Marjorie S. Read

Long-term correction of canine hemophilia B by gene transfer of blood coagulation factor IX mediated by adeno-associated viral vector

Hemophilia B is a severe X-linked bleeding diathesis caused by the absence of functional blood coagulation factor IX, and is an excellent candidate for treatment of a genetic disease by gene therapy. Using an adeno-associated viral vector, we demonstrate sustained expression (>17 months) of factor IX in a large-animal model at levels that would have a therapeutic effect in humans (up to 70 ng/ml, adequate to achieve phenotypic correction, in an animal injected with 8.5 × 1012 vector particles/kg). The five hemophilia B dogs treated showed stable, vector dose-dependent partial correction of the whole blood clotting time and, at higher doses, of the activated partial thromboplastin time. In contrast to other viral gene delivery systems, this minimally invasive procedure, consisting of a series of percutaneous intramuscular injections at a single timepoint, was not associated with local or systemic toxicity. Efficient gene transfer to muscle was shown by immunofluorescence staining and DNA analysis of biopsied tissue. Immune responses against factor IX were either absent or transient. These data provide strong support for the feasibility of the approach for therapy of human subjects.

Correction of hemophilia B in canine and murine models using recombinant adeno-associated viral vectors

Hemophilia B, or factor IX deficiency, is an X-linked recessive disorder occurring in about 1 in 25,000 males. Affected individuals are at risk for spontaneous bleeding into many organs; treatment mainly consists of the transfusion of clotting factor concentrates prepared from human blood or recombinant sources after bleeding has started. Small- and large-animal models have been developed and/or characterized that closely mimic the human disease state. As a preclinical model for gene therapy, recombinant adeno-associated viral vectors containing the human or canine factor IX cDNAs were infused into the livers of murine and canine models of hemophilia B, respectively. There was no associated toxicity with infusion in either animal model. Constitutive expression of factor IX was observed, which resulted in the correction of the bleeding disorder over a period of over 17 months in mice. Mice with a steady-state concentration of 25% of the normal human level of factor IX had normal coagulation. In hemophilic dogs, a dose of rAAV that was approximately 1/10 per body weight that given to mice resulted in 1% of normal canine factor IX levels, the absence of inhibitors, and a sustained partial correction of the coagulation defect for at least 8 months.

Direct intramuscular injection with recombinant AAV vectors results in sustained expression in a dog model of hemophilia

A recombinant adeno-associated virus (rAAV) vector carrying the human factor IX cDNA was tested for safety and therapeutic gene expression in a canine model of human hemophilia B. Intramuscular delivery of rAAV was chosen based on our previous work which described long-term (>1.5 years) reporter gene expression in immunocompetent mice following direct muscle injection. For the current study, rAAV with the human factor IX (hF.IX) cDNA under the control of the cytomegalovirus (CMV) immediate–early promoter was constructed, and rAAV/hF.IX proved capable of transducing hemophilic dog primary fibroblast cultures in a dose-dependent fashion. Direct intramuscular injection of 2.5 × 1012 rAAV/hF.IX virus particles into the hindlimbs of a hemophilia B dog was tolerated without bleeding or systemic reaction, and the animal was asymptomatic throughout the entire study. Transient reduction in the whole blood clotting time (WBCT) occurred during the first week, with the anticipated development of an antihuman F.IX inhibitor antibody which corresponded with the loss of coagulation correction. At 10 weeks after vector administration, immunohistochemical analysis of injected muscle confirmed continued hF.IX expression. Limited areas of focal lymphocytic infiltration and myofiber pathology were detected which directly correlated with positive antibody staining for helper adenovirus contamination. PCR tissue analysis revealed rAAV/hF.IX DNA solely in injected muscle tissue and adjacent lymph node, without dissemination to other organs (including gonads). This first large animal study suggests that intramuscular gene delivery using rAAV vectors is safe and supports continued development of this approach for gene therapy of human diseases, including hemophilia B.

Von Willebrand factor and occlusive arterial thrombosis: A study in normal and von Willebrand's disease pigs with diet-induced hypercholesterolemia and atherosclerosis

The thrombotic response of atherosclerotic arteries to stenosis and injury was studied in 14 pigs, eight normal and six with von Willebrands disease (vWD). Atherosclerosis was produced by feeding a 1% to 2% cholesterol diet for 24 weeks. Both groups of pigs developed severe hypercholesterolemia, greater than five times baseline values. Coronary atherosclerosis was detected in all vWD pigs and in all but one normal pig and was not significantly different between groups. At sacrifice under general anesthesia, a Goldblatt clamp (GC) was positioned around the left anterior descending coronary (LAD) and carotid arteries to produce a stenotic segment, which was pinch-injured with needle holders. A 20 MHz Doppler velocity crystal was placed distal to the GC to detect cyclic flow reductions or permanent cessation of flow velocity indicative of occlusive thrombosis. In the phenotypically normal pigs with diet-induced atherosclerosis, occlusive thrombosis was detected in seven of seven LAD and seven of seven carotid arteries. In atherosclerotic vWD pigs, occlusive thrombosis failed to form in six LAD and 10 carotid arteries (p less than 0.003, Wilcoxon rank sum test). Scanning electron micrographs demonstrated platelet-fibrin microthrombi in both groups of pigs; only phenotypically normal pigs had occlusive thrombi. Von Willebrand factor is essential for the development of occlusive thrombosis and appears to support the progression of a mixed microthrombus to an occlusive thrombus.

Effect of Fibrinogen Concentration on Platelet Adhesion to Glass

Summary The extent to which platelets adhere to glass is dependent on fibrinogen concentration. Platelet adhesion increased progressively in a linear fashion when increasing concentrations of purified fibrinogen (0.5-14.0 mg/100 ml) were added to the blood of a patient with congenital afibrinogenemia.

Activation and adherence of lyophilized human platelets on canine vessel strips in the Baumgartner perfusion chamber

The Baumgartner perfusion technique was used to test the ability of rehydrated lyophilized human platelets to adhere to the thrombogenic surface of a denuded arterial vessel segment and to undergo platelet activation under conditions of high shear. Twenty preparations of washed platelets were stabilized by 1-hour or 2-hour exposure to paraformaldehyde before freeze-drying in a Virtis 600 lyophilizer. The response of these fixed-dried preparations was compared with that of non-fixed platelets in fresh citrated whole blood. The outcome of each perfusion experiment was quantified in photomicrographs by morphometric estimation of the percent area of the vessel segment covered by adherent platelets after immunofluorescent staining with monoclonal antibodies to glycoprotein Ib (CD42) or glycoprotein IIbIIIa (CD41a). Evidence of activation in nonadherent platelets was examined by flow cytometry for CD62 and GP53 expression. In addition, thromboxane B2 was measured by radioimmunoassay as an index of platelet responsiveness to activation conditions during perfusion. The percent vessel coverage observed with lyophilized platelets in recombined whole blood was somewhat less than that of platelets in fresh whole blood (39% vs 73%, respectively). In other perfusion experiments performed with non-denuded vessel segments, the percent coverage was reduced by half or more for both types of platelet preparation. Scanning electron microscopy confirmed that the lyophilized platelets did not adhere to areas of intact endothelium. Furthermore, the lyophilized platelets showed a small-but-significant rise in CD62 or CD63 activation antigen expression and generated thromboxane B2 at about one third the rate of fresh platelets in these perfusion experiments. The thromboxane generation during perfusion was inhibited in fresh or lyophilized platelet preparations by pretreatment with indomethacin or PGE-1. We interpret these data as evidence of the ability of our lyophiilized platelet preparations to respond at least partially to physiologic stimuli and to adhere to appropriate thrombogenic sites to support hemostasis.

Preservation of platelet receptors for platelet aggregating factor/von Willebrand factor by air drying, freezing, or lyophilization: New stable platelet preparations for von Willebrand factor assays

Abstract The long-term preservation of platelet surface receptors for the platelet aggregating von Willebrand factor was achieved by freezing or drying of formaldehyde-fixed human platelets. Three separate procedures for platelet preservation were used: freezing at −70°C, air-drying, and lyophilization. The preserved platelets were compared with the original fixed platelet preparations for reactivity in two types of tests for platelet aggregating von Willebrand factor, the ristocetin test with human plasmas and the platelet aggregating factor test with porcine and bovine plasmas. The new preparations of preserved platelets were equally reactive in both tests and showed no deterioration of activity on storage for periods of over one year. All of these preserved platelet preparations were satisfactory for use in both screening and bioassay procedures for the von Willebrand factor and provide a more convenient reagent than the original fixed platelet preparation.

Plasma levels of platelet aggregating factor/von Willebrand factor in various species

Abstract The relative plasma levels of platelet aggregating factor/von Willebrand factor were determined in human, chimpanzee, rhesus monkey, horse, cow, goat, sheep and pig. The macroscopic aggregation assay procedure was used, employing formaldehyde-fixed human platelets with and without ristocetin. One unit of PAF/vWF is defined as that amount in one ml of reference normal human plasma. The highest PAF/vWF levels were found in the ruminants of the group, goat, 12 units per ml; sheep, 10 units and cow, 6.6 units. The remaining species showed plasma levels as follows: man, rhesus and pig, 1.0 unit; chimpanzee, 0.9 units and horse, 0.6 units. Considerable species specificity was observed in the requirements for assay. Platelets from horse, pig, cow and dog were unreactive or poorly reactive in the tests. Guinea pig, rat, dog, rabbit and opossum plasmas were also unreactive or poorly reactive regardless of the source of platelets, and could not be assayed with the procedures employed. Ristocetin had no effect on aggregation of human platelets with pig, cow, sheep or goat plasmas, all of which aggregate platelets directly, unless the plasmas were greatly diluted, in which case ristocetin accelerated aggregation. A comparison of PAF/vWF levels with relative levels of antihemophilic factor showed that the ruminant group had the highest ratio of PAF/vWF:AHF, pig the lowest. The ratio for rhesus, chimpanzee and horse plasmas was similar to that for human plasmas.

von Willebrand factor and animal models: contributions to gene therapy, thrombotic thrombocytopenic purpura, and coronary artery thrombosis.

Use of animal models of von Willebrand factor (vWF) deficiency, both inherited and induced, continues to advance the knowledge of vWF-related diseases. Three examples are reviewed in this article--von Willebrands disease (vWD), thrombotic thrombocytopenic purpura, and coronary artery thrombosis. The success of gene transfer by liver and bone marrow transplantation in porcine vWD and canine hemophilia A, with a change in phenotype that establishes improved hemostasis, portends imminent testing of gene therapy in these models. With use of recombinant technology, the phenotype of hemophilia B fibroblasts has been transformed to normal, as evidenced by secretion of the normal hemostatically active protein. This result is a prelude to implantation in hemophilic animals. Thrombotic thrombocytopenic purpura is characterized by qualitative and quantitative alterations in vWF. A new animal model induced by the venom factor botrocetin, a cofactor of vWF, closely mimics the human syndrome. A proposed pathophysiologic mechanism for thrombotic thrombocytopenic purpura is outlined. The third contribution is recognition that occlusive coronary thrombosis is a vWF-dependent condition. Without vWF, as in porcine vWD or normal pigs treated with a monoclonal anti-vWF antibody, occlusive thrombi do not develop, even with luminal stenosis. The thrombogenicity of coronary atheromas, including those with fissures of the fibrous cap, is also vWF-dependent.

Primary and secondary hemostatic functionalities of rehydrated, lyophilized platelets

BACKGROUND: The rehydrated, lyophilized (RL) platelet (PLT) is being developed as a hemostatic infusion agent for the control of active bleeding. The key to the method for preparing RL PLTs is a mild aldehyde stabilization that allows for freezing and lyophilizing without cellular rupture. RL PLTs have been shown to be effective at rapidly controlling bleeding in animal models of cardiopulmonary bypass induced PLT dysfunction and washout thrombocytopenia, yet the rehydrated cells have proved to be safe with respect to induction of pathologic intravascular coagulation.