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 8.5x10(12) 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.

In vivo gene therapy of hemophilia B: sustained partial correction in factor IX-deficient dogs.

The liver represents a model organ for gene therapy. A method has been developed for hepatic gene transfer in vivo by the direct infusion of recombinant retroviral vectors into the portal vasculature, which results in the persistent expression of exogenous genes. To determine if these technologies are applicable for the treatment of hemophilia B patients, preclinical efficacy studies were done in a hemophilia B dog model. When the canine factor IX complementary DNA was transduced directly into the hepatocytes of affected dogs in vivo, the animals constitutively expressed low levels of canine factor IX for more than 5 months. Persistent expression of the clotting factor resulted in reductions of whole blood clotting and partial thromboplastin times of the treated animals. Thus, long-term treatment of hemophilia B patients may be feasible by direct hepatic gene therapy in vivo.

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.

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.

Effect of carbon monoxide on atherogenesis in normal pigs and pigs with von Willebrand's disease.

The extent of coronary and aortic atherosclerosis was examined in pigs following balloon-catheter injury of coronary arteries and subsequent feeding of an atherogenic diet for 4 months. The pigs were either exposed intermittently to 100 ppm carbon monoxide or to ambient air alone. Three types of pigs were used: normals, homozygotes for von Willebrand's disease (bleeders), and heterozygotes (carriers). The 3 types of pigs developed coronary artery intimal lesions of similar thickness. Aortic lesions, quantified as percent of aortic surface involved with sudanophilia and raised fibrous plaques, were slightly less extensive in bleeder pigs than in normals. Carbon monoxide exposure did not increase the thickness of coronary artery intimal lesions, nor did it increase the percent of aortic surface involved with sudanophilia or raised fibrous lesions. These results suggest that exposure to low levels of carbon monoxide does not perceptibly enhance atherogenesis induced by hypercholesterolemia. None of 14 bleeder pigs showed evidence of myocardial infarction, despite significant coronary artery narrowing. Of the 24 normal and carrier pigs, 5 showed myocardial infarction. Four of these 5 pigs were exposed to carbon monoxide, while 1 was not exposed. These findings suggest that exposure to low levels of carbon monoxide may increase the incidence of myocardial infarction and that the absence of von Willebrand factor may be protective.

Specific inhibition of platelet agglutination and aggregation by aromatic amidino compounds.

A series of aromatic amidino compounds were investigated for their inhibitory effect on platelet agglutination and platelet aggregation. Agglutination of fresh or fixed platelets was produced by bovine plasma or by human plasma in combination with ristocetin, while aggregation of fresh platelets was induced by ADP, thrombin or collagen. Highly effective inhibitors were found for both types of platelet clumping, but there was no parralelism between the inhibitory activities in the two test system. 5-(5-amidino-2-benzimidazolyl)-2-(4-hydroxybenzene)benzimidazole suppressed agglutination exclusively. Pentamidine, on the other hand, strongly blocked the aggregation reaction, but did not interfere with agglutination, even at high concentrations. Compounds which inhibited aggregation also prevented the liberation of serotonin from the platelets.

Pharmacokinetics of recombinant factor IX after intravenous and subcutaneous administration in dogs and cynomolgus monkeys.

Hemophilia B therapy requires intravenous (IV) infusions of large volumes of factor IX due to the low concentration of factor IX in concentrates (approximately 100 IU/mL). High concentration recombinant factor IX (rFIX) could be a significant advance since it would reduce the large volumes necessary for IV dosing and allow for low-volume subcutaneous (SC) administration. To evaluate high concentration factor IX, we produced formulations with either 2,000 or 4,000 IU/mL and studied the SC bioavailability in beagle dogs, cynomolgus monkeys and hemophilia B dogs along with efficacy in hemophilia B dogs. Beagle dog SC bioavailability was 86.4% using a 2000 IU/mL formulation and 77.0% using a 4000 IU/mL formulation. Monkey bioavailability of a 4000 IU/mL formulation of rFIX was 34.8%. A single SC administration of 200 IU/kg (4000 IU/mL) of rFIX to hemophilia B dogs, produced factor IX clotting activity above 5% for 5 days with a bioavailability of 48.6%. High concentration SC rFIX has an acceptable pharmacokinetic profile in monkeys and dogs, and produces a sustained FIX activity in hemophilic dogs.

Extravascular administration of factor IX: potential for replacement therapy of canine and human hemophilia B.

Current therapy for hemophilia B requires large intravenous doses of factor IX (F.IX) given in the clinic or at home. Although home therapy is possible for many patients, it is often complicated by factors such as the lack of good venous access. Very little is known about extravascular routes for administering proteins like F.IX (57 kD) or other vitamin K-dependent procoagulant factors into the circulation. Questions about the absorption rate from extravascular administration as well as plasma recovery and bioavailability have arisen recently with the growing availability of highly purified procoagulant proteins and increased interest in gene therapy of hemophilia B. Therefore, a group of studies were undertaken to determine the absorption rate, plasma recovery, and bioavailability of high purity, human plasma-derived F.IX concentrates administered via extravascular routes in hemophilia B dogs and in one human hemophilia B subject. Five hemophilia B dogs were given human F.IX via either a subcutaneous (s.c.), intramuscular (i.m.), intraperitoneal (i.p.) or intravenous (i.v.) route. In a subsequent study, a single SC administration of human F.IX was compared to an identical i.v. dose of F.IX in the human hemophilia B subject. All extravascular routes of F.IX administration in both the canine and human gave lower levels of circulating plasma F.IX than the i.v. route, however all routes resulted in measurable F.IX activity. Of the extravascular routes, the i.m. injection in the canine resulted in a bioavailability of 82.8%, while the s.c. injection resulted in a bioavailability of 63.5%. F.IX reached the plasma compartment by all extravascular routes used, confirming that F.IX can be absorbed extravascularly. The duration of measurable F.IX activity following extravascular administration is prolonged beyond that typically seen with i.v. administration. These data show that significant levels of F.IX may be obtained via s.c. injection in canine and human hemophilia B subjects and further highlight the potential of extravascular routes of administration for future experimental and clinical uses of F.IX and other procoagulant proteins.

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 Willebrand's 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.

Morphological study of early phases of platelet adhesion to foreign surfaces: effect of calcium.

The role of calcium in the interfacial reactions occurring during the early phases of blood contact with glass surfaces has been studied using a rheologic technique that permits enumeration of adherent platelets as well as ultrastructural visualization of the blood-glass interface by both transmission and scanning electron microscopy . In the presence of calcium, platelets adhere to the glass surface at a rapid rate. Within 4 minutes, most of the adherent platelets have lost their normal discoid shape and show pseudopod formation and cytoplasmic spreading. These platelets conform with the glass surface and show central apposition of platelet organelles similar to that seen in activated platelets. Fibrin strands and platelet aggregates are also seen. Chelation of calcium using sodium citrate or EDTA results in decreased platelet adhesion or retention to glass. Platelets that are adherent to glass in the presence of citrate and EDTA retain their discoid shape, although an occasional platelet may show pseudopod formation and centralization of organelles. Calcium appears to be an important factor governing the adhesion of platelets to glass and appears to exert this effect on the platelets themselves in their plasma milieu and not on the preceding adsorption of plasma proteins.

Glycoprotein Ib bioassays. Activity levels in Bernard-Soulier syndrome and in stored blood bank platelets.

Glycoprotein Ib (GP-Ib) is a major platelet receptor protein concerned with von Willebrand-factor binding, platelet agglutination, and platelet adhesion, and it is required for normal hemostasis. By the use of botrocetin (venom coagglutinin), both quantitative and semiquantitative assays for GP-Ib activity were developed. The latter assay uses limiting dilutions of botrocetin as a measure of GP-Ib activity. Platelets, stored up to 23 days under blood bank conditions, were assayed by the limiting dilution test. Values of GP-Ib were progressively diminished after 9 to 10 days of storage, reaching levels of less than 10% at 23 days. Platelets from a subject with Bernard-Soulier syndrome showed less than 10% GP-Ib activity. These assays appear to be a specific measure of functional GP-Ib activity, and, when combined with GP-Ib antigen measurement by other methods, they provide a means for further characterizing GP-Ib abnormalities.

Animal models: importance in research on hemorrhage and thrombosis.

Despite some sentiments against animal research, animal models continue to be important in studying hemorrhage and thrombosis. Examples of genetic models are dogs and pigs with von Willebrand's disease. The homozygous von Willebrand pig appears to be resistant to arteriosclerosis, presumably due to impairment of the platelet aggregating function. Among acquired models, pigs are gaining in favor, perhaps because their clotting and platelet characteristics resemble those in humans. Species vary markedly in their normal plasma levels of platelet aggregating factor/von Willebrand factor (PAF/vWF). One promising approach to the study of thrombosis is using platelet anti-aggregating drugs to inhibit PAF/vWF dependent platelet thrombus formation; a drug-induced von Willebrand state seems feasible.

Development and present status of concentrate therapy for hemophilia and von Willebrand's disease.

A brief review of the current status of treatment of two severe bleeder diseases, hemophilia A and von Willebrand's disease, with factor VIII concentrate is given. The functional activities deficient in the two diseases, antihemophilic factor (AHF) or coagulant factor VIII (F. VIII:C) in hemophilia, and the von Willebrand factor(s) (vWF; F.VIII R: Ag) in von Willebrand's disease, are present in a very large protein moiety of plasma, the macromolecular factor VIII complex. The rationale for replacement therapy of hemophilia depends on assessment of the severity of a given hemorrhagic episode followed by single, multiple, or continuous infusions of factor VIII concentrates sufficient to raise the plasma AHF to the hemostatic level (15 - less than 50%, depending on the severity of hemorrhage). Prophylactic regimens prevent hemorrhage but are expensive. Despite many problems in their use, the success of factor VIII concentrates in controlling severe hemorrhage and in preventing crippling by arthropathies is evident in many ways including the demand for concentrates, which now exceed the economic value of two other main products of plasma fractionation, albumin and gamma globulin. The rationale for therapy of von Willebrand's disease is dependent on the formation of the hemostatic platelet plug, which includes adhesion of platelets to a vascular site of bleeding, followed by platelet aggregation to give the hemostatic thrombus. The very large molecular weight arrays of the factor VIII complex contained in cryoprecipitate appear needed for development of the hemostatic plug. A new test for determining the plasma platelet-aggregating vWF, the Botrocetin test, appears superior to the ristocetin test. An activator factor in Bothrops snake venom is used. Some prospects for improving the factor VIII therapeutic preparations through recombinant DNA technology and through altering procedures for fractionation are outlined.