Modification of Antisense Phosphodiester Oligodeoxynucleotides by a 5' Cholesteryl Moiety Increases Cellular Association and Improves Efficacy

• Published in: Proceedings of the National Academy of Sciences - PNAS 1993-02, Vol.90 (3), p.1048-1052
• Main Authors: KRIEG, A. M, TONKINSON, J, MATSON, S, QIUYAN ZHAO, SAXON, M, LI-MING ZHANG, UTPAL BHANJA, YAKUBOV, L, STEIN, C. A
• Format: Article
• Language: English
• Subjects: Animals; Antisense oligonucleotides; B lymphocytes; Biochemistry; Biological and medical sciences; Biotechnology; Cell culture techniques; Cells, Cultured; Cellular biology; Cholesterol - analogs & derivatives; Cholesterol - metabolism; Cholesterols; Cultured cells; Diverse techniques; DNA, Antisense - metabolism; DNA, Antisense - pharmacology; Drug Carriers - metabolism; Endocytosis; Fundamental and applied biological sciences. Psychology; Internalization; LDL receptors; Lipoproteins, LDL - metabolism; Lymphocyte Activation - drug effects; Lymphocytes - metabolism; Mice; Mice, Inbred DBA; Molecular and cellular biology; Oligonucleotides; Oligonucleotides, Antisense - metabolism; Oligonucleotides, Antisense - pharmacology; Receptors, LDL - metabolism; Spleen - cytology; Spleen cells; T lymphocytes
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.90.3.1048

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 μ M 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 μ M. 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.