Kenneth M. Brinkhous

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.

Clotting defect in hemophilia; deficiency in a plasma factor required for platelet utilization.

Summary 1. By special handling and prolonged centrifugation of normal blood, plasmas with a delayed clotting time can be obtained. If centrifugation is prolonged sufficiently, the plasmas become spontaneously incoagulable. 2. Normal plasmas of this type require the presence of platelets and perhaps other formed elements to correct the delayed clotting of hemophilic plasma. 3. These findings indicate that in hemophilia there is a deficiency in a plasma factor required for platelet utilization. It is suggested that this factor is a thrombocytolysin.

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.

Replacing the first epidermal growth factor-like domain of factor IX with that of factor VII enhances activity in vitro and in canine hemophilia B.

Using the techniques of molecular biology, we made a chimeric Factor IX by replacing the first epidermal growth factor-like domain with that of Factor VII. The resulting recombinant chimeric molecule, Factor IXVIIEGF1, had at least a twofold increase in functional activity in the one-stage clotting assay when compared to recombinant wild-type Factor IX. The increased activity was not due to contamination with activated Factor IX, nor was it due to an increased rate of activation by Factor VIIa-tissue factor or by Factor XIa. Rather, the increased activity was due to a higher affinity of Factor IXVIIEGF1 for Factor VIIIa with a Kd for Factor VIIIa about one order of magnitude lower than that of recombinant wild-type Factor IXa. In addition, results from animal studies show that this chimeric Factor IX, when infused into a dog with hemophilia B, exhibits a greater than threefold increase in clotting activity, and has a biological half-life equivalent to recombinant wild-type Factor IX.

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.

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.

Thrombotic Thrombocytopenia Induced in Dogs and Pigs The Role of Plasma and Platelet vWF in Animal Models of Thrombotic Thrombocytopenic Purpura

Thrombotic thrombocytopenia with severe depletion of plasma von Willebrand factor (vWF) was induced in normal large animals (5 dogs and 2 pigs) by botrocetin, a Bothrops factor requiring vWF for platelet agglutination. Botrocetin (90 to 100 U/kg, 2.14 to 2.38 mg/kg, in a single i.v. injection) reduced plasma vWF activity to < 0.1 U/mL for 24 hours. During this period, multimeric analysis of plasma vWF antigen (Ag) revealed the loss of intermediate- and high-molecular-weight forms with a concomitant increase in lower molecular weight forms. A moderate reduction in factor VIII (FVIII) activity was observed. The vWF reduction was accompanied by transient thrombocytopenia and prolonged bleeding times during the deficiency state. Occlusive platelet thrombi were detected by transmission electron microscopy in the microcirculation of lung and spleen but not kidney or brain 30 minutes after the botrocetin injection. Recovery of plasma vWF and platelet count occurred within 48 hours and was associated with the appearance in the plasma of unusually large forms of vWF:Ag multimers. The vWF:Ag multimer distribution was normal at 72 hours. The ultrastructural distribution of vWF in unstimulated normal porcine and canine platelets was examined by using immunogold staining. VWF was detected in the alpha-granules of normal pig platelets but was not observed in platelets from normal dogs. However, both animals developed thrombotic thrombocytopenia when injected with botrocetin. A second group of animals (2 dogs and 3 pigs) with von Willebrand disease (vWD) was given a single botrocetin injection (90 to 100 U/kg). No thrombocytopenia occurred.(ABSTRACT TRUNCATED AT 250 WORDS)