Rna-interference by single-stranded rna molecules

• US 20060166910A1
• Filed: 07/10/2003
• Published: 07/27/2006
• Est. Priority Date: 07/10/2002
• Status: Active Grant

• Current Assignee: Max-Planck-Gesellschaft Zur Foerderung Der Wissenschaften e.V. (Max-Planck-Gesellschaft zur Frderung der Wissenschaften e)
• Original Assignee: Max-Planck-Gesellschaft Zur Foerderung Der Wissenschaften e.V. (Max-Planck-Gesellschaft zur Frderung der Wissenschaften e)
• Inventors: Martinez, Javier, Luhrmann, Reinhard, Tuschl, Thomas, Urlaub, Henning
• Granted Patent: US 8,101,348 B2
• Time in Patent Office: Days
• US Class Current: 514/44.A
• CPC Class Codes: A61K 47/549 Sugars, nucleosides, nucleotides or nucleic acids
• CPC Class Codes: A61K 47/557 the modifying agent being biotin
• CPC Class Codes: C07K 14/4705 stimulating, promoting or activating activity
• CPC Class Codes: C07K 19/00 Hybrid peptides , i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
• 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 2310/351 Conjugate
• CPC Class Codes: C12N 2310/3513 Protein; Peptide
• CPC Class Codes: C12N 2320/30 Special therapeutic applications
• CPC Class Codes: C12N 2320/51 modulating the chemical stability, e.g. nuclease-resistance
• CPC Class Codes: C12N 2330/30 chemically synthesised

Abstract

The present invention relates to sequence and structural features of single-stranded (ss) RNA molecules required to mediate target-specific nucleic acid modifications by RNA-interference (RNAi), such as target mRNA degradation and/or DNA methylation.

Claims

1. Use of single-stranded RNA molecule having a length from 14-50 nucleotides wherein at least the 14-20 5′

most nucleotides are substantially complementary to a target transcript for the manufacture an agent for inhibiting the expression of said target transcript.

2. The use of claim 1 wherein said expression is inhibited by RNA-interference.

3. The use of claim 1 wherein said RNA molecule has a length from 15-29 nucleotides.

4. The use of claim 1, wherein said RNA molecule has a free 5′

hydroxyl moiety or a moiety selected from phosphate groups or analogues thereof.

5. The use of claim 1, wherein said RNA molecule has 5′

-moiety selected from 5′

-monophosphate ((HO)₂(O)P—

O-5′

), 5′

-diphosphate ((HO)₂(O)P—

O—

P(HO)(O)—

O-5′

), 5′

triphophate ((HO)₂(O)P—

O—

(HO)(O)P—

O—

P(HO)(O)—

O-5′

), 5′

guanosine cap (7-methylated or non-methylated) (7m-G-O-5′

-(HO)(O)P—

O—

(HO)(O)P—

O—

P(HO)(O)—

O-5′

), 5′

-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (N—

O-5′

(HO)(O)P—

O—

(HO)(O)P—

O—

P(HO)(O)—

O-5′

), 5′

-monothiophosphate (phosphorothioate;

(HO)₂(S)P—

O-5′

), 5′

-monothiophosphate (phosphorothioate;

(HO)(HS)(S)P—

O-5′

), 5′

PHOSPHOROTHIOLATE ((HO)₂(O)P—

S-5′

);

any additional combination of oxgen/sulfur replaced monophosphate, diphosphate and triphosphates (e.g. 5′

-alpha-thiotriphosphate, 5′

-gamma-thiotriphosphate, etc.), 5′

-phosphoramidates ((HO)₂(O)P—

NH-5′

, (HO)(NH₂)(O)P—

O-5′

), 5′

alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc., e.g. RP(OH)(O)—

O-5′

-, (OH)₂(O)P-5′

-CH₂—

), 5′

alkyletherphosphonates (R=alkylether=methoxymethyl (MeOCH₂—

), ethoxymethyl, etc., e.g. RP(OH)(O)—

O-5′

-).

6. The use of claim 1, wherein said RNA molecule is completely complementary to said target transcript optionally with exception of nucleotides that extend beyond position 20 (counted from the 5′

terminus).

7. The use of claim 1, wherein said RNA molecule comprises at least one modified nucleotide analogue.

8. The use of claim 7, wherein the modified nucleotide analogues are selected from sugar-backbone- and nucleobase-modified ribonucleotides and combinations thereof.

9. The use of claim 1 for the inhibition of target gene expression in vitro.

10. The use of claim 1 for the inhibition of target gene expression in vivo.

11. The use of claim 1, wherein said RNA molecule is formulated as a pharmaceutical composition which contains a pharmaceutically acceptable carrier.

12. The use of claim 11, wherein said carrier is selected from cationic liposomes and cationic lipid formulations.

13. The use of claim 1, wherein said RNA molecule is associated with biodegradable polymers or microparticles.

14. The use of claim 13, wherein said association comprises a covalent coupling.

15. The use of claim 14, wherein said covalent coupling occurs via the 3′

-terminus of the RNA molecule.

16. The use of claim 10 for diagnostic applications.

17. The use of claim 10 for therapeutic applications.

18. The use of claim 17 for the prevention or treatment of disease associated with overexpression of at least one target transcript.

19. The use of claim 18, wherein the diseases are selected from tumor diseases, inflammatory diseases, infectious diseases, e.g. viral infections, degenerative diseases and autoimmune diseases.

20. A pharmaceutical composition for inhibiting the expression of a target transcript by RNAi comprising an active agent a single-stranded RNA molecule having a length from 14-50 nucleotides, wherein at least the 14-20 5′

most nucleotides are substantially complementary to said target transcript.

21. A method for the prevention or treatment of disease associated with overexpression of at least one target gene comprising administering a subject in need thereof a single-stranded RNA molecule having a length from 14-50 nucleotides, wherein at least the 14-20 5′

most nucleotides are substantially complementary to a transcript of said target gene in an amount which is therapeutically effective RNAi.

22. Purified human RISC having a molecular weight of from up to about 150-160 kDa.

23. The RISC of claim 22 comprising at least one member of the Argonaute family of proteins.

24. The RISC of claim 22 containing eIF2C1 and/or eIFC2 and optionally at least one eIFC3, eIFC4, HILI and HIWI.

25. The RISC of claim 22, further containing an RNA component.

26. A host cell or non-human host organism capable of overexpressing RISC.

27. A method of enhancing RNAi in a cell or an organism comprising causing said cell or organism to overexpress at least one component of RISC.

28. The method of claim 27 for screening applications.

29. The method of claim 27 for therapeutic applications.

30. An antisense siRNA precursor molecule in the form of a hairpin stem-loop structure comprising 19 to 29 base pairs in stem, wherein at least 14 nucleotides in the stem are substantially complementary to a target transcript.

31. The siRNA precursor molecule of claim 30 having a 3′

overhanging end.