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  • 1
    Keywords: CANCER ; CANCER CELLS ; CELLS ; IN-VITRO ; tumor ; AGENTS ; Germany ; human ; PHASE-I ; VITRO ; DEATH ; TUMORS ; MICE ; TIME ; GENE-TRANSFER ; MARKER ; ANTIGEN ; DENDRITIC CELLS ; H-1 ; cytokines ; MATURATION ; virus ; VECTORS ; VECTOR ; CELL-DEATH ; MARKERS ; MELANOMA ; LYMPHOCYTES ; CANCER-CELLS ; MINUTE VIRUS ; IMMUNE-RESPONSE ; T-LYMPHOCYTES ; CTL ; AUTONOMOUS PARVOVIRUSES ; T lymphocytes ; CROSS-PRESENTATION ; HEAT-SHOCK PROTEINS ; CYTOLYTIC T-LYMPHOCYTES ; STANDARD ; AGENT ; VARIANT ; oncolytic virus ; ONCOLYTIC PARVOVIRUS ; HLA-A2 MELANOMAS ; cell death ; dendritic cell ; APOPTOTIC CELLS ; APC ; FUTURE-DIRECTIONS ; HUMAN-MELANOMA CELLS ; SERUM-FREE CONDITIONS
    Abstract: Oncotropic and oncolytic viruses have attracted high attention as antitumor agents because they preferentially kill cancer cells in vitro and reduce the incidence of spontaneous, induced, or implanted animal tumors. Some autonomous parvoviruses (H-1, minute virus of mice) and derived recombinant vectors are currently under preclinical evaluation. Still not fully understood, their antitumor properties involve more than just tumor cell killing. Because wild-type parvovirus-mediated tumor cell lysates (TCLs) may trigger antigen-presenting cells (APCs) to augment the host immune repertoire, we analyzed phagocytosis, maturation, and cross-presentation of H-1-induced TCLs by human dendritic cells (DCs). We first established H-1-mediated oncolysis in two HLA-A2(+) and A2(-) variant melanoma cell clones. Monocyte-derived immature DCs phagocytosed H-1-infected TCLs as well as ultraviolet-induced apoptotic TCLs and better than freeze-thaw-induced necrotic TCLs. Immature DCs incubated with H-1-induced TCLs acquired specific maturation markers comparable to a standard cytokine cocktail. Furthermore, A2(+) DCs pulsed with H-1-infected A2(-) TCLs cross-presented melanoma antigens to specific cytotoxic T lymphocytes (CTLs) and released proinflammatory cytokines. This shows for the first time that tumor cell killing by a wild-type oncolytic virus directly stimulates human APCs and CTLs. Because H-1-infected tumors enhance the immune repertoire, the clinical perspectives of parvoviral vectors are even more promising
    Type of Publication: Journal article published
    PubMed ID: 16076257
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  • 2
    Keywords: IN-VIVO ; MESSENGER-RNA ; CELL-LINE ; MELANOMA ; CANCER-THERAPY ; CONDITIONALLY REPLICATING ADENOVIRUS ; BEARING MICE ; TYROSINASE ENHANCER/PROMOTER ; CYTOSINE DEAMINASE GENE ; RECOMBINANT ADENOVIRUSES
    Abstract: Key challenges facing cancer therapy are the development of tumor-specific drugs and potent multimodal regimens. Oncolytic adenoviruses possess the potential to realize both aims by restricting virus replication to tumors and inserting therapeutic genes into the virus genome, respectively. A major effort in this regard is to express transgenes in a tumor-specific manner without affecting virus replication. Using both luciferase as sensitive reporter and genetic prodrug activation, we show that promoter control of E1A facilitates highly selective expression of transgenes inserted into the late transcription unit. This, however, required multistep optimization of late transgene expression. Transgene insertion via IRES, splice acceptor (SA) or viral 2A sequences resulted in replication-dependent expression. Unexpectedly, analyses in appropriate substrates and with matching control viruses revealed that IRES and SA, but not 2A facilitated indirect transgene targeting via tyrosinase promoter control of E1A. Transgene expression via SA was more selective (up to 1.500-fold) but less effective than via IRES. Notably, we also revealed transgene-dependent interference with splicing. Hence, the prodrug convertase FCU1 (a CD-UPRT fusion protein) was expressed, only, after optimizing the sequence surrounding the SA site and mutating a cryptic splice site within the transgene. The resulting tyrosinase promoter-regulated and FCU1-encoding adenovirus combined effective oncolysis with targeted prodrug activation therapy of melanoma. Thus, prodrug activation showed potent bystander killing and increased cytotoxicity of the virus up to 10-fold. We conclude that armed oncolytic viruses can be improved substantially by comparing and optimizing strategies for targeted transgene expression, thereby implementing selective and multimodal cancer therapies.
    Type of Publication: Journal article published
    PubMed ID: 20939692
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  • 3
    Keywords: ACCURACY ; virus ; TRIAL ; PURIFICATION ; GENE-THERAPY ; VIRAL VECTORS ; GENOMES ; CAPSIDS ; SEROTYPE-2
    Abstract: A recombinant adeno-associated virus serotype 2 Reference Standard Material (rAAV2 RSM) has been produced and characterized with the purpose of providing a reference standard for particle titer, vector genome titer, and infectious titer for AAV2 gene transfer vectors. Production and purification of the reference material were carried out by helper virus-free transient transfection and chromatographic purification. The purified bulk material was vialed, confirmed negative for microbial contamination, and then distributed for characterization along with standard assay protocols and assay reagents to 16 laboratories worldwide. Using statistical transformation and modeling of the raw data, mean titers and confidence intervals were determined for capsid particles ({X}, 9.18 x 10(11) particles/ml; 95% confidence interval [CI], 7.89 x 10(11) to 1.05 x 10(12) particles/ml), vector genomes ({X}, 3.28 x 10(10) vector genomes/ml; 95% CI, 2.70 x 10(10) to 4.75 x 10(10) vector genomes/ml), transducing units ({X}, 5.09 x 10(8) transducing units/ml; 95% CI, 2.00 x 10(8) to 9.60 x 10(8) transducing units/ml), and infectious units ({X}, 4.37 x 10(9) TCID50 IU/ml; 95% CI, 2.06 x 10(9) to 9.26 x 10(9) TCID50 IU/ml). Further analysis confirmed the identity of the reference material as AAV2 and the purity relative to nonvector proteins as greater than 94%. One obvious trend in the quantitative data was the degree of variation between institutions for each assay despite the relatively tight correlation of assay results within an institution. This relatively poor degree of interlaboratory precision and accuracy was apparent even though attempts were made to standardize the assays by providing detailed protocols and common reagents. This is the first time that such variation between laboratories has been thoroughly documented and the findings emphasize the need in the field for universal reference standards. The rAAV2 RSM has been deposited with the American Type Culture Collection and is available to the scientific community to calibrate laboratory-specific internal titer standards. Anticipated uses of the rAAV2 RSM are discussed
    Type of Publication: Journal article published
    PubMed ID: 20486768
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  • 4
    Keywords: EXPRESSION ; IN-VIVO ; MOUSE MODEL ; DUCHENNE MUSCULAR-DYSTROPHY ; SYSTEMIC DELIVERY ; ALPHA-SARCOGLYCAN ; GLYCOPROTEIN COMPLEX ; LINKED DILATED CARDIOMYOPATHY ; DEFICIENT CARDIOMYOPATHY ; SKELETAL-MUSCLES
    Abstract: Dystrophin plays an important role in muscle contraction, linking the intracellular cytoskeleton to the extracellular matrix. Mutations of the dystrophin gene leading to a complete loss of the protein cause Duchenne muscular dystrophy (DMD), frequently associated with severe cardiomyopathy. Early clinical trials in DMD using gene transfer to skeletal muscle are underway, but gene transfer to dystrophic cardiac muscle has not yet been tested in humans. The aim of this study was to develop an optimized protocol for cardiac gene therapy in the mouse model of dystrophin deficiency (mdx), using a cardiac promoter for expression of a microdystrophin (mu Dys) transgene packaged into an adeno-associated virus serotype 9 vector (AAV9). In this study adult mdx mice were intravenously injected with 1 x 10(12) genomic particles of AAV9 vectors carrying a cDNA encoding mu Dys under the control of either a ubiquitously active cytomegalovirus (CMV) promoter or a cardiac-specific CMV-enhanced myosin light chain (MLC0.26) promoter. After 10 months, both AAV9 vectors led to sustained mu Dys expression in cardiac muscle, but the MLC promoter conferred about 4-fold higher protein levels. AAV9-CMV-MLC0.26-mu Dys resulted in significant protection of cardiac morphology and function as assessed by histopathology, echocardiography, and left ventricular catheterization. In conclusion, we established an AAV9-mediated gene transfer approach for efficient and specific long-term mu Dys expression in the hearts of mdx mice, resulting in a sustained therapeutic effect. Thus, this approach might be a basis for further translation into a treatment strategy for DMD-associated cardiomyopathy.
    Type of Publication: Journal article published
    PubMed ID: 22248393
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  • 5
    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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  • 6
    Keywords: ADENOASSOCIATED VIRUS ; RETINITIS-PIGMENTOSA ; LEBER CONGENITAL AMAUROSIS ; RHODOPSIN KINASE PROMOTER ; LONG-TERM PRESERVATION ; RETINAL GENE-THERAPY ; CONE PHOTORECEPTORS ; VISUAL FUNCTION ; MOUSE RETINA ; ROD
    Abstract: Acute Intermittent Porphyria (AIP) results from haplo-insufficient activity of porphobilinogen deaminase (PBGD) and is characterized clinically by life-threatening, acute neurovisceral attacks. To date, liver transplantation is the only curative option for AIP. The aim of the present preclinical nonhuman primate (NHP) study was to determine the safety and transduction efficacy of an adeno-associated viral vector encoding PBGD (rAAV5-cohPBGD) administered intravenously as part of a safety program to start a clinical study in patients with AIP. Macaques injected with either 1x1013 or 5x1013 vector genomes/kg of clinical grade rAAV5-cohPBGD, were monitored using standardized clinical parameters and vector shedding was analyzed. Liver transduction efficacy, biodistribution, vector integration and histopathology at day 30 post-vector administration were determined. There was no evidence of acute toxicity and no adverse effects were observed. The vector achieved efficient and homogenous hepatocellular transduction reaching transgenic PBGD expression levels equivalent to 50% of the naturally expressed protein. No cellular immune response was detected against the human PBGD or AAV capsid proteins. Integration site analysis in transduced liver cells revealed an almost random integration pattern supporting the good safety profile of rAAV5-cohPBGD. Together, data obtained in NHPs indicate that rAAV5-cohPBGD represents a safe therapy to correct the metabolic defect present in AIP patients.
    Type of Publication: Journal article published
    PubMed ID: 24070415
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  • 7
    Keywords: CLINICAL-TRIAL ; DELIVERY ; CENTRAL-NERVOUS-SYSTEM ; GENE-THERAPY ; CONTROLLED-TRIAL ; NIGROSTRIATAL SYSTEM ; SUBSTANTIA-NIGRA ; ADENOASSOCIATED VIRAL VECTOR ; 6-OHDA LESION ; GDNF
    Abstract: Standard integration-proficient lentiviral vectors (IPLVs) are effective at much lower doses than other vector systems and have shown promise for gene therapy of Parkinson's disease (PD). Their main drawback is the risk of insertional mutagenesis. The novel biosafety-enhanced integration-deficient lentiviral vectors (IDLVs) may offer a significant enhancement in biosafety, but have not been previously tested in a model of a major disease. We have assessed biosafety and transduction efficiency of IDLVs in a rat model of PD, using IPLVs as a reference. Genomic insertion of lentivectors injected into the lesioned striatum was studied by linear amplification-mediated polymerase chain reaction (PCR), followed by deep sequencing and insertion site analysis, demonstrating lack of significant IDLV integration. Reporter gene expression studies showed efficient, long-lived, and transcriptionally targeted expression from IDLVs injected ahead of lesioning in the rat striatum, although at somewhat lower expression levels than from IPLVs. Transgenic human glial cell line-derived neurotrophic factor (hGDNF) expression from IDLVs was used for a long-term investigation of lentivector-mediated, transcriptionally targeted neuroprotection in this PD rat model. Vectors were injected before striatal lesioning, and the results showed improvements in nigral dopaminergic neuron survival and behavioral tests regardless of lentiviral integration proficiency, although they confirmed lower expression levels of hGDNF from IDLVs. These data demonstrate the effectiveness of IDLVs in a model of a major disease and indicate that these vectors could provide long-term PD treatment at low dose, combining efficacy and biosafety for targeted central nervous system applications.
    Type of Publication: Journal article published
    PubMed ID: 24635742
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  • 8
    Abstract: Unlike for other digestive cancer entities, chemotherapy, radiotherapy, and targeted therapies have, so far, largely failed to improve patient survival in pancreatic adenocarcinoma (PDAC), which remains the fourth leading cause of cancer-related death in Europe and the United States. In this context, gene therapy may offer a new avenue for patients with PDAC. In this review, we explore the research currently ongoing in French laboratories aimed at defeating PDAC using nonviral therapeutic gene delivery, targeted transgene expression, or oncolytic virotherapy that recently or will soon bridge the gap between experimental models of cancer and clinical trials. These studies are likely to change clinical practice or thinking about PDAC management, as they represent a major advance not only for PDAC but may also significantly influence the field of gene-based molecular treatment of cancer.
    Type of Publication: Journal article published
    PubMed ID: 26603492
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  • 9
    Abstract: Bacterial cytosine deaminase (CD) converts the non-toxic prodrug 5-fluorocytosine (5-FC) into 5-fluorouracil (5-FU), which is toxic for mammalian cells. Therefore, the CD gene is used in cancer gene therapy to achieve high local concentration of a toxic metabolite without significant systemic toxicity. To allow the detection of CD expression at the protein level, we raised both polyclonal rabbit antisera and a monoclonal antibody (mAb) against a histidine-tagged CD fusion protein. The specificity of the polyclonal antisera and the mAb was confirmed by immunohistochemistry, immunoblot analysis, and immunoprecipitation using CD-expressing tumor cell lines. Furthermore, the antibodies can be used for ELISA assays and flow cytometry. Finally, the CD protein could be demonstrated in frozen tissue sections of CD-modified tumors in a rat tumor model using the anti-CD serum. With these antibodies, CD expression can now be monitored throughout in vitro and in vivo gene transfer studies, including clinical protocols relying on the CD suicide gene strategy.
    Type of Publication: Journal article published
    PubMed ID: 9295134
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  • 10
    Keywords: RECEPTOR ; CANCER CELLS ; CELLS ; EXPRESSION ; tumor ; CELL ; COMBINATION ; evaluation ; Germany ; human ; IN-VIVO ; MODEL ; THERAPY ; GENE ; EFFICIENCY ; COMPONENTS ; TISSUE ; TUMORS ; gene therapy ; DOMAIN ; animals ; treatment ; GLYCOPROTEIN ; virus ; VECTORS ; VECTOR ; MEMBRANE ; genetics ; COMPONENT ; EFFICIENT ; FIBER ; AD ; GENE-THERAPY ; heredity ; ifosfamide ; TUMOR-GROWTH ; VIRIONS ; development ; INTERNALIZATION ; USA ; animal ; microbiology ; LIMIT ; B ADENOVIRUSES ; CELLULAR RECEPTOR ; EPIDEMIC KERATOCONJUNCTIVITIS ; FUSOGENIC MEMBRANE-GLYCOPROTEINS ; REPLICATING ADENOVIRUS ; SUICIDE GENE-THERAPY
    Abstract: The clinical course of sarcoma warrants the development of new therapeutic options, such as gene therapy. However, the lack of coxsackievirus-adenovirus receptor (CAR) on sarcoma cells limits the efficacy of adenovirus type 5 (Ad5)-based gene therapy. In this study we evaluated 20 different adenoviral types and 1 Ads vector with RGD-containing fiber for their internalization efficiency in sarcoma cells. We demonstrated that adenovirus types 35, 3, 7, 11, 9, and 22 and Ad5LucRGD virions (ranked in descending order) have significantly higher internalization efficiency in the tested sarcoma cells when compared with Ads. On the basis of these results we developed a conditionally replication-competent adenoviral vector, Ad5 Delta 24.Ki center dot COX, and compared its oncolytic efficacy with that of Ads/35 Delta 24.Ki center dot COX, an Ads-based vector with the Ad35 fiber shaft and knob domains. Because both vectors differed only in the fiber, we were able to assess whether the adenoviral type with the most efficient internalization resulted also in enhanced treatment efficacy. We evaluated the antineoplastic activity of the oncolytic adenoviral vectors alone or in combination with the expression of measles virus fusogenic membrane glycoproteins and/or ifosfamide. The findings of our xenograft model were as follows: animals that received Ads/35-based therapy had significantly smaller tumors than animals treated with the homologous Ads-based vectors. In addition, we demonstrated that the combination of virotherapy, intratumoral expression of fusogenic membrane glycoproteins, and ifosfamide was clearly superior compared with treatment with individual components alone or as combinations of two components. In conclusion, Ad35-based vectors are promising for the treatment of sarcoma
    Type of Publication: Journal article published
    PubMed ID: 17184155
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