Growth inhibition of human papillomavirus 16 DNA-positive mouse tumor by antisense RNA transcribed from U6 promoter.

Over 95% of cervical carcinomas are human papillomavirus (HPV) DNA positive, and the expression of the E6 and E7 genes is required for the maintenance of malignant phenotype. Here, antisense sequences targeted against the HPV16 E6 and E7 genes were cloned as U6/antisense chimeric genes under the control of the U6 RNA gene promoter. Cationic liposome-mediated transfection of these plasmids into human 293 cells and HPV16 DNA-positive mouse C3 tumor cells were performed, and the chimeric U6/antisense transcripts were detected. The U6 promoter could express RNA up to 360 nucleotides in length. In a cotransfection study, the E7 antisense plasmid (but not the sense control) caused the down-regulation of E7 gene expression. Similarly, decreased E7 protein levels were observed in the C3 cells that were transfected with E7 antisense plasmid. In vitro growth inhibition was studied by delivering liposome-antisense plasmid complex to C3 tumor cells. Up to 50% growth inhibition was achieved with antisense plasmids, whereas only about 15% growth inhibition was observed with sense plasmids. Transfection into human cervical carcinoma C33A cells and mouse melanoma BL6 cells, which contain no HPV DNA, showed no growth inhibition. These results indicate that growth inhibition resulted from specific E6/E7 down-regulation of the tumors or cells. Furthermore, intratumor injection of DNA-liposome complex containing either E6 or E7 antisense plasmid resulted in significant growth inhibition of C3 tumors but not BL6 tumors, grown in a syngeneic mouse model. This promising result indicates that E6/E7 antisense sequences expressed in the context of the U6 gene might be useful for gene therapy of cervical carcinoma.