A rigorous, structured, biphasic five-year program has been designed to provide in depth study of molecular biology and virology. Phase I will include didactic study in the fields of molecular biology, biochemistry, microbiology and virology, to provide the framework for the proposed laboratory based research. Also during phase I, work will begin in the laboratory of Dr. Thomas R. Cech of the Department of Chemistry and Biochemistry, the University of Colorado, who will serve as the phase I sponsor. The phase I laboratory work will provide intense training in basic research in the fields of molecular biology and ribozymology. Dr. Robert T. Schooley of the Infectious Diseases Division, the University of Colorado Health Sciences Center, will serve an the phase II sponsor. During phase II, the techniques developed in phase I will be applied to develop anti-HIV-1 ribozymes. Despite many advances in the understanding of the molecular pathogenesis of the human immunodeficiency virus type 1 (HIV-1), effective treatment of HIV-1 infection in still not available. Ribozymes (ribonucleotide enzymes) are RNA molecules that possess sequence specific endoribonuclease activity, and have potential utility an tools to study the molecular pathogenesis of HIV-1 and as anti-HIV-1 therapeutic agents. Anti-HIV-1 ribozymes employed in existing studies have been designed by a lengthy, systematic approach. The studies in this proposal will develop a more direct method to identify biologically active ribozymes. This method will use selective pressure applied to a biological system to determine which ribozymes in a pool of randomly generated hammerhead variants, cleave substrate most effectively in vivo. Phase I of this proposal will study the feasibility and limitations of in vivo ribozyme selection in E. coli by targeting ribozymes against the E. coli lac operon. In the later stages of phase I, this selection procedure will be extended to select biologically active anti-HIV-1 ribozymes in E. coli on the basis of their ability to inhibit expression of a HIV-1-lacZ fusion transcript. Phase II will employ the findings of phase I to select anti-HIV-1 ribozymes in eukaryotic cells by interference with the cytopathic effects of HIV-1, or by inhibition of HIV-1 LTR activation. initially, anti-HIV-1 ribozymes will be selected against laboratory strains of HIV-1, later studies will determine if active ribozymes can be selected against diverse clinical isolates of HIV-1. Although the studies outlined herein are designed to select anti-HIV-1 ribozymes, the selection methods developed as a result of this work are expected to have much broader applications for the development of ribozyme technology.
| Campbell, T B; McDonald, C K; Hagen, M (1997) The effect of structure in a long target RNA on ribozyme cleavage efficiency. Nucleic Acids Res 25:4985-93 |
| Campbell, T B; Cech, T R (1996) Mutations in the Tetrahymena ribozyme internal guide sequence: effects on docking of the P1 helix into the catalytic core and correlation with catalytic activity. Biochemistry 35:11493-502 |
| Campbell, T B; Cech, T R (1995) Identification of ribozymes within a ribozyme library that efficiently cleave a long substrate RNA. RNA 1:598-609 |
| Campbell, T B; Sullenger, B A (1995) Alternative approaches for the application of ribozymes as gene therapies for retroviral infections. Adv Pharmacol 33:143-78 |
