RECOMBINANT ADENOVIRUS-MEDIATED GENE-TRANSFER TO GENITOURINARY EPITHELIUM IN-VITRO AND IN-VIVO

Transitional cell carcinoma (TCC) of the bladder is associated with ch aracterized lesions in dominant and recessive oncogenes. The understan ding of the molecular basis of tumorigenesis in these instances makes possible the application of gene therapy strategies for TCC. In th is regard, the ability to directly access the epithelium of the genitouri nary (CU) tract via the urethra provides a practical means to implemen t these various gene therapy approaches. We thus explored vector strat egies to accomplish direct in vivo transduction of CU epithelium. Init ially, three human (HT 1197, HT 1376, T24) and one mouse (MET-2) TCC c ell lines were transduced using a recombinant adenoviral vector expres sing the firefly luciferase reporter gene, rAd-CMV-Luc. In these studi es, reporter gene expression was found to be significantly elevated ab ove background for all four cell lines. Of note, the TCC cell lines HT 1197 and HT 1376 showed expression levels comparable with the cervica l carcinoma cell line HeLa, a cell line previously shown to be highly susceptible to recombinant adenovirus-mediated gene transduction. An i n vitro time course for T24 and MET-2 using rAd-CMV-Luc showed peak ex pression 1 day after transduction for the T24 line and 3 days after tr ansduction for the MET-2 line, with detectable levels of expression pe rsisting for at least 7 days. As a next step, human and mouse primary tissue deriving from the CU epithelium were transduced using rAd-CMV-L uc. In this assay, luciferase expression levels significantly above ba ckground were observed in both instances. Thus, using a recombinant ad enoviral vector, highly efficient in vitro gene transfer to cells deri ving from the GU epithelium can be accomplished. To establish the prac tical utility of this vector for the application of gene therapy for T CC, direct in vivo transfer was evaluated in a murine model. For this analysis, rAd-CMV-Luc and the recombinant adenovirus containing the re porter gene for beta galactosidase (rAd-CMV-beta gal) were delivered i ntravesically by the uretheral route to BALB/c mice and Sprague-Dawley rats, respectively. The experimental animals that received the rAd-CM V-Luc were evaluated i, 3, and 7 days posttransduction. High levels of luciferase activity were observed in the bladders from treated mice. Expression peaked 1 day posttransduction but was detected up to the se venth day after vector delivery. In a similar manner, a Sprague-Dawley rat received the rAd-CMV-beta gal virus intravesically and was assaye d for reporter gene expression 48 hours posttransfusional. Staining fo r reporter gene expression showed beta-galalactosidase activity within the cells of interest. Therefore, recombinant adenovirus can accompli sh direct in vivo transduction of bladder epithelium when delivered by a physiologic route. Recombinant adenovirus thus represents a promisi ng gene transfer vector to use for strategies to accomplish anticancer gene therapy for TCC.