Therapeutic compositions

• US 8,809,516 B2
• Filed: 09/25/2012
• Issued: 08/19/2014
• Est. Priority Date: 03/07/2003
• Status: Active Grant

• Current Assignee: Alnylam Pharmaceuticals Incorporated
• Original Assignee: Alnylam Pharmaceuticals Incorporated
• Inventors: Manoharan, Muthiah, Rajeev, Kallanthottathil G., Bumcrot, David
• Primary Examiner(s): VIVLEMORE, TRACY ANN
• Application Number:
  US13/626,196
• Publication Number:
  US 20130144048A1
• Time in Patent Office: 693 Days
• Field of Search: None
• 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 agent, comprising:

a sense strand sequence having 4-12 asymmetrical 2′

-O-alkyl modifications, at least 4 of which occur within the 6 terminal nucleotides of the 5′

end; and

an antisense sequence having at least 4 asymmetrical phosphorothioate modifications.

2. The iRNA agent of claim 1, wherein the sense and antisense sequences of the iRNA agent are on different RNA strands.

3. The iRNA agent of claim 1, wherein the sense and antisense sequences of the iRNA agent are on the same RNA strand.

4. The iRNA agent of claim 1, wherein the sense and antisense sequences of the iRNA agent are fully complementary to each other.

5. The iRNA agent of claim 1, further comprising a cholesterol moiety, wherein the cholesterol moiety is coupled to the sense sequence of the iRNA agent.

6. The iRNA agent of claim 1, wherein the iRNA agent is at least 21 nucleotides in length, and the duplex region of the iRNA agent is about 19 nucleotides in length.

7. The iRNA agent of claim 1, the iRNA agent having a duplex region of about 19 nucleotides in length and one or two 3′

overhangs of about 2 nucleotides in length.

8. The iRNA agent of claim 1, wherein the antisense sequence contains 4-20 asymmetrical phosphorothioate modifications.

9. The iRNA agent of claim 1, wherein each of the 2′

-O-alkyl modifications are 2′

-O-methyl modifications.

10. The iRNA agent of claim 1, wherein the sense strand further comprises at least one asymmetric modification selected from the group consisting of 2′

-5′

-linkages, L sugars, modified sugars, nucleobase modifications, cation groups, Zwitterionic groups, and conjugate groups.

11. The iRNA agent of claim 10, wherein the modification is 2′

-5′

linkages, and the 2′

-5′

linkage is phosphorothioate.

12. The iRNA agent of claim 10, wherein the modification is L sugars, and the L sugar is L ribose or L-arabinose sugar.

13. The iRNA agent of claim 10, wherein the modification is modified sugars, and the modified sugar is a locked nucleic acid, hexose nucleic acid or cyclohexane nucleic acid.

14. The iRNA agent of claim 10, wherein the conjugate group is naproxen, biotin, ibuprofen, folic acid, peptides or carbohydrates.

15. The iRNA agent of claim 1, wherein the antisense strand further comprises at least one asymmetric modification selected from the group consisting of 2′

-5′

-linkages, L sugars, modified sugars, nucleobase modifications, cation groups, Zwitterionic groups, and conjugate groups.

16. The iRNA agent of claim 15, wherein the modification is 2′

-5′

linkages, and the 2′

-5′

linkage is phosphorothioate.

17. The iRNA agent of claim 15, wherein the modification is L sugars, and the L sugar is L ribose or L-arabinose sugar.

18. The iRNA agent of claim 15, wherein the modification is modified sugars, and the modified sugar is a locked nucleic acid, hexose nucleic acid or cyclohexane nucleic acid.

19. The iRNA agent of claim 1, wherein the sense strand has 4 asymmetrical 2′

-O-alkyl modifications within the 6 terminal nucleotides of the 5′

end, and at least one 2′

-O-alkyl modification in another portion of the sense strand.

20. The iRNA agent of claim 15, wherein antisense strand does not contain any 2′

-O-alkyl modifications, and the sense strand does not contain any phosphorothioate modifications.

22. A method of making the iRNA agent of claim 1, comprising the steps of providing the modified sense strand sequence, and the modified antisense sequence, and hybridizing the sense and antisense strands to form the iRNA agent.

21. An iRNA agent, comprising:

a sense strand sequence having 4-12 asymmetrical 2′

-O-alkyl modifications, at least 4 of which occur within the 6 terminal nucleotides of the 3′

end; and

an antisense sequence having at least 4 asymmetrical phosphorothioate modifications.