729. Investigation of miRNA Targeting Sequences for Selective Regulated Gene Therapy in Cardiomyocytes

CARDIOVASCULAR GENE & CELL THERAPY abnormal collagen structure, or by irregular MV thickening that reduces close opposition of the leaflets. Collagen abnormalities may involve reductions in collagen III and/or increases in collagenases, which may be induced by activation of inflammatory pathways by binding of GAGs to TLR4. Since neonatal gene therapy is not totally effective at preventing MV disease in MPS VII, inhibition of TLR4, TNFa, SYK, or cathepsin K are reasonable targets to achieve a moreprofound effect on MV disease.

727. MicroRNAs 99b, 181a and 181b Potentiate Pluripotent hES Cell Endothelial Differentiation and Comprise Part of an Endothelial CellMicroRNA Signature Panel

Nicole M. Kane,1 Lynsey Howard,1 Raya Khanin,3 John McClure,1 Christopher R. Breen,1 Graeme Milligan,2 Andrew H. Baker.1 1 Institute of Cardiovascular & Medical Sciences, University of Glasgow, Glasgow, United Kingdom; 2Institute of Neuroscience & Psychology, University of Glasgow, Glasgow, United Kingdom; 3 Computational Biology Center, Memorial Sloan Kettering Cancer Center, New York. MicroRNAs (miRNAs) have emerged as important regulators of cell differentiation, development and disease by post-transcriptionally regulating gene expression through induction of mRNA degradation or by blocking translation. However, despite recent studies demonstrating the dysregulation of miRNAs in a number of cardiovascular complications, there is relatively little evidence demonstrating their functional role in early human development of vascular cells, how they control endothelial cell (EC) fate commitment and the mechanisms involved in these differentiation processes (1). Here, we differentiated human embryonic stem (hES) cells to ECs using a serum- and feeder- free protocol (2). Loss of miRNA biogenesis by lentiviral-mediated suppression of DICER impaired hES-EC differentiation indicating functional role for miRNA in EC commitment. We next performed a miRNA microarray to screen hES-EC miRNA profiles during defined early stages of differentiation and identified and validated several novel EC-associated miRNAs expressed in a time and EC differentiation-dependent manner, namely 99b, 181a and 181b. We report these miRNAs are expressed at similar levels in hES-EC cells differentiated for 10 days and adult venous ECs. To define the role of miRNAs 99b, 181a and 181b in EC differentiation we employed self-inactivating (SIN) vesicular stomatitis virus-pseudotyped lentiviral vectors to modulate miRNA expression in pluripotent hES cells prior to directed EC differentiation. miR overexpression was achieved by inducing expression of the pre-miR sequence under the control of the constitutive spleen focus forming viral promoter. miR knockdown was achieved by expressing specific shRNAs to produce short, single-stranded sequences that competitively bind their endogenous microRNA target and inhibit its function, or a scramble sequence control, under the control of the H1 promoter. Cells were infected with a multiplicity of infection (MOI) of 25 and modulation of each miRNA confirmed by TaqMan. miRNA overexpression potentiated the transcript and protein expression of EC-specific markers, Pecam1 and VE Cadherin, at day 10 and 14 of differentiation as compared to uninfected and scramble sequence controls. Conversely, knockdown did not evoke suppression of endothelial marker protein expression indicating that the expression of miRNAs 99b, 181a and 181b are capable of potentiating differentiation but are not the sole regulators inducing and governing differentiation. These results indicate that miRNAs 99b, 181a and 181b comprise part of an endothelial-miRNA signature panel as evidenced by expression profiles in adult and hES-EC cells. These miRNAs are also capable of potentiating EC differentiation from pluripotent hES cells, but are not essential for successful EC differentiation. 1. Kane et al., Pharmacol Ther 129, 29 (2011). 2. Kane et al., Arterioscler Thromb Vasc Biol 30, 1389 (2010). S278

728. In Vivo Functional Selection Using an AAV Library Encoding the Mouse Secretome Identifies Ghrelin as a Powerful Cardioprotective Molecule

Giulia Ruozi,1 Serena Zacchigna,1 Antero Macedo,1 Matteo Dalferro,1 Antonella Falcione,1 Lorena Zentilin,1 Mauro Giacca.1 1 Molecular Medicine Laboratory, International Centre for Genetic Engineering and Biotechnology - ICGEB, Trieste, Italy. The need to develop novel biotherapeutics for myocardial ischemia and heart failure is increasingly pressing. In our efforts to identify novel genes controlling prenatal cardiomyocyte proliferation, regulating cardiomyocyte survival or promoting new blood vessel formation that might be exploited therapeutically, we have devised an innovative, in vivo functional selection strategy. According to this strategy, a cDNA library corresponding to the mouse secretome (1600+ genes) is directly selected in vivo under ischemic conditions upon subsequent rounds of delivery with vectors derived from the Adeno-Associated Virus (AAV). These vectors appear particularly suitable for in vivo screening, as they can be produced at high titers, transduce the heart at high efficiency and drive persistent transgene expression. To support the feasibility of the in vivo functional selection strategy, we initially tested a pool of 50 AAV2 vectors expressing different hormones and growth factors. After intracardiac injection of this vector pool, animals were subjected to myocardial infarction, and, after 15 days, persisting vector DNA was recovered, re-cloned into AAV and used for subsequent cycles of selection. From this initial set of experiments, we were already able to observe an unexpected, marked enrichment for surviving cardiomyocytes transduced with AAV2-ghrelin, a neuro-hormone involved in endocrine metabolism. The cardioprotective potential of ghrelin was further characterized by a series of morphological and functional assays. In vitro transduction with AAV2-Ghrelin resulted in the marked survival of cardiomyocytes after treatment with the pro-apoptotic drugs isoproterenol and doxorubicin. In vivo, AAV2Ghrelin preserved cardiac function and reduced infarct size upon myocardial infarction, as assessed by echocardiography, histological and morphometric analysis. Interestingly, this effect was paralleled by a decreased number of apoptotic cells at early time points after infarction, and by the reduced expression of a few novel markers of heart failure, such as miR-21 and MMP-2. In conclusion, these results support the feasibility of our innovative in vivo selection approach and identify ghrelin as a novel and powerful molecule, with a protective function in cardiac apoptosis and providing benefit after cardiac damage.

729. Investigation of miRNA Targeting Sequences for Selective Regulated Gene Therapy in Cardiomyocytes Carolyn Haggerty,1 Caroline Fattah,1 Stuart A. Nicklin.1 Institute of Cardiovascular & Medical Sciences, University of Glasgow, Glasgow, United Kingdom.

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The heart is a target for gene therapy in disorders including heart failure and myocardial infarction. Regulated gene delivery would improve efficacy and safety either via a tissue or a disease-specific approach. These approaches could be mediated by microRNAs (miRNAs) which post-transcriptionally control gene expression in a spatial, temporal and disease-regulated manner. Cardiac remodelling and hypertrophy are associated with a signature of up and down-regulated miRNAs. Initially expression cassettes under the control of a disease-regulated miRNA, miR-133, tested the concept that its down-regulation would regulate transgene expression in hypertrophied cardiomyocytes. Expression cassettes were generated in which destabilized luciferase was transcribed from the chicken beta-actin cytomegalovirus (CAG-CMV) promoter and miR-133 targeting sequences (TS) inserted into the 3’ untranslated region. Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy

CARDIOVASCULAR GENE & CELL THERAPY Following transfection into H9c2 cardiomyocytes, CAG-CMV (no miR-133_TS) induced luciferase expression in the presence or absence of AngII. In the absence of AngII, CAG-CMV+miR133_TS only reduced luciferase expression by 50%. To further ensure selective cardiac transgene expression, tissue-specific regulation using miRNA targeting sequences was also investigated. Two groups of miRNA_TS expression constructs, with either a mixture of tissue-specific miRNAs; miR-122 (liver), miR-126 (endothelial) and miR-206 (skeletal muscle) or only miR-206, with a variable number of miRNA_TS copies (x3, x6 and x9), were tested for both approaches (Figure 1).

Constructs were transfected into cells selected for high or low expression of specific miRNA: HepG2 (miR-122), A549 (miR-126), L6 & C2C12 (miR-206), HeLa’s and H9c2 cardiomyocytes were used as negative and positive controls respectively. Luciferase expression in HeLa cells was not affected by the presence of miRNA_TS, however all miRNA_TS constructs in H9c2 cardiomyocytes had reduced luciferase due to high miR-206 levels in this myoblast cell line. Increased luciferase repression was observed with constructs containing more target sequence copies. In L6 cells, miRNA-206_TS x9 significantly decreased luciferase expression by 81% (+/-13, P<0.005) with a similar effect for miR-mix_TS x9, 67% (+/-20, P<0.05) reduction, which may be attributed to high miR-206 levels in these cells. The miR-mix_TS x9 construct produced the greatest luciferase repression, 94% (+/-3, P<0.001), in C2C12 cells presumably due to very high miR-206 and high miR-122 levels. In summary, insertion of tissue-specific miRNA targeting sequences into an expression cassette provided regulated luciferase expression in a tissue-specific manner and to a lesser extent in a disease-regulated manner for hypertrophied cardiomyocytes. Incorporating miRNA target sequences into expression cassettes may enable the development of regulated tissue-specific gene therapy in cardiovascular disease.

730. Myocardial Infarction-Induced Acute Inflammation Impairs Therapeutic Efficacy of Bone Marrow Cells

Xiaoyin Wang,1 Viola C. Lam,2 Daniel J. Haddad,1 Yan Zhang,3 Brian C. Clifford,1 Kranthi Pinnamaneni,1 Maya J. Bartel,3 Richard E. Sievers,3 Larry Carbone,4 Scott Kogan,5 Yerem Yeghiazarians,3,6 Michelle Hermiston,2 Matthew L. Springer.1,3,6 1 Cardiovascular Research Institute, University of California, San Francisco, San Francisco, CA; 2Dept. of Pediatrics, University of California, San Francisco, San Francisco, CA; 3Division of Cardiology, University of California, San Francisco, San Francisco, CA; 4Laboratory Animal Resource Center, University of California, San Francisco, San Francisco, CA; 5Dept. of Laboratory Medicine, University of California, San Francisco, San Francisco, CA; 6Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, CA.

been less robust. We have demonstrated that donor MI impairs BMC therapeutic efficacy in mice. However, the mechanism of this impairment remains unknown. Acute MI leads to a localized host inflammatory response. We hypothesized that MI leads to an increased or activated inflammatory state in the bone marrow that impairs the therapeutic efficacy of BMCs. To investigate this, we implanted BMCs from healthy or post-MI donor mice under ultrasound guidance. Donors and recipients were subjected to MI by permanent left coronary artery ligation. MI recipients were always identical from experiment to experiment. Donor bone marrow at 3 days post-MI showed several characteristics consistent with increased inflammation, including fewer CD11c+ dendritic cells, increased MHC Class II expression on B cells, and more Ly6Chigh inflammatory myeloid cells. Donor BMCs harvested at varying times post-MI were increasingly impaired over the first 3 days but were less impaired when harvested after day 5, consistent with the time course of an inflammatory response (see figure). Recipient EF is shown measured at 28 days post-recipient-MI in groups injected with BMCs harvested at various times after donor MI. Serum from donors at 1 and 3 days post-MI but not 7 days showed an increase in several cytokines and related proteins including G-CSF, IL-1β, IL-1Ra, IL-6, MCP-1, TIMP-1 and TREM-1. Notably, anti-inflammatory treatment of donor mice post-MI with dexamethasone prevented the BMC impairment. BMCs harvested from sham-operated mice in which skin and bone were still wounded with no artery ligation were not substantially impaired. Our findings suggest that MI-induced acute inflammation impairs BMC therapeutic efficacy.

731. AAV8-Mediated Long Term Expression of Human LCAT Significantly Improves Lipid Profiles in hCETP; Ldlr+/- Mice

Heather H. Zhou,1 Zhu Chen,2 Donald Chu,1 Jose Castro-Perez,2 Weihua Ni,1 Aiwu Zhang,1 Mihajlo L. Krsmanovic,2 Dan Xie,2 Vinit Shah,2 Steven J. Stout,2 David G. McLaren,2 Alice C. Stefanni,2 Sang Ho Lee,3 Andrew S. Plump,2 Brian K. Hubbard,2 Thomas F. Vogt.1 1 Translational Models, Merck Research Laboratories, Rahway; 2 Atherosclerosis, Cardiovascular Diseases, Merck Research Laboratories, Rahway; 3In Vivo Pharmacology, Merck Research Laboratories, Rahway.

Lecithin:cholesterol acyltransferase (LCAT) is a major enzyme in HDL metabolism and potentially promotes reverse cholesterol transport and ameliorates atherosclerosis. To assess therapeutic potential of increased LCAT functionality, we employed adeno-associated viral vector serotype 8 (AAV8)-mediated gene transfer to achieve sustained high level expression of human LCAT in hCETP;Ldlr+/- mice during a 32-week study. The AAV8-hLCAT treated mice had a concomitant and marked increase in HDL throughout the 32-week study when compared to AAV8-luciferase or saline treated control mice. Plasma lipid analyses at study termination revealed increased HDL particle size as well as HDL cholesterol level. Ex vivo LCAT activity had marked increase with humanized substrate specificity. Furthermore, LDL cholesterol, triglycerides and apoB were significantly reduced in AAV8-hLCAT treated mice. Lipid analysis via LC/MS method demonstrated that plasma HDL cholesterol ester increased in a fashion that fits human LCAT specificity, whereas no major lipid change in liver, bile and feces was revealed. The observed lipid profiles are novel among mouse models of LCAT overexpression, resemble previous findings in higher species, and are potentially antiatherosclerotic. Our data suggest that achievement of sustained overexpression of LCAT in the liver may provide a therapeutic opportunity for dyslipidemia and atherosclerosis.

Delivery of bone marrow cells (BMCs) to infarcted myocardium has been reported to be therapeutic in most animal experiments, but results in similar clinical trials of autologous BMC therapy have Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy

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