DELIVERY OF AN ANTI-HIV-1 RIBOZYME INTO HIV-INFECTED CELLS VIA CATIONIC LIPOSOMES

Cationic liposome-mediated intracellular delivery of a fluorescein-lab eled chimeric DNA-RNA ribozyme targeted to the HIV-15' LTR was investi gated, using THP-1, THP-1/HIV-1(IIIB) or HeLa/LAV cells. Different flu orescence patterns were observed when the cells were exposed to Lipofe ctamine, Lipofectin or DMRIE:DOPE (1:1) complexed to the ribozyme. Wit h Lipofectamine intense cell-associated fluorescence was found. Incuba tion with Lipofectin resulted in less intense diffuse fluorescence, wh ile with DMRIE an intense but sporadic fluorescence was observed. Diff erentiated THP-1/HIV-1(IIIB) cells were more susceptible to killing by liposome-ribozyme complexes than THP-1 cells. Under non-cytotoxic con ditions (a 4-h treatment) complexes of 5, 10 or 15 mu M Lipofectin or DOTAP:DOPE (1:1) and ribozyme, at lipid:ribozyme ratios of 8:1 or 3:1, did not affect p24 production in THP-1/HIV-1(IIIB) cells in spite of the intracellular accumulation of the ribozyme. A 24-h exposure of THP -1/HIV-1(IIIB) cells to 5 mu M Lipofectin or DOTAP:DOPE (1:1) complexe d with either the functional or a modified control ribozyme reduced vi rus production by approximately 30%. Thus, the antiviral effect of the liposome-complexed ribozyme was not sequence-specific. In contrast, t he free ribozyme at a relatively high concentration inhibited virus pr oduction by 30%, while the control ribozyme was ineffective, indicatin g a sequence-specific effect. Both Lipofectin and DOTAP complexed with ribozyme were toxic at 10 and 15 mu M after a 24-h treatment. A 4-h t reatment of HeLa/LAV cells with Lipofectin at 5, 10 or 15 mu M was not toxic to the cells, but also did not inhibit p24 production. In contr ast, treatment of HeLa CD4(+) cells immediately after infection with H IV-1(IIIB) at the same lipid concentrations and lipid:ribozyme ratios was cytotoxic. Our results indicate that the delivery of functional ri bozyme into cells by cationic liposomes is an inefficient process and needs extensive improvement before it can be used in ex vivo and in vi vo applications. (C) 1998 Elsevier Science B.V. All rights reserved.