USE OF ADENOVIRAL VAI SMALL RNA AS A CARRIER FOR CYTOPLASMIC DELIVERYOF RIBOZYMES

The in vivo effectiveness of therapeutic RNAs, like antisense molecule s and ribozymes, relies on several features: RNA molecules need to be expressed at high levels in the correct cellular compartment as stable and active molecules. The exploitation of ''natural'' small RNA codin g genes as expressing cassettes gives high chances to fulfill these re quirements. We have investigated the utilization of the adenoviral VAI RNA as a cytoplasmatic carrier for expressing ribozymes against HIV-1 . The conserved 5' leader sequence of HIV was chosen as a target, beca use it is present in all the viral transcripts and is highly conserved . Hammerhead ribozymes were substituted to different portions of the V AI RNA and the resulting chimera were tested in the in vivo system of Xenopus laevis oocytes for their level of accumulation, cellular compa rtmentalization, and assembly in specific ribonucleoparticles containi ng the La antigen. Interesting differences in the activity of the diff erent chimera were found in both in vitro cleavage assays and S100 ext racts of injected oocytes where the catalytic activity of the ribozyme s in the RNP context can be analyzed.