Trans-splicing ribozymes for targeted gene delivery

Ribozymes are potential tools for genetic manipulation, and various natural ly occurring catalytic RNAs have been dissected and used as the basis for t he 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 r ibozymes have been employed to repair mutant mRNAs in vivo. We have designe d modified trans-splicing ribozymes with improved biological activity. Thes e allow accurate splicing of a new 3' exon sequence into a chosen site with in a target RNA, and in frame fusion of the exon can result in expression o f a new gene product. These trans-splicing ribozymes contain catalytic sequ ences 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 t o the target RNA and precise alteration of the guide sequences required for substrate recognition. Both modifications are required for improved biolog ical activity of the ribozymes. Whereas cleaving ribozymes must efficiently deplete a chosen mRNA species to be effective in vivo, even inefficient tr ans-splicing can allow the useful expression of a new gene activity, depend ent on the presence of a chosen RNA. We have targeted trans-splicing ribozy mes against mRNAs of chloramphenicol acetyltransferase, human immunodeficie ncy virus, and cucumber mosaic virus, and demonstrated trans-splicing and d elivery of a marker gene in Escherichia coli cells. The improved trans-spli cing ribozymes may be tailored for virtually any target RNA, and provide a new tool for triggering gene expression in specific cell types. (C) 1999 Ac ademic Press.