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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
David G. Spiller,
Richard V. Giles,
John Grzybowski,
David M. Tidd, and
Richard E. Clark
From the University Department of Haematology, Royal Liverpool
University Hospital, Liverpool, UK; and the School of Biological
Sciences, University of Liverpool, Liverpool, UK.
The hybrid gene BCR-ABL that typifies chronic myeloid
leukemia (CML) represents an attractive target for therapy with
antisense oligodeoxyribonucleotides (ODN). A central obstacle in the
therapeutic application of ODN is their poor cellular uptake. Adding
various lipophilic conjugates to the ODN backbone has been reported to improve uptake, and electroporation of target cells has also been shown
to enhance intracellular ODN delivery. We have shown that (1)
BCR-ABL-directed ODN will specifically decrease the level of
BCR-ABL mRNA, provided that cells are first permeabilized with Streptolysin-O (SL-O), and (2) chimeric
methylphosphonodiester:phosphodiester ODN directed against 9 bases
either side of the BCR-ABL junction are more efficient ODN
effectors than structures 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 following groups
at the 5 end: cholesterol, vitamin E, polyethylene glycol of
average molecular weight 2,000 or 5,000, N-octyl-oligo-oxyethylene, or
dodecanol. ODN associated with Lipofectin was also studied. Comparison
was made in untreated, electroporated, and SL-O permeabilized KYO1
cells. Uptake was examined by fluorescence microscopy and flow
cytometry, using ODN structures that were 3 labeled with fluorescein. The effect on target BCR-ABL mRNA expression was analyzed by Northern blotting. Several conjugated structures associated avidly with the cell membrane without achieving significant
intracellular uptake or molecular effect. Similarly, ODN:Lipofectin
complexes moderately increased cell association, without enhancing
intracellular levels of ODN or inducing detectable molecular effect. In
SL-O permeabilized or electroporated cells, uptake was approximately 1 to 2 logs greater than in untreated cells, and rapid nuclear localization was seen, especially with unmodified chimeric ODN. In SL-O
permeabilized cells treated with ODN directed 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 mRNA levels
were decreased to 4.0% ± 1.4% of control levels by b2a2 directed
ODN, although very little nontargeted suppression was seen with
b3a2-targeted ODN (93.4% ± 4.2% of control). Greater cell to cell
variation in ODN uptake was seen for SL-O permeabilized cells when
compared with electroporated cells, suggesting that, after SL-O
permeabilization, relatively unpermeabilized and overpermeabilized populations may coexist. No structure had any effect on the level of
irrelevant (p53, MYC, and GADPH) mRNA levels.
We conclude that the conjugation of chimeric ODN with one of the
above-mentioned lipophilic groups or the complexing of ODN with
Liopfectin does not improve either intracellular delivery of ODN or the
molecular effect. In contrast, both electroporation and SL-O
permeabilization (1) considerably enhanced uptake of chimeric ODN (even
for structures without a conjugate group) and (2) achieved significant
suppression of target mRNA levels.
Blood, Vol. 91 No. 12 (June 15), 1998:
pp. 4738-4746
© 1998 by The American Society of Hematology.
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