The overall goal of this proposal is to explore the ability of group I and group II introns to repair genetic instructions inside mammalian cells. These introns have been of great scientific interest because they are able to perform catalysis and because a subclass of these RNA enzymes can act as mobile genetic elements. Moreover, their ability to modify RNA and DNA sequences through forward and reverse-splicing reactions makes these introns of particular interest to translational researchers. In the previous funding cycle of this grant application, we demonstrated that the Tetrahymena thermophila group I intron can perform self- and trans-splicing reactions to alter the sequences of transcripts in mammalian cells. More recently, we demonstrated that the Lactococcus lactis group II intron can reverse-splice and insert itself into DNA in transfected human cells. These proof of concept studies suggest that such catalytic RNAs may represent molecules that can be employed to modify genetic instructions for therapeutic ends. These studies also underscore the necessity for further evaluation of these catalytic RNAs in a clinically relevant setting if they are to become therapeutically useful. Herein we propose to perform more detailed analyses of group I and group II intron activity in mammalian cells focusing upon repair of mutant p53 transcripts and genes as a model experimental system. The p53 gene has been chosen as a target for genetic repair in these studies because it is a tumor suppressor gene that is often mutated in human cancers. Moreover, because p53 is a transcription factor, simple and sensitive assays exist to detect p53 activity in mammalian cells. Finally, we have previously demonstrated that a trans-splicing group I ribozyme can repair mutant p53 transcripts and induce p53 activity in a variety of different human cancer cells; thus such an experimental approach will logically build upon our previous work. The completion of these studies will yield a more detailed understanding of group I and group II intron-mediated catalysis in mammalian cells as well as establish the needed experimental foundation from which the logical development of therapeutic group I and group II ribozymes can proceed.
| Watanabe, T; Sullenger, B A (2000) Induction of wild-type p53 activity in human cancer cells by ribozymes that repair mutant p53 transcripts. Proc Natl Acad Sci U S A 97:8490-4 |
| Zarrinkar, P P; Sullenger, B A (1999) Optimizing the substrate specificity of a group I intron ribozyme. Biochemistry 38:3426-32 |
| Long, M B; Sullenger, B A (1999) Evaluating group I intron catalytic efficiency in mammalian cells. Mol Cell Biol 19:6479-87 |
| Zarrinkar, P P; Sullenger, B A (1998) Probing the interplay between the two steps of group I intron splicing: competition of exogenous guanosine with omega G. Biochemistry 37:18056-63 |
| Jones, J T; Sullenger, B A (1997) Evaluating and enhancing ribozyme reaction efficiency in mammalian cells. Nat Biotechnol 15:902-5 |
| Jones, J T; Lee, S W; Sullenger, B A (1996) Tagging ribozyme reaction sites to follow trans-splicing in mammalian cells. Nat Med 2:643-8 |
