237. Gene Expression Profiles in Diabetic Cardiomyopathy Following SERCA2a Gene Transfer

GENE THERAPY OF CARDIAC MUSCLE 236. Ultrasound-Targeted Microbubble Destruction Augments Cardiac AAV-Mediated Gene Transfer after Systemic Administration in Adult Rats Oliver J. Müller,1 Stefanie Schinkel,2 Hugo A. Katus,1 Jürgen A. Kleinschmidt,2 Raffi Bekeredjian.1 1 Internal Medicine III, University of Heidelberg, Heidelberg, Germany; 2Applied Tumorvirology F010, German Cancer Research Center, Heidelberg, Germany. Systemic gene transfer using AAV-6 serotype-vectors has been shown to be an efficient route of application in mice. However, systemic application requires high doses of vector or pre-treatment with VEGF to allow vascular permeabilization. The aim of our study was to analyse whether ultrasound-targeted microbubble destruction can augment virus uptake in the heart after intravenous administration. Lipid stabilized microbubbles loaded with 4x10(10) genomic particles of AAV6 encoding for a luciferase reporter gene under the control of a CMV-enhanced 1.5 kb myosin-light chain promoter were infused into the jugular vein of adult (250g) Sprague-Dawley rats. During the infusion high mechanical index ultrasound was administered to the heart. After 4 weeks, organs were harvested and analyzed for reporter gene expression. In contrast to low cardiac expression after systemic transfer of the vector solution without ultrasound, ultrasound-mediated destruction of microbubbles augmented cardiac reporter activities by more than one order of magnitude. Specificity of gene transfer was also increased since background activities in extracardiac organs such as liver and lung were similar with or without ultrasound. In conclusion, ultrasound-targeted microbubble destruction augments cardiac gene transfer with low titer AAV-6 serotype vectors in rats. This approach may be suitable also for increasing efficiency and specificity of AAV-mediated gene transfer in larger species and holds promise for human gene therapy.

237. Gene Expression Profiles in Diabetic Cardiomyopathy Following SERCA2a Gene Transfer Djamel Lebeche,1 Susumu Sakata,2 Yuri Sakata,2 Naoya Sakata,2 Elie R. Chemaly,1 Miyako Takaki,2 Roger J. Hajjar.1 1 Carrdiovascular Research Center, Massachusetts General Hospital, Charlestown, MA; 2Department of Physiology II, Nara Medical University, Kashihara, Nara, Japan. Diabetes is associated with impaired cardiac function in both humans and animals. As a consequence of myocardial dysfunction, the expression of several genes within the heart is altered. Our aim is to investigate the changes in gene expression profiles accompanying diabetic cardiomyopathy and to identify molecular and cellular pathways and genes that may contribute to cardiac remodeling as a result of the disease and/or its phenotypic rescue by restoration of the sarcoplasmic reticulum calcium ATPase pump (SERCA2a) through adenoviral gene transfer. Using Otsuka Long-Evans Tokushima Fatty (OLETF) rat model of type II diabetes and the Agilent rat chip, which contains oligonucleotide probe sets for ∼22,000 rat genes, we performed analyses of gene expression by comparing differential changes in age and sex-matched control versus diabetic hearts and diabetic hearts that received gene transfer of SERCA2a (which we previously showed to induce functional improvement in heart failure). Microarray results of selected genes were verified with quantitative real-time PCR and immunoblotting. After normalization and filtration of the data and using a 2-fold cutoff, we found that diabetes has differentially induced the expression of 743 genes (216 up- and 527 down-regulated) which take part in many cellular processes. Our analysis indicates that diabetic Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright  The American Society of Gene Therapy

cardiomyopathy appears to be, in general, associated with a downregulation of transcripts. Diabetic cardiomyopathic hearts have reduced levels of SERCA2a. Adenoviral gene transfer targeted to these hearts differentially induced the expression of 76 genes and appears, in general, to reverse the transcriptional profile induced by diabetes. Functional gene ontology classification of these genes indicated that SERCA2a restoration is associated with global changes in the cytoskeleton, in cellular energetics and metabolism, in calcium cycling, and in intracellular signaling and transcription regulation pathways. There are also 17 genes of unknown function that are specifically regulated by SERCA overexpression in diabetic hearts. The biological role of some of these novel genes are currently being pursued in other projects. Altogether this investigation provided valuable insight into the pathophysiology of cardiac remodeling and the role of SERCA2a normalization of multiple pathways in diabetes.

238. Knock down Phospholamban To Improve Heart Function by AAV-Mediated shRNA Xiaochun Lu,1 Guang Yang,1 Xiaoying Li,1 Xiaobing Wu.2 1 Geriatrics Cardiology, PLA General Hospital, Beijing, China; 2 National Virus Gene Lab, National Institute for the Control of Pharmaceutical and Biological Products, Beijing, China. Background:Congestive heart failure is the syndrome of final stage in most heart diseases, which prognosis is worst. Alterations of calcium-regulatory are the certain access, whereas function and regulation of sarcoplasmic reticulim Ca2+-ATPase are the definitive factors to maintain the calcium equilibrium in cardiomyocyts, and abnormalities of calcium cycling in the failing heart. Phospholamban is a key gene, which control the homoeostasis of Ca2+ by regulating sarcoplasmic reticulum Ca2+ -pump(SERCA2a) in cardiac muscle. Overexpression of an antisense phospholamban construct or a dominant-negative mutant of phospholamban has been shown to improve both systolic and diastolic function. RNA interference (RNAi) is a form of posttranscriptional control in which the doublestranded RNA (dsRNA) leads to specific degradation of mRNAs with complementary sequence. To assess value in the gene therapy of congestive heart failure, A recombinant Adeno-Associated Virus, which delivery the short 21nt stem hairpin RNA duplexes to knock down phospholamban in cardiomyocyts, was constructed and employed in animal experiment. Methods: We constructed three AAV plasmids, named pSNAVpRi1, pSNAV-pRi2 and pSNAV-pRi3, in which short 21nt stem hairpin RNA duplexes under the control of the U6 snRNA promoter were cloned into pSNAV-1 by PCR or a big adaptor. Transfections of these siRNA plasmids were performed with Lipofectamine into cultured rat cardiac myocytes. Western blot were performed to compare the difference between the expression quantity of phospholamban. Then two recombinant Adeno-Associated Virus, were constructed according to the strategy a recombinant helper virus infecting a carrier cell, rAAV-pRi2 and rAAV-pRi3. Abdominal aortic coarctation rat model that aortery coarctated diameter 0.7mm just above double renal arteris were prepared. After heart failure affirmed, the rAAV were injected into pericardial cavity through diaphragm. The hemodynamics parameters were record by left ventricle ar terial cannulation through carotid artery 10 and 30 days after rAAV injected. Results: three AAV plasmids pSNAV-pRi1, pSNAV-pRi2 and pSNAV-pRi3, could inhibit phospholamban expression about 10%, 14.5% and 23.6%. Then we produced the recombinant adenoassociated virus rAAV-pRi2 and rAAV-pRi3. The hemodynamics state of rAAV- pRi2 and rAAV- pRi3 group was equal to that of control group in 10 days after virus delivery , but better in 30 days. In 30 days after virus delivery, two AAV vector could inhibit phospholamban expression about 74.56% and 91.46%, whereas the SERCA2a mRNA level were no different in control group and two S91