Modification of the genetic program of human alveolar macrophages by adenovirus vectors in vitro is feasible but inefficient, limited in part by the low level of expression of the coxsackie/adenovirus receptor

Robust expression of genes transferred by adenovirus (Ad) vectors depends u pon efficient entry of vectors into target cells. Cells deficient in the co xsackie/adenovirus receptor (CAR) are difficult targets for Ad-mediated gen e transfer. We hypothesized that low levels of CAR expression may be respon sible, in part, for the relative inefficiency of Ad-mediated gene transfer to human alveolar macrophages (AMs). CAR gene expression was detected in hu man AMs by reverse transcription-polymerase chain reaction and at low level s by Northern analysis. Indirect immunofluorescence showed specific, low-in tensity surface staining for CAR, but at levels below those found on the po sitive-control A549 human lung epithelial cell line. Consistent with this, AMs expressed Ad vector transgenes 100 to 1,000-fold less efficiently than A549 cells, as assessed using the beta-galactosidase reporter (chemilumines cence assay) and green fluorescent protein (fluorescence microscopy and flo w cytometry). At high multiplicity of infection, AMs from an HIV+ individua l could be transduced with an AdIFN gamma vector to secrete detectable huma n interferon-gamma. Ad transgene expression by AMs was blocked by capsid fi ber protein, suggesting that CAR is required in the pathway for productive Ad entry into alveolar macrophages. To confirm that Ad transgene expression by AMs is limited by low levels of CAR expression, cells were infected wit h an Ad vector containing the CAR complementary DNA (cDNA). Enhanced expres sion of CAR protein was demonstrated by indirect immunofluorescence, and th e CAR cDNA-transduced cells showed 5-fold enhancement of subsequent Ad tran sgene expression. These observations demonstrate that human AMs can be targ ets for Ad-mediated gene transfer, but that efficiency of transgene express ion is limited, at least in part, by low levels of CAR expression.