CpG oligonucleotide analogs containing hydrophobic T analogs with enhanced immunostimulatory activity
The invention relates to oligonucleotides including at least one lipophilic substituted nucleotide analog and a pyrimidine-purine dinucleotide. The invention also relates to pharmaceutical compositions and methods of use thereof.
1. An oligonucleotide comprising:
the sequence R 1 CGR 2 , wherein R 1 and R 2 are selected from the group consisting of a lipophilic substituted nucleotide analog (L), a nucleotide, and a linkage;
wherein at least one of R 1 and R 2 is a lipophilic substituted nucleotide analog (L), wherein the nucleobase ring of L is selected from the group consisting of 5-chloro-uracil, 5-bromo-uracil, 5-ethyl-uracil, and (E)-5-(2-bromovinyl)-uracil; wherein at least two nucleotides of the oligonucleotide have a stabilized linkage; and
wherein the stabilized linkage is a phosphorothioate, a phosphorodithioate, a methylphosphonate, a methylphosphonothioate, a boranophosphonate, a phosphoramidate, or a dephospho linkage, either as an enantiomeric mixture or as an enantiomeric pure S- or R-configuration.
2. The oligonucleotide of claim 1 , wherein R 1 is L and R 2 is a nucleotide.
3. An oligonucleotide comprising:
the sequence R 1 CGR 2 , wherein R 1 and R 2 are selected from the group consisting of a lipophilic substituted nucleotide analog (L), and a linkage;
wherein at least one of R 1 and R 2 is a lipophilic substituted nucleotide analog (L), wherein the nucleobase ring of L is selected from the group consisting of 5-chloro-uracil, 5-bromo-uracil, 5-ethyl-uracil, and (E)-5-(2-bromovinyl)-uracil; and
wherein R 1 is a L and R 2 is a linkage, such that the oligonucleotide comprises a structure 5′LCG3′, wherein L is the 5′ terminal nucleotide.
4. The oligonucleotide of claim 1 , wherein the oligonucleotide is 7-100 nucleotides in length.
5. The oligonucleotide of claim 1 , wherein the oligonucleotide comprises one to four unmethylated CG dinucleotides.
6. The oligonucleotide of claim 1 , wherein the oligonucleotide comprises a non-nucleotidic modification, wherein the non-nucleotidic modification is selected from the group consisting of C 6 -C 48 -polyethyleneglycol, C 3 -C 20 -alkane-diol, C 3 -C 18 -alkylamino linker, C 3 -C 18 -alkylthiol linker, cholesterol, bile acid, saturated or unsaturated fatty acid, folate, a hexadecyl-glycerol or dihexadecyl-glycerol group, an octadecyl-glycerol or dioctadecylglycerol group, and a vitamin E group.
7. The oligonucleotide of claim 1 , wherein the stabilized linkage is a phosphorothioate.
8. The oligonucleotide of claim 1 , wherein the CG of R 1 CGR 2 has a phosphodiester linkage.
9. The oligonucleotide of claim 8 , wherein all other nucleotides have a phosphorothioate linkage.