Trans-cleaving hammerhead or hairpin
ribozymes were expressed in transgenic mice and in cell lines, using a cassette containing a second cis-cleaving
hammerhead ribozyme positioned 3' of the trans-cleaving hammerhead or
hairpin ribozyme. Cis-cleavage could be detected readily in transgenic mice, demonstrating in vivo release of the desired short trans-cleaving
ribozyme transcript with a defined 3'-end. In transgenic organs, all cis-cleavage products containing a
hairpin ribozyme were found at significantly higher steady-state levels than products containing a
hammerhead ribozyme. Furthermore, an organ difference - kidney > liver > lung > spleen - regarding steady-state levels of both 5' and 3' cleavage products was found. In pools of stably transfected human T cells (HUT78), the efficacy of the 3' cis-cleavage was found to affect both the steady-state level and the
antiviral efficiency of a trans-cleaving
hairpin ribozyme targeting HIV-1. Insertion of a point mutation, efficiently inhibiting the cis-cleavage mechanism, led to higher overall steady-state levels of the noncleaved full-length transcript but, at the same time, also abolished the
hairpin ribozyme protection against HIV-1
infection. We conclude that the cis-cleavage affects hammerhead and
hairpin ribozyme steady-state levels differently and that it has a strong impact on trans-targeting efficiency.