RNA-INTERFERENCE BY SINGLE-STRANDED RNA MOLECULES

• US 20120246747A1
• Filed: 12/19/2011
• Published: 09/27/2012
• Est. Priority Date: 07/10/2002
• Status: Abandoned Application

• 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: TUSCHL, Thomas, Martinez, Javier, Patkaniowska, Agnieszka, Urlaub, Henning, Lührmann, Reinhard
• Application Number:
  US13/329,710
• Publication Number:
  US 20120246747A1
• Time in Patent Office: Days
• US Class Current: 800/13
• 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. Purified human RISC having a molecular weight of from up to about 150-160 kDa.

2. The RISC of claim 1 comprising at least one member of the Argonaute family of proteins.

3. The RISC of claim 1 containing eIF2C1 and/or eIFC2 and optionally at least one of eIFC3, eIFC4, HILI and HIWI.

4. The RISC of claim 1, further containing an RNA component, particularly a single-stranded RNA molecule.

5. The RISC of claim 4, wherein the single-stranded RNA molecule has a length from 14-50 nucleotides wherein at least the 14-20 5′

most nucleotides are substantially complementary to a target transcript.

6. The RISC of claim 4, wherein said RNA molecule has a length from 15-29 nucleotides.

7. The RISC of claim 4, wherein said RNA molecule has a free 5′

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

8. The RISC of claim 7, wherein said RNA molecule has a 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′

-triphosphate ((HO)2(O)P—

O—

(HO)(O) P—

O—

P(HO)(O)—

O-5′

), 5′

-guanosine cap (7-methylated or non-methylated) (7m-G-0-5′

-(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′

-monodithiophosphate (phosphorodithioate;

(HO)(HS)(S)P—

O-5′

), 5′

-phosphorothiolate ((HO)2(O)P—

S-5′

);

any additional combination of oxygen/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′

-).

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

terminus).

10. The RISC of claim 1, wherein said RNA molecule comprises at least one modified nucleotide analogue, which is preferably selected from sugar-backbone- and nucleobase-modified ribonucleotides and combinations thereof.

11. The RISC of claim 1, wherein said RNA molecule is associated with biodegradable polymers or microparticles, preferably wherein said association comprises a covalent coupling, in particular a covalent coupling via the 3′

-terminus of the RNA molecule.

12. A host cell or non-human host organism capable of overexpressing RISC according to claim 1.

13. 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 according to claim 1.

14. The RISC molecule according to claim 1 for use as a target for diagnosis and/or therapy.

15. The RISC according to claim 1 for use as a diagnostic and/or therapeutic agent itself, as a molecular-biological reagent or as component in a screening procedure for identification and/or characterization of pharmaceutical agents.