Oligonucleotide compounds comprising non-nucleotide overhangs
The invention relates to siRNA compounds comprising one non-nucleotide moiety covalently attached to at least one of the sense or antisense strands to down-regulate the expression of human genes. The invention also relates to pharmaceutical compositions comprising such compounds and a pharmaceutically acceptable carrier and to methods of treating and/or preventing the incidence or severity of various diseases or conditions associated with the target genes and/or symptoms associated with such diseases or conditions.
1. A double-stranded nucleic acid molecule comprising a sense strand and an antisense strand, wherein the antisense strand comprises a non-nucleotide overhang covalently attached via a phosphodiester or a phosphorothioate linkage to the 3′ or to the 2′ position of the sugar residue of the 3′ terminal nucleotide,
wherein the non-nucleotide overhang is selected from the group consisting of C3Pi, C3Ps, C3Pi-C3OH, C3Pi-C3Pi, C3Pi-C3Ps, C3Ps-C3OH, C3Ps-C3Pi, C3Ps-C3Ps, C3Pi-C3Pi-C3OH, C3Ps-C3Ps-C3OH, C3Pi-C3Ps-C3OH, C3Ps-C3Pi-C3OH, C3Pi-C3Pi-C3Pi, C3Ps-C3Ps-C3Ps, C3Pi-C3Ps-C3Ps, C3Ps-C3Pi-C3Ps, C3Ps-C3Ps-C3Pi, C3Pi-C3Pi-C3Ps, C3Ps-C3Pi-C3Pi, C3Pi-C3Ps-C3Pi, C3Pi-rAb, C3Pi-dAb, C3Ps-rAb, C3Ps-dAb, rAbPi-C3OH, rAbPi-C3Pi, rAbPs-C3OH, rAbPs-C3Pi, dAbPi-C3OH, dAbPi-C3Pi, dAbPs-C3OH and dAbPs-C3Pi; and wherein each Pi is a phosphodiester linkage and each Ps is a phosphorothioate linkage;
or a pharmaceutically acceptable salt of such molecule.
2. The double-stranded nucleic acid molecule of claim 1 having structure (A1)
(A1)
5′
(N)x-Z 3′ (antisense strand)
3′
Z′-(N′)y-z″ 5′ (sense strand)
wherein each of N and N′ is an unmodified ribonucleotide, a modified ribonucleotide, or an unconventional moiety;
wherein each of (N)x and (N′) y is an oligonucleotide in which each consecutive N or N′ is joined to the next N or N′ by a covalent bond;
wherein Z comprises a non-nucleotide overhang covalently attached via a phosphate based linkage to the 3′ position or the 2′ position of the sugar residue of the 3′ terminal nucleotide in the antisense strand;
wherein Z comprises a non-nucleotide overhang selected from the group consisting of C3Pi, C3Pi-C3OH, C3Pi-C3Pi, C3Pi-C3Ps, C3Ps, C3Ps-C3OH, C3Ps-C3Pi, C3Ps-C3Ps, C3Pi-C3Pi-C3OH, C3Ps-C3Ps-C3OH, C3Pi-C3Ps-C3OH, C3Ps-C3Pi-C3OH, C3Pi-C3Pi-C3Pi, C3Ps-C3Ps-C3Ps, C3Pi-C3Ps-C3Ps, C3Ps-C3Pi-C3Ps, C3Ps-C3Ps-C3Pi, C3Pi-C3Pi-C3Ps, C3Ps-C3Pi-C3Pi, C3Pi-C3Ps-C3Pi, C3Pi-rAb, C3Pi-dAb, C3Ps-rAb, C3Ps-dAb, rAbPi-C3OH, rAbPi-C3Pi, rAbPs-C3OH, rAbPs-C3Pi, dAbPi-C3OH, dAbPi-C3Pi, dAbPs-C3OH and dAbPs-C3Pi;
wherein each Pi is a phosphodiester linkage and each Ps is a phosphorothioate linkage;
wherein Z′ is present or absent; but if present, Z′ is an inorganic phosphate or a non-nucleotide overhang selected from the group consisting of an abasic moiety, an inverted abasic moiety and an alkyl moiety or a derivative thereof or a combination thereof; covalently attached via a phosphate based linkage to the 3′ position or the 2′ position of the sugar residue of the 3′ terminal nucleotide in the sense strand;
wherein z″ may be present or absent, but if present is a capping moiety covalently attached at the 5′ terminus of (N′) y ;
wherein each of x and y is independently an integer between 18 and 40;
wherein the sequence of (N′)y has complementarity to the sequence of (N)x; and wherein the sequence of (N)x has complementarity to a consecutive sequence in a target RNA;
or a pharmaceutically acceptable salt of such molecule.
3. The molecule of claim 2 , wherein x=y=19.
4. The molecule of claim 2 , wherein Z′ is present.
5. The molecule of claim 2 , wherein Z comprises a non-nucleotide overhang selected from the group consisting of C3Pi-C3OH, C3Pi-C3Pi, C3Pi-C3Ps, C3Ps-C3Pi, C3Ps-C3OH and C3Ps-C3Ps.
6. The molecule of claim 4 , wherein Z′ is a non-nucleotide overhang selected from the group consisting of C3OH, C3Pi and C3Ps, wherein Pi is a phosphodiester linkage and Ps is a phosphorothioate linkage.
7. A composition comprising the molecule of claim 1 or a pharmaceutically acceptable salt of such molecule, and a pharmaceutically acceptable carrier.
8. A method for treating a subject suffering from a disease or disorder associated with expression of a target gene comprising administering to the subject the molecule of claim 1 or a pharmaceutically acceptable salt of such molecule, in an amount effective to down-regulate expression of the target gene, thereby treating the subject.
9. The molecule of claim 1 , wherein the non-nucleotide overhang is covalently attached to the 3′ position of the sugar residue of the 3′ terminal nucleotide such that the 3′ terminal nucleotide has one of the following structures:
wherein B is a nucleotide base and R is H, OH or a 2′ sugar modification.
10. The molecule of claim 1 , wherein the non-nucleotide overhang is selected from the group consisting of C3Pi-C3OH, C3Pi-C3Pi, C3Pi-C3Ps, C3Ps-C3OH, C3Ps-C3Pi and C3Ps-C3Ps.
11. The molecule of claim 1 , wherein the non-nucleotide overhang is selected from the group consisting of —C3Pi-C3Pi-C3OH, —C3Ps-C3Ps-C3OH, —C3Pi-C3Ps-C3OH, —C3Ps-C3Pi-C3OH, —C3Pi-C3Pi-C3Pi, —C3Ps-C3Ps-C3Ps, —C3Pi-C3Ps-C3Ps, —C3Ps-C3Pi-C3Ps, —C3Ps-C3Ps-C3Pi, —C3Pi-C3Pi-C3Ps, —C3Ps-C3Pi-C3Pi and —C3Pi-C3Ps-C3Pi.
12. The molecule of claim 1 , wherein the non-nucleotide overhang is selected from the group consisting of —C3Pi-rAb, —C3Pi-dAb, —C3Ps-rAb and —C3Ps-dAb.
13. The molecule of claim 1 , further comprising a non-nucleotide overhang covalently attached to the 3′ terminal nucleotide in the sense strand, wherein the non-nucleotide overhang covalently attached to the 3′ terminal nucleotide in the sense strand is selected from the group consisting of propanol, a C3 alkyl moiety linked to a phosphodiester and a C3 alkyl moiety linked to a phosphorothioate.
14. The molecule of claim 13 , wherein the non-nucleotide overhang covalently attached to the 3′ terminal nucleotide in the sense strand is propanol; and wherein the 3′ terminal sugar residue has the following structure:
wherein B is a nucleotide base and R is H, OH or a 2′ sugar modification.
15. The molecule of claim 13 , wherein the non-nucleotide overhang covalently attached to the 3′ terminal nucleotide in the sense strand is C3 alkyl moiety linked to a phosphodiester; and wherein the 3′ terminal sugar residue has the following structure:
wherein B is a nucleotide base and R is H, OH or a 2′ sugar modification.
16. The molecule of claim 13 , wherein the non-nucleotide overhang covalently attached to the 3′ terminal nucleotide in the antisense strand is selected from the group consisting of C3Pi-C3OH, C3Pi-C3Pi, C3Pi-C3Ps, C3Ps-C3OH, C3Ps-C3Pi and C3Ps-C3Ps; and wherein each Pi is a phosphodiester linkage and each Ps is a phosphorothioate linkage.
17. The molecule of claim 16 , wherein the non-nucleotide overhang covalently attached to the 3′ terminal nucleotide in the antisense strand is C3Pi-C3OH; and wherein the 3′ terminal sugar residue has the following structure:
wherein B is a nucleotide base and R is H, OH or a 2′ sugar modification.
18. The molecule of claim 16 , wherein the non-nucleotide overhang covalently attached to the 3′ terminal nucleotide in the antisense strand is C3Pi-C3Pi; and wherein the 3′ terminal sugar residue has the following structure:
wherein B is a nucleotide base and R is H, OH or a 2′ sugar modification.
19. The double stranded nucleic acid molecule of claim 2 , wherein each of x and y is independently an integer between 18 and 27.
20. The double stranded nucleic acid molecule of claim 19 , wherein x=y.
21. The double stranded nucleic acid molecule of claim 4 , wherein Z′ is a non-nucleotide overhang is selected from the group consisting of C3OH, C3Pi, C3Pi-C3OH, C3Pi-C3Pi, C3Pi-C3Ps, C3Ps, C3Ps-C3OH, C3Ps-C3Pi, C3Ps-C3Ps, C3Pi-C3Pi-C3OH, C3Ps-C3Ps-C3OH, C3Pi-C3Ps-C3OH, C3Ps-C3Pi-C3OH, C3Pi-C3Pi-C3Pi, C3Ps-C3Ps-C3Ps, C3Pi-C3Ps-C3Ps, C3Ps-C3Pi-C3Ps, C3Ps-C3Ps-C3Pi, C3Pi-C3Pi-C3Ps, C3Ps-C3Pi-C3Pi, C3Pi-C3Ps-C3Pi, C3Pi-rAb, C3Pi-dAb, C3Ps-rAb, C3Ps-dAb, rAbPi-C3OH, rAbPi-C3Pi, rAbPs-C3OH, rAbPs-C3Pi, dAbPi-C3OH, dAbPi-C3Pi, dAbPs-C3OH and dAbPs-C3Pi.
22. The double stranded nucleic acid molecule of claim 4 , wherein Z′ is an inorganic phosphate.