Group D adenoviruses infect primary central nervous system cells more efficiently than those from group C

Abstract

Group C adenovirus-mediated gene transfer to central nervous system cells is inefficient. We found that wild-type group D viruses, or recombinant adenovirus type 2 (Ad2) (group C) modified to contain Ad17 (group D) fiber, were more efficient in infecting primary cultures of neurons. Together with studies on primary vascular endothelial cells and tissue culture cell lines, our results indicate that there is not a universally applicable adenovirus serotype for use as a gene transfer vector.

FIG. 1

Schematic representation of the construction of Ad2 virus containing Ad17 fiber sequences. The final construct contains Ad2 backbone sequences with all but the first 17 amino acids of the fiber molecule removed. Replaced in frame is the Ad17 fiber from amino acid 18 to the COOH terminus. A simian virus 40 (SV40) polyadenylation signal was added to the 3′ end of the fiber sequence as indicated. The E4 ORF6 sequences are indicated for orientation. CMV, cytomegalovirus; β-gal, β-galactosidase; MLT, major late transcription.

FIG. 2

Transgene expression after Ad2βgal2(17f) and Ad2βgal2 infection of primary cortical cultures. Cells were infected at an MOI of 10 for 2 h at 37°C, virus solution was removed, and cells were cultured in standard media for an additional 48 h. (A) β-Galactosidase histochemistry; (B) β-galactosidase activity assay. LU, relative light units. Data represent averages ± standard errors of the means.

FIG. 2

Transgene expression after Ad2βgal2(17f) and Ad2βgal2 infection of primary cortical cultures. Cells were infected at an MOI of 10 for 2 h at 37°C, virus solution was removed, and cells were cultured in standard media for an additional 48 h. (A) β-Galactosidase histochemistry; (B) β-galactosidase activity assay. LU, relative light units. Data represent averages ± standard errors of the means.

FIG. 3

Colocalization of β-galactosidase activity and cell-specific markers. Primary cortical cultures were infected with Ad2βgal2(17f) or Ad2βgal2 as described for Fig. 2. Two days after infection cells were histochemically stained for β-galactosidase followed by immunostaining with antibodies specific for astrocytes (glial fibrillary acidic protein [GFAP]) and oligodendrocytes (OLIGO). The photographs are selective and do not represent the cultures in general, which contained more than 90% neurons (17).

FIG. 4

β-Galactosidase activity in primary vascular endothelia or various cell lines infected with Ad2βgal2(17f) or Ad2βgal2 (MOI = 50). Data represent averages ± standard errors of the means. RLU, relative light units.