Compounds and methods for enhanced cellular uptake
Described herein are conjugated modified oligonucleotides that are complementary to a target RNA. The conjugate facilitates cellular uptake of the modified oligonucleotide, resulting improved potency.
1. A compound comprising the structure:
wherein each N of N m is, independently, a modified or unmodified nucleoside and m is from 1 to 5; X 1 and X 2 are each, independently, a phosphodiester linkage or a phosphorothioate linkage; and MO is a modified oligonucleotide; and wherein when m is greater than 1, each modified or unmodified nucleoside of N m is connected to adjacent modified or unmodified nucleosides of N m by a phosphodiester internucleoside linkage or a phosphorothioate internucleoside linkage.
2. The compound of claim 1 , wherein at least one of X 1 and X 2 is a phosphodiester linkage.
3. The compound of claim 1 , wherein each of X 1 and X 2 is a phosphodiester linkage.
4. The compound of claim 1 , wherein m is 1, 2, or 3.
5. The compound of claim 1 , wherein N m is N′ p N″, wherein each N′ is, independently, a modified or unmodified nucleoside and p is from 0 to 4; and N″ is a nucleoside comprising an unmodified sugar moiety; and wherein each pair of adjacent N′ nucleosides are connected by a phosphodiester internucleoside linkage or a phosphorothioate internucleoside linkage and N″ is connected to an adjacent N′ by a phosphodiester internucleoside linkage or a phosphorothioate internucleoside linkage.
6. The compound of claim 5 , wherein p is 0, 1, 2, 3, or 4.
7. The compound of claim 6 , wherein p is 1, 2, 3, or 4 and each N of N m is linked by a phosphodiester internucleoside linkage.
8. The compound of claim 5 , wherein each N′ comprises an unmodified sugar moiety.
9. The compound of claim 5 , wherein N″ is a β-D-deoxyriboadenosine or a β-D-deoxyriboguanosine.
10. The compound of claim 1 , wherein the sugar moiety of each N of N m is independently selected from a β-D-ribose, a β-D-deoxyribose, a 2′-O-methoxy sugar, a 2′-O-methyl sugar, a 2′-fluoro sugar, and a bicyclic sugar moiety.
11. The compound of claim 1 , wherein the nucleobase sequence of the modified oligonucleotide is complementary to a target RNA.
12. The compound of claim 11 , wherein the target RNA is selected from a microRNA, a messenger RNA, a pre-messenger RNA, and a long non-coding RNA.
13. The compound of claim 12 , wherein the modified oligonucleotide is hybridized to a second modified oligonucleotide, wherein the nucleobase sequence of the second modified oligonucleotide is complementary to the nucleobase sequence of the modified oligonucleotide.
14. The compound of claim 11 , wherein the target RNA is a human target RNA.
15. The compound of claim 1 , wherein the nucleobase sequence of the modified oligonucleotide is at least 90% identical to the nucleobase sequence of a microRNA.
16. The compound of claim 15 , wherein the microRNA is a human microRNA.
17. The compound of claim 1 , wherein the modified oligonucleotide consists of 8 to 25 linked nucleosides.
18. The compound of claim 1 , wherein the modified oligonucleotide comprises at least one nucleoside with a modified sugar moiety.
19. The compound of claim 18 , wherein each modified sugar moiety is independently selected from a 2′-O-methyl sugar moiety, a 2′-O-methoxyethyl sugar moiety, a 2′-fluoro sugar moiety, and a bicyclic sugar moiety.
20. The compound of claim 19 , wherein each bicyclic sugar moiety is independently selected from a cEt sugar moiety and an LNA sugar moiety.
21. The compound of claim 1 , wherein each nucleoside of the modified oligonucleotide comprises a modified sugar moiety.
22. The compound of claim 1 , wherein the modified oligonucleotide comprises a plurality of nucleosides with a modified sugar moiety, and a plurality of nucleosides with an unmodified sugar moiety.
23. The compound of claim 1 , wherein the modified oligonucleotide comprises a plurality of non-bicyclic nucleosides and a plurality of bicyclic nucleosides.
24. The compound of claim 1 , wherein at least one linkage of the modified oligonucleotide is a modified internucleoside linkage.
25. A process of making a compound comprising the structure
wherein each N of N m is, independently, a modified or unmodified nucleoside and m is from 1 to 5; X 1 and X 2 are each, independently, a phosphodiester linkage or a phosphorothioate linkage; wherein when m is greater than 1, each modified or unmodified nucleoside of N m is connected to adjacent modified or unmodified nucleosides of N m by a phosphodiester internucleoside linkage or a phosphorothioate internucleoside linkage; and MO is a modified oligonucleotide, the process comprising the steps of:
providing a solid support containing a conjugate as shown in formula IV;
deprotecting the DMTr group under conditions effective to produce a reactive hydroxyl;
performing sequential phosphoramidite coupling steps to form N m ;
performing sequential phosphoramidite coupling steps to form the modified oligonucleotide; and
releasing the conjugated modified oligonucleotide from the solid support.