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  • 1
    Keywords: EXPRESSION ; Germany ; human ; IN-VIVO ; THERAPY ; GENE ; GENE-EXPRESSION ; PROTEIN ; EFFICIENCY ; HEART ; gene therapy ; MICE ; TRANSDUCTION ; gene transfer ; GENE-TRANSFER ; INDEX ; RAT ; animals ; RATS ; CONTRAST ; BIOLOGY ; MOLECULAR-BIOLOGY ; PARTICLES ; virus ; gene expression ; HUMANS ; VECTORS ; VECTOR ; genetics ; EFFICIENT ; DELIVERY ; specificity ; GENE-THERAPY ; RECOMBINANT ADENOASSOCIATED VIRUS ; VIRAL VECTORS ; GREEN FLUORESCENT PROTEIN ; contrast media ; adeno-associated virus ; LUCIFERASE ; heredity ; AAV ; ultrasound ; MICROBUBBLES ; molecular biology ; molecular ; ADULT ; TRANSGENE EXPRESSION ; THERAPIES ; REPORTER GENE ; analysis ; animal ; microbiology ; ENGLAND ; VASCULAR-PERMEABILITY ; MYOCARDIAL CONTRAST ECHOCARDIOGRAPHY ; systemic ; viral ; MEDICINE ; biotechnology ; mechanical ; viruses ; ANTERIOR ; ADENOVIRUS VECTORS ; AUGMENTS ; PORCINE HEART ; REPLICATION-DEFICIENT ; TARGETED MICROBUBBLE DESTRUCTION ; ultrasonics
    Abstract: Adeno-associated virus (AAV)-6 or -9-pseudotyped vectors are suitable for efficient cardiac gene transfer after intravenous injection in mice. However, a systemic application in larger animals or humans would require very high doses of viral particles. Therefore, the aim of our study was to test if ultrasound-targeted microbubble destruction could augment cardiac transduction of AAV vectors after intravenous administration in rats. To analyze efficiency and specificity of gene transfer, microbubbles loaded with AAV-6 or -9 harboring a luciferase or enhanced green fluorescent protein (EGFP) reporter gene were infused into the jugular vein of adult Sprague-Dawley rats. During the infusion, high mechanical index ultrasound was administered to the heart. Control rats received the same amount of virus without microbubbles, but with ultrasound. 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 microbubbles, ultrasound-targeted destruction of microbubbles significantly increased cardiac reporter activities between 6- and 20-fold. Analysis of spatial distribution of transgene expression using an AAV-9 vector encoding for EGFP revealed transmural expression predominantly in the left ventricular anterior wall. In conclusion, ultrasound targeted microbubble destruction augments cardiac transduction of AAV vectors in rats. This approach may be suitable for efficient, specific and noninvasive AAV-mediated gene transfer in larger animals or humans
    Type of Publication: Journal article published
    PubMed ID: 18615116
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  • 2
    Keywords: RECEPTOR ; CELLS ; tumor ; GENE ; MOLECULAR CHARACTERIZATION ; LINES ; LIGAND ; RAT ; CELL-LINES ; IMMUNE-RESPONSES ; SELECTION ; ADENOASSOCIATED VIRUS VECTORS ; MOUSE-LIVER ; biotechnology ; VIRAL-VECTORS ; HUMAN GENE-THERAPY ; PERSISTENT EXPRESSION ; RECOMBINANT VECTORS ; TYPE-2 CAPSIDS
    Abstract: Libraries based on the insertion of random peptide ligands into the capsid of adeno-associated virus type 2 (AAV2) have been widely used to improve the efficiency and selectivity of the AAV vector system. However, so far only libraries of 7-mer peptide ligands have been inserted at one well-characterized capsid position. Here, we expanded the combinatorial AAV2 display system to a panel of novel AAV libraries, displaying peptides of 5, 7, 12, 19, or 26 amino acids in length at capsid position 588 or displaying 7-mer peptides at position 453, the most prominently exposed region of the viral capsid. Library selections on two unrelated cell types-human coronary artery endothelial cells and rat cardiomyoblasts-revealed the isolation of cell type-characteristic peptides of different lengths mediating strongly improved target-cell transduction, except for the 26-mer peptide ligands. Characterization of vector selectivity by transduction of nontarget cells and comparative gene-transduction analysis using a panel of 44 human tumor cell lines revealed that insertion of different-length peptides allows targeting of distinct cellular receptors for cell entry with similar efficiency, but with different selectivity. The application of such novel AAV2 libraries broadens the spectrum of targetable receptors by capsid-modified AAV vectors and provides the opportunity to choose the best suited targeting ligand for a certain application from a number of different candidates
    Type of Publication: Journal article published
    PubMed ID: 22171602
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  • 3
    Keywords: RECEPTOR ; EXPRESSION ; Germany ; IN-VIVO ; THERAPY ; TOOL ; liver ; GENE ; GENE-EXPRESSION ; EFFICIENCY ; HEART ; gene therapy ; MICE ; gene transfer ; GENE-TRANSFER ; RAT ; BINDING ; virus ; MOUSE ; TRANSGENIC MICE ; gene expression ; PROMOTER ; DELIVERY ; specificity ; TYPE-2 ; HEPARAN-SULFATE PROTEOGLYCAN ; ENHANCER ; INCREASE ; in vivo ; ADULT MICE ; cytomegalovirus ; VIRUS VECTORS
    Abstract: Objective: Vectors based on recombinant adeno-associated virus 2 (AAV-2) are a promising tool for cardiac gene transfer. However, potential therapeutic applications need to consider the predominant transduction of the liver once AAV-2 vectors enter the systemic circulation. We therefore aimed to increase efficiency and specificity of cardiac vector delivery by combining transcriptional and cell surface targeting. Methods: For analysis of transcriptional targeting, recombinant AAV vectors were generated harboring a luciferase reporter gene under control of the cytomegalovirus (CMV) promoter or the 1.5-kb cardiac myosin light chain promoter fused to the CMV immediate-early enhancer (CMVenh/MLC1.5). Luciferase activities were determined in representative organs three weeks after intravenous injection of the vector into adult mice. Transductional targeting was studied using luciferase-reporter constructs crosspackaged into capsids of AAV serotypes I to 6 and modified AAV-2 capsids devoid of binding their primary receptor heparan sulfate proteoglycan. Results: Intravenous injections of AAV-2 vectors harboring the CMVenh/MLC1.5 promoter enabled a specific and 50-fold higher reporter gene expression in left ventricular myocardium, of adult mice compared to vectors containing the CMV promoter. Comparison of AAV-2 vector genomes crosspackaged into capsids of AAV-1 to -6 showed that AAV-1, -4, -5, and -6 capsids increased cardiac transduction efficiency by about 10-fold. However, transduction of other organs such as the liver was also increased after systemic administration. In contrast, AAV-2-based vectors with ablated binding to their primary receptor heparan sulfate proteoglycan enabled a significantly increased efficiency of cardiac gene transfer and reduced transduction of the liver. Conclusions: Combining transcriptional targeting by the CMVenh/MLC1.5 promoter and AAV vectors devoid of binding the AAV-2 primary receptor results in an efficient cardiac gene transfer with a significantly reduced hepatic transduction. Q 2005 European Society of Cardiology. Published by Elsevier B.V. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 16448634
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  • 4
    Keywords: EXPRESSION ; IN-VIVO ; HEART ; TRANSDUCTION ; BLOOD-FLOW ; virus ; VECTORS ; DELIVERY ; LUCIFERASE ; AAV ; VEGF ; TRANSFECTION ; HEART-FAILURE ; ADENOVIRUS VECTORS ; CORONARY VEINS ; MYOCARDIAL-ISCHEMIA ; retroinfusion
    Abstract: Cornerstone for an efficient cardiac gene therapy is the need for a vector system, which enables selective and long-term expression of the gene of interest. In rodent animal models adeno-associated viral (AAV) vectors like AAV-6 have been shown to efficiently transduce cardiomyocytes. However, since significant species-dependent differences in transduction characteristics exist, large animal models are of imminent need for preclinical evaluations. We compared gene transfer efficiencies of AAV-6 and heparin binding site-deleted AAV-2 vectors in a porcine model. Application of the AAVs was performed by pressure-regulated retroinfusion of the anterior interventricular cardiac vein, which has been previously shown to efficiently deliver genes to the myocardium (3.5 x 10(10) viral genomes per animal; n = 5 animals per group). All vectors harbored a luciferase reporter gene under control of a cytomegalovirus (CMV)-enhanced 1.5 kb rat myosin light chain promoter (CMV-MLC2v). Expression levels were evaluated 4 weeks after gene transfer by determining luciferase activities. To rule out a systemic spillover peripheral tissue was analyzed by PCR for the presence of vector genomes. Selective retroinfusion of AAV serotype 6 vectors into the anterior cardiac vein substantially increased reporter gene expression in the targeted distal left anterior descending (LAD) territory ( 65 943 +/- 31 122 vs control territory 294 +/- 69, P 〈 0.05). Retroinfusion of AAV- 2 vectors showed lower transgene expression, which could be increased with coadministration of recombinant human vascular endothelial growth factor (1365 +/- 707 no vascular endothelial growth factor (VEGF) vs 38 760 +/- 2448 with VEGF, P 〈 0.05). Significant transgene expression was not detected in other organs than the heart, although vector genomes were detected also in the lung and liver. Thus, selective retroinfusion of AAV- 6 into the coronary vein led to efficient long-term myocardial reporter gene expression in the targeted LAD area of the porcine heart. Coapplication of VEGF significantly increased transduction efficiency of AAV-2
    Type of Publication: Journal article published
    PubMed ID: 17943147
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  • 5
    Keywords: EXPRESSION ; TRIAL ; CALCIUM ; MYOCARDIAL-INFARCTION ; DILATED CARDIOMYOPATHY ; FEASIBILITY ; LEFT-VENTRICULAR DYSFUNCTION ; cardiomyocytes ; BASIC SCIENCE ; CA2+-BINDING PROTEIN S100A1 ; CONTRACTILE PERFORMANCE ; ENERGY-METABOLISM ; FAILING MYOCARDIUM
    Abstract: As a prerequisite for clinical application, we determined the long-term therapeutic effectiveness and safety of adeno-associated virus (AAV)-S100A1 gene therapy in a preclinical large animal model of heart failure. S100A1, a positive inotropic regulator of myocardial contractility, becomes depleted in failing cardiomyocytes in humans and animals, and myocardial-targeted S100A1 gene transfer rescues cardiac contractile function by restoring sarcoplasmic reticulum calcium (Ca(2+)) handling in acutely and chronically failing hearts in small animal models. We induced heart failure in domestic pigs by balloon occlusion of the left circumflex coronary artery, resulting in myocardial infarction. After 2 weeks, when the pigs displayed significant left ventricular contractile dysfunction, we administered, by retrograde coronary venous delivery, AAV serotype 9 (AAV9)-S100A1 to the left ventricular, non-infarcted myocardium. AAV9-luciferase and saline treatment served as control. At 14 weeks, both control groups showed significantly decreased myocardial S100A1 protein expression along with progressive deterioration of cardiac performance and left ventricular remodeling. AAV9-S100A1 treatment prevented and reversed these functional and structural changes by restoring cardiac S100A1 protein levels. S100A1 treatment normalized cardiomyocyte Ca(2+) cycling, sarcoplasmic reticulum calcium handling, and energy homeostasis. Transgene expression was restricted to cardiac tissue, and extracardiac organ function was uncompromised. This translational study shows the preclinical feasibility of long-term therapeutic effectiveness of and a favorable safety profile for cardiac AAV9-S100A1 gene therapy in a preclinical model of heart failure. Our results present a strong rationale for a clinical trial of S100A1 gene therapy for human heart failure that could potentially complement current strategies to treat end-stage heart failure
    Type of Publication: Journal article published
    PubMed ID: 21775667
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  • 6
    Keywords: CANCER ; THERAPY ; MICE ; DELIVERY ; SELECTION ; VIRAL VECTORS ; PHAGE DISPLAY ; LIBRARIES ; ADENOASSOCIATED VIRUS TYPE-2 ; TROPISM ; LIVER TRANSDUCTION ; SEROTYPE-8
    Abstract: Efficiency and specificity of viral vectors are vital issues in gene therapy. Insertion of peptide ligands into the adeno-associated viral (AAV) capsid at receptor binding sites can re-target AAV2-derived vectors to alternative cell types. Also, the use of serotypes AAV8 and -9 is more efficient than AAV2 for gene transfer to certain tissues in vivo. Consequently, re-targeting of these serotypes by ligand insertion could be a promising approach but has not been explored so far. Here, we generated AAV8 and -9 vectors displaying peptides in the threefold spike capsid domain. These peptides had been selected from peptide libraries displayed on capsids of AAV serotype 2 to optimize systemic gene delivery to murine lung tissue and to breast cancer tissue in PymT transgenic mice (PymT). Such peptide insertions at position 590 of the AAV8 capsid and position 589 of the AAV9 capsid changed the transduction properties of both serotypes. However, both peptides inserted in AAV8 did not result in the same changes of tissue tropism as they did in AAV2. While the AAV2 peptides selected on murine lung tissue did not alter tropism of serotypes 8 and -9, insertion of the AAV2-derived peptide selected on breast cancer tissue augmented tumor gene delivery in both serotypes. Further, this peptide mediated a strong but unspecific in vivo gene transfer for AAV8 and abrogated transduction of various control tissues for AAV9. Our findings indicate that peptide insertion into defined sites of AAV8 and -9 capsids can change and improve their efficiency and specificity compared to their wild type variants and to AAV2, making these insertion sites attractive for the generation of novel targeted vectors in these serotypes
    Type of Publication: Journal article published
    PubMed ID: 21850255
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  • 7
    Keywords: CELLS ; CELL ; Germany ; liver ; SITE ; PROTEIN ; PROTEINS ; TISSUE ; HEART ; MICE ; GENE-TRANSFER ; INFECTION ; MOTIFS ; CONTRAST ; SIMULATION ; BINDING ; ACID ; ACIDS ; virus ; IDENTIFICATION ; SUBUNIT ; MUTATION ; REGION ; CAPSID PROTEIN ; SUBUNITS ; AMINO-ACIDS ; INFECTIVITY ; adeno-associated virus ; ADENOASSOCIATED VIRUS ; TYPE-2 ; PROTEIN INTERACTIONS ; CLUSTERS ; AAV ; ATOMIC-STRUCTURE ; AUTOMATED DOCKING ; CELL-SURFACE GLYCOSAMINOGLYCANS ; HERPES-SIMPLEX VIRUS ; INITIAL INTERACTION ; insertional mutagenesis ; PROTEOGLYCAN ; RECOMBINANT VIRUS ; SULFATE GLYCOSAMINOGLYCANS ; TRANSDUCTION EFFICIENCY
    Abstract: Infection of cells with adeno-associated virus (AAV) type 2 (AAV-2) is mediated by binding to heparan sulfate proteoglycan and can be competed by heparin. Mutational analysis of AAV-2 capsid proteins showed that a group of basic amino acids (arginines 484, 487, 585, and 588 and lysine 532) contribute to heparin and HeLa cell binding. These amino acids are positioned in three clusters at the threefold spike region of the AAV-2 capsid. According to the recently resolved atomic structure for AAV-2, arginines 484 and 487 and lysine 532 on one site and arginines 585 and 588 on the other site belong to different capsid protein subunits. These data suggest that the formation of the heparin-binding motifs depends on the correct assembly of VP trimers or even of capsids. In contrast, arginine 475, which also strongly reduces heparin binding as well as viral infectivity upon mutation to alanine, is located inside the capsid structure at the border of adjacent VP subunits and most likely influences heparin binding indirectly by disturbing correct subunit assembly. Computer simulation of heparin docking to the AAV-2 capsid suggests that heparin associates with the three basic clusters along a channel-like cavity flanked by the basic amino acids. With few exceptions, mutant infectivities correlated with their heparin- and cell-binding properties. The tissue distribution in mice of recombinant AAV-2 mutated in 8484 and 8585 indicated markedly reduced infection of the liver, compared to infection with wild-type recombinant AAV, but continued infection of the heart. These results suggest that although heparin binding influences the infectivity of AAV-2, it seems not to be necessary
    Type of Publication: Journal article published
    PubMed ID: 14512555
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  • 8
    Keywords: PEPTIDE ; RECEPTOR ; CELLS ; CELL ; Germany ; IN-VIVO ; GENERATION ; SYSTEM ; TOOL ; GENE ; GENOME ; PROTEINS ; EFFICIENCY ; gene therapy ; TRANSDUCTION ; gene transfer ; GENE-TRANSFER ; LIGAND ; MOTIFS ; REDUCTION ; SEQUENCE ; TARGET ; virus ; VECTOR ; PEPTIDES ; REPLICATION ; SELECTION ; specificity ; VIRAL VECTORS ; ADENOASSOCIATED VIRUS ; TYPE-2 ; AAV ; PHAGE DISPLAY ; AAV2 CAPSID GENE ; LEVEL ; PEPTIDE LIBRARIES ; TOOLS ; CONTAMINATION ; GENE-THERAPY VECTORS ; peptide display library ; vector targeting
    Abstract: Random peptide ligands displayed on viral capsids are emerging tools for selection of targeted gene transfer vectors even without prior knowledge of the potential target cell receptor. We have previously introduced adeno-associated viral (AAV)-displayed peptide libraries that ensure encoding of displayed peptides by the packaged AAV genome. A major limitation of these libraries is their contamination with wild-type (wt) AAV. Here we describe a novel and improved library production system that reliably avoids generation of wt AAV by use of a synthetic cap gene. Selection of targeted AAV vectors from wt-containing and the novel wt-free libraries on cell types with different permissivity for wt AAV2 replication suggested the superiority of the wt-free library. However, from both libraries highly specific Peptide sequence motifs were selected which improved transduction of cells with moderate or low permissivity for AAV2 replication. Strong reduction of HeLa cell transduction compared to wt AAV2 and only low level transduction of non-target cells by some selected clones showed that not only the efficiency but also the specificity of gene transfer was improved. In conclusion, our study validates and improves the unique potential of virus display libraries for the development of targeted gene transfer vectors. Copyright (c) 2006 John Wiley & Sons, Ltd
    Type of Publication: Journal article published
    PubMed ID: 16955542
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  • 9
    Keywords: brain ; COMPLEX ; COMPLEXES ; ADULT ; CARDIOMYOPATHY ; ALPHA-SARCOGLYCAN ; CARDIAC GENE-TRANSFER ; cytomega
    Abstract: delta-Sarcoglycan is a member of the dystrophin-associated glycoprotein complex linking the cytoskeleton to the extracellular matrix. Similar to patients with defects in the gene encoding delta-sarcoglycan (Sgcd), knockout mice develop cardiomyopathy and muscular dystrophy. The aim of our study was to develop an approach for preventing cardiomyopathy in Sgcd-deficient mice by cardiac expression of the intact cDNA upon systemic delivery of adeno-associated viral (AAV) vectors. We packaged the Sgcd cDNA under transcriptional control of a myosin light chain-promoter fused with a cytomegalovirus enhancer into AAV-9 capsids. Vectors carrying either the Sgcd cDNA or an enhanced green fluorescent protein (EGFP) reporter gene were intravenously injected into adult Sgcd knockout mice. After 6 months, immunohistochemistry revealed almost complete reconstitution of the sarcoglycan subcomplex in heart but not skeletal muscle of mice with the Sgcd vector. Furthermore, Sgcd gene transfer resulted in prevention of cardiac fibrosis and significantly increased running distance measured by voluntary wheel running. Left ventricular function remained stable in mice expressing Sgcd while it deteriorated in EGFP controls within 6 months, paralleled by increased expression of brain natriuretic peptide, a molecular marker of heart failure. Our study establishes an approach to specifically treat hereditary cardiomyopathies by targeting gene expression into the myocardium upon systemic application of AAV vectors
    Type of Publication: Journal article published
    PubMed ID: 19218289
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  • 10
    Keywords: EXPRESSION ; INHIBITOR ; gene therapy ; MICE ; VECTORS ; adeno-associated virus ; ADENOASSOCIATED VIRUS ; CROHNS-DISEASE ; CHEMOKINE RECEPTOR ; HEART-FAILURE ; MONOCYTE CHEMOATTRACTANT PROTEIN-1 ; INTERLEUKIN-10 ; troponin I ; ADENOVIRUS VECTORS ; CARDIAC TROPONIN-I ; Myocarditis ; TRANSFER SUPERIOR ; VIRAL-VECTORS
    Abstract: Aims Overexpression of therapeutic genes with potential disease-limiting effects, specifically at the site of inflammation, remains a major clinical challenge. In this study, we investigate the potential of adeno-associated virus (AAV)-9-mediated cardiac expression of the anti-inflammatory mediators interleukin (IL)-10 and a dominant-negative inhibitor of monocyte chemoattractant protein-1 (MCP1-7ND) on prevention of autoimmune myocarditis. Methods and results Autoimmune myocarditis was induced by immunizing A/J mice with subcutaneous injection of 120 mu g cardiac Troponin I (cTnI) on Days 0, 7, and 14. Two weeks prior to initial immunization, each mouse received a single systemic dose of 10(12) AAV9 vectors carrying the coding sequence of IL-10 or MCP1-7ND transcriptionally targeted to the heart. Mice were sacrificed 28 days after initial immunization for further analysis. Only expression of IL-10 resulted in a highly significant decrease in myocardial inflammation and fibrosis, as well as an increased ejection fraction compared with controls. Further analyses of cytokine profiles of cTnI-stimulated splenocytes from IL-10 and MCP1-7ND-treated mice revealed significant alterations compared with controls. In addition, transcript levels of chemokine receptor CCR4 and T-cell activation gene were significantly reduced in hearts of IL-10-treated mice as determined by quantitative real-time PCR. Conclusion Our study suggests that cardiac expression of IL-10 with AAV9 vectors is a promising therapeutic approach for autoimmune myocarditis
    Type of Publication: Journal article published
    PubMed ID: 21354997
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