611. Group I Intron-Mediated Trans-Splicing Revision of Mutant p53 RNA Transcripts

Abstract

Mutations in the gene encoding the p53 tumor suppressor are reported to be associated with more than half of reported cancer cases. Restoration of wild-type p53 function, as mediator of apoptosis and cell-cycle arrest, has been a popular cancer treatment strategy. Unregulated overexpression of wild-type p53 through conventional gene replacement therapy has been shown to induce deleterious phenotypic changes in normal tissue. We hypothesize that correction of native mutant RNA transcripts by a group I ribozyme can restore the wild-type function of p53 while maintaining endogenous physiologic expression levels. We have developed and characterized the activity of three group I ribozymes that specifically revise mutant p53 transcripts in vitro. Most p53 mutations affect its DNA binding domain, and certain mutations in p53 affect the transactivation of well-characterized p53-responsive genes, such as WAF1/p21, which is involved in cell-cycle inhibition, and the proapoptotic Bax. In the 2774 ovarian cancer cell line, the mutated p53 is unable to transactivate either gene. Ribozyme-mediated revision of the mutant p53 transcript in 2774 cells results in significant stimulation of expression for both p21 and Bax. At 72 hours post-transfection, apoptosis in ribozyme-transfected 2774 cells was increased 2.5 fold over that of mock trasfectants using a fluorescent TUNEL assay. This is achieved even though only about 10% of the mutant transcripts were repaired in the 2774 cells with transfection efficiency in the range of only 40–50% using our plasmid constructs. Certain mutations in p53 also affect the transcription of the multi-drug resistance gene (MDR1), whose gene product renders several structurally unrelated chemotherapeutic agents ineffective. Ribozyme-transfected tumor cells, which contained exogenous truncated p53 message, and cotransfected with the human MDR1 promoter upstream of the luciferase gene as a reporter plasmid were used to assay for functional p53 activity. Group I intron-mediated trans-splicing correction of truncated p53 to wild-type function results in the reduction of MDR1 expression by at least half in Saos-2 sarcoma cells. These results suggest that group I intron-mediated trans-splicing revision of cancer cells with mutant p53 RNA transcripts could be an effective method for reestablishing apoptosis and chemosensitivity in malignant tumors. We are currently using adenoviral ribozyme constructs to increase tumor cell transduction efficiency and mutant RNA transcript repair efficiency in preparation of a clinically applicable cancer treatment modality.