IMPROVING THE INTRACELLULAR DELIVERY AND MOLECULAR EFFICACY OF ANTISENSE OLIGONUCLEOTIDES IN CHRONIC MYELOID-LEUKEMIA CELLS - A COMPARISON OF STREPTOLYSIN-O PERMEABILIZATION, ELECTROPORATION, AND LIPOPHILIC CONJUGATION

The hybrid gene BCR-ABL that typifies chronic myeloid leukemia (CML) r epresents an attractive target for therapy with antisense oligodeoxyri bonucleotides (ODN). A central obstacle in the therapeutic application of ODN is their poor cellular uptake. Adding various lipophilic conju gates to the ODN backbone has been reported to improve uptake, and ele ctroporation of target cells has also been shown to enhance intracellu lar ODN delivery. We have shown that (1) BCR-ABL-directed ODN will spe cifically decrease the level of BCR-ABL mRNA, provided that cells are first permeabilized with Streptolysin-O (SL-O), and (2) chimeric methy lphosphonodiester:phosphodiester ODN directed against 9 bases either s ide of the BCR-ABL junction are more efficient ODN effecters than stru ctures composed solely of phosphodiester or phosphorothioate linkages. In this study, we compared the efficacy of lipophilic conjugation, SL -O permeabilization and electroporation on the intracellular delivery and molecular effect of BCR-ABL-directed ODN. b2a2- and b3a2-directed chimeric ODN were synthesized either unmodified or with one of the fol lowing groups at the 5' end: cholesterol, vitamin E, polyethylene glyc ol of average molecular weight 2,000 or 5,000, N-octyl-oligo-oxyethyle ne, or dodecanol. ODN associated with Lipofectin was also studied. Com parison was made in untreated, electroporated, and SL-O permeabilized KYO1 cells. Uptake was examined by fluorescence microscopy and flow cy tometry, using ODN structures that were 3' labeled with fluorescein. T he effect on target BCR-ABL mRNA expression was analyzed by Northern b lotting. Several conjugated structures associated avidly with the cell membrane without achieving significant intracellular uptake or molecu lar effect. Similarly, ODN:Lipofectin complexes moderately increased c ell association, without enhancing intracellular levels of ODN or indu cing detectable molecular effect. In SL-O permeabilized or electropora ted cells, uptake was approximately 1 to 2 logs greater than in untrea ted cells, and rapid nuclear localization was seen, especially with un modified chimeric ODN. In SL-O permeabilized cells treated with ODN di rected to the b2a2 and b3a2 junctions respectively, b2a2 BCR ABL mRNA levels at 4 hours were reduced to 2.6% +/- 2.1% and 38.4% +/- 1.3% of control values. In cells permeabilized by electroporation, BCR-ABL mRN A levels were decreased to 4.0% +/- 1.4% of control levels by b2a2 dir ected ODN, although very little nontargeted suppression was seen with b3a2-targeted ODN (93.4% +/- 4.2% of control). Greater cell to cell va riation in ODN uptake was seen for SL-O permeabilized cells when compa red with electroporated cells, suggesting that, after SL-O permeabiliz ation, relatively unpermeabilized and overpermeabilized populations ma y coexist. No structure had any effect on the level of irrelevant (p53 , MYC, and GADPH) mRNA levels. We conclude that the conjugation of chi meric ODN with one of the above mentioned lipophilic groups or the com plexing of ODN with Liopfectin does not improve either intracellular d elivery of ODN or the molecular effect. In contrast, both electroporat ion and SL-O permeabilization (1) considerably enhanced uptake of chim eric ODN (even for structures without a conjugate group) and (2) achie ved significant suppression of target mRNA levels. (C) 1998 by The Ame rican Society of Hematology.