114. Integrins Are Required for Adenovirus 5 Transduction in Cells of Lymphocyte Origin

Abstract

Listen

Translate article

Adenovirus serotype 5 (Ad5) is the most widely used vector in cancer gene therapies. The high efficiency and broad range of host transduction, easy genome manipulation, non-integration into the host genome, and the well-characterized molecular biology of adenovirus have made them an excellent choice for cancer therapeutics. Ad5 infects cells by binding to the coxsackie-adenovirus receptor (CAR) followed by internalization mediated by binding of viral penton RGD motifs to cell surface integrins (avb3, avβ5). Low levels of Ad5 infection of both human and murine lymphocytes have been linked to the absence of CAR on these cells. We have previously reported that low levels of integrins on canine lymphoma cells are a potential obstacle to Ad5 infection. In the current report we have evaluated CAR and integrins at the molecular level by using quantitative reverse transcriptase PCR (Q-RT-PCR) in a broad range of canine tumor cells including lymphomas (OSW, 17-71), mast cells (BR, MPT1), melanocytes (CML7, CML10), histiocytoma (DH82) and mammary adenocarcinomas (CMT28). Normal canine fibroblasts, peripheral blood mononuclear cells (PBMC) and HEK293 cells were used as controls. All of the cell lines were exposed to Ad5G/L virus at multiplicities of infection (MOI) of 10, 100, and 1000 viral particles per cell. Ad5G/L is a replication incompetent virus vector that infect cells utilizing the classical CAR and integrin infection mechanism. Ad5G/L expresses both GFP and luciferase after infecting cells. Fluorescence microscopy demonstrated that Ad5G/L successfully infected CMT28, DH82, HEK293, CML7, CML10, and NCF at 48 hrs. post infection at all 3 multiplicities of infection. There was limited or no Ad5G/L infection in the lymphoma cell lines, PBMCs, and MPT1 mast cells. The mast cell line BR was the exception, showing relatively high levels of infection. Q-RT-PCR results showed low level of integrins in MPT1, 17-71, OSW, and PBMC cells in comparison to CMT28, DH82, and BR cells. Interestingly, CAR mRNA levels were comparatively higher in MPT1, 17-71, OSW, and PBMC cells. These data indicate that lack of integrins and not CAR is the primary reason for the inability of Ad5G/L infection to infect cells of lymphocyte origin. The defect in cell surface integrins must be addressed if Adenoviral vectors are to be used to transfer genes to lymphocytes or tumors in cells of lymphocyte origin.