Nucleic Acids Research Advance Access published online on November 27, 2006
Nucleic Acids Research, doi:10.1093/nar/gkl867
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Chemistry |
Use of thermolytic protective groups to prevent G-tetrad formation in CpG ODN type D: structural studies and immunomodulatory activity in primates
Laboratory of Immunology, Division of Therapeutic Proteins, Center for Drug Evaluation and Research Food and Drug Administration, 8800 Rockville Pike, Bethesda, MD 20892, USA 1 Laboratory of Chemistry, Division of Therapeutic Proteins, Center for Drug Evaluation and Research, Food and Drug Administration 8800 Rockville Pike, Bethesda, MD 20892, USA 2 Department of Physiology, School of Medicine, University of Pennsylvania 3700 Hamilton Walk, Philadelphia, PA 19104-6085, USA
*To whom correspondence should be addressed at 8800 Rockville Pike, Building 29A, Rm 3B19, Bethesda, MD 20892, USA. Tel: +1 301 827 1702; Fax: +1 301 480 3256; Email: daniela.verthelyi{at}fda.hhs.gov
Received August 23, 2006. Revised September 25, 2006. Accepted October 4, 2006.
CpG oligodeoxynucleotides (ODN) show promise as immunoprotective agents and vaccine adjuvants. CpG ODN type D were shown to improve clinical outcome in rhesus macaques challenged with Leishmania major. These ODN have a self-complementary core sequence and a 3' end poly(G) track that favors G-tetrad formation leading to multimerization. Although multimerization appears necessary for localization to early endosomes and signaling via Toll-like receptor 9 (TLR-9), it can result in product polymorphisms, aggregation and precipitation, thereby hampering their clinical applications. This study shows that functionalizing the poly(G) track of D ODN with thermolytic 2-(N-formyl-N-methyl)aminoethyl (fma) phosphate/thiophosphate protecting groups (pro-D ODN) reduces G-tetrad formation in solution, while allowing tetrad formation inside the cell where the potassium concentration is higher. Temperature-dependent cleavage of the fma groups over time further promoted formation of stable G-tetrads. Peripheral blood cells internalized pro-D ODN efficiently, inducing high levels of IFN
, IL-6, IFN
and IP-10 and triggering dendritic cell maturation. Administration of pro-D35 to macaques challenged with L.major significantly increased the number of antigen-specific IFN-secreting PBMC and reduced the severity of the skin lesions demonstrating immunoprotective activity of pro-D ODN in vivo. This technology fosters the development of more efficient immunotherapeutic oligonucleotide formulations for the treatment of allergies, cancer and infectious diseases.
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
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