Duplex oligonucleotide complexes and methods for gene silencing by RNA interference

• US 8,252,755 B2
• Filed: 09/20/2007
• Issued: 08/28/2012
• Est. Priority Date: 09/22/2006
• Status: Active Grant

• Current Assignee: Dharmacon Incorporated (Revvity, Inc.)
• Original Assignee: Dharmacon Incorporated (Revvity, Inc.)
• Inventors: Yamada, Christina, Khvorova, Anastasia, Kaiser, Rob, Anderson, Emily, Leake, Devin
• Primary Examiner(s): MCDONALD, JENNIFER SUE PITRAK
• Application Number:
  US11/858,829
• Publication Number:
  US 20080085869A1
• Time in Patent Office: 1,804 Days
• Field of Search: None
• US Class Current: 514/44
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07H 19/00 Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
• CPC Class Codes: C12N 15/11 DNA or RNA fragments; Modified forms thereof DNA or RNA not used in recombinant technology, C07H21/00; Non-coding nucleic acids having a biological activity
• 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 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 2310/322 2'-R Modification
• CPC Class Codes: C12N 2310/346 having a combination of backbone and sugar modifications
• CPC Class Codes: C12N 2310/3515 Lipophilic moiety, e.g. cholesterol
• CPC Class Codes: C12N 2310/3521 Methyl
• CPC Class Codes: C12N 2320/32 Special delivery means, e.g. tissue-specific
• CPC Class Codes: C12N 2330/30 chemically synthesised

Abstract

Provided herein are duplex oligonucleotide complexes which can be administered to a cell, tissue or organism to silence a target gene without the aid of a transfection reagent(s). The duplex oligonucleotide complexes of the disclosure include a conjugate moiety that facilitates delivery to a cell, tissue or organism.

Claims

1. A duplex oligonucleotide complex comprising:

a. a sense strand that is 19 nucleotides 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 and one mismatched nucleotide with the sense strand; and

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;

3. The duplex oligonucleotide complex of claim 1 wherein:

the opposite nucleotide 6 on the sense strand is the same nucleotide that occurs on position 14 of the antisense strand.

5. The duplex oligonucleotide of claim 3 wherein the 2 nucleotide overhang at the 3′

end of the antisense strand is UU.

6. The duplex oligonucleotide of claim 5 wherein all nucleotides in the sense and antisense strands 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.

7. The duplex oligonucleotide of claim 1, wherein between about 40% and about 90% of the nucleotides of the sense strand and between about 40% and about 90% of the nucleotides of the antisense strand are chemically modified.

2. A kit comprising a container, said container comprising:

(1) a duplex oligonucleotide complex comprising;

a. a sense strand that is 19 nucleotides 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 and one mismatched nucleotide with the sense strand; and

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;

4. The kit of claim 2, wherein:

the opposite nucleotide 6 on the sense strand is the same nucleotide that occurs on position 14 of the antisense strand.