RIBOZYME-MEDIATED IN-VITRO CLEAVAGE OF TRANSCRIPTS ARISING FROM THE MAJOR TRANSFORMING GENES OF HUMAN PAPILLOMAVIRUS TYPE-16

Human papillomaviruses (HPV) have been strongly implicated as importan t cofactors in the development of several human malignancies, particul arly anogenital carcinomas. Products arising from the E6 and E7 open r eading frames (ORFs) from HPV-16, a type commonly associated with huma n cervical carcinoma, are essential for viral transformation. Unfortun ately, a highly effective treatment for this infection is not availabl e. To develop a novel treatment for this disease, ribozymes were desig ned to cleave all transcripts encoding HPV-16 E6 and E7 ORFs in proxim ity to their translational start sites (''AUG''). Cleavage sites for R z110 and Rz558 occur immediately 3' to nucleotides 110 and 558 of the viral genomic DNA, respectively. Oligonucleotides corresponding to the se ribozymes were synthesized and inserted into a eucaryotic viral vec tor derived from the nonpathogenic parvovirus, adeno-associated virus. Ribozyme transcription from this vector, termed CWRT7:SVN, is under c ontrol of both the highly active Rous sarcoma virus long terminal repe at and bacteriophage T7 promoters. T7 transcripts of the E6 and E7 rib ozymes efficiently cleaved their cognate targets in vitro under a vari ety of conditions, including physiological temperature. These results may provide the basis for the development of a ribozyme-based, gene th erapeutic treatment for HPV-associated diseases.