RNA-interference by single-stranded RNA molecules

• US 10,023,865 B2
• Filed: 09/07/2016
• Issued: 07/17/2018
• 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:
  US15/258,603
• Publication Number:
  US 20170067050A1
• Time in Patent Office: 678 Days
• Field of Search: None
• 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, wherein said RISC is a ribonucleoprotein complex containing a single stranded RNA component, and wherein said RNA component comprises at least one modified nucleotide analogue which is selected from sugar backbone and nucleobase-modified ribonucleotides and combinations thereof, wherein said RNA component is a single-stranded RNA molecule wherein the single-stranded RNA molecule has a length from 19-29 nucleotides, and wherein said RISC has a molecular weight of less than about 160 kDa.

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

3. The RISC of claim 2 wherein said at least one member of the Argonaute family of proteins is elF2C1 and/or elFC2.

4. The RISC of claim 3, further comprising at least one of elFC3, elFC4, HILI or HIWI.

5. The RISC of claim 1, wherein at least the 14-20 5′

most nucleotides are substantially complementary to a target transcript.

6. 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.

7. The RISC of claim 6, wherein said RNA molecule has a 5′

-moiety selected from the group consisting of 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-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′

-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, 5′

-phosphoramidates ((HO)₂(O)P—

NH-5′

, (HO)(NH₂)(O)P—

O-5′

), 5′

-alkylphosphonates, and 5′

-alkyletherphosphonates.

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

terminus.

9. The RISC of claim 1, wherein said covalent coupling is via the 3′

-terminus of the RNA molecule.

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

11. 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.

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

13. The RISC according to claim 7, 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′

-CH₂—

), and said 5′

-alkyletherphosphonate is RP(OH)(O)—

O-5′

-.

14. The RISC according to claim 1, wherein the 3′

end of said RNA component is stabilized against degradation.

15. The RISC according to claim 14, wherein the 3′

end of said RNA component is stabilized against degradation by incorporating non-nucleotide chemical derivatives selected from the group consisting of aminolinkers, thiol linkers, carboxyl linkers, and non-nucleotidic spacers.

16. The RISC according to claim 1, wherein the 3′

end of said RNA component is modified with biotin or fluoresceine.