Rna interference mediating small rna molecules

• US 20040259247A1
• Filed: 07/26/2004
• Published: 12/23/2004
• Est. Priority Date: 12/01/2000
• Status: Abandoned Application

• Current Assignee: Massachusetts Institute of Technology, Max-Planck-Gesellschaft Zur Foerderung Der Wissenschaften e.V. (Max-Planck-Gesellschaft zur Frderung der Wissenschaften e), University Of Massachusetts, Whitehead Institute for Biomedical Research
• Original Assignee: Massachusetts Institute of Technology, Max-Planck-Gesellschaft Zur Foerderung Der Wissenschaften e.V. (Max-Planck-Gesellschaft zur Frderung der Wissenschaften e), University Of Massachusetts, Whitehead Institute for Biomedical Research
• Inventors: Wilm, Matthias, Lendeckel, Winfried, Elbashir, Sayda Mahgoub, Tuschl, Thomas, Luhrmann, Reinhard
• Application Number:
  US10/433,050
• Publication Number:
  US 20040259247A1
• Time in Patent Office: Days
• US Class Current: 435/375
• CPC Class Codes: A01K 2217/075 inducing loss of function, i.e. knock out
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: A61K 9/0019 Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner non-active ingredients are additionally classified in A61K47/00
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/00 Drugs for immunological or allergic disorders
• CPC Class Codes: A61P 37/02 Immunomodulators
• CPC Class Codes: A61P 37/06 Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C12N 15/1079 Screening libraries by altering the phenotype or phenotypic trait of the host reporter assays C12N15/1086
• 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 2310/321 2'-O-R Modification
• CPC Class Codes: C12N 2310/3521 Methyl
• CPC Class Codes: C12N 2310/53 partially self-complementary or closed
• CPC Class Codes: C12N 2330/30 chemically synthesised

Abstract

Double-stranded RNA (dsRNA) induces sequence-specific post-transcriptional gene silencing in many organisms by a process known as RNA interference (RNAi). Using a Drosophila in vitro system, we demonstrate that 19-23 nt short RNA fragments are the sequence-specific mediators of RNAi. The short interfering RNAs (siRNAs) are generated by an RNase III-like processing reaction from long dsRNA. Chemically synthesized siRNA duplexes with overhanging 3′ ends mediate efficient target RNA cleavage in the lysate, and the cleavage site is located near the center of the region spanned by the guiding siRNA. Furthermore, we provide evidence that the direction of dsRNA processing determines whether sense or antisense target RNA can be cleaved by the produced siRNP complex.

Claims

1. Isolated double-stranded RNA molecule, wherein each RNA strand has a length from 19-25 nucleotides, wherein said RNA molecule is capable of target-specific nucleic acid modifications.

2. The RNA molecule of claim 1 wherein at least one strand has a 3′

-overhang from 1-5 nucleotides.

3. The RNA molecule of claim 1 or 2 capable of target-specific RNA interference and/or DNA methylation.

4. The RNA molecule of any one of claims 1-3, wherein each strand has a length from 19-23, particularly from 20-22 nucleotides.

5. The RNA molecule of any one of claims 2-4, wherein the 3′

-overhang is from 1-3 nucleotides.

6. The RNA molecule of any one of claims 2-5, wherein the 3′

-overhang is stabilized against degradation.

7. The RNA molecule of any one of claims 1-6, which contains at least one modified nucleotide analogue.

8. The RNA molecule of claim 7, wherein the modified nucleotide analogue is selected from sugar- or backbone modified ribonucleotides.

9. The RNA molecule according to claim 7 or 8, wherein the nucleotide analogue is a sugar-modified ribonucleotide, wherein the 2′

-OH group is replaced by a group selected from H, OR, R, halo, SH, SR¹, NH₂, NHR, NR₂ or CN, wherein R is C₁-C₆ alkyl, alkenyl or alkynyl and halo is F, Cl, Br or I.

10. The RNA molecule of claim 7 or 8, wherein the nucleotide analogue is a backbone-modified ribonucleotide containing a phosphothioate group.

11. The RNA molecule of any one of claims 1-10, which has a sequence having an identity of at least 50 percent to a predetermined mRNA target molecule.

12. The RNA molecule of claim 11, wherein the identity is at least 70 percent.

13. A method of preparing a double-stranded RNA molecule of any one of claims 1-12 comprising the steps:

(a) synthesizing two RNA strands each having a length from 19-25 nucleotides, wherein said RNA strands are capable of forming a double-stranded RNA molecule,

(b) combining the synthesized RNA strands under conditions, wherein a double-stranded RNA molecule is formed, which is capable of target-specific nucleic acid modifications.

14. The method of claim 13, wherein the RNA strands are chemically synthesized.

15. The method of claim 13, wherein the RNA strands are enzymatically synthesized.

16. A method of mediating target-specific nucleic acid modifications in a cell or an organism comprising the steps:

(a) contacting said cell or organism with the double-stranded RNA molecule of any one of claims 1-12 under conditions wherein target-specific nucleic acid modifications can occur, and

(b) mediating a target-specific nucleic acid modification effected by the double-stranded RNA towards a target nucleic acid having a sequence portion substantially corresponding to the double-stranded RNA.

17. The method of claim 16, wherein the nucleic acid modification is RNA interference and/or DNA methylation.

18. The method of claim 16 and 17 wherein said contacting comprises introducing said double-stranded RNA molecule into a target cell in which the target-specific nucleic acid modification can occur.

19. The method of claim 18 wherein the introducing comprises a carrier-mediated delivery or injection.

20. Use of the method of any one of claims 16-19 for determining the function of a gene in a cell or an organism.

21. Use of the method of any one of claims 16-19 for modulating the function of a gene in a cell or an organism.

22. The use of claim 20 or 21, wherein the gene is associated with a pathological condition.

23. The use of claim 22, wherein the gene is a pathogen-associated gene.

24. The use of claim 23, wherein the gene is a viral gene.

25. The use of claim 22, wherein the gene is a tumor-associated gene.

26. The use of claim 22, wherein the gene is an autoimmune disease-associated gene.

27. Pharmaceutical composition containing as an active agent at least one double-stranded RNA molecule of any one of claims 1-12 and a pharmaceutical carrier.

28. The composition of claim 27 for diagnostic applications.

29. The composition of claim 27 for therapeutic applications.

30. A eukaryotic cell or a eukaryotic non-human organism exhibiting a target gene-specific knockout phenotype wherein said cell or organism is transfected with at least one double-stranded RNA molecule capable of inhibiting the expression of an endogeneous target gene or with a DNA encoding at least one double-stranded RNA molecule capable of inhibiting the expression of at least one endogeneous target gene.

31. The cell or organism of claim 30 which is a mammalian cell.

32. The cell or organism of claim 31 which is a human cell.

33. The cell or organism of any one of claims 30-32 which is further transfected with at least one exogeneous target nucleic acid coding for the target protein or a variant or mutated form of the target protein, wherein said exogeneous target nucleic acid differs from the endogeneous target gene on the nucleic acid level such that the expression of the exogeneous target nucleic acid is substantially less inhibited by the double stranded RNA molecule than the expression of the endogeneous target gene.

34. The cell or organism of claim 33 wherein the exogeneous target nucleic acid is fused to a further nucleic acid sequence encoding a detectable peptide or polypeptide.

35. Use of the cell or organism of any of claims 30-34 for analytic procedures.

36. The use of claim 35 for the analysis of gene expression profiles.

37. The use of claim 35 for a proteome analysis.

38. The use of any one of claims 35-37 wherein an analysis of a variant or mutant form of the target protein encoded by an exogeneous target nucleic acid is carried out.

39. The use of claim 38 for identifying functional domains of the target protein.

40. The use of any one of claims 35-39 wherein a comparison of at least two cells or organisms is carried out selected from:

(i) a control cell or control organism without target gene inhibition,

(ii) a cell or organism with target gene inhibition and

(iii) a cell or organism with target gene inhibition plus target gene complementation by an exogeneous target nucleic acid.

41. The use of any one of claims 35-40 wherein the analysis comprises a functional and/or phenotypic analysis.

42. Use of a cell of any one of claims 30-34 for preparative procedures.

43. The use of claim 41 for the isolation of proteins or protein complexes from eukaryotic cells.

44. The use of claim 43 for the isolation of high molecular weight protein complexes which may optionally contain nucleic acids.

45. The use of any one of claims 35-44 in a procedure for identifying and/or characterizing pharmacological agents.

46. A system for identifying and/or characterizing a pharmacological agent acting on at least one target protein comprising:

(a) a eukaryotic cell or a eukaryotic non-human organism capable of expressing at least one target gene coding for said at least one target protein,

(b) at least one double-stranded RNA molecule capable of inhibiting the expression of said at least one endogeneous target gene, and

(c) a test substance or a collection of test substances wherein pharmacological properties of said test substance or said collection are to be identified and/or characterized.

47. The system of claim 46 further comprising:

(d) at least one exogeneous target nucleic acid coding for the target protein or a variant or mutated from of the target protein wherein said exogeneous target nucleic acid differs from the endogeneous target gene on the nucleic acid level such that the expression of the exogeneous target nucleic acid is substantially less inhibited by the double stranded RNA molecule than the expression of the endogeneous target gene.