Duplex oligonucleotides with enhanced functionality in gene regulation

• US 8,188,060 B2
• Filed: 02/10/2009
• Issued: 05/29/2012
• Est. Priority Date: 02/11/2008
• Status: Active Grant

• Current Assignee: Dharmacon Incorporated (Revvity, Inc.)
• Original Assignee: Dharmacon Incorporated (Revvity, Inc.)
• Inventors: Khvorova, Anastasia, Leake, Devin, Robertson, Barbara, Vermeulen, Annaleen, Yamada, Christina
• Primary Examiner(s): Chong, Kimberly
• Application Number:
  US12/368,856
• Publication Number:
  US 20090209626A1
• Time in Patent Office: 1,204 Days
• Field of Search: 435/6, 435/91.31, 435/455, 435/458, 514/44, 536/23.1, 536/24.5
• US Class Current: 514/44
• CPC Class Codes: A61P 25/08 Antiepileptics; Anticonvulsants
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2310/321 2'-O-R Modification
• CPC Class Codes: C12N 2310/322 2'-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 2310/3521 Methyl
• CPC Class Codes: C12N 2310/3533 Halogen
• CPC Class Codes: C12N 2310/533 having a mismatch or nick in at least one of the strands
• CPC Class Codes: C12N 2320/52 modulating the physical stability, e.g. GC-content

Abstract

Disclosed are methods of enhancing functionality of duplex oligonucleotides and compositions made by the methods. The duplex oligonucleotides include siRNAs, miRNA mimics, and piRNA mimics which contain modified nucleotides and mismatches between the two strands of the molecule at specific nucleotide positions.

Claims

1. A method for inhibiting expression of a target mRNA in a cell, the method comprising delivering to said cell a duplex oligonucleotide complex comprising:

a. a sense strand that is 19 nucleotide in length, wherein nucleotides 1 and 2 and all C nucleotides and all U nucleotides on said sense strand are 2′

O- methyl modified;

b. an antisense strand that is 21 nucleotides in length, wherein;

i. all C nucleotides and all U nucleotides on said antisense strand are 2′

F modified;

ii. the antisense strand has 100% complementarity with a target mRNA;

iii. the sense strand and the antisense strand form a duplex having a 2 nucleotide overhang at the 3′

end of the antisense strand, said 2 nucleotide overhang comprising phosphorothioate linkages;

c. a cholesterol molecule attached to the 3′

end of the sense strand via a C5 linker molecule wherein the cholesterol- linker-sense strand has the structure;

2. The method of claim 1 wherein said duplex oligonucleotide complex is delivered to said cell by reverse transfection.

3. The method of claim 1 wherein said duplex oligonucleotide complex is delivered to said cell in vivo.

4. The method of claim 1 wherein the nucleotide 6 on the sense strand of said duplex oligonucleotide complex is the same nucleotide that occurs on position 14 of the antisense strand, the nucleotide 13 on the sense strand is the same nucleotide that occurs on position 7 on the antisense strand, and the nucleotide 19 on the sense strand is the same as the opposite nucleotide 1 on the antisense strand.

5. The method of claim 4 wherein the 2 nucleotide overhang at the 3′

end of the antisense strand of said duplex oligonucleotide complex is UU.

6. The method of claim 5 wherein all nucleotides of said duplex oligonucleotide complex other than the nucleotides 1 and 2 of the sense strand and all C nucleotides and all U nucleotides of the sense and antisense strands comprise a 2′

-OH.