CD9 promotes adeno-associated virus type 2 infection of mammary carcinoma cells with low cell surface expression of heparan sulphate proteoglycans.

Recombinant adeno-associated virus (AAV) is a promising non-pathogenic vector in the emerging field of gene therapy. For AAV serotype 2 (AAV-2) infection, experimental evidence points to an involvement of heparan sulphate proteoglycans (HSPG), but also to the existence of additional receptors. We investigated a potential role of the tetraspanin CD9 in AAV-2 infection of breast cancer cells mainly because it binds to the heparin-binding EGF-like growth factor, suggesting that it may also interact with a heparin-binding virus. Among breast cancer cell lines, expression of HSPG or potential AAV-2 (co)-receptors was not found to correlate with transduction efficiency. In complete accordance with the role of CD9, blocking with anti-CD9 antibodies resulted in drastically decreased AAV-2 transduction efficiencies in cell lines with low expression of HSPG. Furthermore, specific inhibition of CD9 expression with siRNA resulted in fewer transgene-positive cells, whereas overexpression of CD9 in the breast cancer cell line T47D as well as in BT8Ca and BT12Ca rat glioma cells (with low background expression of HSPG and CD9) increased the number of AAV-transduced cells. The minimal epitope recognized by antibody 72F6, which most efficiently blocked AAV-mediated transgene expression, was deduced from the specific binding to peptides immobilized on colour-encoded microspheres consisting of the amino acid sequence PKKDV located in the large extracellular loop of CD9. Our results clearly point to an involvement of CD9 in the attachment, uptake or processing of AAV-2 by target cells expressing low amounts of HSPG, which may help to define cell populations accessible in AAV-based therapeutic applications.

Chemotherapeutic agents enhance AAV2-mediated gene transfer into breast cancer cells promoting CD40 ligand-based immunotherapy.

PURPOSE: Supplementing conventional treatment with gene therapy to induce an immune response might be beneficial to cancer patients. In this study, we evaluated the efficiency of transduction of breast cancer cells with recombinant adeno-associated virus (rAAV) and effects of cytotoxic agents used in chemotherapy. Furthermore, the capacity of tumor cells expressing transgenic CD40 ligand (CD40L) to stimulate dendritic cells was measured. METHODS: Breast cancer cell lines were infected with a rAAV encoding the enhanced green fluorescent protein (EGFP) or murine CD40L and transgene expression was analyzed by flow cytometry. Stimulation of isolated human dendritic cells by CD40L-expressing tumor cells was quantified by measuring secreted interleukin 12. RESULTS: Infection with an EGFP-encoding rAAV resulted in variable fractions (14-93%, mean 42%) of transgene-expressing cells. Pre-incubation of MM 157, MM 231, and MCF7 cells with epirubicin or carboplatin substantially increased AAV-mediated transgene expression. rAAV/CD40L was used to generate CD40L-transgenic tumor cells, which specifically activated immature dendritic cells, as confirmed by blocking with an antibody binding to CD40L. CONCLUSIONS: The efficiency of rAAV-mediated gene transfer into breast cancer cells is significantly higher than previously reported and can be further enhanced by co-administration of chemotherapeutic agents. We also confirmed that breast cancer cells can activate human dendritic cells after infection with a CD40L-encoding rAAV.

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.