Rna interference mediating small rna molecules

Patent metadata

Field	Value
Publication number	US-2015141492-A1
Application number	US-201414476465-A
Country	US
Kind code	A1
Filing date	Sep 3, 2014
Priority date	Dec 1, 2000
Publication date	May 21, 2015
Grant date	—

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.

First claim

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 capable of target-specific RNA interference and/or DNA methylation.

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

5 . The RNA molecule of claim 2 , wherein the 3′-over-hang is from 1-3 nucleotides.

6 . The RNA molecule of claim 2 , wherein the 3′-over-hang is stabilized against degradation.

7 . The RNA molecule of claim 1 , 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 , 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, SR′, NH2, NHR, NR2 or CN, wherein R is C1-C6 alkyl, alkenyl or alkynyl and halo is F, Cl, Br or 1.

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

11 . The RNA molecule of claim 1 , 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 claim 1 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 claim 1 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 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 claim 16 for determining the function of a gene in a cell or an organism.

21 . Use of the method of claim 16 for modulating the function of a gene in a cell or an organism.

22 . The use of claim 20 , 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 claim 1 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 claim 30 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 claim 30 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 claim 35 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 claim 35 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 claim 35 wherein the analysis comprises a functional and/or phenotypic analysis.

42 . Use of a cell of claim 30 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 claim 35 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 ide

Assignees

• Max Planck Ges Zur Förderung Der Wissenschaften E V
• Massachusetts Inst Technology
• Whitehead Biomedical Inst
• Univ Massachusetts

Inventors

• Tuschl Thomas
• Elbashir Sayda Mahgoub
• Lendeckel Winfried

Classifications

• A61P43/00

  Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00 · CPC title

• A61P37/06

  Immunosuppressants, e.g. drugs for graft rejection · CPC title

• A61P37/02

  Immunomodulators · CPC title

• A61P31/12

  Antivirals · CPC title

• A61P35/00

  Antineoplastic agents · CPC title