INHIBITION OF HPV-16 E6 E7 IMMORTALIZATION OF NORMAL KERATINOCYTES BYHAIRPIN RIBOZYMES/

HPV-16 E6 and E7 genes are required to efficiently immortalize a broad spectrum of cell types including cervical keratinocytes, Therefore, t he E6/E7 genes can be considered relevant targets for anti cancer ther apy, We produced several engineered hairpin (HP) ribozymes to specific ally disrupt HPV-16 E6/E7 mRNA, After extensive biochemical characteri zation, one anti-E6 HP ribozyme (R434) was selected for in vivo testin g because of its superior catalytic capabilities, When expressed in ci s, R434 efficiently inhibited E6 in vitro translation, Cis-expression of the HP ribozyme with HPV-16 E6/E7 genes in normal human keratinocyt es reduced the growth rate and prevented immortalization. RNA analysis by reverse transcription-PCR showed that E6/E7 transcripts were cleav ed in post-transfected cells and virtually were eliminated after long term expression, Of interest, an inactive version of the HP also was a ble to significantly affect the immortalizing ability of E6/E7, probab ly through passive hybridization. The combination of passive and cleav ing antisense RNA therefore is established as an effective inhibitor o f HPV-16 E6/E7 immortalization.