THERAPEUTIC COMPOSITIONS

• US 20110201798A1
• Filed: 07/16/2010
• Published: 08/18/2011
• Est. Priority Date: 03/07/2003
• Status: Active Grant

• Current Assignee: Alnylam Pharmaceuticals Incorporated
• Original Assignee: Alnylam Pharmaceuticals Incorporated
• Inventors: MANOHARAN, Muthiah
• Granted Patent: US 8,420,799 B2
• Time in Patent Office: Days
• US Class Current: 536/24.5
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 2267/0362 Animal model for lipid/glucose metabolism, e.g. obesity, type-2 diabetes
• CPC Class Codes: A01K 67/0275 Genetically modified vertebrates, e.g. transgenic
• CPC Class Codes: A61K 31/713 Double-stranded nucleic acids or oligonucleotides
• CPC Class Codes: A61K 47/554 the modifying agent being a steroid plant sterol, glycyrrhetic acid, enoxolone or bile acid
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: C07H 21/02 with ribosyl as saccharide radical
• CPC Class Codes: C07K 14/775 Apolipopeptides
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 15/1137 against enzymes viral enzymes C12N15/1131; receptors C12N15/1138
• CPC Class Codes: C12N 15/1138 against receptors or cell surface proteins
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2310/315 Phosphorothioates
• CPC Class Codes: C12N 2310/321 2'-O-R Modification
• CPC Class Codes: C12N 2310/344 Position-specific modifications, e.g. on every purine, at the 3'-end
• CPC Class Codes: C12N 2310/3515 Lipophilic moiety, e.g. cholesterol
• CPC Class Codes: C12N 2310/533 having a mismatch or nick in at least one of the strands
• CPC Class Codes: C12N 2320/32 Special delivery means, e.g. tissue-specific
• CPC Class Codes: C12N 2320/51 : modulating the chemical stability, e.g. nuclease-resistance
• CPC Class Codes: C12N 2320/53 : reducing unwanted side-effects
• CPC Class Codes: C12N 2330/30 : chemically synthesised

Abstract

This application relates to therapeutic siRNA agents and methods of making and using the agents.

Claims

1. An iRNA duplex agent comprising a first and a second sequence, having at a selected or constrained sequence, a first monomer in the first sequence, and a second monomer in the second sequence, wherein the monomers are selected such that the stability of the pairing of the monomers contributes to a duplex between the first and second sequence that differs from the stability of the pairing between the first or second sequence and a target sequence.

2. The iRNA duplex agent of claim 1, wherein the constrained or selected site is within 3, 4, 5, or 6 positions from either the 3′

end or the 5′

end.

3. The iRNA duplex agent of claim 1, wherein the monomer is selected such that the first and second monomers are naturally occurring ribonucleotides or modified ribonucleotides having naturally occurring bases, wherein the ribonucleotides, when occupying complementary sites that do not pair and have no substantial level of H-bonding, form a non canonical Watson-Crick pairing.

4. The iRNA duplex agent of claim 3, wherein the ribonucleotides form a non canonical Watson-Crick pairing and the non canonical Watson-Crick pairing is selected from the group consisting of U-U, G-G, G-Atrans, G-Acis and G-U.

5. The iRNA duplex agent of claim 4, wherein the non canonical Watson-Crick pairing is G-U.

6. The iRNA duplex agent of claim 1, wherein the pairing of the monomers contributes to a duplex between the first and second sequence having decreased stability.

7. The iRNA duplex agent of claim 1, further comprising at least one mismatch.

8. The iRNA duplex agent of claim 1, wherein the iRNA agent comprises a TT overhang sequence.

9. The iRNA duplex agent of claim 1, wherein the monomer is selected from 2-amino A, 2-thio U, or 2-thio T.

10. The iRNA duplex agent of claim 1, wherein the first sequence is an anti-sense strand and the second sequence is a sense strand, wherein the monomer at the selected site in the anti-sense stand includes a U or a modified base which pairs with A, the corresponding monomer in the target is an A, and the sense strand is a monomer which will not pair or which will form a non-canonical pairing.

11. The iRNA agent of claim 1, represented by

12. The iRNA agent of claim 11, wherein at least 1 or more pairs in P₋

5 through P<sub>−

1</sub> are, independently, A;

U, G;

U, I;

C, or mismatched pairs.

13. The iRNA agent of claim 11, wherein at least 2 of the pairs in P₋

1 through P<sub>−

4</sub>, are G;

U.

14. The iRNA agent of claim 11, wherein at least 1 or more pairs in P₅ through P₁ are, independently, G:

C, A;

T, A;

U, 2-amino-A;

U, 2-thio U or 5 Me-thio-U;

A, G-clamp;

G, guanadinium-G-clamp;

G;

psuedo uridine;

A, or a pair in which one or both subunits has a sugar modification.

15. The iRNA agent of claim 11, wherein least 2 of the pairs in P₁ through P₄, are G:

C.

16. The iRNA agent of claim 11, wherein at least 1 or more pairs in P₋

5 through P<sub>−

1</sub> are, independently, A;

U, G;

U, I;

C, or mismatched pairs, and wherein at least 1 or more pairs in P₅ through P₁ are, independently, G;

C, A;

T, A;

U, 2-amino-A;

U, 2-thio U or 5 Me-thio-U;

A, G-clamp;

G, guanadinium-G-clamp;

G;

psuedo uridine;

A, or a pair in which one or both subunits has a sugar modification.

17. A method of reducing the level of off-target silencing in an iRNA agent of claim 1 by promoting disassociation of the duplex formed by the sense strand and the off-target sequence by selecting one or more of the monomers at the selected constrained or selected sites such that the iRNA agent duplex has a comparatively higher free energy of association than a duplex formed between a sense targeting sequence and its off-target sequence.

18. The method of claim 17, wherein the monomers are selected such that the first and second monomers are naturally occurring ribonucleotides or modified ribonucleotides having naturally occurring bases, wherein the ribonucleotides, when occupying complementary sites that do not pair and have no substantial level of H-bonding, form a non canonical Watson-Crick pairing.

19. The method of claim 17, wherein the constrained or selected site is within 3, 4, 5, or 6 positions from either the 3′

end or the 5′

end.

20. The method of claim 18, wherein the ribonucleotides form a non canonical Watson-Crick pairing and the non canonical Watson-Crick pairing is selected from the group consisting of U-U, G-G, G-Atrans, G-Acis and G-U.

21. The method of claim 20, wherein the non canonical Watson-Crick pairing is G-U.