Short interfering nucleic acid (siNA) molecules containing a 2′ internucleoside linkage
View Patent ↗The present invention relates to RNAi molecules, and compositions thereof, comprising a 2′ internucleoside linkage connecting the nucleotide at position 1 and the nucleotide at position 2 at the 5′ end of the antisense strand. Specifically, the invention relates to single- and double-stranded short interfering nucleic acid (siNA) molecules that are capable of mediating RNA interference comprising 5′ modified nucleotides that comprise, among other potential modifications, a 2′ internucleoside linkage. The invention further relates to 5′ modified nucleotides used as reagents to generate the RNAi molecules of the invention and methods of using the disclosed RNAi molecules.
1. A short interfering nucleic acid (siNA) molecule capable of mediating RNA interference comprising an antisense strand that is complementary to a nucleic acid target, wherein said antisense strand comprises a 5′ modified nucleotide having Formula II:
wherein:
A is —OC(R 3 ) 2 —, —C(R 3 ) 2 O—, —C(R 3 ) 2 —, —C(R 3 ) 2 C(R 3 ) 2 — or —CR 3 ═CR 3 —;
B is any heterocyclic base moiety;
D 1 and D 1′ are independently selected from hydroxyl, —OR 4 , —SR 4 , or —N(R 4 ) 2 ;
E is O, S, —NR 5 , —N—N(R 4 ) 2 or —N—OR 4 ;
J is an internucleoside linking group linking the 5′ modified nucleotide of Formula II to the sugar moiety of an adjacent nucleotide of the siNA molecule;
R 1 and R 1′ are independently selected from H, hydroxyl, halogen, C 1-6 alkyl, —OR 6 , —N(R 6 ) 2 , or together form ═O or ═CH 2 ;
R 2 is H, C 1-6 alkyl or C 2-6 alkenyl;
R 3 and R 5 are independently selected from H, hydroxyl, halogen, C 1-10 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, aryl,
wherein R′ is selected from H or C 1-4 alkyl (which is optionally substituted with one to three substituents independently selected from —SR 11 , aryl, heteroaryl, amino, hydroxyl, oxo or —NH—C═(NH)NH 2 , wherein the aryl and heteroaryl are optionally substituted with hydroxyl), and R″ is selected from H, C 1-18 alkyl or aryl;
R 4 is independently selected from H, C 1-10 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, aryl,
wherein R′ is selected from H or C 1-4 alkyl (which is optionally substituted with one to three substituents independently selected from —SR 11 , aryl, heteroaryl, amino, hydroxyl, oxo or —NH—C═(NH)NH 2 , wherein the aryl and heteroaryl are optionally substituted with hydroxyl), and R″ is selected from H, C 1-18 alkyl or aryl;
R 6 is independently selected from H, C 1-6 alkyl (which is optionally substituted with —OR 7 , —SR 7 , —N(R 8 ) 2 , or (═O)—NR 9 or from one to three halogen), C 2-6 alkenyl, C 2-6 alkynyl, aryl,
wherein R′ is selected from H or C 1-4 alkyl (which is optionally substituted with one to three substituents independently selected from —SR 10 , aryl, heteroaryl, amino, hydroxyl, oxo, —NH—C═(NH)NH 2 , wherein the aryl and heteroaryl are optionally substituted with hydroxyl), and R″ is selected from H, C 1-18 alkyl or aryl;
R 7 is methyl, —CF 3 , —N(R 8 ) 2 or —CH 2 —N(R 8 ) 2 ;
R 8 is independently selected from H or C 1-6 alkyl;
R 9 is (R 8 ) 2 , —R 8 —(CH 2 ) 2 —N(R 8 ) 2 or —R 8 —C(═NR 8 )[N(R 8 ) 2 ]; and,
R 10 is H or C 1-4 alkyl,
wherein the siNA molecule is a single stranded antisense molecule, or wherein the siNA molecule is a double-stranded molecule comprising a sense strand that is complementary to the antisense strand; and
wherein the single-stranded molecule is between 15 and 30 nucleotides in length or wherein the antisense strand and the sense strand are each independently 15 to 30 nucleotides in length.
2. The siNA molecule of claim 1 , wherein R 3 is independently selected from H, hydroxy, F, Cl, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, aryl,
wherein R′ is selected from H, C 1-4 alkyl,
and R″ is selected from H, C 1-4 alkyl or aryl.
3. The siNA molecule of claim 1 , wherein A is —OCH 2 —, —CH 2 CH 2 — or —CH═CH—.
4. The siNA molecule of claim 1 , wherein D 1 and D 1′ is independently selected from hydroxyl, —OCH 3 or —OCH 2 CH 3 .
5. The siNA molecule of claim 1 , wherein B is uracil, thymine, cytosine, 5-methylcytosine, adenine or guanine.
6. The siNA molecule of claim 1 , wherein E is O.
7. The siNA molecule of claim 1 , wherein J is a phosphodiester internucleoside linking group or a phosphorothioate internucleoside linking group.
8. The siNA molecule of claim 1 , wherein R 1 is H or hydroxyl, and R1′: is H, hydroxyl, halogen or —OR 6 .
9. The siNA molecule of claim 1 , wherein R 1 , R 1′ and R 2 are each H.
10. The siNA molecule of claim 1 , wherein the 5′ modified nucleotide is
11. The siNA molecule claim 10 , wherein B is thymine.
12. The siNA molecule of claim 1 , wherein the siNA molecule is a single-stranded, antisense molecule.
13. The siNA molecule of claim 12 , wherein the siNA molecule comprises one or more additional chemically-modified nucleotides.
14. The siNA molecule of claim 1 , wherein the siNA molecule is a double-stranded molecule comprising a sense strand that is complementary to the antisense strand.
15. The siNA molecule of claim 14 , wherein the siNA molecule comprises one or more additional chemically-modified nucleotides.
16. A composition comprising the siNA molecule of claim 1 in a pharmaceutically acceptable carrier or diluent.