Trans-splicing Ribozyme Research Articles

Trans-splicing ribozymes for targeted gene delivery

Ribozymes are potential tools for genetic manipulation, and various naturally occurring catalytic RNAs have been dissected and used as the basis for the design of new endoribonuclease activities. While such cleaving ribozymes may work well in vitro, they have not proved to be routinely effective in depleting living cells of the chosen target RNA. Recently, trans-splicing ribozymes have been employed to repair mutant mRNAs in vivo. We have designed modified trans-splicing ribozymes with improved biological activity. These allow accurate splicing of a new 3′ exon sequence into a chosen site within a target RNA, and in frame fusion of the exon can result in expression of a new gene product. These trans-splicing ribozymes contain catalytic sequences derived from a self-splicing group I intron, which have been adapted to a chosen target mRNA by fusion of a region of extended complementarity to the target RNA and precise alteration of the guide sequences required for substrate recognition. Both modifications are required for improved biological activity of the ribozymes. Whereas cleaving ribozymes must efficiently deplete a chosen mRNA species to be effective in vivo, even inefficient trans-splicing can allow the useful expression of a new gene activity, dependent on the presence of a chosen RNA. We have targeted trans-splicing ribozymes against mRNAs of chloramphenicol acetyltransferase, human immunodeficiency virus, and cucumber mosaic virus, and demonstrated trans-splicing and delivery of a marker gene in Escherichia coli cells. The improved trans-splicing ribozymes may be tailored for virtually any target RNA, and provide a new tool for triggering gene expression in specific cell types.

Evaluating and enhancing ribozyme reaction efficiency in mammalian cells.

The ability of ribozymes to cleave specific transcripts and repair defective RNAs in the test tube has engendered speculation about their potential clinical utility. Therapeutic development has been hindered by an inability to evaluate and optimize the efficiency of RNA catalysis in vivo. We describe an experimental system that has allowed us to assess and enhance the efficiency with which a trans-splicing group I ribozyme reacts with a targeted RNA in mammalian cells. These results demonstrate that the ribozyme can convert up to 49% of a specific substrate RNA to product in the cellular environment and that the efficiency of this reaction is apparently a function of the ribozyme's ability to find and bind to the substrate RNA in the cell. These observations suggest that trans-splicing ribozymes may become useful reagents to repair a therapeutically significant fraction of mutant RNAs associated with a variety of genetic diseases.