Abstract

The overall goal of this proposed research project is to develop ribozyme technology to the point that it is close to therapeutic reality. This will be done using the hairpin ribozyme which is a small trans-acting RNA enzyme that is highly suitable for cellular RNA inactivation studies. Previous work has elucidated rules by which the hairpin ribozyme can be engineered to efficiently cleave HIV sequences and has developed an in vitro selection method through which ribozyme activity against a given target sequence can be optimized. These findings have been used to generate a ribozyme targeted to a conserved HIV pol sequence that strongly inhibits HIV-1 replication in cultured lymphocytes.
The Specific Aims of the current proposal are: (1) Rigorously define the site and mechanism of antiviral action of anti-HIV hairpin ribozymes, and assess their substrate selectivity in vivo; (2) Develop and evaluate a novel class of anti-HIV targets, using new information defining substrate specificity of the hairpin ribozyme. It is expected that this work will result in (i) definitive proof that engineered hairpin ribozymes inhibit HIV replication by site- specific RNA cleavage at the targeted site, (ii) the exploration of several novel strategies for the development, intracellular activation, and metabolic regulation of highly active ribozymes, and (iii) the development and rapid testing of a number of potential new anti-HIV therapeutic agents.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI029892-08
Application #
2413575
Study Section
AIDS and Related Research Study Section 4 (ARRD)
Project Start
1990-07-01
Project End
1999-04-30
Budget Start
1997-05-01
Budget End
1998-04-30
Support Year
8
Fiscal Year
1997
Total Cost
Indirect Cost

  Institution

Name
University of Vermont & St Agric College
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
066811191
City
Burlington
State
VT
Country
United States
Zip Code
05405

  Publications

Yu, Q; Pecchia, D B; Kingsley, S L et al. (1998) Cleavage of highly structured viral RNA molecules by combinatorial libraries of hairpin ribozymes. The most effective ribozymes are not predicted by substrate selection rules. J Biol Chem 273:23524-33
Berzal-Herranz, A; Burke, J M (1997) Ligation of RNA molecules by the hairpin ribozyme. Methods Mol Biol 74:349-55
Sargueil, B; Pecchia, D B; Burke, J M (1995) An improved version of the hairpin ribozyme functions as a ribonucleoprotein complex. Biochemistry 34:7739-48
Joseph, S; Burke, J M (1993) Optimization of an anti-HIV hairpin ribozyme by in vitro selection. J Biol Chem 268:24515-8