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  • 1
    Keywords: CANCER ; CELLS ; tumor ; human ; MODEL ; MODELS ; PROTEIN ; TUMORS ; MICE ; PATIENT ; RESPONSES ; DNA ; INDUCTION ; ANTIGEN ; T-CELL ; T-CELLS ; TOLERANCE ; E7 ; papillomavirus ; SUPPRESSION ; IMMUNE-RESPONSES ; virus ; TRANSGENIC MICE ; MALIGNANCIES ; CERVICAL-CANCER ; human papillomavirus ; HPV ; E6 ; VACCINE ; STRATEGIES ; IMMUNE-RESPONSE ; vaccination ; SFV ; CANCER PATIENTS ; CTL ; LANGERHANS CELLS ; FUSION PROTEIN ; ANIMAL-MODELS ; IMMUNIZATION ; MALIGNANCY ; CAPACITY ; DNA vaccine ; PRIME ; ANIMAL-MODEL ; INNATE ; cytotoxic T lymphocyte ; HPV-transgenic mice ; immune tolerance
    Abstract: Despite promising preclinical results of various therapeutic anticancer immunization strategies, these approaches may not be effective enough to eradicate tumors in cancer patients. While most animal models are based on fast-growing transplantable tumors, malignancies in, for example, cervical cancer patients in general develop much more slowly, which may lead to immune suppression and/or immune tolerance. As a consequence, the immunomodulating signal of any therapeutic immunization regimen should be sufficiently potent to overcome this immunocompromised condition. In previous studies, we demonstrated that an experimental vaccine against human papillomavirus (HPV)-induced cervical cancer, based on Semliki Forest virus (SFV), induces robust HPV-specific cellular immune responses in mice. Now we studied whether this strategy is potent enough to also prime a cellular immune response in immune-tolerant HPV transgenic mice, in which CTL activity cannot be induced using protein or DNA vaccines. We demonstrate that, depending on the route of immunization, SFV-expressing HPV16 E6 and E7 indeed has the capacity to induce HPV16 E7-specific cytotoxic T cells in HPV-transgenic mice
    Type of Publication: Journal article published
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  • 2
    Keywords: tumor ; THERAPY ; SITE ; SITES ; GENE ; TUMORS ; gene therapy ; PATIENT ; DNA ; MECHANISM ; BIOLOGY ; TARGET ; virus ; VECTOR ; NUMBER ; genetics ; leukemia ; REPLICATION ; TARGETS ; GENE-THERAPY ; heredity ; INTEGRATION ; FRA3B ; VIRAL INTEGRATION ; LIFE ; BREAKPOINTS ; analysis ; fragile sites ; LEUKEMIA-VIRUS ; LMO2
    Abstract: Following gene therapy of SCID-X1 using murine leukemia virus (MLV) derived vector, two patients developed leukemia owing to an activating vector integration near the LMO2 gene. We found that these integrations reside within FRA11E, a common fragile site known to correlate with chromosomal breakpoints in tumors. Further analysis showed that fragile sites attract a nonrandom number of MLV integrations, shedding light on its integration mechanism and risk-to-benefit ratio in gene therapy
    Type of Publication: Journal article published
    PubMed ID: 16511518
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  • 3
    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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  • 4
    Keywords: EXPRESSION ; IN-VIVO ; TRANSGENIC MICE ; PROMOTER ; MOUSE MODEL ; HEMATOPOIETIC-CELLS ; insertional mutagenesis ; BETA-GLOBIN INSULATOR ; B-CELL LINES ; EFFICIENTLY TRANSDUCE
    Abstract: Insertional mutagenesis represents a serious adverse effect of gene therapy with integrating vectors. However, although uncontrolled activation of growth-promoting genes in stem cells can predictably lead to oncological processes, this is far less likely if vector transcriptional activity can be restricted to fully differentiated cells. Diseases requiring phenotypic correction only in mature cells offer such an opportunity, provided that lineage/stage-restricted systems can be properly tailored. In this study, we followed this reasoning to design lentiviral vectors for the gene therapy of chronic granulomatous disease (CGD), an immune deficiency due a loss of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in phagocytes, most often secondary to mutations in gp91(phox). Using self-inactivating HIV1-derived vectors as background, we first expressed enhanced green fluorescent protein (eGFP) from a minimal gp91(phox) promoter, adding various natural or synthetic transcriptional regulatory elements to foster both specificity and potency. The resulting vectors were assessed either by transplantation or by lentiviral transgenesis, searching for combinations conferring strong and specific expression into mature phagocytic cells. The most promising vector was modified to express gp91(phox) and used to treat CGD mice. High-level restoration of NADPH activity was documented in granulocytes from the treated animals. We propose that this lineage-specific lentiviral vector is a suitable candidate for the gene therapy of CGD.
    Type of Publication: Journal article published
    PubMed ID: 21544095
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  • 5
    Keywords: THERAPY ; MICE ; virus ; MOUSE ; MUSCLE ; TRANSGENE EXPRESSION ; TUMORIGENESIS ; MEDIATED GENE-TRANSFER ; VECTOR INTEGRATION ; RAAV VECTORS
    Abstract: Delivery of recombinant adeno-associated virus (rAAV) vectors to the newborn liver is followed by a rapid loss of episomal vector copies because of hepatocyte proliferation. In selected hepatocytes, integration of rAAV genomes can lead to a sustained expression of the transgene. The safety of in vivo gene therapy with single-stranded AAV vectors has been questioned in a study reporting a high incidence of hepatocellular carcinoma, associated with provirus integration events in mice that receive an single-stranded AAV injection at birth. To investigate the tumour-initiating potential of the newly established self-complementary AAV (scAAV) vectors in the liver, groups of newborn rats received intravenous injection of a scAAV vector encoding the green fluorescent protein (GFP), or were injected with phosphate-buffered saline (PBS) or diethylnitrosamine (DEN), a well-known liver tumour initiator. The rats were fed on a diet containing 2-acetylaminofluorene, a potent liver tumour-promoting agent to accelerate the carcinogenic process. After 2 months, the animals were killed and their livers analysed. Preneoplastic nodules were identified by glutathion S-transferase-p (GSTp) staining, and GFP expression was detected by immunohistochemistry. Vector genome integration events were analysed. The numbers of GSTp-positive foci were comparable in the PBS and the scAAV-GFP groups and significantly higher in the DEN group. The proportion of GSTp-positive foci that also expressed GFP was low and in the range expected for random occurrence. No specific integration hot spots were detected by linear amplification-mediated-PCR in transduced liver. In conclusion, scAAV transduction of newborn rat liver does not trigger preneoplastic lesions suggesting an absence of liver tumourigenesis.
    Type of Publication: Journal article published
    PubMed ID: 23364314
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  • 6
    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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  • 7
    Abstract: Several events of insertional mutagenesis in pre-clinical and clinical gene therapy studies have created intense interest in assessing the genomic insertion profiles of gene therapy vectors. For the construction of such profiles, vector-flanking sequences detected by inverse PCR, linear amplification-mediated-PCR or ligation-mediated-PCR need to be mapped to the host cell's genome and compared to a reference set. Although remarkable progress has been achieved in mapping gene therapy vector insertion sites, public reference sets are lacking, as are the possibilities to quickly detect non-random patterns in experimental data. We developed a tool termed QuickMap, which uniformly maps and analyzes human and murine vector-flanking sequences within seconds (available at www.gtsg.org). Besides information about hits in chromosomes and fragile sites, QuickMap automatically determines insertion frequencies in +/- 250 kb adjacency to genes, cancer genes, pseudogenes, transcription factor and (post-transcriptional) miRNA binding sites, CpG islands and repetitive elements (short interspersed nuclear elements (SINE), long interspersed nuclear elements (LINE), Type II elements and LTR elements). Additionally, all experimental frequencies are compared with the data obtained from a reference set, containing 1 000 000 random integrations ('random set'). Thus, for the first time a tool allowing high-throughput profiling of gene therapy vector insertion sites is available. It provides a basis for large-scale insertion site analyses, which is now urgently needed to discover novel gene therapy vectors with 'safe' insertion profiles.
    Type of Publication: Journal article published
    PubMed ID: 19387483
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  • 8
    Keywords: CELLS ; IN-VIVO ; GENE ; TRANSDUCTION ; BIOLOGY ; NEUTRALIZING ANTIBODIES ; genetics ; DELIVERY ; EVOLUTION ; PEPTIDES ; VIRAL VECTORS ; adeno-associated virus ; HEPARAN-SULFATE PROTEOGLYCAN ; endothelial cell ; ADENOASSOCIATED VIRUS TYPE-2 ; TROPISM ; vector targeting ; cardiovascular system ; random peptide display library ; THERAPY VECTORS
    Abstract: We have demonstrated the potential of random peptide libraries displayed on adeno-associated virus (AAV) 2 to select for AAV2 vectors with improved efficiency for cell type-directed gene transfer. AAV9, however, may have advantages over AAV2 because of a lower prevalence of neutralizing antibodies in humans and more efficient gene transfer in vivo. Here we provide evidence that random peptide libraries can be displayed on AAV9 and can be utilized to select for AAV9 capsids redirected to the cell type of interest. We generated an AAV9 peptide display library, which ensures that the displayed peptides correspond to the packaged genomes and performed four consecutive selection rounds on human coronary artery endothelial cells in vitro. This screening yielded AAV9 library capsids with distinct peptide motifs enabling up to 40-fold improved transduction efficiencies compared with wild-type (wt) AAV9 vectors. Incorporating sequences selected from AAV9 libraries into AAV2 capsids could not increase transduction as efficiently as in the AAV9 context. To analyze the potential on endothelial cells in the intact natural vascular context, human umbilical veins were incubated with the selected AAV in situ and endothelial cells were isolated. Fluorescence-activated cell sorting analysis revealed a 200-fold improved transduction efficiency compared with wt AAV9 vectors. Furthermore, AAV9 vectors with targeting sequences selected from AAV9 libraries revealed an increased transduction efficiency in the presence of human intravenous immunoglobulins, suggesting a reduced immunogenicity. We conclude that our novel AAV9 peptide library is functional and can be used to select for vectors for future preclinical and clinical gene transfer applications
    Type of Publication: Journal article published
    PubMed ID: 21956692
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  • 9
    Keywords: ACTIVATION ; HUMAN GENOME ; HEMATOPOIETIC-CELLS ; GENE-THERAPY ; TRANSCRIPTS ; CHRONIC GRANULOMATOUS-DISEASE ; RETROVIRAL INTEGRATION ; INTEGRATION SITE SELECTION ; EPIDERMOLYSIS-BULLOSA ; PROSPECTS
    Abstract: Transplantation of epithelia derived from keratinocyte stem cells transduced by retroviral vectors is a potential therapy for epidermolysis bullosa (EB), a family of inherited skin adhesion defects. The biosafety characteristics of retroviral vectors in keratinocytes are, however, poorly defined. We developed self-inactivating (SIN) vectors derived from the Moloney murine leukemia (MLV) and the human immunodeficiency (HIV) viruses expressing therapeutic levels of LAMB3, a transgene defective in junctional EB, and tested their integration profile in human primary keratinocytes. The SIN-HIV vector showed the expected preference for transcribed genes while the SIN-MLV vector integrated preferentially in regulatory elements, but showed a significantly lower tendency to target cell growth-related genes, transcription start sites and epigenetically defined promoters compared with a wild-type MLV vector in an epithelial cell context. A quantitative gene expression assay in individual keratinocyte clones showed that MLV-derived vectors deregulate expression of targeted genes at a lower frequency than in hematopoietic cells, and that the SIN-MLV design has the lowest activity compared to both MLV and SIN-HIV vectors. This study indicates that SIN-MLV vectors may have a better safety profile in keratinocyte than in hematopoietic cells, and be a reasonable alternative to lentiviral vectors for gene therapy of inherited skin disorders.
    Type of Publication: Journal article published
    PubMed ID: 23615186
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  • 10
    Keywords: IN-VIVO ; MODEL ; TISSUE ; antibodies ; MOUSE ; IDENTIFICATION ; GENE-THERAPY ; TROPISM ; DISPLAY PEPTIDE LIBRARIES ; VIRUS TYPE-2
    Abstract: Adeno-associated viral (AAV) vectors yield high potential for clinical gene therapy but, like for other vectors systems, they frequently do not sufficiently transduce the target tissue and their unspecific tropism prevents their application for multifocal diseases such as disseminated cancer. Targeted AAV vectors have been obtained from random AAV display peptide libraries but so far, all vector variants selected from AAV libraries upon systemic administration in vivo retained some collateral tropism, frequently the heart. Here we explored, if this impediment can be overcome by microRNA-regulated transgene cassettes as the combination of library-derived capsid targeting and micro-RNA control has not been evaluated so far. We used a tumor-targeted AAV capsid variant (ESGLSQS) selected from random AAV-display peptide libraries in vivo with remaining off-target tropism toward the heart and regulated targeted transgene expression in vivo by complementary target elements for heart-specific microRNA (miRT-1d). Although this vector still maintained its strong transduction capacity for tumor target tissue after intravenous injection, transgene expression in the heart was almost completely abrogated. This strong and completely tumor-specific transgene expression was used for therapeutic gene transfer in an aggressive multifocal, transgenic, polyoma middle T-induced, murine breast cancer model. A therapeutic suicide gene, delivered systemically by this dual-targeted AAV vector to multifocal breast cancer, significantly inhibited tumor growth after one single vector administration while avoiding side effects compared with untargeted vectors.
    Type of Publication: Journal article published
    PubMed ID: 26446851
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