An anti-αVß3 antibody inhibits coronary artery atherosclerosis in diabetic pigs.

BACKGROUND AND AIMS: Diabetes is a major risk factor for the development of atherosclerosis. Hyperglycemia stimulates vascular smooth muscle cells (VSMC) to secrete ligands that bind to the αVß3 integrin, a receptor that regulates VSMC proliferation and migration. This study determined whether an antibody that had previously been shown to block αVß3 activation and to inhibit VSMC proliferation and migration in vitro, inhibited the development of atherosclerosis in diabetic pigs. METHODS: Twenty diabetic pigs were maintained on a high fat diet for 22 weeks. Ten received injections of anti-ß3 F(ab)2 and ten received control F(ab)2 for 18 weeks. RESULTS: The active antibody group showed reduction of atherosclerosis of 91 ± 9% in the left main, 71 ± 11%, in left anterior descending, 80 ± 10.2% in circumflex, and 76 ± 25% in right coronary artery, (p < 0.01 compared to lesions areas from corresponding control treated arteries). There were significant reductions in both cell number and extracellular matrix. Histologic analysis showed neointimal hyperplasia with macrophage infiltration, calcifications and cholesterol clefts. Antibody treatment significantly reduced number of macrophages contained within lesions, suggesting that this change contributed to the decrease in lesion cellularity. Analysis of the biochemical changes within the femoral arteries that received the active antibody showed a 46 ± 12% (p < 0.05) reduction in the tyrosine phosphorylation of the ß3 subunit of αVß3 and a 40 ± 14% (p < 0.05) reduction in MAP kinase activation. CONCLUSIONS: Blocking ligand binding to the αVß3 integrin inhibits its activation and attenuates increased VSMC proliferation that is induced by chronic hyperglycemia. These changes result in significant decreases in atherosclerotic lesion size in the coronary arteries. The results suggest that this approach may have efficacy in treating the proliferative phase of atherosclerosis in patients with diabetes.

Canine models of inherited bleeding disorders in the development of coagulation assays, novel protein replacement and gene therapies.

Animal models of inherited bleeding disorders are important for understanding disease pathophysiology and are required for preclinical assessment of safety prior to testing of novel therapeutics in human and veterinary medicine. Experiments in these animals represent important translational research aimed at developing safer and better treatments, such as plasma-derived and recombinant protein replacement therapies, gene therapies and immune tolerance protocols for antidrug inhibitory antibodies. Ideally, testing is done in animals with the analogous human disease to provide essential safety information, estimates of the correct starting dose and dose response (pharmacokinetics) and measures of efficacy (pharmacodynamics) that guide the design of human trials. For nearly seven decades, canine models of hemophilia, von Willebrand disease and other inherited bleeding disorders have not only informed our understanding of the natural history and pathophysiology of these disorders but also guided the development of novel therapeutics for use in humans and dogs. This has been especially important for the development of gene therapy, in which unique toxicities such as insertional mutagenesis, germ line gene transfer and viral toxicities must be assessed. There are several issues regarding comparative medicine in these species that have a bearing on these studies, including immune reactions to xenoproteins, varied metabolism or clearance of wild-type and modified proteins, and unique tissue tropism of viral vectors. This review focuses on the results of studies that have been performed in dogs with inherited bleeding disorders that closely mirror the human condition to develop safe and effective protein and gene-based therapies that benefit both species.

Pharmacokinetics and pharmacodynamics of turoctocog alfa and N8-GP in haemophilia A dogs.

The objective of the present study was to evaluate the pharmacokinetic (PK) and pharmacodynamic (PD) profiles of the new recombinant FVIII compound turoctocog alfa and a Glyco-PEGylated FVIII derivative thereof (N8-GP) in Haemophilia A dogs. Six haemophilic dogs divided into two groups were included in the study. Each dog was administered a dose of 125 U kg(-1) , blood samples were collected at predetermined time points for both pharmacokinetic (FVIII measured by one-stage aPTT assay) and pharmacodynamic [whole blood clotting time (WBCT)] evaluations. After intravenous administration to haemophilic dogs, the plasma concentration at the first sampling point was comparable for turoctocog alfa and N8-GP, and the clearance was estimated to be 6.5 and 3.9 mL h(-1) kg(-1) for turoctocog alfa and N8-GP respectively. Both turoctocog alfa and N8-GP were able to reduce the WBCT time to normal levels (<20 min), however, the reduced clearance was reflected in the WBCT, which returned to baseline at a later time point for N8-GP as compared with dogs dosed with turoctocog alfa. The clearance was 40% reduced for N8-GP as compared with turoctocog alfa. Simulations of a multiple dosing regimen in dogs, suggest that to maintain WBCT <20 min N8-GP can be dosed at reduced intervals, e.g. with 4 days between doses, whereas turoctocog alfa will have to be dosed with 2½ day between doses. Data thereby supports N8-GP as an alternative to standard rFVIII replacement therapy, with a more convenient dosing regimen.

Improved kinetics of rIX-FP, a recombinant fusion protein linking factor IX with albumin, in cynomolgus monkeys and hemophilia B dogs.

BACKGROUND: Prophylaxis of hemophilia B, at present, requires multiple infusions of human factor (F)IX concentrates per week. A FIX molecule with a prolonged half-life has the potential to greatly improve the convenience of, and adherence to, prophylaxis. OBJECTIVES: The aim of our studies was to investigate the pharmacokinetic (PK) and pharmacodynamic (PD) profile of a recombinant fusion protein linking coagulation FIX with albumin (rIX-FP). METHODS: Cynomolgus monkeys and hemophilia B dogs received single intravenous doses of rIX-FP (50-500 IU kg(-1)). rIX-FP plasma levels were determined by an activity-based assay (dogs only) and anti-FIX ELISA methods. Additionally, activated partial thromboplastin time (APTT) was determined in hemophilia B dogs. Data were compared with a direct study comparator (recombinant FIX [rFIX]) or previously published data. RESULTS: The terminal half-life of rIX-FP was prolonged in both species compared with FIX reference data. In hemophilia B dogs, human FIX antigen levels remained above 0.05 IU mL(-1) more than three times longer after rIX-FP (7.3 days) compared with rFIX (2.3 days), whereas respective calculations based on activity levels confirmed the observed superior profile. Prolonged PDs of rIX-FP were demonstrated with APTT<60 s sustained around four times longer with rIX-FP (5.9 days) than rFIX (1.5 days). CONCLUSIONS: These studies indicate that the recombinant albumin fusion technology successfully improves the PK profile of FIX. Clinical studies will test whether the improved kinetics result in a significant half-life extension in patients with hemophilia B.

Pharmacokinetics and ex vivo whole blood clot formation of a new recombinant FVIII (N8) in haemophilia A dogs.

N8, a new recombinant factor VIII (rFVIII) compound developed for the treatment of haemophilia A, is produced in Chinese hamster ovary (CHO) cells and formulated without human- or animal-derived materials. The aim of the present study was to compare the pharmacokinetics (PK) and the procoagulant effect, measured by ex vivo whole blood clot formation, of N8 and a commercial rFVIII in a cross-over study in haemophilia A dogs. N8 and Advate® (100 IU kg⁻¹) were administered intravenously to three haemophilia A dogs. Blood was sampled between 0 and 120 h postdose and FVIII:C analysed. PK parameters maximum plasma concentration, area under the curve, half-life (t(½)), clearance, mean residence time (MRT) and volume of distribution and incremental recovery were calculated. Whole blood clotting time (WBCT) and thromboelastography (TEG®) were used to determine the haemostatic potential. No adverse reactions were observed with N8 or Advate ®. N8 and Advate® exhibited similar PK parameters, with t(½) 7.7-11 h and MRT 11-14 h. Both rFVIII compounds corrected the prolonged WBCT (> 48 min) to the range of normal dogs (8-12 min), i.e. N8 to 7.5-10.5 min and Advate® to 7.5-11.5 min. N8 and Advate® also normalized the whole blood clot formation according to TEG®. The native whole blood clotting assays (WBCT, TEG®) appeared to be more sensitive to low concentrations of FVIII than assays in citrated plasma samples. In conclusion, comparison of N8 and Advate ® in haemophilia A dogs revealed similar safety, similar PK and similar effects in whole blood clot formation assays.

Pharmacokinetics, pharmacodynamics and safety of recombinant canine FVIIa in a study dosing one haemophilia A and one haemostatically normal dog.

Recombinant human FVIIa (rhFVIIa) corrects the coagulopathy in hemophilia A and B as well as FVII deficiency. This is also the case in dogs until canine anti-human FVIIa antibodies develop (~2 weeks). Recombinant canine factor VIIa (rcFVIIa), successfully over-expressed by gene transfer in haemophilia dogs, has provided long-term haemostasis (>2 years). However, pharmacokinetics (PK), pharmacodynamics (PD) and safety of rcFVIIa after pharmacological administration have not been reported. We therefore wanted to explore the safety, PK and PD of rcFVIIa in dogs. A pilot study was set up to evaluate the safety as well as PK and PD of rcFVIIa after a single intravenous dose of 270 µg kg(-1) to one HA and one haemostatically normal dog and to directly compare rcFVIIa with rhFVIIa in these two dogs. Single doses of rcFVIIa and rhFVIIa were well tolerated. No adverse events were observed. Pharmacokinetic characteristics including half-life (FVIIa activity: 1.2-1.8 h; FVIIa antigen 2.8-3.7 h) and clearance were comparable for rcFVIIa and rhFVIIa. Kaolin-activated thromboelastography approached normal in the HA dog with the improvement being most pronounced after rcFVIIa. This study provided the first evidence that administering rcFVIIa intravenously is feasible, safe, well tolerated and efficacious in correcting the haemophilic coagulopathy in canine HA and that rcFVIIa exhibits pharmacokinetic characteristics comparable to rhFVIIa in haemophilic and haemostatically competent dogs. This strengthens the hypothesis that rcFVIIa can be administered to dogs to mimic the administration of rhFVIIa to humans.

Prevention of spontaneous bleeding in dogs with haemophilia A and haemophilia B.

Dogs with haemophilia A or haemophilia B exhibit spontaneous bleeding comparable with the spontaneous bleeding phenotype that occurs in humans with severe haemophilia. The phenotypic and genotypic characteristics of haemophilic dogs have been well-described, and such dogs are suitable for testing prophylactic protein replacement therapy and gene transfer strategies. In dogs with haemophilia, long-term effects on spontaneous bleeding frequency (measured over years) can be used as an efficacy endpoint in such studies. Although complete correction of coagulopathy has not been achieved, published data show that prophylactic factor replacement therapy and gene transfer can markedly reduce the frequency of spontaneous bleeding in haemophilic dogs. Further studies are currently ongoing.

A phase II prospective open-label escalating dose trial of recombinant interleukin-11 in mild von Willebrand disease.

von Willebrand factor (VWF) is a multimeric glycoprotein that mediates platelet adhesion and is decreased in von Willebrand disease (VWD). 1-8 deamino-d-arginine vasopressin (DDAVP), the most common treatment for VWD, is limited by tachyphylaxis and inconvenience, and in 20% of the patients, unresponsiveness. Recombinant human interleukin-11 (rhIL-11), a gp-130 signalling cytokine with haematopoietic and anti-inflammatory activity, increases VWF antigen and its activity in heterozygous VWF(+/-) mice and dogs. To determine the biological efficacy and safety of rhIL-11 in non-bleeding human subjects with mild VWD, we conducted a phase II prospective open-label trial of rhIL-11 at 10, 25 and 50 mug kg(-1) subcutaneously (s.c.), given daily for 7 days in nine subjects with mild VWD. VWF and factor VIII (FVIII) levels increased gradually and progressively after s.c. rhIL-11, which was sustained through 7 days of dosing to 1.5- to 3-fold over baseline. Following intravenous DDAVP, 0.3 mug kg(-1), on day 7 there was a further boost in VWF and FVIII levels, suggesting that the mechanism of rhIL-11 differs from that of DDAVP. Platelet VWF mRNA expression measured by quantitative PCR increased from two- to eightfold over baseline, suggesting that the mechanism of rhIL-11 effect may be upregulation of VWF mRNA. VWF and FVIII levels returned to baseline by day 14. rhIL-11 was well tolerated with less than grade-1 hypertension, hypokalaemia and fluid retention. Recombinant IL-11 increases VWF levels in humans with mild VWD, justifying future clinical trials to determine its potential in preventing or reducing bleeding in this patient population.

Protease-resistant insulin-like growth factor (IGF)-binding protein-4 inhibits IGF-I actions and neointimal expansion in a porcine model of neointimal hyperplasia.

IGF-I has been shown to play a role in the progression of atherosclerosis in experimental animal models. IGF-binding protein-4 (IGFBP-4) binds to IGF-I and prevents its association with receptors. Overexpression of a protease-resistant form of IGFBP-4 has been shown to inhibit the ability of IGF-I to stimulate normal smooth muscle cell growth in mice. Based on these observations, we prepared a protease-resistant form of IGFBP-4 and infused it into hypercholesterolemic pigs. Infusion of the protease-resistant mutant inhibited lesion development by 53.3 +/- 6.1% (n = 6; P < 0.01). Control vessels that received an equimolar concentration of IGF-I and the protease-resistant IGFBP-4 showed no reduction in lesion size compared with control lesions that were infused with vehicle. Infusion of a nonmutated form of IGFBP-4 did not significantly inhibit lesion development. Proliferating cell nuclear antigen analysis showed that the mutant IGFBP-4 appeared to inhibit cell proliferation. The area occupied by extracellular matrix was also reduced proportionally compared with total lesion area. Immunoblotting revealed that the mutant IGFBP-4 remained intact, whereas the wild-type IGFBP-4 that was infused was proteolytically cleaved. Further analysis of the lesions revealed that a marker protein, IGFBP-5, whose synthesis is stimulated by IGF-I, was decreased in the lesions that received the protease-resistant, IGFBP-4 mutant, whereas there was no change in lesions that received wild-type IGFBP-4 or the mutant protein plus IGF-I. These findings clearly illustrate that infusion of protease-resistant IGFBP-4 into the perilesion environment results in inhibition of cell proliferation and attenuation of the development of neointima. The findings support the hypothesis that inhibiting IGFBP-4 proteolysis in the lesion microenvironment could be an effective means for regulating neointimal expansion.

Helper-dependent adenoviral gene therapy mediates long-term correction of the clotting defect in the canine hemophilia A model.

BACKGROUND: Adenoviral vector-mediated gene therapy might have potential for long-term correction of the monogenic disease hemophilia A. OBJECTIVE: In this study, we tested the efficacy of administering a helper-dependent adenoviral vector (HDV) designed for maximal liver-restricted canine factor VIII (cFVIII) expression on three out-bred hemophilia A dogs. METHODS: Three FVIII-deficient animals from the University of North Carolina colony were injected with 1 x 10(12) (Dog A), and 3 x 10(12) (Dog B and C) vp kg(-1) helper-dependent adenoviral vector, and we performed systematic analysis of toxicity, persistence of therapeutic gene expression, and molecular analysis of gene transfer. RESULTS: We observed acute dose-dependent elevation in liver enzymes and thrombocytopenia after injection, although both were transient and resolved within 2 weeks. The whole blood clotting time (WBCT), plasma FVIII concentration, FVIII activity, and activated partial thromboplastin time in all animals improved significantly after treatment, and two animals receiving a higher dose reached near normal WBCT with low-level FVIII activity until terminal sacrifice at 3 months, and 2 years. Importantly, the treated dogs suffered no bleeding events after injection. Moreover, we observed persistent vector-specific DNA and RNA in liver tissue collected from one high-dose animal at days 18 and 79, and could not detect the formation of inhibitory antibodies. CONCLUSION: Although vector-associated toxicity remains an obstacle, a single injection of HDV led to long-term transgene expression and vector persistence in two FVIII-deficient animals with conversion of their severe phenotype to a moderate one.

Haemophilic factors produced by transgenic livestock: abundance that can enable alternative therapies worldwide.

Haemophilia replacement factors, both plasma-derived and recombinant, are in relatively short supply and are high-cost products. This has stymied the study and development of alternative methods of administration of haemophilia therapy even in the most economically advanced countries, owing to the large amounts of material needed because bioabsorption and bioavailability of haemophilic factors can be less than 10% when using non-intravenous routes of delivery. There is therefore a need to increase access to therapy worldwide by decreasing the cost and increasing the abundance so that therapy can be achieved through simplified, alternative delivery methods. Transgenic livestock have been used to produce haemophilic factors in milk. Only the pig mammary gland has been shown to carry out the post-translational processing necessary to enable both the biological activity and long circulation half-life needed for therapeutic glycoproteins. Furthermore, the large amounts of recombinant protein that can be produced from pig milk make feasible the use of alternative delivery methods such as oral, intratracheal, subcutaneous, and intramuscular administration.

Intravenous administration of an AAV-2 vector for the expression of factor IX in mice and a dog model of hemophilia B.

Previous experiments have demonstrated the stable expression of factor IX (FIX) protein in mice and canine models of hemophilia B following portal vein gene transfer with a recombinant adeno-associated virus (rAAV) vector encoding FIX. Here, we present the results of studies that further optimized the rAAV vector transgene cassette used to express FIX and explored the use of the less-invasive intravenous (i.v.) route of vector administration for the treatment of hemophilia B. First, a liver-specific promoter was evaluated in conjunction with cis-acting regulatory elements in mice. Constructs that included both the beta-globin intron and the woodchuck hepatitis virus post-transcriptional regulatory element resulted in the highest level of FIX expression in vivo. Using this optimized vector, we demonstrate that i.v. injection was feasible for hepatic gene transfer in mice, achieving 70-80% of portal vein expression levels of FIX. In further studies using the Chapel Hill strain of hemophilia B dogs, we demonstrate for the first time FIX expression and partial correction of the bleeding disorder following i.v. administration of an AAV vector.

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.

Lack of germline transmission of vector sequences following systemic administration of recombinant AAV-2 vector in males.

A potential consequence of systemic administration of viral vectors is the inadvertent introduction of foreign DNA into recipient germ cells. To evaluate the safety of in vivo recombinant adeno-associated virus (rAAV) mediated gene transfer approaches for hemophilia B, we explored the risk of germline transmission of vector sequences following intramuscular (IM) injection of rAAV in four species of male animals (mouse, rat, rabbit and dog). In vector biodistribution studies in mice and rats, there is a dose-dependent increase in the likelihood that vector sequences can be detected in gonadal DNA using a sensitive PCR technique. However, in dogs DNA extracted from semen is negative for vector sequences. To address this discrepancy, studies were done in rabbits, and both semen and testicular DNAs were analyzed for the presence of vector sequences. These studies showed that no AAV vector sequences were detected in DNA extracted from rabbit semen samples collected at time points ranging from 7 to 90 days following IM injection of 1 x 10(13) vector genomes rAAV (vg) per kg. In contrast, DNA extracted from gonadal tissue was positive for vector sequences, but the positive signals diminished in number and strength with time. By FISH analysis, AAV signals were localized to the testis basement membrane and the interstitial space; no intracellular signal was observed. We observed similar findings following hepatic artery administration of rAAV in rats and dogs, suggesting that our findings are independent of the route of administration of vector. Attempts to transduce isolated murine spermatogonia directly with AAV-lacZ were unsuccessful. In clinical studies human subjects injected IM with an AAV vector at doses up to 2 x 10(12) vg/kg have shown no evidence of vector sequences in semen. Together, these studies suggest that rAAV introduced into skeletal muscle or the hepatic artery does not transduce male germ cells efficiently. We conclude that the risk of inadvertent germline transmission of vector sequences following IM or hepatic artery injection of AAV-2 vectors is extremely low.

Intratracheal administration of recombinant human factor IX (BeneFix) achieves therapeutic levels in hemophilia B dogs.

The purpose of this paper was to establish proof of concept for administration of human recombinant F.IX (rF.IX) by inhalation for therapy of hemophilia B. The pharmacokinetics of intratracheal (IT) administration of rF.IX was studied in nine hemophilia B dogs randomized into 3 groups that received 200 IU/kg IT, 1,000 IU/kg IT, or 200 IU/kg intravenously (IV). IT rF.IX produced therapeutic levels of F.IX antigen and activity and the pharmacokinetic parameters were consistent with a slow release from a depot site within the lungs. Bioavailability compared to IV administration was 11% for 200 IU/kg IT and 4.9% for 1,000 IU/kg. The whole blood clotting time began to shorten at 2 h but F.IX bioactivity was not detected until 8 h post infusion in both IT groups. In all groups, F.IX activity was detected through 72 h post administration. These data demonstrate that biologically active rF.IX can reach the systemic circulation when given IT. Aerosolization of rF.IX may provide a needle-free therapeutic option for delivery of rF.IX to hemophilia B patients.

Splenic clearance mechanisms of rehydrated, lyophilized platelets.

A variety of platelet substitutes (e.g., rehydrated, lyophilized (RL) platelets, thromboerythrocytes, plateletsomes, infusible platelet membranes, synthocytes, fibrinogen-coated microcapules) are potentially useful as hemostatic agents in transfusion medicine. However, as "foreign" particles, platelet substitutes interact to varying extents with elements of the reticulo-endothelial system for clearance, reducing hemostatic efficacy. Experiments were performed to better understand the interaction of RL platelets with elements of the innate and acquired immune systems. The infusion of heterologous RL platelets into rats resulted in rapid clearance from the free circulation with half-life values of minutes. The clearance of RL platelets was inhibited when macrophages were rendered apoptotic with gadolinium. Transmission EM analysis of splenic tissue after infusion of lyophilized cells, as well as in vitro mixing studies with splenic macrophages and RL platelets, indicated that macrophage-mediated phagocytosis mechanisms were operant in RL platelet clearance by the reticulo-endothelial system. Studies with IV IgG, as a competitive inhibitor of the macrophage Fc receptor, provides evidence that RL platelet destruction is in part mediated by platelet surface bound IgG. This hypothesis was further supported by the finding that RL platelets react with IgG class antibodies that are pre-existing in naïve animals. These studies provide a rational basis for prolonging the circulation time of RL platelets and other platelet substitutes.

Role of nuclear factor-kappa B (NF-kappa B) in inflammation, periodontitis, and atherogenesis.

Atherosclerosis, the major cause of death and disability in the United States, is a chronic disease with inflammatory components. The first objective of this review is to explain how activation of NF-kappa B contributes to atherosclerosis. The second objective is to describe a potential link between inflammation, activation of NF-kappa B, and periodontitis. The nuclear transcription factor NF-kappa B controls the expression of many genes linked to atherogenesis including those involved with inflammation. We hypothesize that one unifying mechanism in this complex disease is the activation of NF-kappa B. The mechanism(s) that activates NF-kappa B in atherogenesis is unknown and the effect of inhibiting NF-kappa B activation on atherogenesis is untested. Periodontal disease has now been established as a risk factor for atherosclerosis and its thrombotic complications. It is unknown if periodontal disease contributes to the initiation or progression of atherosclerosis. We hypothesize that the chronic and intense inflammatory response accompanying periodontal disease produces an excess burden of circulating mediators of inflammation that initiate or exacerbate the inflammatory components of atherogenesis. Further understanding of the mechanisms involved in the activation of NF-kappa B in atherosclerosis could lead to important therapeutic applications especially as it relates to the impact of periodontitis.

Intracellular function in rehydrated lyophilized platelets.

This study aimed to evaluate the effect of cross-linking and lyophilization on intracellular signalling processes in rehydrated, lyophilized (RL) platelets, which are under development as a platelet substitute for transfusion. Exposure of RL platelets to thrombin resulted in enhanced phosphorylation of several proteins, including 18 kDa and 42 kDa kinase substrates that were shown to be the substrates of myosin light chain and protein kinase C respectively. Cross-linking and lyophilization depleted the platelets of free cytoplasmic ADP and ATP, but had less effect on protein-bound nucleotides. The surface membrane of RL platelets was found to be permeable to poly dT probes less than approximately 3 kDa in size; larger nucleotide probes and proteins did not penetrate the surface membrane. Taken together, our results indicate that RL platelets retain some of the haemostatic stimulus-response functions of fresh platelets and are capable of feedback amplification in coagulation.

Sustained expression of therapeutic level of factor IX in hemophilia B dogs by AAV-mediated gene therapy in liver.

We demonstrate that a single intraportal vein injection of a recombinant adeno-associated virus (rAAV) vector encoding canine factor IX (cFIX) cDNA under the control of a liver-specific enhancer/promoter leads to a long-term correction of the bleeding disorder in hemophilia B dogs. Stable expression of the therapeutic level of cFIX (5% of normal level) was detected in the plasma of a dog injected with an AAV vector at a dose of 4.6 x 10(12) particles/kg for over 7 months. Both whole-blood clotting time (WBCT) and activated partial thromboplastin time (aPTT) of the treated dogs have been greatly decreased since the treatment. No anti-canine factor IX antibodies have been detected in the treated animals. Importantly, no bleeding has been observed in the dog that expresses a therapeutic level of cFIX for 7 months following vector administration. Moreover, no persistent significant hepatic enzyme abnormalities were detected in the treated dogs. Thus, a single intraportal injection of a rAAV vector expressing cFIX successfully corrected the bleeding disorder of hemophilia B dogs, supporting the feasibility of using AAV-based vectors for liver-targeted gene therapy of genetic diseases.