Conjugates for use in hepatocyte free uptake assays

• US 7,884,086 B2
• Filed: 09/07/2005
• Issued: 02/08/2011
• Est. Priority Date: 09/08/2004
• Status: Active Grant

• Current Assignee: Ionis Pharmaceuticals, Inc.
• Original Assignee: ISIS Pharmaceuticals Incorporated
• Inventors: Monia, Brett P., McKay, Robert, Baker, Brenda F., Sioufi, Namir, Bennett, C. Frank
• Primary Examiner(s): VIVLEMORE, TRACY ANN
• Application Number:
  US11/221,001
• Publication Number:
  US 20060084094A1
• Time in Patent Office: 1,980 Days
• Field of Search: None
• US Class Current: 514/44.00R
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 15/1137 against enzymes viral enzymes C12N15/1131; receptors C12N15/1138
• CPC Class Codes: C12N 2310/11 Antisense
• 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 2310/323 modified ring structure
• CPC Class Codes: C12N 2310/3341 5-Methylcytosine
• CPC Class Codes: C12N 2310/341 Gapmers, i.e. of the type ===---===
• 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 2310/3525 MOE, methoxyethoxy
• CPC Class Codes: C12N 2320/11 for the determination of target sites, i.e. of active nucleic acids
• CPC Class Codes: C12N 2320/32 Special delivery means, e.g. tissue-specific
• CPC Class Codes: C12Y 301/03048 Protein-tyrosine-phosphatase (3.1.3.48)

Abstract

The present invention provides methods of identifying oligomeric compounds, such as siRNA and double-stranded RNA compounds, having bioactivity in vivo, and kits.

Claims

1. A method for reducing the level of target mRNA in a cell comprising:

contacting the cell with a single-stranded sense oligonucleotide consisting of 10 to 40 linked nucleosides; and

contacting the cell with a single-stranded antisense oligonucleotide consisting of 10 to 40 linked nucleosides at least one hour after contacting the cell with the sense oligonucleotide;

wherein the antisense oligonucleotide is complementary to the target mRNA, the sense and antisense oligonucleotides are fully complementary to each other, the contacting occurs in the absence of a transfection reagent, and the sense oligonucleotide is a symmetric gapped oligomeric compound comprising

a 5′

region consisting of three to five 2′

-O—

(CH₂)₂—

OCH₃ modified nucleosides;

a central region consisting of nine to twenty β

-D-ribonucleosides; and

a 3′

region consisting of three to five 2′

-O—

(CH₂)₂—

OCH₃ modified nucleosides; and

thereby reducing the level of target mRNA in the cell.

2. The method of claim 1, wherein the cell is a rodent primary hepatocyte or a primate primary hepatocyte.

3. The method of claim 1 wherein the cell is a mammalian tissue-derived cell.

4. The method of claim 1 wherein the cell is contacted with the antisense oligonucleotide at least two hours after the cell is contacted with the sense oligonucleotide.

5. The method of claim 1 wherein each of the nucleosides of the antisense oligonucleotide comprises a β

-D-ribofuranose sugar group.

6. The method of claim 1 wherein the 3′

-terminus of at least one of the sense and antisense oligonucleotides, independently, comprises a stabilizing or conjugate group.

7. The method of claim 6 wherein the stabilizing group is a capping group or a dTdT dimer.

8. The method of claim 1 wherein at least one of the sense and antisense oligonucleotides comprises a 5′

-phosphate group.

9. The method of claim 1 wherein the 5′

-terminus of at least one of the sense and antisense oligonucleotides, independently, comprises a stabilizing or conjugate group.

10. The method of claim 9 wherein the stabilizing group is a capping group.

11. The method of claim 1 wherein each of the internucleoside linking groups of at least one of the sense oligonucleotide and the antisense oligonucleotide is, independently, a phosphorothioate.

12. The method of claim 1 wherein the sense and antisense oligonucleotides are capable of hybridizing to form a duplex having 3′

-dTdT overhangs.

13. The method of claim 1 wherein the sense and antisense oligonucleotides are capable of hybridizing to form a duplex having blunt ends.

14. The method of claim 1 wherein at least one of the sense and antisense oligonucleotides comprises at least one terminal cap moiety.

15. The method of claim 14 wherein the terminal cap moiety is attached to one or both of the 3′

-terminal and 5′

-terminal ends of the at least one oligonucleotide.

16. The method of claim 14 wherein the terminal cap moiety is an inverted deoxy abasic moiety.

17. The method of claim 1 wherein the antisense oligonucleotide comprises a 2′

-substituent selected from the group consisting of —

F, —

O—

CH₂CH₂—

O—

CH₃, —

O—

CH₃, —

O—

CH₂—

CH═

CH₂ or —

O—

CH₂—

CH—

CH₂—

NH(R_j), where R_j is H or C₁-C₁₀ alkyl.