Ribozymes pick out elongation factors as potential anti-virals

Scientists from the University of California, San Diego report in the July 18 issue of the Proceedings of the National Academy of Sciences, on the isolation of two factors required for viral protein translation that might provide new targets for anti-viral drugs. Using "inverse genetics," senior author Flossie Wong-Staal and co-workers found that interferring with eukaryotic initiation factors 2Bg and 2g prevented viral protein expression in human cells.

As described by its developers Immusol Inc. (San Diego), inverse genomics uses a library of randomized, hairpin-shaped ribozymes to sift through the entire human genome and rapidly isolate those genes that are potentially useful as drug targets. Ribozymes are RNA molecules that were originally discovered in a function to catalyze their own cleavage, but can be engineered to cleave and inactivate other RNA molecules in a specific, sequence-dependent fashion. By cleaving a target RNA, ribozymes inhibit the translation of RNA into protein, thus stopping the expression of a specific gene. Immusol's approach to ribozyme technology employs hairpin ribozyme, because of their stability and activity in the cellular environment.

Use of ribozyme technologies to determine gene function

In Wong-Staal's experiments, a library of ribozymes was transfected into Hela cells stably expressing the herpes virus thymidine kinase gene whose translation was mediated by the hepatitis C virus internal ribosome entry site (IRES). Cells harboring ribozymes that interacted with IRES were isolated by selecting for cells resistant to gangciclovir. Ganciclovir is a toxic purine nucleoside analog that is incorporated into nucleic acids by viral thymidine kinases; disruption of viral protein expression confers resistance to the analog, providing a positive selection for inhibitors of viral protein synthesis.

Using this approach, Wong-Staal isolated two ribozymes that conferred ganciclovir resistance that she showed also inhibited IRES-mediated translation of hepatitis proteins. Sequence analysis revealed that the target of the ribozymes were two eukaryotic initiation factors.

"We believe this discovery will enable the development of a new generation of highly-effective Hepatitis C viral therapeutics, those that target human cellular genes instead of viral genes," noted Wong-Staal. "We expect that anti-viral drugs targeting human cellular genes will hinder the emergence of drug-resistant viruses, a common shortcoming of drugs that target viral genes." Wong-Staal is on leave of absence from the university to direct the genomics program at Immusol.

Immusol's mission is to generate and develop therapeutic entities, and to establish collaborations for discovering and validating gene-based drug targets. Immusol also applies its ribozyme gene technology to selectively inactivate candidate drug target genes that have been identified by the more traditional approaches to drug target gene discovery used by pharmaceutical companies. By providing rapid and specific analysis of gene function in vivo, this "Target Validation" technology determines the potential of a specific gene as a therapeutic target

For more information: Immusol Inc., 10790 Roselle St., San Diego, CA 92121. Tel: 858-824-1100.

Edited by Laura DeFrancesco