Modification of antisense phosphodiester oligodeoxynucleotides by a 5' cholesteryl moiety increases cellular association and improves efficacy.
Phosphodiester oligodeoxynucleotides bearing a 5' cholesteryl (chol) modification bind to low density lipoprotein (LDL), apparently by partitioning the chol-modified oligonucleotides into the lipid layer. Both HL60 cells and primary mouse spleen T and B cells incubated with fluorescently labeled chol-modified oligonucleotide showed substantially increased cellular association by flow cytometry and increased internalization by confocal microscopy compared to an identical molecule not bearing the chol group. Cellular internalization of chol-modified oligonucleotide occurred at least partially through the LDL receptor; it was increased in mouse spleen cells by cell culture in lipoprotein-deficient medium and/or lovastatin, and it was decreased by culture in high serum medium. To determine whether chol-modified oligonucleotides are more potent antisense agents, we titered antisense unmodified phosphodiester and chol-modified oligonucleotides targeted against a mouse immunosuppressive protein. Murine spleen cells cultured with 20 microM phosphodiester antisense oligonucleotides had a 2-fold increase in RNA synthesis, indicating the expected lymphocyte activation. Antisense chol-modified oligonucleotides showed an 8-fold increase in relative potency: they caused a 2-fold increase in RNA synthesis at just 2.5 microM. The increased efficacy was blocked by heparin and was further increased by cell culture in 1% (vs. 10%) fetal bovine serum, suggesting that the effect may, at least in part, be mediated via the LDL receptor. Antisense chol-modified oligonucleotides are sequence specific and have increased potency as compared to unmodified oligonucleotides.
Krieg
A M
AM
Department of Internal Medicine, University of Iowa, Iowa City 52242.
Tonkinson
J
J
Matson
S
S
Zhao
Q
Q
Saxon
M
M
Zhang
L M
LM
Bhanja
U
U
Yakubov
L
L
Stein
C A
CA
D000818:Animals; D002478:Cells, Cultured; D002784:Cholesterol / Q000031:analogs & derivatives* / Q000378:metabolism*; D016373:DNA, Antisense / Q000378:metabolism* / Q000494:pharmacology; D004337:Drug Carriers / Q000378:metabolism*; D004705:Endocytosis; D008077:Lipoproteins, LDL / Q000378:metabolism; D008213:Lymphocyte Activation / Q000187:drug effects; D008214:Lymphocytes / Q000378:metabolism; D051379:Mice; D008811:Mice, Inbred DBA; D016376:Oligonucleotides, Antisense / Q000378:metabolism* / Q000494:pharmacology; D011973:Receptors, LDL / Q000378:metabolism; D013154:Spleen / Q000166:cytology
D016373:DNA, Antisense; D004337:Drug Carriers; D008077:Lipoproteins, LDL; D016376:Oligonucleotides, Antisense; D011973:Receptors, LDL; D002784:Cholesterol