1117. Gene Therapy in a Rat Model of Pulmonary Hypertension

CARDIOVASCULAR: ANGIOGENESIS AND VASCULAR GENE THERAPY humans. Apolipoprotein (apo) A-I is the principal apolipoprotein of HDL and a strong correlation exists between apo A-I and HDL cholesterol levels. The ATP binding cassette transporter I (ABCA1) is defective in patients with Tangier disease and familial HDL deficiency. Indirect evidence suggests that ABCA1 expression in the liver is an important determinant of HDL cholesterol levels. Therefore, the effect of adenoviral human apo A-I (AdA-I), murine ABCA1 (AdABCA1) and combined AdA-I and AdABCA1 transfer on the lipoprotein profile and on progression of atherosclerosis in C57BL/6 apo E deficient mice was evaluated. Methods: Forty-four apo E deficient mice were randomized to 5 different groups: a baseline reference group, an Adnul control progression group, an AdABCA1 intervention group, an AdA-I intervention group and a combined AdA-I and AdABCA1 intervention group. Gene transfer was performed at the age of 3 months with 5 x 1010 particles of E1E3E4-deleted adenoviral vectors expressing the transgenes under control of a hepatocyte-specific promoter. Lipoprotein profiles were determined by gel filtration on a Superdex HR column. The baseline reference group was sacrificed at the age of 3 months for histological analysis, whereas gene transfer mice were sacrificed 56 days after transfer. Results: Non-HDL and HDL cholesterol levels at baseline in apo E deficient mice were 290 ± 37 mg/dl and 25 ± 1.4 mg/dl, respectively. The lipoprotein profile was unaltered after transfer with Adnul. Human apo A-I levels were stable for the entire duration of the experiment and were 96 ± 12 mg/dl and 120 ± 5.4 mg/dl after AdAI and combined AdA-I and AdABCA1 transfer, respectively. Transfer with AdA-I resulted in a 1.8-fold (p<0.0001) increase of HDL cholesterol at day 10 and day 35 and a 1.6-fold (p<0.05) increase at day 56 whereas non-HDL cholesterol levels were unaltered. HDL cholesterol in mice treated with AdABCA1 increased 1.2-fold (p<0.05) and 1.3-fold (p<0.01) at day 35 and day 56, respectively. However, non-HDL cholesterol also increased 1.2-fold (p<0.05). Combined transfer with AdA-I and AdABCA1 resulted in a similar increase of HDL cholesterol as observed after isolated AdA-I transfer. Intimal area was 7200 ± 2500 µm2 at baseline and increased 3.0-fold (p<0.05) in the Adnul progression group. Gene transfer with AdABCA1 resulted in an accelerated progression of atherosclerosis. The intimal area was 6.0-fold (p=0.0002) and 2.0-fold (p<0.05) larger in comparison with the baseline and the Adnul group, respectively. The intimal area after transfer with AdA-I was not significantly different compared to baseline. The opposing effects of AdA-I and AdABCA1 transfer were confimed in the combined transfer group. The intimal area was 2.3-fold (p<0.05) lower compared to the isolated AdABCA1 transfer. Conclusion: AdA-I and AdABCA1 transfer have opposing effects on the progression of early atherosclerosis in apoE deficient mice notwithstanding an increase of HDL cholesterol induced by both vectors.

1116. Gene Transfert of SERCA2a Inhibits Smooth Muscle Proliferation and Reduce Neointima Formation after Balloon Injury in the Rat Larissa Lipskaia,1 Federica Del Monte,2 Roger J. Hajjar,2 AnneMarie Lompre.1 1 INSERM U621, CHU Pitié-Salpêtrière, Paris, France; 2 Cardiovascular Research Center, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA. Proliferation of vascular smooth muscle cells (VSMC) is associated with major alterations in SR Ca2+ handling and loss of SERCA 2a. We have restored SERCA 2a expression by adenoviral gene transfer in a model of neointima formation induced by balloon injury of the left carotid artery of the rat using a 2F Fogarty catheter. The effect of gene delivery was evaluated after 2 weeks with morphometry and immunohistochemical staining. There was no significant S430

difference in the thickness of the media between groups. The intima/ media ratio was significantly lower in SERCA 2a infected than in injured non-infected (0.29 ± 0.043, n=5 vs 0.85 ± 0.179, n=5 p< 0.05) carotids, and was comparable to that observed in control carotids (0.196 ± 0.025, n=11, NS). The SERCA 2a-infected carotids displayed a differentiated phenotype. The pathways leading to inhibition of proliferation was analysed in rat aortic VSMC cultured in presence of serum. Forced expression of SERCA 2a was sufficient to arrest proliferation at the G1 phase. SERCA 2a expression resulted in inactivation of calcineurin. The transcriptional activity of its target, NFAT was greatly reduced in SERCA 2a-expressing cells. Furthermore, SERCA 2a increased pCREB transcriptional activity and expression of its target p21Waf1/Cip1 without any changes in p27 KIP . In conclusion, we provide evidence that SERCA 2a gene transfer can prevent neointimal formation that occurs after balloon injury in the rat. We demonstrate that SERCA 2a controls VSMC proliferation by regulating the calcium-dependent transcription factors NFAT and CREB.

1117. Gene Therapy in a Rat Model of Pulmonary Hypertension Stacy L. Porvasnik,1,2 Tankut Onal,1 Yoshi Sakai,1,2 Raquel Torres,5 Glenn A. Walter,4 Barry J. Byrne,1,2,3 Carolyn T. Spencer.1 1 Pediatric Cardiology, University of Florida, Gainesville, FL; 2 Powell Gene Therapy Center, University of Florida, Gainesville, FL; 3Cellular and Molecular Therapy, University of Florida, Gainesville, FL; 4Physiology, University of Florida, Gainesville, FL; 5AMRIS, University of Florida, Gainesville, FL. Primary pulmonary hypertension is a life threatening disease characterized by increased pulmonary vascular resistance. This results in right ventricle (RV) hypertrophy, dilation, and potential RV failure due to increased right ventricular afterload. Current treatment strategies involve the use of various pulmonary vasodilators, including intravenous administration of prostacyclin (PGI2) which improves symptoms, morbidity and mortality of patients. The goal of this project is to develop a clinically relevant gene therapy treatment strategy for patients with primary pulmonary hypertension. Preliminary studies have involved the characterization of a monocrotaline (MCT)-induced rat model of pulmonary hypertension (PH) to be used for evaluation of recombinant adenoassociated viral (rAAV) vector-mediated correction. Using this model, we can recapitulate many of the PH-associated symptoms such as; endothelial cell damage and pulmonary vascular injury, after a single MCT injection. Pulmonary artery pressure was significantly different between the 60 mg/kg test group and the PBS control group (0.44 ± 0.1 mmHg vs 0.22 ± 0.04 mmHg; p=0.0006). The right ventricle weight per body weight also differed between the two groups (0.727 ± 0.1 vs 0.605 ± 0.087). Using a single injection of MCT to induce PH, we currently are assessing intratracheal delivery of rAAV serotype 5 vectors expressing the prostacyclin synthase gene to produce sufficient levels of prostacyclin for prevention of PH. Four week after intratracheal delivery of 3 x 1011 P rAAV5-hPGIS, a single 60 mg/kg dose of MCT was administered. Currently the animals are being weighed weekly and will have MRI images, echocardiograms and pulmonary artery pressures measured by catheterization to evaluate the success of treatment. Preliminary data of MRI images suggest no RV enlargement at seven weeks post gene delivery and three weeks post induction of PH. Long term over expression of PGIS using AAV has the potential to be a useful treatment strategy in PH.

Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright  The American Society of Gene Therapy