Effectiveness of three ribozymes for cleavage of an RNA transcript from human papillomavirus type 18

We tested three hammerhead ribozymes for their ability to bind and cleave RNA transcripts derived from the E6 and E7 genes of human papillomavirus (HPV) type-18. Targets were located at nucleotides (nt) 123, 309, and 671 of the viral transcript. In vitro each ribozyme hybridized to its target site when the ribozyme:target ratio was 20:1 or greater and achieved maximal hybridization within 1 hour. HPV RNA from the HeLa cervical cancer cell line was cleaved effectively by each ribozyme. When HPV RNA and a ribozyme were expressed simultaneously in Escherichia coli, each ribozyme produced a significant reduction in the intracellular concentration of HPV RNA. In each assay the ribozyme directed to nt 309 was the most effective. A noncatalytic antisense molecule was used as a control and did not digest HPV RNA or reduce its concentration. The data imply that three different ribozymes each have potential for use in gene therapy of human tumors that express HPV-18 but that the ribozyme targeted to nt 309 is likely to be most effective.