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Volume 272, Number 43, Issue of October 24, 1997 pp. 27099-27106
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Efficient Transfer of Synthetic Ribozymes into Cells Using Hemagglutinating Virus of Japan (HVJ)-Cationic Liposomes
APPLICATION FOR RIBOZYMES THAT TARGET HUMAN T-CELL LEUKEMIA VIRUS TYPE I tax/rex mRNA

(Received for publication, April 3, 1997, and in revised form, August 13, 1997)

Isao Kitajima , Naohiro Hanyu , Yasuko Soejima , Ryuki Hirano , Satoko Arahira , Shoji Yamaoka ^¶ , Ryo Yamada , Ikuro Maruyama and Yasufumi Kaneda ^**

From the  Department of Laboratory and Molecular Medicine, University of Kagoshima, 8-35-1 Sakuragaoka, Kagoshima 890, the ^¶ Department of Viral Oncology, Institute for Virus Research, Kyoto University, 53 Kawahara-cho, Seigoin, Sakyou-ku, Kyoto 606-01,  Tsukuba Research & Development Division, Hitachi Chemical Co., Tsukuba, 48 Wadai, Tsukuba City 300-42, and the ^** Institute for Molecular and Cellular Biology, Osaka University, 1-3 Yamadaoka, Suita Osaka 565, Japan

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-I rex (RR) and tax (TR) mRNA were synthesized. Both ribozymes were sequence-specific in the in vitro cleavage analysis of run-off transcripts from tax/rex cDNA. Intracellular activities of the ribozymes were studied in HTLV-I tax cDNA-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 95% 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 tax antisense oligodeoxynucleotides reduced Tax expression by about 20%, whereas RR and tax sense oligodeoxynucleotides had no effect. These results suggest that the ribozymes' effect against tax mRNA was sequence-specific, and HVJ-cationic liposomes can be useful for intracellular introduction of ribozymes.

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