 54 treatment episodes (20%). The mechanism for thrombocytopenia demonstrated in a previous study involves porcine von Willebrand factor, which may copurify with porcine factor VIII and cause platelet aggregation and/or clearance from the circulation. The phenomenon of thrombocytopenia appears to be related to the administration of high doses of porcine factor VIII in some cases; however, in other patients, it may develop inconsistently at modest doses. It may be that there are idiosyncratic differences between patients that determine the dose of porcine factor VIII likely to cause thrombocytopenia. As yet, there is no way to predict which patient will develop thrombocytopenia while being treated with porcine factor VIII.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

New perspectives on the coagulation cascade.

The view that clotting is basically a succession of zymogen activations remains valid but is in need of some modifications, in part because the intrinsic and extrinsic coagulation pathways are more closely interrelated than had been thought. The new knowledge is beginning to influence treatment of the hereditary bleeding disorders--and to point the way toward genetic cures.

Blood Coagulation↗
PubMed1990

Factor IX New London: substitution of proline for glutamine at position 50 causes severe hemophilia B.

We describe a novel point mutation in the fourth exon of human factor IX (encoding the first EGF-like domain) in which cytosine is substituted for adenosine at position 10,401, resulting in the substitution of proline for glutamine at position 50 in the polypeptide chain. Sequence analysis of all eight exons, all exon-intron junctions, 160 base pairs (bp) of DNA 5' to the proposed translation start site, and 60 bp 3' to the translation termination site shows no other difference from the normal factor IX gene, with the exception of a previously described benign polymorphism at position 148 in the protein (Ala----Thr). The affected subject has severe hemophilia B with no detectable factor IX activity despite normal factor IX antigen levels. We purified the abnormal factor IX by immunoaffinity chromatography and demonstrated that its activation by factor Xla is markedly delayed compared with normal factor lX. Once activated, the abnormal factor lX binds antithrombin III in a 1:1 molar ratio, and the activated protein demonstrates catalytic activity, suggesting an intact active site. The mutation creates a new Bst Yl restriction endonuclease cleavage site. Restriction with Bst Yl shows the mutation in maternal DNA and offers the possibility of direct carrier status analysis and prenatal diagnosis in kindreds with this mutation. We designate this new mutation factor lXNew London. This is the only reported mutation in the first EGF-like domain that causes severe hemophilia B.

Amino Acid Sequence↗
PubMed1984

Purification of glycopeptides of human plasma proteins by high-performance liquid chromatography.

The combination of gel permeation chromatography and high-performance liquid chromatography proves to be very effective for the purification of high-molecular-weight glycopeptides containing a single glycan, that have been difficult to separate by other procedures. In order to facilitate comparison of the chromatographic properties of glycopeptides derived from a variety of proteins and having different structures, identical procedures were used for their purification. The method was applied to a series of human plasma proteins, including immunoglobulin D, ceruloplasmin, hemopexin, beta-2-glycoprotein I, 3.1S alpha-2-leucine-rich glycoprotein, and alpha-1-B-glycoprotein. All the purified glycopeptides were placed in the protein structure of these plasma proteins. In several cases the carbohydrate structure has been determined by collaborating groups. Immunoglobulin D is the first example of a glycoprotein whose entire primary structure has been defined by utilizing a a single protein source. Furthermore, hemopexin and 3.1S alpha-2-leucine-rich glycoprotein were both found to contain GalN oligosaccharide, which had not previously been identified in these proteins. The method was also used to identify the oligosaccharide that is missing in a carbohydrate variant of ceruloplasmin.

Amino Acid Sequence↗