RNA-interference by single-stranded RNA molecules

• US 9,476,044 B2
• Filed: 07/22/2014
• Issued: 10/25/2016
• 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: Tuschl, Thomas, Martinez, Javier, Patkaniowska, Agnieszka, Urlaub, Henning, Luehrmann, Reinhard
• Primary Examiner(s): SHIN, DANA H
• Application Number:
  US14/337,710
• Publication Number:
  US 20150096064A1
• Time in Patent Office: 826 Days
• Field of Search: None
• US Class Current: 1/1
• 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 less than about 160 kDa, wherein said RISC is a ribonucleoprotein complex containing an RNA component and one member of the Argonaute family of proteins, and wherein said RNA component is a single-stranded RNA molecule which has a length of 19-29 nucleotides comprising at least one modified nucleotide analogue, which is selected from sugar-backbone- and nucleobase-modified ribonucleotides and combinations thereof.

2. The RISC of claim 1 wherein said one member of the Argonaute family of proteins is eIF2C1 or eIF2C2.

3. The RISC of claim 1 wherein the RNA molecule comprises at least one mismatch with the target transcript at the 3′

-terminus.

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

-moiety selected from the group consisting of 5′

-monophosphate ((HO).sub.2(O)P—

O-5′

), 5′

-diphosphate ((HO).sub.2(O)P—

O—

P(HO)(O)—

O-5′

), 5′

-triphosphate ((HO).sub.2(O)P—

O—

(HO)(O)P—

O—

P(HO)(O)—

O-5′

), 5′

-guanosine cap (7-methylated or non-ethylated) (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).sub.2(S)P—

O-5′

), 5′

-monodithiophosphate (phosphorodithioate;

(HO)(HS)(S)P—

O-5′

), 5′

-phosphorothiolate ((HO).sub.2(O)P—

S-5′

);

any additional combination of oxygen/sulfur replaced monophosphate, diphosphate and triphosphates, 5′

-phosphoramidates ((HO).sub.2(O)P—

NH-5′

, (HO)(NH.sub.2)(O)P—

O-5′

), 5′

-alkylphosphonates, and 5′

-alkyletherphosphonates.

6. The RISC of claim 1, wherein said RNA molecule is completely complementary to said target transcript.

7. A composition comprising the RISC according to claim 1 in combination with a pharmaceutical carrier.

8. The RISC according to claim 5, wherein said sulfur replaced monophosphate, diphosphate or triphosphate is 5′

-alpha-thiotriphosphate or 5′

-gamma-thiotriphosphate, said 5′

-alkylphosphonate is RP(OH)(O)—

O-5′

- or (OH)2(O)P-5′

-CH.sub.2-), and said 5′

-alkyletherphosphonate is RP(OH)(O)—

O-5′

-.

9. The RISC according to claim 1, wherein said RISC has a molecular weight of 120-150 kDa.

10. The RISC of claim 1, wherein said one member of the Argonaute family of proteins is selected from the group consisting of eIF2C3, eIF2C4, HILI and HIWI.

11. A method of enhancing RNAi in a cell or an organism comprising causing said cell or organism to overexpress the RISC according to claim 1.