title: Efficient gene transfer of CD40 ligand into ovarian carcinoma cells with a recombinant adeno-associated virus vector.
abstract: Recombinant adeno-associated virus type 2 (rAAV) has many properties of an ideal vector for gene therapy: broad spectrum of susceptible cells, efficient gene transfer, persistent transgene expression in vivo, and no indiction of vector-related toxicity. Ovarian carcinoma cell lines, however, were previously reported to be quite resistant to rAAV transduction. Using an optimized adenovirus-free packaging system, highly purified rAAV vectors coding for the enhanced green fluorescent protein (AAV/EGFP) and for mCD40 ligand (AAV/CD40L) were generated. Their transduction efficiency in ovarian carcinoma cell lines was assessed with and without irradiation prior to infection. As measured by flow cytometry, transgene expression in up to 92% of cells was achieved with AAV/EGFP. gamma-irradiation (20 Gy) significantly increased the transduction rates up to 3.5-fold in cell lines with low susceptibility to AAV infection. The aquired capability of AAV/CD40L transduced tumor cells to activate dendritic cells was demonstrated in a second step. Dendritic cells were generated from human peripheral blood monocytes and maturized by stimulation with IL-4 and GM-CSF. Co-cultivation of mCD40L transgenic tumor cells with these dendritic cells resulted in strong ELISA-determined expression of IL-12 as an indicator of dendritic cell activation. We conclude that transduction of tumor cells with rAAV encoding mCD40L is a promising strategy for tumor immunotherapy which may be further developed to a vaccination approach with transgenic ovarian carcinoma cells generated by ex vivo transduction.
authors.affiliation: University of Ulm Medical School, Department of Obstetrics and Gynecology, Ulm, Germany.
authors.forename: Bernd
authors.initials: B
authors.lastname: Koppold
authors.forename: Georg
authors.initials: G
authors.lastname: Sauer
authors.forename: Hildegard
authors.initials: H
authors.lastname: Büning
authors.forename: Michael
authors.initials: M
authors.lastname: Hallek
authors.forename: Rolf
authors.initials: R
authors.lastname: Kreienberg
authors.forename: Helmut
authors.initials: H
authors.lastname: Deissler
authors.forename: Christian
authors.initials: C
authors.lastname: Kurzeder
mesh_terms: D000818:Animals; D023201:CD40 Ligand / Q000235:genetics* / Q000502:physiology; D002277:Carcinoma / Q000235:genetics / Q000276:immunology* / Q000628:therapy; D045744:Cell Line, Tumor; D003131:Combined Modality Therapy; D003713:Dendritic Cells / Q000276:immunology / Q000378:metabolism; D000229:Dependovirus / Q000235:genetics*; D005260:Female; D005720:Gamma Rays; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D006801:Humans; D018664:Interleukin-12 / Q000276:immunology / Q000378:metabolism; D051379:Mice; D010051:Ovarian Neoplasms / Q000235:genetics / Q000276:immunology* / Q000628:therapy; D014161:Transduction, Genetic / Q000379:methods
chemical_list: C485184:enhanced green fluorescent protein; D023201:CD40 Ligand; D049452:Green Fluorescent Proteins; D018664:Interleukin-12