Efficient Transfer of Synthetic Ribozymes into Cells Using Hemagglutinating Virus of Japan (HVJ)-Cationic Liposomes

We investigated the usefulness of ribozymes in inhibiting the expression of human T-cell leukemia virus type I (HTLV-I) gene. Two hammerhead ribozymes that were against HTLV-Irex(RR) and tax(TR) mRNA were synthesized. Both ribozymes were sequence-specific in the in vitrocleavage analysis of run-off transcripts fromtax/rexcDNA. Intracellular activities of the ribozymes were studied in HTLV-I taxcDNA-transfected rat embryonic fibroblasts (Rat/Tax cells), which expressed the Tax but not Rex. Ribozymes were delivered into cells using anionic or cationic liposomes fused with hemagglutinating virus of Japan (HVJ). Cellular uptake of ribozymes complexed with HVJ-cationic liposomes was 15–20 times higher cellular uptake than naked ribozymes, and 4–5 times higher than that of ribozymes complexed with HVJ-anionic liposomes. HVJ-cationic liposomes promoted accumulation of ribozymes in cytoplasm and accelerated transport to the nucleus. Tax protein levels were decreased about 957 and were five times lower when the same amount of TR was introduced into the cells using HVJ-cationic, rather than HVJ-anionic liposomes. Inactive ribozyme and taxantisense oligodeoxynucleotides reduced Tax expression by about 207, whereas RR and taxsense oligodeoxynucleotides had no effect. These results suggest that the ribozymes' effect against taxmRNA was sequence-specific, and HVJ-cationic liposomes can be useful for intracellular introduction of ribozymes.