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

Abstract

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 AAVbased therapeutic applications.

Identification and Quantification of a New Family of Peptide Endocannabinoids (Pepcans) Showing Negative Allosteric Modulation at CB1 Receptors

“…To determine the minimal sequence required for antibody binding, a bead-based peptide array was prepared as previously described (22)(23)(24). The array consisted of the RVD-Hp␣ peptide (RVDPVNFKLLSH), a carboxamide version of the RVDHp␣ peptide, and 22 truncated peptides.…”

Targeting of tetraspanin proteins — potential benefits and strategies

“…Adenoviruses encoding CD9 or CD82 were administered, via the trachea, into mice orthotopically preimplanted with Lewis lung carcinoma cells, resulting in a dramatic reduction in metastasis 65 . Curiously, host-cell expression of CD9 itself may facilitate adeno-associated virus serotype 2 infection 164 .…”

Comparison of DNA Delivery and Expression Using Frequently Used Delivery Methods

“…Ad transduced all cell types examined with varying degrees of efficiency, correlating with the ubiquitous expression of the CAR receptor at different levels in cells (Lyons et al 2006). The finding that AAV2 was unable to transduce all cancer cell types examined, is consistent with a lack of expression of its primary receptor, heparin sulfate proteoglycans (HSPG), which may be absent or deleted on certain tumour types (Summerford & Samulski 1998), and/or CD9, which can mediate AAV-2 infection in certain cancer cells in the absence of HSPG (Kurzeder et al, 2007). The HSPG and CD9 status of the tumour cell lines used in this study is unknown.…”