114. Adenoviral Vectors for Gene Therapy with Improved Entry of Malignant Glioma

Abstract

An adenovirus serotype 35 (Ad35) vector, which is composed of whole Ad35 belonging to subgroup B, has gained interest as a promising vehicle for gene transfer. This is because Ad35 vectors can circumvent the preexisting immunity against Ad serotype 5 (Ad5). In addition, Ad35 vectors efficiently transduce a wide spectrum of human cells in a coxsackievirus-adenovirus receptor (CAR)-independent manner. This spectrum includes human CD34+ cells and dendritic cells, which are refractory to Ad5 vectors. However, cellular receptors for Ad35 have yet to be identified. Recently, human CD46 (hCD46), a ubiquitously expressed complement regulatory protein, has been identified as a cellular receptor for most subgroup B Ad, including Ad35 (Nat. Med., 2003, 9, S1408-1412, J. Virol. 2003, 77, 9183-9191). We have recently developed a replication-incompetent, recombinant Ad35 vector, and we have reported the properties of Ad35 vector-mediated transduction in vitro and in vivo (Gene Ther. 2003, 10, 1041-1048; Mol. Ther., 2003, 8, 813-821). In the present study, in order to fully evaluate the relationship between the expression levels of hCD46 and Ad35 vector-mediated transduction efficiencies, we examined the role of hCD46 on Ad35 vector-mediated transduction in detail. First, we performed Ad35 vector-mediated transduction into CHO cell lines that stably expressed the hCD46 BC1 or BC2 isoform. After infection with the Ad35 vector, these hCD46-expressing CHO cells showed increases of up to 17-fold in their transduction efficiencies compared with wild-type CHO cells, which are refractory to Ad35 infection. Next, the expression levels of hCD46 in various types of human cells and Ad35 vector-mediated transduction efficiencies were examined. FACS analysis revealed that almost all of the cultured human cells, which mediated efficient transduction with the Ad35 vector, expressed high levels of hCD46, and that the expression level of hCD46 was much lower in SF295 cells (glioblastoma multiforme), which are refractory to the Ad35 vector, than in the other cells. Almost 100% of human bone marrow-CD34+ cells, which are susceptible to the Ad35 vector, also expressed high levels of hCD46. Furthermore, the role of hCD46 was examined using hCD46-transgenic (hCD46-Tg) mice. Normal mouse CD46 expression is limited to the testes, whereas hCD46-Tg mice express hCD46 in a pattern closely mimicking that observed in humans. Transduction of the Ad35 vector into primary hepatocytes and peritoneal macrophages from hCD46-Tg mice resulted in significantly higher transgene expression than that from wild-type mice. Finally, to confirm that hCD46 could act as a cellular receptor in vivo, the Ad35 vector was intraperitoneally administered into hCD46-Tg mice. All the organs examined in the hCD46-Tg mice exhibited higher levels of transgene expression than were exhibited by wild-type mice. These findings demonstrated that the Ad35 vector recognizes hCD46 as a functional receptor in vitro and in vivo. Further in vivo characterization of the Ad35 vector using hCD46-Tg mice would be an important step toward gene therapy applications of the Ad35 vector.