Dwight A. Bellinger

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.

Porphyromonas gingivalis Bacteremia Induces Coronary and Aortic Atherosclerosis in Normocholesterolemic and Hypercholesterolemic Pigs

Objectives—The aim of this study was to determine whether recurrent intravenous injections with Porphyromonas gingivalis (P gingivalis), mimicking periodontitis-associated bacteremia, promotes coronary artery and aortic atherosclerosis in pigs. Methods and Results—Pigs (n=36) fed low- or high-fat chow were divided into P gingivalis–sensitized and P gingivalis–challenged groups or P gingivalis–sensitized controls and saline-treated controls. Pigs were sensitized with 109 killed P gingivalis subcutaneously. Four weeks later all sensitized pigs in the group to be challenged started intravenous injections thrice weekly for 5 months with 106 to 107 colony forming units of P gingivalis while controls received saline. Anti–P gingivalis antibody, serum cholesterol, and complete blood counts were assayed monthly. Pigs were euthanized 2 weeks after the last injection, and coronary arteries and aortas were analyzed by histomorphometry and immunohistochemistry. Anti–P gingivalis antibody was increased by P gingivalis exposure. P gingivalis–challenged pigs developed a significantly greater amount of coronary and aortic atherosclerosis than controls in the normocholesterolemic group and nearly significant in the hypercholesterolemic group. P gingivalis was detected by polymerase chain reaction in arteries from most (94%, 16 of 17) P gingivalis–challenged pigs but not controls. Conclusions—Recurrent P gingivalis bacteremia induces aortic and coronary lesions consistent with atherosclerosis in normocholesterolemic pigs and increases aortic and coronary atherosclerosis in hypercholesterolemic pigs.

Influence of Vector Dose on Factor IX-Specific T and B Cell Responses in Muscle-Directed Gene Therapy

Intramuscular injection of an adeno-associated virus (AAV) vector has resulted in vector dose-dependent, stable expression of canine factor IX (cF.IX) in hemophilia B dogs with an F.IX missense mutation (Herzog et al., Nat. Med. 1999;5:56-63). The use of a species-specific transgene allowed us to study risks and characteristics of antibody formation against the therapeutic transgene product. We analyzed seven dogs that had been injected at a single time point at multiple intramuscular sites with varying vector doses (dose per kilogram, dose per animal, dose per site). Comparison of individual animals suggests an increased likelihood of inhibitory anti-cF.IX (inhibitor) development with increased vector doses, with dose per site showing the strongest correlation with the risk of inhibitor formation. In six of seven animals, such immune responses were either absent or transient, and therefore did not prevent sustained systemic expression of cF.IX. Transient inhibitory/neutralizing anti-cF.IX responses occurred at vector doses of 2 x 10(12)/site, whereas a 6-fold higher dose resulted in a longer lasting, higher titer inhibitor. Anti-cF.IX was efficiently blocked in an eighth animal that was injected with a high vector dose per site, but in addition received transient immune suppression. Inhibitor formation was characterized by synthesis of two IgG subclasses and in vitro proliferation of lymphocytes to cF.IX antigen, indicating a helper T cell-dependent mechanism. Anti-cF.IX formation is likely influenced by the extent of local antigen presentation and may be avoided by limited vector doses or by transient immune modulation.

Peripheral transvenular delivery of adeno-associated viral vectors to skeletal muscle as a novel therapy for hemophilia B

Muscle represents an important tissue target for adeno-associated viral (AAV) vector-mediated gene transfer of the factor IX (FIX) gene in hemophilia B (HB) subjects with advanced liver disease. Previous studies of direct intramuscular administration of an AAV-FIX vector in humans showed limited efficacy. Here we adapted an intravascular delivery system of AAV vectors encoding the FIX transgene to skeletal muscle of HB dogs. The procedure, performed under transient immunosuppression (IS), resulted in widespread transduction of muscle and sustained, dose-dependent therapeutic levels of canine FIX transgene up to 10-fold higher than those obtained by intramuscular delivery. Correction of bleeding time correlated clinically with a dramatic reduction of spontaneous bleeding episodes. None of the dogs (n = 14) receiving the AAV vector under transient IS developed inhibitory antibodies to canine FIX; transient inhibitor was detected after vector delivery without IS. The use of AAV serotypes with high tropism for muscle and low susceptibility to anti-AAV2 antibodies allowed for efficient vector administration in naive dogs and in the presence of low- but not high-titer anti-AAV2 antibodies. Collectively, these results demonstrate the feasibility of this approach for treatment of HB and highlight the importance of IS to prevent immune responses to the FIX transgene product.

Reduction in Atherosclerotic Lesion Size in Pigs by αVβ3 Inhibitors Is Associated With Inhibition of Insulin-Like Growth Factor-I–Mediated Signaling

Insulin-like growth factor-I (IGF-I) is a potent stimulant of smooth muscle cell (SMC) migration and proliferation and has been implicated in the development of experimental atherosclerotic lesions. Because optimal stimulation of SMC in vitro by IGF-I requires ligand occupancy of alphaVbeta3, these studies were conducted to determine whether alphaVbeta3 antagonists would result in a change in lesion size and whether they could alter IGF-I-mediated actions. Clamps were placed on the carotid and femoral arteries of normal pigs that had been fed a high-cholesterol diet for 4 weeks. alphaVbeta3 inhibitors (SC-69000, SC-65811) (10(-6) mol/L) or saline were infused for 2 weeks into the peristenotic area. Lesion area, the number of SMC layers, and proliferating cell nuclear antigen positive cells were determined in a 1.2-mm segment of each artery. Lesion areas were 304 788+/-113 453 micron(2) (saline), compared with 149 799+/-35 456 micron(2) (SC-69000) (P<0.01). Lesion areas in arteries treated with SC-64258, a compound that does not bind to alphaVbeta3, were 310 284+/-160 467 micron(2), P=not significant. In a second experiment, lesion areas were 110 391+/-17 347 micron(2) (saline) and 59 533+/-17 568 micron(2) (SC-65811, P<0.001). Neointimal SMC layers were reduced by SC-65811 from 7.4+/-4.5 to 3.0+/-0.4 (P<0.001). To determine whether IGF-I action was altered, IGF binding protein-5, which is synthesized in response to IGF-I, was analyzed. IGF-I binding protein-5 mRNA abundance was reduced by 67+/-8% in the 6 lesions treated with SRL-69000 compared with saline controls (P<0.001). We conclude that alphaVbeta3 antagonists block the development of lesions in pigs that have been induced by a high-cholesterol diet and stenosis, and the effect of these compounds is associated with their ability to inhibit IGF-I-mediated signaling.

Absence of a desmopressin response after therapeutic expression of factor VIII in hemophilia A dogs with liver-directed neonatal gene therapy

Hemophilia A (HA) is a bleeding disorder caused by factor VIII (FVIII) deficiency. FVIII replacement therapy can reduce bleeding but is expensive, inconvenient, and complicated by development of antibodies that inhibit FVIII activity in 30% of patients. Neonatal hepatic gene therapy could result in continuous secretion of FVIII into blood and might reduce immunological responses. Newborn HA mice and dogs that were injected i.v. with a retroviral vector (RV) expressing canine B domain-deleted FVIII (cFVIII) achieved plasma cFVIII activity that was 139 ± 22% and 116 ± 5% of values found in normal dogs, respectively, which was stable for 1.5 yr. Coagulation tests were normalized, no bleeding had occurred, and no inhibitors were detected. This is a demonstration of long-term fully therapeutic gene therapy for HA in a large animal model. Desmopressin (DDAVP; 1-deamino-[d-Arg8]vasopressin) is a drug that increases FVIII activity by inducing release of FVIII complexed with von Willebrand factor from endothelial cells. It has been unclear, however, if the FVIII is synthesized by endothelial cells or is taken up from blood. Because the plasma cFVIII in these RV-treated dogs derives primarily from transduced hepatocytes, they provided a unique opportunity to study the biology of the DDAVP response. Here we show that DDAVP did not increase plasma cFVIII levels in the RV-treated dogs, although von Willebrand factor was increased appropriately. This result suggests that the increase in FVIII in normal dogs after DDAVP is due to release of FVIII synthesized by endothelial cells.

von Willebrand's disease prevents occlusive thrombosis in stenosed and injured porcine coronary arteries.

We studied the role of von Willebrand factor in coronary thrombosis in normal, heterozygous, and homozygous von Willebrands disease pigs by producing coronary stenosis with a Goldblatt clamp positioned around the left anterior descending coronary artery. Flow velocity was assessed by a 20-MHz Doppler velocity probe distal to the Goldblatt clamp. Myocardial extracellular potassium levels were measured by potassium-sensitive electrodes in myocardium supplied by the left anterior descending artery. Whereas stenosis sufficient to block reactive hyperemia to a 20-second occlusion produced an elevation of myocardial extracellular potassium, it produced neither spontaneous cyclic flow reductions nor permanent cessation of coronary blood flow velocity. Injury of the coronary artery at the stenosis site with spring-loaded forceps produced cyclic flow reductions or permanent cessation of flow in eight of nine phenotypically normal pigs. On the other hand, flow variations occurred in none of the 10 von Willebrands disease pigs, including four given purified von Willebrand factor at a dose that failed to correct the bleeding time (p<0.001, X2 test). Permanent cessation of flow was caused by an occlusive platelet-fibrin-red-blood-cell thrombus. Scanning electron micrographs from pigs with cyclic flow variations and from von Willebrands disease pigs showed injured endothelium covered by adherent platelets, red and white blood cells, and fibrin. These data suggest an important role of native von Willebrand factor in sudden occlusive arterial thrombosis following stenosis and intimal injury.

Platelet-targeted gene therapy with human factor VIII establishes haemostasis in dogs with haemophilia A

It is essential to improve therapies for controlling excessive bleeding in patients with haemorrhagic disorders. As activated blood platelets mediate the primary response to vascular injury, we hypothesize that storage of coagulation Factor VIII within platelets may provide a locally inducible treatment to maintain haemostasis for haemophilia A. Here we show that haematopoietic stem cell gene therapy can prevent the occurrence of severe bleeding episodes in dogs with haemophilia A for at least 2.5 years after transplantation. We employ a clinically relevant strategy based on a lentiviral vector encoding the ITGA2B gene promoter, which drives platelet-specific expression of human FVIII permitting storage and release of FVIII from activated platelets. One animal receives a hybrid molecule of FVIII fused to the von Willebrand Factor propeptide-D2 domain that traffics FVIII more effectively into α-granules. The absence of inhibitory antibodies to platelet-derived FVIII indicates that this approach may have benefit in patients who reject FVIII replacement therapies. Thus, platelet FVIII may provide effective long-term control of bleeding in patients with haemophilia A.

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.