ABSTRACT
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.
Subject(s)
Dependovirus/genetics , Factor IX/genetics , Genetic Therapy/methods , Genetic Vectors/administration & dosage , Hemophilia B/therapy , Animals , Dogs , Factor IX/analysis , Gene Expression , Genetic Vectors/genetics , Hepatic Veins , Humans , Infusions, Intravenous , Injections, Intravenous , Liver/metabolism , Mice , Tail/blood supply , Transduction, Genetic/methodsABSTRACT
Inhibition of proliferative neointima formed by vascular smooth muscle cells is a potential target in preventing angioplasty-induced restenosis. We have created a potent antiproliferative by fusing the active regions of the p27 and p16 cell cycle inhibitors. Intravascular delivery of a replication-deficient adenoviral vector (AV) encoding this p27-p16 fusion protein, named W9, inhibited balloon injury-induced neointimal hyperplasia in rabbit carotid arteries. In a therapeutically more relevant model, AV-W9 was delivered to balloon-injured porcine coronary arteries in vivo using an infusion catheter. Of the three coronary arteries, two were injured with a 15-mm balloon catheter and either were left untreated or were treated with 10(12) viral particles of either AV-W9 or a control null virus. AV-W9 treatment significantly inhibited neointimal hyperplasia in this porcine arterial balloon injury model compared with untreated or control virus-treated vessels. The average intimal area of the AV-W9-treated group 10 days after balloon injury and treatment was 0.42+/-0.36 mm(2), whereas the AV-null group demonstrated an intimal area of 0.70+/-0.52 mm(2). At day 10 the average intimal thickness of the AV-W9-treated vessels was 9.1 microm (n=5, x 20 magnification) compared with 21.2 microm (n=5, x 20 magnification) in control virus-treated vessels. This trend was also observed at 28 days after balloon injury and gene transfer during which AV-W9-treated vessels demonstrated an average intimal thickness of 4.7 microm (n=8, x 20 magnification) compared with 13.3 microm (n=3, x 20 magnification) in control virus-treated vessels and 7.3 microm (n=5, x 20 magnification) in the sham-treated vessels. The AV-W9 treatment was safe and well tolerated. These data suggest that AV-W9 gene therapy may be useful in preventing angioplasty-induced intimal hyperplasia in the coronary artery.
Subject(s)
Angioplasty, Balloon, Coronary/adverse effects , Cell Cycle Proteins/genetics , Coronary Disease/prevention & control , Cyclin-Dependent Kinase Inhibitor p16/genetics , Genetic Therapy/methods , Hyperplasia/prevention & control , Tumor Suppressor Proteins , Adenoviridae/genetics , Animals , Cardiac Catheterization , Cells, Cultured , Coronary Disease/etiology , Coronary Disease/pathology , Cyclin-Dependent Kinase Inhibitor p27 , Cyclin-Dependent Kinases/antagonists & inhibitors , Disease Models, Animal , Female , Genetic Vectors/administration & dosage , Genetic Vectors/genetics , Humans , Infusions, Intra-Arterial , Male , Muscle, Smooth, Vascular/cytology , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/metabolism , Rabbits , Recombinant Fusion Proteins/administration & dosage , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Swine , Transduction, Genetic/methods , Treatment Outcome , Tunica Intima/pathologyABSTRACT
Adenoviral vectors have shown promise in a variety of preclinical vascular disease models. Intravascular infusion is one methodology to introduce the adenoviral vector into the affected area of the blood vessel. The biocompatibility of the infusion catheter with the adenoviral vector is key for successful local transfer. It has been recently suggested that catheter-based delivery of adenoviral vectors may result in the loss of vector infectivity. We demonstrate here a catheter capable of delivering adenoviral vectors without the loss of viral particle or infectious titers. First- (DeltaE1) and second- (DeltaE1/DeltaE4) generation adenoviral vectors were tested for their biocompatibility with the Crescendo microporous infusion catheter, which is designed for local infusion of therapeutic agents to human coronary or peripheral arteries. We found that incubation of either the DeltaE1 or the DeltaE1/DeltaE4 viral vectors for up to 30 min in the catheter at 37 degrees C did not result in a loss of viral particles or of viral infectivity. Here, we show that the Crescendo catheter is biocompatible with adenoviral vectors and suitable for vascular gene therapy.
