Efficient gene transfer of CD40 ligand into ovarian carcinoma cells with a recombinant adeno-associated virus vector.
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.
Koppold
Bernd
B
University of Ulm Medical School, Department of Obstetrics and Gynecology, Ulm, Germany.
Sauer
Georg
G
Büning
Hildegard
H
Hallek
Michael
M
Kreienberg
Rolf
R
Deissler
Helmut
H
Kurzeder
Christian
C
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
C485184:enhanced green fluorescent protein; D023201:CD40 Ligand; D049452:Green Fluorescent Proteins; D018664:Interleukin-12