Lm. Alvarezsalas et al., INHIBITION OF HPV-16 E6 E7 IMMORTALIZATION OF NORMAL KERATINOCYTES BYHAIRPIN RIBOZYMES/, Proceedings of the National Academy of Sciences of the United Statesof America, 95(3), 1998, pp. 1189-1194
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.