WO2007048629A2

WO2007048629A2 - Modulation of rna silencing efficiency by argonaute proteins

Info

Publication number: WO2007048629A2
Application number: PCT/EP2006/010373
Authority: WO
Inventors: Volker Patzel; Stefan H. E. Kaufmann
Original assignee: MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.
Priority date: 2005-10-28
Filing date: 2006-10-27
Publication date: 2007-05-03
Also published as: WO2007048629A3

Abstract

The present invention relates to methods and compositions for modulating RNA silencing efficiency by providing selective RISC (RNA-induced silencing complex) formation.

Description

Modulation of RNA silencing efficiency by Argonaute proteins

Specification

Background

In mammalian systems, RNAi is mainly triggered by siRNAs and microRNAs (miRNAs)¹^. SiRNA and miRNA duplexes are composed of complementary RNA of preferably 21-23 nucleotides (nts) in length with sense and antisense orientation to the mRNA target. In siRNA duplexes, sense- and antisense- siRNA (as-siRNA) are perfectly base-paired. MiRNA duplexes exhibit imperfect pairing between the mature miRNA (antisense) and the opposing strand termed miRNA (sense). One of the two strands, the guide strand, is included into the RISC, whereas the other strand, the passenger strand, is excluded and destroyed. Only if the antisense strand is chosen as guide, it directs activated RISC to the mRNA target inducing gene silencing. RISC is a multiprotein complex containing as core a protein of the Argonaute (Ago) family. MiRNA-associated RISC contains Ago-1 , 2, 3 or 4, whereas siRNA- induced mRNA cleavage is exclusively associated with Ago-2-containing RISC⁵.

In mammalian cells, siRNA-triggered RNAi starts with formation of the RISC-loading complex (RLC) including siRNA duplex recognition and definition of guide and passenger strand. Subsequent steps encompass duplex unwinding, RISC formation and activation, mRNA targeting, cleavage, and release of the cleaved target sequence prior to targeting of further mRNA molecules⁶⁷. Lower thermodynamic duplex stabilities at the 5' antisense compared to the 5' sense terminus favor selection of as-siRNAs as guide strands and, thus, formation of silencing competent RISCs^8"10. Specific base preferences and GC contents, the absence of internal repeats, and accessible target sites were reported to favor siRNA activities^{11 18}. However, the meaning of many of these correlations for the silencing pathway and, thus siRNA design remains unclear.

The current model argues that siRNAs, shRNAs (small hairpin RNAs) or miRNAs (micro RNAs) share the later stages of the silencing pathway. A recent study reported fatality in mice due to competition of artificial regulatory RNAs (shRNAs) with cellular regulatory RNAs (miRNAs) for limiting cellular factors resulting in oversaturation of vital cellular pathways³⁷.

It is thus an object of the present invention to design double stranded RNAs, in particular siRNAs, shRNAs and miRNAs, which actively avoid interference with processing and action of cellular regulatory RNA. The solution provided by the present invention may be of importance for

1. in vivo applications and siRNA or/and shRNA based therapeutics, or/and 2. ex vivo target validation in living cells. In target validation, interference of artificial siRNAs or/and shRNAs with regulation of gene expression may be disadvantageous.

Summary of the invention

The invention is based on data which demonstrate that selective enhancement and/or suppression of RNA silencing pathways may lead to a modulated, e.g. an increased or reduced RNA silencing activity in target cells, organisms or cell-free systems.

Here we demonstrate for the first time, that RNAi triggered by perfectly base- paired siRNA duplexes (Fig. 4: RNAs 2-9, 2-4, IL1 , IL2, and IL3; Hg. 10: duplex 2, 3, 4, and 5) depends on Argonaute-2 RISCs and that duplexes with distinct mismatches trigger Argonaute-1 dependent silencing (such as distinct mismatches at positions 1 , 4 and 15 with respect to the antisense siRNA strand; see .e.g. Fig. 4: RNAs 2-9-2, IL4; Fig. 10: duplex 6 and 7).

It is further demonstrated that a perfectly base-paired duplex which gives rise to a GU wobble base pair between the 5¹ region (position 2) of the antisense siRNA and the mRNA target results in Argonaute-1 and Argonaute-2 independent silencing (Fig. 4: RNA 3-8; Fig. 10: duplex 1 ).

All these data indicate that the structure/design of the effector duplex (siRNA or shRNA) determines the choice of Argonaute for RISC and thereby the mechanism of gene silencing. Therefore the duplex structure represents the basis to design artificial regulatory RNAs which do not interfere with Argonaute-1 or/and Argonaute-2 dependent cellular gene silencing mechanisms.

A first aspect to the invention relates to a method for preparing a double stranded RNA molecule with target gene specific silencing activity which selectively interacts with an RNA-induced silencing complex (RISC) containing a predetermined species of Argonaute protein, comprising the steps

(a) identifying a double stranded RNA molecule directed to the mRNA of a target gene, wherein said RNA molecule comprises:

(i) a double stranded portion of 9-35 nucleotides and optionally at least one 3' overhang,

(ii) an antisense strand which has a sufficient degree of complementarity to the mRNA of the target gene for RISC formation, and

(iii) a sense strand which has a predetermined degree of complementarity to the antisense strand to provide a double stranded RNA molecule for specific interaction with an RISC containing a predetermined species of Argonaute protein, and

(b) preparing the selected double stranded RNA molecule or a precursor thereof or a DNA molecule encoding said RNA molecule or precursor.

A further aspect relates to a method for regulating the expression of a target gene in a cell, an organism or a cell-free system, comprising the steps of: - A -

(i) a double stranded portion of 9-35 nucleotides and optionally at least one 3' overhang, (ii) an antisense strand which has a sufficient degree of complementarity to the mRNA of the target gene for RISC formation, and

(iii) a sense strand which has a predetermined degree of complementarity to the antisense strand to provide a double stranded RNA molecule for specific interaction with a RISC containing a predetermined species of Argonaute protein, and

(b) introducing the molecule of (a) into said cell, organism or cell-free system under conditions under which target-specific nucleic acid silencing selectively occurs with a RISC containing a predetermined species of Argonaute protein.

Still a further aspect of the invention relates to a method for modulating target gene specific silencing activity in a cell, an organism or a cell-free system, comprising selecting increasing and/or suppressing the activity of at least one polypeptide of the gene silencing machinery selected from Argonaute proteins such as Ago-1 (elF2C1), Ago-2 (elF2C2), Ago-3 (elF2C3), Ago-4 (elF2C4), PIWIL 1 (HIWI), PIWIL 2 (HILI), PIWIL 3 and PIWIL 4 (HIWI 2), preferably Ago-1 and/or Ago-2, and other proteins of the gene silencing machinery such as Dicer proteins, e.g. Dicer 1 (DcM), or Dicer 2 (Dcr2); DGCR8 (Drosha, Pasha), R2D2 (dsRBD), NR, FmM/Fxr, Vig, Tsn, Dmp68, Gemin3, Gemin4, Exportin-5 and Loquacious, and providing a double stranded RNA molecule directed to the mRNA of a target gene in said cell, organism or cell-free system. Preferably, the proteins are human proteins.

The invention also relates to compositions of matter comprising double stranded RNA molecules or precursors thereof or DNA molecules encoding said RNA molecules or precursors obtainable by the methods as indicated above.

Furthermore, the invention relates to a composition for target gene specific silencing comprising: (a) a double stranded RNA molecule directed to the mRNA of a target gene, a precursor thereof or a DNA molecule encoding the double stranded RNA molecule or the precursor thereof, and (b) (i) at least one polypeptide of the gene silencing machinery selected from Argonaute proteins, preferably Ago-1 and/or Ago-2, and other proteins of the gene silencing machinery or

(ii) a nucleic acid encoding the polypeptide of (i), wherein component (b) is present in an amount or form to provide a selective activity increase of the polypeptide (i) or nucleic acid.

Furthermore, the invention relates to a composition for target gene specific silencing comprising:

(a) a double stranded RNA molecule directed to the mRNA of a target gene, a precursor thereof or a DNA molecule encoding the double stranded RNA molecule or the precursor thereof, and (b) a double stranded RNA molecule directed to the mRNA encoding at least one polypeptide of the gene silencing machinery selected from Argonaute proteins, preferably Ago-1 and/or Ago-2, and other proteins of the gene silencing machinery, a precursor of the RNA molecule or a DNA molecule encoding the double stranded RNA molecule or the precursor thereof.

Furthermore, the invention comprises a double stranded RNA molecule directed against an mRNA of a polypeptide of the gene silencing machinery, or a precursor thereof or a DNA molecule encoding said RNA molecule or precursor.

Furthermore, the invention relates to the use of a polypeptide of the gene silencing machinery or a nucleic acid coding therefor or of a double stranded RNA with gene silencing activity directed against a polypeptide of the gene silencing machinery, or a precursor of the double stranded RNA molecule or a DNA molecule encoding the RNA molecule or the precursor thereof for the manufacture of a medicament for the prophylaxis or treatment of disorders associated with dysfunctional gene expression including, but not limited to, infectious diseases, particularly viral, bacterial or protozoal diseases.

The compounds and compositions of the present invention are suitable as reagents, diagnostics or medicaments.

Detailed description

The present invention relates to the field of RNA silencing which describes a gene regulatory mechanism that limits the transcript level by suppressing transcription, i.e. transcriptional gene silencing (TGS) or by activating a sequence-specific RNA degradation process (post-transcriptional gene silencing (PTGS)). PTGS includes translational attenuation and/or RNA interference. Three phenotypically different but mechanistically similar forms of RNAi, cosuppression or PTGS in plants, quelling in fungi, and RNAi in the animal kingdom, have been described. More recently, micro-RNA formation, heterochromatinization, etc., have been revealed as other facets of naturally occurring RNAi processes of eukaryotic cells. RNA silencing is mediated by RISC formation. An RISC may contain as a core different proteins of the Argonaute family.

According to the present invention, double stranded RNA molecules with RNA silencing activity are provided which selectively interact with a RISC containing a determined species of Argonaute protein, e.g. Ago-1 , Ago-2, Ago-3, Ago-4, PIWIL1 , PIWIL 2, PIWIL 3 or PIWIL 4, preferably Ago-1 or Ago-2. Preferably, the RISC is a mammalian RISC, e.g. a human RISC and the Argonaute proteins are mammalian, e.g. human proteins.

The double stranded RNA molecule with gene silencing activity comprises a double stranded portion of e.g. 9-35 nucleotides, preferably 14-25 nucleotides and more preferably 18-22 nucleotides and optionally at least one, e.g. one or two 3¹ overhangs which have a length of e.g. 1-10, preferably 1-5, such as 1 , 2, 3, 4 or 5 nucleotides.

The double stranded RNA molecule comprises an antisense strand which has a sufficient degree of complementarity to the mRNA of the target gene for RISC formation. For example, the degree of complementarity may be at least 50%, preferably at least 70% and more preferably at least 90%, e.g. 100% to the mRNA of a target gene. In this context, it should be noted that complementarity according to the present application is defined as comprising Watson-Crick base pairs, i.e. A-U, U-A, G-C and C-G base pairs and Wobble base pairs, i.e. G-U and U-G base pairs.

The double stranded RNA molecule also comprises a sense strand which has a sufficient degree of complementarity to the antisense strand to provide a double stranded RNA molecule which is suitable for interaction with a RISC. The sense strand and the antisense strand have usually a length between 9 and 40 nucleotides, preferably between 15 and 30 nucleotides and more preferably between 19 and 25 nucleotides.

According to the present invention, selective interaction with an RISC containing a predetermined species of Argonaute protein is achieved by selecting a predetermined degree of complementarity between sense and antisense strand has to be selected. For example, in a double stranded RNA molecule which is intended to selectively interact with an Ago-2 containing RISC the sense strand is selected to have a degree of complementarity with the antisense strand of 100%, wherein complementarity comprises Watson- Crick base pairs and Wobble base pairs, e.g. the sense strand and the antisense strand have a 100% complementarity of Watson-Crick base pairs only or a 100% complementarity of Watson-Crick base pairs plus at least one Wobble base pair, and wherein complementarity preferably comprises Watson-Crick base pairs and no Wobble base pairs. The effect of a Wobble base pair on selective interaction with an Ago-2 containing RISC may depend upon the position of the Wobble base pair in the double stranded RNA molecule.

If, on the other hand, a double stranded RNA molecule which selectively interacts with a non Ago-2-containing RISC, e.g. an Ago- T containing RISC is prepared, the sense strand is selected to have a degree of complementarity of less than 100% to the antisense strand, wherein complementarity comprises Watson-Crick base pairs and Wobble base pairs, e.g. only Watson-Crick base pairs or Watson-Crick base pairs and at least one Wobble base pair. In this embodiment, double stranded portion of the sense and antisense strand comprises at least one mismatch, preferably 1 , 2, 3, 4 or even more mismatches. If, for example, the antisense strand has a length of 1-23, preferably 20-22 nucleotides, the at least one mismatch is preferably located between position 13 and 17, more preferably between position 14 and 16 of the antisense strand (when the 5' end of the antisense strand is designated as position 1 ). The at least one mismatch may also be located at position 1 , 4 or/and 15 of the antisense strand.

According to the invention it was found, that perfectly base paired siRNA duplexes preferably enter the Ago-2-dependent silencing pathway, i.e. result in formation of Ago-2-containing RISC leading to target cleavage (RNAi) whereas imperfectly base paired siRNA duplexes preferably enter the Ago- 1 -dependent silencing pathway, i.e. result in formation of Ago-1 -containing RISC leading to translational attentuation.

In yet another embodiment of the present invention, a double stranded RNA molecule is prepared which has Argonaute-1 or/and Argonaute-2 independent cellular gene silencing activity. In particular, a double-stranded RNA molecule is prepared which has Agb-1 and Ago-2 independent cellular gene silencing activity. More particular, the double stranded RNA molecule selectively interacts with a RISC different from a RISC selected from Ago-1 containing RISC and Ago-2 containing RISC. Even more particular, the double stranded RNA molecule selectively interacts with a RISC selected from Ago-3 containing RISC and Ago-4 containing RISC. Argonaute-1 or/and Argonaute-2 independent cellular gene silencing activity may result from at least one Wobble base pair formed between the antisense strand and the target mRNA. Thus, in this embodiment, the antisense strand of the double stranded RNA preferably is capable of forming at least one Wobble base pair (U-G, G-U) with the mRNA of the target gene. More preferably, the antisense strand forms 1 , 2, 3, 4 or even more Wobble base pairs with the mRNA of the target gene. The base capable of forming the at least one Wobble base pair is preferably located at position 1 , 2, 3, 4 or/and 5, more preferably at position 2 (when the 5¹ position of the antisense strand is designated as position 1), in particular if the antisense strand has, for example, a length of 1-23, preferably 20-22 nucleotides. In this embodiment, the double stranded portion preferably comprises no mismatches, i.e. the sense strand is preferably selected to have a degree of complementarity with the antisense strand of 100%.

The double stranded RNA may be an siRNA, shRNA or an miRNA or a precursor thereof. The term "precursor" relates to an RNA species which is processed in the cell to a double stranded RNA with target-gene specific silencing activity. Preferred examples of precursors of siRNA molecules are small hairpin (sh) molecules, i.e. single stranded RNA molecules having a stem-loop structure wherein the stem corresponds to the double stranded RNA and the loop portion is cleaved off. Further examples of siRNA precursors are long double stranded RNA molecules which are processed within a cell, particularly an eukaryotic cell in order to give double stranded RNA molecules as indicated above.

Preferred examples of precursors of miRNA molecules are primary miRNA molecules or precursor miRNA molecules which are processed by Drosha or Dicer respectively to mature miRNA molecule comprising an antisense and a sense strand. Further, the invention relates to DNA molecules encoding the double stranded RNA molecule or a precursor thereof. Preferably, the DNA molecule comprises a sequence which - when transcribed using a suitable DNA-dependent RNA polymerase - gives the double stranded RNA molecule or a precursor thereof. Thus, the sequences encoding the double stranded RNA molecule or the RNA molecule precursor are preferably operatively linked to suitable expression control sequences.

The strands of the double stranded RNA molecule or the precursor may be chemically and/or enzymatically synthesized, for example, the antisense

RNA strand and the sense RNA strands may be synthesized and the strands may be combined to form the double stranded RNA molecule. Alternatively, the precursor of the double stranded RNA molecule may be synthesized and subjected to a processing step, whereby the double stranded RNA molecule is formed. Alternatively, the DNA molecule encoding the double stranded RNA molecule or the precursor thereof may be synthesized and the resulting DNA molecule may be transcribed whereby the double stranded

RNA molecule or the precursor thereof is formed and wherein the precursor may be subjected to a processing step whereby the double stranded RNA molecule is formed.

The RNA molecules may contain 3¹ overhangs which are stabilized against degradation, e.g. by incorporating deoxyribonucleotides such as dT, and/or at least one modified nucleotide analogue, which may be selected from sugar-, backbone- or nucleobase-modified ribonucleotides, i.e. ribonucleotides, containing a non-naturally occurring nucleobase instead of a naturally occurring nucleobase such as uridines or cytidines modified at the 5-position, e.g. 5-(2-amino)propyl uridine, 5-bromo uridine; adenosines and guanosines modified at the 8-position, e.g. 8-bromo guanosine; deaza nucleotides, e.g. 7-deaza-adenosine; O- and N-alkylated nucleotides, e.g. N6-methyl andenosine are suitable. In preferred sugar-modified ribonucleotides the 2' OH-group is replaced by a group selected from H, OR, R, halo, SH, SR, NH₂, NHR, N(R)₂ or CN₁ wherein R is Ci-C₆-alkyl, alkenyl or alkynyl and halo is F₁ Cl, Br or I. In preferred backbone-modified ribonucleotides the phoshoester group connecting to adjacent ribonucleotides is replaced by a modified group, e.g. of phosphothioate group. It should be noted that the above modifications may be combined.

Further, the double stranded RNA molecule may comprise modifications at the 5¹ end or 3¹ terminus of at least one strand. These modifications are preferably selected from lipid groups, e.g. cholesterol groups, vitamins, etc.

The double stranded RNA molecule, or the precursor thereof or the DNA molecule encoding the RNA molecule or the precursor, may be used for the regulation of the expression of a target gene in cell, an organism or a cell- free system or to produce a cell, organism or cell-free system comprising a double stranded RNA molecule which selectively interacts with a RISC containing a predetermined species of Argonaute protein. For this purpose, the molecule is introduced into the cell, organism or cell-free system under conditions under which target-specific nucleic acid silencing selectively occurs with a RISC containing a predetermined species of Argonaute protein. Preferably, the silencing selectively occurs with a RISC containing Ago-1 or alternatively with a RISC containing Ago-2.

The cell is preferably a eukaryotic cell, more preferably an animal cell, still more preferably a mammalian cell such as a human cell. The organism is preferably a eukaryotic organism, e.g. a mammal including a human. The cell-free system is preferably an extract or a fractionated extract from a eukaryotic cell, e.g. a mammalian cell such as a human cell.

The target gene may be a reporter gene, a pathogen-associated gene, e.g. a viral, protozoal or bacterial gene, or an endogenous gene, e.g. an endogenous mammalian, particularly human gene. The endogenous gene may be associated with a disorder, particularly with a hyperproliferative disorder, e.g. cancer, or with a metabolic disorder, e.g. a disorder associated with carbohydrate, energy, lipid, nucleotide, or amino acid metabolism or a disorder associated with the biosynthesis or metabolism of glycans, polyketides and and nonribosomal peptides, cofactors and vitamins or secondary metabolites, with the biodegradation of xenobiotics or with a neurodegenerative disorder such as Alzheimer, Parkinson, Huntington, ALS, MS etc. Thus, the present invention is suitable for the manufacture of reagents, diagnostics and therapeutics.

For pharmaceutical applications, the invention provides also a pharmaceutical composition comprising as an active agent at least one double stranded RNA molecule as described herein, or a precursor thereof or a DNA molecule encoding the double stranded RNA molecule or the precursor and a pharmaceutical carrier. The composition may be used for diagnostic and therapeutic applications in human medicine or in veterinary medicine.

For diagnostic or therapeutic applications the composition may be in form of a solution, e.g. an injectible solution, a cream, ointment, tablet, suspension or the like. The composition may be administered in any suitable way, e.g. by injection, by oral, topical, nasal, rectal application etc. The carrier may be any suitable pharmaceutical carrier. Preferably, a carrier is used of increasing the efficacy of RNA molecules to enter the target cells. Suitable examples of such carriers are liposomes, particularly cationic liposomes.

A further aspect of the invention relates to the modulating of a target gene specific silencing activity in a cell, an organism or a cell-free system, wherein the activity of at least one polypeptide of the gene silencing machinery is selectively modulated, e.g. increased and/or suppressed. These polypeptides are preferably selected from Argonaute proteins such as Ago- 1 , Ago-2, Ago-3 and Ago-4, such as Ago-1 (elF2C1), Ago-2 (elF2C2), Ago3 (elF2C3), Ago4 (elF2C4), PIWIL 1 (HIWI), PIWIL 2 (HFLI), PIWIL 3 and PIWIL 4 (HIWI 2), more preferably Ago-1 and/or Ago-2, and other proteins of the gene silencing machinery such as Dicer proteins, e.g. DcM , or Dcr2; DGCR8 (Drosha, Pasha), R2D2 (dsRBD), NR, Fmr1/Fxr, Vig, Tsn, Dmp68, Gemin3, Gemin4, Exportin-5 and Loquacious. Preferably, the polypeptide is an Argonaute or Dicer protein such as Ago-1 , Ago2, Dcr1 or Dcr2. In this context, reference is made to the publications Sasaki et al., (Genomics 82 (2003), 323-330) and Sontheimer (Nat. Ref. MoI. Cell. Biol. (2002), 127-38) which are herein incorporated by reference. These publications contain a detailed description of polypeptides of the gene silencing machinery and complexes containing these polypeptides.

By means of this selective activity increase and/or suppression, the efficacy of target-gene specific silencing may be considerably increased. Thus, administration of double stranded molecules directed to the mRNA of a target gene, organism or a cell-free system (as indicated above) may be more effective.

The RNA molecule may have a target gene silencing activity of at least 90%, 92%, 94%, 96% or 98% (based on the target gene expression in the absence of the RNA molecule). The gene silencing activity may be determined at concentrations of e.g. 0.001 nM, 0.01 nM, 0.1 nM, 0.5 nM, 1 nM, 5 nM, 10 nM or 50 nM in a suitable test system, e.g. as described in the Examples.

In one embodiment, the activity of at least one polypeptide of the gene silencing machinery is selectively increased. This embodiment preferably relates to a selective increase in the activity of Ago-2. The activity increase may be accomplished for example by overexpression of the polypeptide, e.g. in a target cell or a target organism and/or by adding an excess of the polypeptide, e.g. to a cell-free system. A selective activity increase of Ago-2, for example, leads to a significant increase of gene silencing activity.

In a further embodiment, the activity of at least one polypeptide of the gene silencing machinery is selectively suppressed. This suppression may be accomplished by gene-specific silencing of the polypeptide in the target cell, organism or cell-free system. The gene-specific silencing may comprise, for example, administering double stranded RNA molecules, e.g. siRNA molecules or miRNA molecules, precursors thereof or DNA molecules encoding^" said RNA molecules or precursors thereof directed to the mRNA encoding the at least one polypeptide of the gene silencing machinery which is to be suppressed. This embodiment particularly relates to a suppression of Ago-1 activity which may be accomplished by administering double stranded RNA molecules, precursors thereof or DNA molecules encoding said RNA molecules or precursors thereof directed against Ago-1 mRNA. Preferably, the RNA molecules directed against Ago-1 mRNA are selected such that they specifically interact with an Ago-1 containing RISC as explained above.

Further, the invention relates to a composition for target gene specific silencing comprising (a) a molecule suitable for gene-specific silencing of a target gene, e.g. a double stranded RNA molecule directed to the mRNA of a target gene, a precursor thereof or a DNA molecule encoding the double stranded RNA molecule or the precursor thereof and (b) (i) at least one polypeptide of the gene silencing machinery as indicated above or (ii) a nucleic acid encoding this polypeptide, wherein component (b) is present in an amount or form to provide a selective activity increase of the polypeptide or the nucleic acid.

The composition may be an expression system comprising as component (a) a DNA molecule encoding a double stranded RNA molecule directed to the mRNA of the target gene or a precursor thereof and as component (b) a DNA molecule encoding the polypeptide of the gene silencing machinery wherein DNA molecules (a) and (b) are operatively linked to expression control sequences, either on a single expression vehicle or on a plurality of expression vehicles such as plasmid vectors, viral vectors etc. Alternatively, the composition may be a mixture or kit comprising as component (a) a double stranded RNA molecule directed to the mRNA of the target gene or a precursor thereof and as compound (b) a purified or partially purified polypeptide of the gene silencing machinery or a DNA molecule encoding said polypeptide operatively linked to an expression control sequence. In this embodiment, the polypeptide of the gene silencing machinery is preferably Ago-2 and/or Diceri (DcM ).

Furthermore, the invention provides a composition for target gene specific silencing which comprises a double stranded RNA molecule directed to the mRNA of a target gene, a precursor thereof or a DNA molecule encoding the double stranded RNA molecule or the precursor thereof in combination with (b) a double stranded RNA molecule directed to the mRNA encoding at least one polypeptide of the gene silencing machinery, a precursor of the RNA molecule or a DNA molecule encoding the double stranded RNA molecule or the precursor thereof. More preferably, this composition comprises a combination of (a) a double stranded RNA molecule directed to the mRNA of the target gene and (b) a double stranded RNA molecule directed to the mRNA of a protein of the gene silencing machinery. In this embodiment, the polypeptide of the gene silencing machinery is preferably Ago-1.

The compositions as described above may be a reagent, e.g. a research tool, a diagnostic or a medicament as described above.

The invention also relates to a cell or non-human organism transformed or transfected with the composition or an expression system comprising the composition which comprises at least one expression vehicle.

The invention also relates to a double stranded RNA molecule with gene silencing activity directed against an mRNA of a polypeptide of the gene silencing machinery as indicated above, e.g. Ago-1 , or Ago-2 or the precursor thereof or a DNA molecule encoding said RNA molecule or precursor.

The double stranded RNA molecule is preferably chosen such it selectively interacts with a RISC containing the predetermined species of protein, e.g. Argonaute protein. For example, an Ago-2 selective double stranded RNA molecule, e.g. a perfectly base paired double stranded RNA molecule may be used to suppress silencing activity associated with Ago-2 containing RISC. Especially preferred is the use of Ago-1 selective double stranded RNA molecules, wherein the antisense strand and the sense strand comprise at least one mismatch within the double stranded portion of the RNA molecule, for selective inhibition of gene silencing activity associated with Ago-1 containing RISC. The above compounds are suitable for use as a reagent, a diagnostic or a medicament.

Particularly preferred examples of such compounds are as follows:

1. Ago-2-dependent (perfectly base-paired) Ago-1 -directed siRNA:

Sense: 5'-UGUAUGAUGGAAAGAAGAAdTdT-3' Antisense: 5^I-UUCUUCUUUCCAUCAUACAdCdA-3¹

2. Ago-2-dependent (perfectly base-paired) Ago-2-directed siRNA:

Sense: 5'-GGAGAGUUAACAGGGAAAUdTdT-S¹ Antisense: 5'-AUUCCCUGUUAACUCUCCdTdC-3'

3. Ago-1 -dependent (imperfectly base-paired) Ago-1 -directed siRNA:

Sense: 5'-UGUACGAUGGAAAGAAGACdTdT-S¹ Antisense: 5^I-UUCUUCUUUCCAUCAUACAdCdA-3^I

Finally, the invention relates to the use of a composition capable of enhancing gene silencing activity, e.g. by selectively enhancing Ago-2 associated gene silencing activity, for the manufacture of a medicament for the prophylaxis or treatment of disorders associated with dysfunctional gene expression including infectious diseases, particularly viral, bacterial or protozoal diseases. This aspect of the invention is based on the finding that over-expression of Ago-2 and/or Dcr1 leads to a reduced susceptibility of eukaryotic, e.g. mammalian cells against infection with pathogens, e.g. bacteria. Thus, increasing the amount of a polypeptide of the gene silencing machinery, e.g. by administering the polypeptide or a nucleic acid coding therefor, or alternatively by suppressing specific gene silencing activities, e.g. Ago-1 associated gene silencing activities, a specific desired gene silencing activity, e.g. Ago-2 associated gene silencing activity may be increased leading to desirable pharmacological results.

Further, the present invention shall be explained in more detail by the following figures and examples.

Description of drawings

Figure 1 : In silico-selected jagged-1 -directed guide-siRNA. a, Jagged-1 target sequence and overlapping as-siRNA sequences. The common core sequence is shaded in grey. Nts forming a conserved tetra-loop are framed, b, Predicted secondary structures of as-siRNA, structural signatures, calculated target accessibilities, and siRNA activities, c, Jagged-1 expression (MFI) in cells transfected with 1 (π) or 10(») pmol siRNA duplexes, (a) and (b), Identical sequence stretches are colour-coded. Error bars represent standard deviations (SD) of 3 to 6 experiments.

Figure 2: Activities of in silico selected siRNA. a, Knock-down of GFP and b Luciferase gene expression. G: GFP-directed siRNA; L: Luciferase-directed siRNA; US: unstable; RC: random-coiled; IL: intern loop; 2SL: 2 stem loops; h/m/l: high/medium/low energy; C: Control siRNA; /: Mock-transfected cells; error bars represent SD of 3 to 6 experiments. Correlations r between free 3' nts or ΔG of structures in (a) and (b) and gene silencing. *21/2=10.5 nts were assigned to 3' ends of unstructured RNA; ^Opseudo-free 3' nts resulting from opening of 3'-adjacent stems divided by 2.

Figure 3: Programming active as-siRNA/guide-RNA structures by base exchanges, a, A>G and C>U exchanges (red) can program structures and/or ΔG of guide-RNA thereby inducing wobble-base pairing with the target but preserving target complementarity, b, Jagged-1 expression (MFI) in 293T cells transfected with siRNA duplexes containing parental and programmed as-siRNA strands. Duplexes form only Watson-Crick bp. ^*Partition structures. RC: Randomly-coiled as-siRNA. Error bars represent SD of 3-4 measurements, c, Dimensions of guide-siRNA sequence spaces without (Di) and including base exchanges (D₂) for a given target RNA of L nts in length containing guanine (G) and uracil (U) bases.

Figure 4: SiRNA duplex structures determine Argonaute dependence of RISC. Jagged-1 expression (MFI) in Ago-1 (■), Ago-2 (π), and Ago-1+2 (■) knock-down or native (π) 293T cells. D-wob, duplex-intrinsic base wobbling (blue); t-wob: target base wobbling (red); d-mis, duplex-intrinsic mismatching. *Partition structures. Error bars represent SD of 3-4 measurements.

Figure 5: Structures of guide-siRNA are correlated with RNA i. A, Mfe structures of guide siRNA corresponding to each 13 active and inactive published siRNA^{911 1330 32} targeting 5 mRNA targets were predicted. Predictions based on the canonical AUCG base alphabet and for consistency with physical structures were preferably considered siRNA with XY or dXdY 3'-overhangs for analysis. Structures were characterized by numbers of terminal free nts, loop size, bp, and ΔG of secondary structure formation. Error bars represent averages or numbers of structures; maxima and minima are indicated, b, consensus structures derived from active and inactive guide-si RNA species.

Figure 6: RNAse T1 probing of guide-siRNA structures 4-7, 0-0, and 2-9. Guide-siRNA strands were 5'-labeled with ³²P using polynucleotide kinase (MBI Fermentas, St. Leon-Rot, Germany) and [γ³²P]-ATP. Labeled RNA was denatured at 90⁰C for 2 min, slowly cooled down to room temperature to allow for intramolecular structure formation, and exposed for 15 min at room temperature to 0.1 , 0.001 , and 0.001 U/μl Rnase T1 (Ambion, Austin, USA) respectively. Rnase T1 specifically cleaves single stranded RNA after guanosine residues. Cleavage products were separated by denaturing 15% PAGE prior to autoradiography of dried gels. Predicted mfe structures, cleavage sites, and sites protected upon base pairing are indicate. ►: strong cleavage, E> : weak cleavage, (G): protected G. C: Control lanes.

Figure 7: Prediction and proof of target structure accessibilities. Predicted mfe structures and accessibility profiles of local mRNA targets a, T, b, T-a, and c, T-i. Bases targeted by siRNA or asODN are indicated at the structures. Accessibility profiles, representing accessibility probabilities of individual bases derived from the complete Boltzmann ensemble of secondary structures were calculated using the program TARGETscout. The accessible loop structure L1 in (b) is highlighted in light blue, d, Jagged-1 expression (MFI) in cells transfected with 100 (■) or 500 (D) pmol asODN corresponding in sequence to guide-siRNA targeting T, T-a, and T-i. Error bars represent standard deviations of 3 experiments.

Figure 8: Classifying guide-RNA structures, a, Classification of guide- structures according to accessibility of 573' ends and ΔG. Random coils (RC are most active, followed by stem-loop structures with free 5¹ and 3' nts (X-Y) and internal loop (IL) or 2-stem-loop (2SL) structures with pseudo-accessible ends. Structures lacking free 5' and/or 3' nts (0-X, 0-0, X-O) are inactive. Unstable structures (US) can fall into potential holes of active or inactive structures according to ambient conditions, b, Probability P of structure formation in dependency of ΔG. Considering 2 states, mfe folding and RC, then P is given by exp(- ΔG/RT)/(1+exp( AGfRT)). R: Universal gas constant; T: Absolute temperature.

Figure 9: Model describing the determination of RNA silencing by RNA secondary structures. SiRNA duplexes are recognized, unwound, and guide- strands are incorporated into RISC. Perfectly matching duplexes induce formation of Ago-2-containing RISC, mismatching duplexes induce formation of Ago-1-dependend complexes. Guide-strands linked to RISC can form stable secondary structures. Guide-RNA structures determine strength of silencing correlating with accessibility of terminal nts increasing form complexes I to VII. MRNA-targeting initiates via free ends of guide- structures, base-matching with guide-RNA 5'domains monitors for target specificity. Upon targeting, wobble pairings with guide-RNA 5" regions induce reprogramming or resolving of RISC* leading to Ago-1/2-independent silencing or antisense effects.

Figure 10: Programming Argonaut-dependence of RISC by siRNA structure design. Wobble base pairing between target and the 5'terminus of the guide- strands prevents Ago-1 and Ago-2 dependency (1). Conventional duplexes (2) and those inducing target wobbling through central regions of the guide strand (3,4,5) mediate Ago-2-dependent gene silencing. Mismatches within siRNA duplexes (6,7) result in Ago-1 -dependent silencing. Inhibition of jagged-1 gene expression (MFI) 72h post transfection of 293T cells with jagged-1 -specific siRNA (Control) or cotransfection including Ago-1 , Ago-2, or Ago-1 +2-specific siRNA. SD of 3 independent measurements.

Figure 11 : Co-delivery of Ago-1 -specific siRNA enhances gene knock down mediated by target-specific siRNA, the activity of which depends on Ago-2 protein. Inhibition of jagged-1 gene expression 72h post transfection of 293T cells with jagged-1 -specific siRNA or co-transfection including Ago-1 -specific siRNA. SD of 3 independent measurements.

Figure 12: Co-delivery of Argonaute-specific siRNA can modulate knock down of the Luciferase gene expression mediated by 10 pmol/well Luciferase-specific siRNAs (siRNA-1 and siRNA-2). Silencing activity decreases with the delivery of Ago-2-dependend (perfectly base-paired duplex) Ago-2-specific siRNA (si-Ago-2). Silencing activity is increased if only 5 pmol/well Luciferase-specific siRNA is delivered together with 5 or 0.5 pmol/well of a Ago-2-dependend (perfectly base-paired duplex) Ago-1 - specific siRNA si-Ago-1-2). An Ago-1 -dependent (imperfectly base paired duplex) Ago-1 -directed siRNA (si-Ago-1-1) which does not compete for cellular Ago-2 protein supports knock down most efficiently. Luciferase gene expression was detected 48h post transfection of 293T cells with a Luciferase gene expression vector (pGL2) and different Luciferase- and Ago-specific siRNAs. SD of 3 independent measurements.

Figure 13: Over-expression of Argonaute-2 protein enhances specific siRNA-mediated knock-down of Luciferase gene expression. Gene expression was monitored 48h post transfection of 293T cells with 500ng / 24-well pGL2 Luciferase expression vector, a, Cell were additionally transfected with 500ng / 24-well Argonaute-2 expression vector (pAgo-2) and/or 2,5 pmol / 24-well Luciferase-specific siRNA (Luci-siRNA). b, analog to a but total amounts per well of plasmid DNA and siRNA were adjusted using control DNA (pControl) and RNA (Control-siRNA). SD of 3 independent measurements.

Figure 14: Replication of S. thyphimurium (CFU) 5h post infection in control (transfected with control siRNA) and RNAi-disabled (RNAi KO) HEK293T cells transfected with Ago-1+Ago-2-directed siRNAs.

Figure 15: Replication of GFP-expressing S. thyphimurium (a, GFP expression; c, CFU) and L. monocytogenes (b, GFP expression; d, CFU) in control cells compared to Ago-1 , Ago-2, and Diceri knock-down HEK293T cells 6 h post infection.

Figure 16: Transfection of HEK293T cells with Argonaute-2 expressing plasmid DNA decreases susceptibility to S. thyphimurium. a, Bacterial growth (cfu) in infected cells, b and c, Bacterial proliferation in infected cells monitored by bacterial EGFP expression 12 and 36 h post infection.

EXAMPLES

1. Methods

SiRNA/asODN preparation and design. SiRNA were selected using the algorithm siRNAscouf (STZ Nucleic Acids Design, Berlin, Germany) targeting coding sequences. SiRNA single-strands were synthesized at Xeragon or Dharmacon as 21-mers, sense strands with dTdT 3'-ends antisense strands with dXdY 3'-ends including dT or dU nts (jagged-⁴!) or XY

3'-ends {Luciferase and GFP). SiRNA strands were annealed according to manufacturer's instruction resulting in 19 bp duplexes with 2 nt 3' overhangs. Qualities and quantities of ssRNA and duplexes were monitored using a bioanalyzer (Agilent Technologies, Palo Alto, USA). Jagged- 7-directed siRNA not included in Figures: t-a: sense:

5'-GAAACAGUAGCUGCCUGCCdTdT-S', antisense:

5'-GGCAGGCAGCUACUGUUUCdGdG-S'; t-l: sense: 5'-ACUUGCAUCGAUGGUGUCAdTdT-S', antisense:

5'-UGACACCAUCGAUGCAAGUdGdC-S'. Luciferase-directed siRNA: L-RC: sense: 5'-GAGGAGUUGUGUUUGUGGAdTdT-3', antisense: δ'-UCCACAAACACAACUCCUCCG-S'; L-3-10: sense:

5'-UCGGGGAAGCGGUUGCAAAdTdT-S', antisense: δ'-UUUGCAACCGCUUCCCCGACU-S'; L-US: sense:

5'-ACGACAAGGAUAUGGGCUCdTdT-S', antisense: δ'-GAGCCCAUAUCCUUGUCGUAU-S'; L-2SL: sense:

5'-CGUUCGGUUGGCAGAAGCUdTdT-S', antisense: δ'-AGCUUCUGCCAACCGAACGGA-S'; L-5-6-h: sense: 5'-AAAACGGAUUACCAGGGAUdTdT-S', antisense: δ'-AUCCCUGGUAAUCCGUUUUAG-S'; L-5-6-m: sense:

5'-AUGUGUCAGAGGACCUAUGdTdT-S', antisense:

5'-CAUAGGUCCUCUGACACAUAA-3^I; L-5-6-I: sense:

5'-AUCUACCUCCCGGUUUUAAdTdT-3^I, antisense: δ'-UUAAAACCGGGAGGUAGAUGA-S'; L-IL: sense:

5'-AUUCUGAUUACACCCGAGGdTdT-3', antisense:

5'-CCUCGGGUGUAAUCAGAAUAG-S'; L-5-0: sense:

5'-AACGCUUCCAUCUUCCAGGdTdT-3', antisense: δ'-CCUGGAAGAUGGAAGCGUUUU-S'; L-O-O: sense: 5'-UACAUUCUGGAGACAUAGCdTdT-S', antisense: δ'-GCUAUGUCUCCAGAAUGUAGC-S'. GFP-directed siRNA: G-US1 : sense:

5'-AGCGCACCAUCUUCUUCAAdTdT-3', antisense: δ'-UUGAAGAAGAUGGUGCGCUCC-S'; G-US2: sense: 5'-AACGUCUAUAUCAUGGCCGdTdT-S', antisense:

5'-CGGCCAUGAUAUAGACGUUGU-3^I, G-5-6-T1: sense:

5'-CGGCAUCAAGGUGAACUUCdTdT-S¹, antisense: δ'-GAAGUUCACCUUGAUGCCGUU-S'; G-5-6-T2: sense: 5'-AGAAGCGCGAUCACAUGGUdTdT-S', antisense: δ'-ACCAUGUGAUCGCGCUUCUCG-S'; G-2SL: sense:

5'-GCCCUGGCCCACCCUCGUGdTdT-3', antisense:

5'-CACGAGGGUGGGCCAGGGCAC-3¹; G-IL: sense:

5'-UGGAGUACAACUACAACAGdTdT-S', antisense: δ'-CUGUUGUAGUUGUACUCCAGC-S'; G-1-0: sense:

5'-ACAACGUCUAUAUCAUGGCdTdT-S', antisense:

5^>-GCCAUGAUAUAGACGUUGUGG-3¹. Ago-1/2-specific siRNA were selected with siRNAscout having a minimum of cross-homology to the Ago-

2/1 mRNA respectively. Ago-1 : sense: 5'-UGUAUGAUGGAAAGAAGAAdTdT-3', antisense:

5'-UUCUUCUUUCCAUCAUACAdCdA-3'; Ago-2: sense:

5'-GGAGAGUUAACAGGGAAAUdTdT-3', antisense:

5'-AUUUCCCUGUUAACUCUCCdTdC-S'. AsODN were selected using the algorithm TARGETscotvf (STZ Nucleic Acids Design, Berlin, Germany) and synthesized at Thermo Electron (UIm, Germany) with each 2 5' and 3' terminal phosphothioate bonds.

Construction and purification of plasmids. A fragment containing the human jagged-1 cDNA (accession no. AF003837) was excised by SaAnHI and Sa/I digestion from vector pBabe-Jagged-1 and subsequently cloned into the pcDNA3.1(+) plasmid (Invitrogen, Carlsbad, USA) using the unique SamHI and Xho\ restriction sites resulting in jagged-1 expression vector pcDNA-Jagged-1. All plasmids were prepared using the Endofree Plasmid Maxi Kit™ (Qiagen, Hilden, Germany). For RNA co-transfection, plasmid DNA was further purified under RNAse-free conditions by repetitive phenol extraction.

Evaluation of siRNA/asODN activity in tissue culture. GFP positive HEK 293T cells were analyzed for jagged-1 expression 72 h after co-transfection of siRNA (0.1-100 pmol) or asODN (100 or 500 pmol), jagged-1 expression vector pcDNA-Jagged-1 , and pEGFP-C1 (BD Biosciences Clontech, Palo Alto, USA) using Lipofectamine 2000 according to manufacturer's instructions (Invitrogen). Cells seeded in 24 well plates were detached using PBS containing 2 mM EDTA and subsequently stained with biotin- conjugated anti-jagged-1 (R&D Systems, Abingdon, UK) and allophycocyanin-conjugated streptavidin (BD Biosciences Pharmingen, San Diego, USA). Jagged-1 expression was analyzed on a FACS Calibur™ (BD Biosciences lmmunocytometry Systems, San Jose, USA), and quantified by gating on GFP positive cells and determining the median fluorescence intensity (MFI) of jagged-1 staining. Alternatively, the percentage of jagged-1 positive cells was measured. Apparent values of half maximal inhibition (IC₅₀ values) were determined from MFI values using the program GraFit (Erithacus Software, Horley, UK). To detect Ago-1 and 2 protein dependence of jagged-1 silencing, 293T cells were pre-transfected with 480 pmol Ago-1- and/or 480 pmol Ago-2-siRNA in 75 cm² tissue culture flasks. After 48h, cells were co-transfected and processed as described above with 20 pmol jagged-1 siRNA additionally including 50 pmol Ago-1- and/or 50 pmol Ago-2-siRNA per 24 well. Expression of Firefly luciferase in 293T cells was analyzed 48h post co-transfection of 20 pmol siRNA and pGL2-Basic (Promega, San Luis Obispo, USA). Activities of GFP-directed siRNA were monitored in 293T cells by fluoroscan using the Fluorskan Ascent fluorometer (Thermo Labsystems, Helsinki, Finland) 48 h post co- transfection of 20 pmol siRNA and pEGFP-C1.

Thermodynamic duplex profiling. Free energy values representing internal average stabilities of pentamer subsequences within siRNA duplexes were calculated using the program OligoWalk²⁸.

RNA secondary structure prediction. Mfe structures were predicted based on default parameters of mfold2.0 (ref. 19). Partition structures were predicted based on mfold2.0 default parameters implemented into the dynamic programming algorithm of the Vienna RNA package²⁹. For sequences selected in this study, mfe and partition structures are identical except for as-siRNA 2-4, IL1 , and IL2. For these sequences partition structures are better compatible with our model.

2. Results and Discussion

We interrogated the potential role of secondary structures of as-siRNA in RNAi. Secondary structures of as-siRNAs relating to active and inactive siRNA duplexes were predicted using mfolcP⁹ and McCaskill's partition function²⁰. The vast majority (69%) of as-siRNA structures encompass stem- loops with or without single-stranded 5' and 3' ends. Active structures contained more terminal free nts, mainly at the 3' ends, compared to inactive and random structures (see Fig. 5). Structures without free nts at either terminus were only observed among inactive sequences and about 1 in 5 active or inactive as-siRNA failed to form stable structures. We hypothesize that single-stranded ends of as-siRNA structures are required for efficient induction of RNAi.

We developed a structure-based siRNA selection program and identified a set of overlapping (1 or 2 nt shifts) as-siRNA sequences directed against the human jagged-1 gene relating to structures containing a conserved stem- loop element and 11 terminal unpaired nucleotides, the latter of which can be assigned either to the 5' end, to the 3¹ end or to both termini at varying distribution (Fig. 1). Selected structures were suitable to systematically evaluate the impact of terminal free nts of as-siRNA structures on RNAi, independent of Gibbs free energies (ΔG) of structure formation and target- related influences. Structures were termed according to numbers of free nts assigned to the termini, e.g. structure 6-5 comprises 6 5' and 5 3' unpaired nts. Notably, favorable structures 4-7 and 2-9 were predicted to frame unfavorable structure 0-0 (not a putative structure 3-8) without free nts at any terminus (Fig. 1b). Transitions from structure 4-7 to 0-0 to 2-9 were confirmed by enzymatic RNA secondary structure probing in vitro (see Fig. 6). The local mRNA target region T corresponding to the selected as-siRNAs was predicted inaccessible and unfavorable for targeting by complementary nucleic acids. To investigate target structure roles independently of as- siRNA structures, we selected as-siRNA structures t-a and t-i of the type 0- 10, both identical in geometry and unfavorable in terms of silencing but directed against an accessible (T-a) or an inaccessible (T-i) target region (Fig. 1b and Fig. 7).

Activities of duplexes containing the selected as-siRNA strands were tested in human cells in transient assays. Target gene expression was monitored (Fig. 1c) and doses resulting in 50% inhibition (IC₅₀) oijagged-1 expression were calculated. Strongest inhibition (IC₅₀ ~0.1 nM) was determined for structures 6-5, 4-7, and 2-9 containing ≥ 5 free 3' and ≥ 2 free 5' nts (Fig. 1b). Poor effects (IC₅O ~10² nM) were observed for structures 0-11 , t-a, t-i, and 10-1. Structure 0-0 did not show any activity (IC₅O ~10³ nM). Thus, free 3' and 5' ends of as-siRNA structures were critical for RNAi. A single-nt shift from siRNA 4-7 to siRNA 0-0 resulted in 7,000-fold higher IC₅₀ and by a further single-nt shift towards siRNA 2-9, full activity was restored. These differences are independent of target structure but reflect structural changes of as-siRNAs. We found only poor correlations between IC₅O values and thermodynamic duplex profiles, base preferences, and low 5'-antisense duplex stabilities, reported previously to correlate with siRNA activity^8"12. Target accessibilities did not correlate with RNAi either. We observed a correlation between concomitant occurrence of > 1 5' and > 3 3' unpaired nts within stem-loop structures of as-siRNAs and RNAi. For as-siRNA structures containing > 2 unpaired 5' nts, numbers of free 3' nucleotides strongly correlated (correlation coefficient r = 0.94) with siRNA activity (1/IC₅₀). Other structural parameters of as-siRNAs did not correlate with RNAi (Fig. 1b).

Extrapolating the observed relationship, completely unstructured as-siRNA strands should be most favorable and ΔG could show reciprocal correlation to the silencing activity. Mature miRNAs represent natural counterparts of as-siRNAs and systematic analyses revealed that structures of mature human miRNA²¹ are thermodynamically less stable (higher energy value) compared to structures based on random or human coding sequences (data not shown). Unstable structures (ΔG > 0) and RNA which cannot form any stable or unstable secondary structure (unstructured RNA; ΔG = +infinite) due to missing base-pairing possibilities are with 32% more abundant in miRNA compared to random structures (24%). Structures with internal loops (IL) or two stem-loops (2SL) appear more frequently among miRNA. Contrary to statistics, structures without free terminal nts were not observed among miRNA. Thus, termini and/or folding energies of mature miRNA structures appear crucial for miRNA action. We assessed unstructured sequences which can be described by a random coil polymer conformation (RC), unstable structures (US), IL and 2SL structures, and stem-loop structures directed against the mRNAs of the firefly luciferase (L) and the green fluorescent protein (GFP) (Fig. 2). Type 5-6 stem-loop structures identical in geometry but differing in ΔG (L-5-6-h, -m, and -I) or identical in geometry and energy but directed against different target regions (G-5-6-T1 and -T2) showed similar activities indicating that shapes of structures and not ΔG or mRNA targets determine siRNA activities. Strongest silencing was observed for unstructured sequence L-RC and unstable structures L-US and G-US1 followed by favorable stem-loop structures, however, unstable structure G-US2 failed to induce silencing. Structures G-1-0, L-5-0, and L-O- 0 were inactive. IL and 2SL structures showed moderate to good activities although they had no or only few free terminal nts. Their ΔG values allocate to 2 stems which can break up separately. Thus, closed ends of IL and 2SL structures are regarded as pseudo-accessible rather than inaccessible explaining the activity of these miRNA-assigned types of structures. Inhibition of gene expression correlated strongly (r = 0.89) with the numbers of free 3' nts but only moderately (r = 0.57) with ΔG (Fig. 2). Thus, regardless of guide strand preferences for sense- or as-siRNAs, structures of as-siRNAs represent major determinants of RNA silencing. In the following we refer to antisense strands when talking about guide-RNA.

We classify guide-RNA structures as follows: strongest silencing is induced by sequences which do not form secondary structures; second best are stem-loop structures with ≥ 2 free 5¹ and > 4 free 3' nts, followed by IL and 2SL structures, and stem-loop structures with short free ends. Stem-loops without free 5' and/or 3' nts are inactive indicating that accessible ends provide the condition for activity (see Fig. 8a). Algorithms predict unstable guide-siRNAs with frequencies of -25% at physiological salt conditions. If conditions change, such as in the cellular milieu or resulting from interactions with proteins of RISC, unstable foldings may become stable and must not be considered unstructured/active. Independent of the environment, around ΔG = 0 (folding probability = 1/2) there is a corridor of uncertainty as to whether structures are folded or not (see Supplementary Fig. 4b online). Only at |ΔG| ≥ 1.3 or > 2.8 kcal/mol structures are un-/folded with a probability of ≥ 90 or ≥99 %. This may explain that some unstable guide-siRNA structures (G-US1 , L-RC₁ L-US) are active and others (G-US2, 8-2-US, 7-3-US1/2) are not. For the latter, unfavorable mfe structures were predicted. Considering both, activity and predictability of RNA structures, most successful strategies will focus on identification of guide-RNAs which fail to form secondary structures and, secondly, sequences forming favorable mfe structures but no unfavorable suboptimal foldings.

For given mRNA targets of L nts in length, L-21 complementary 21 mer guide strands statistically containing -0.14% of most active unstructured sequences are possible. We investigated the possibility to expand the space of complementary guide-siRNAs in order to increase the absolute frequencies of active guide-structures (Fig. 3). We performed A to G (A>G) and C to U (OU) base exchanges within inactive guide-siRNA 0-0 and active species 2-9 as well as corresponding U>C and G>A exchanges within the sense strands. Such changes preserve target homology, induce wobble base pairing with the target, and can alter guide-RNA structures or DG and, hence, silencing activities (Fig. 3a). A OU exchange at position 2 of the guide-strand changed unfavorable structure 0-0 to favorable structure 3-8 resulting in enhanced silencing (Fig. 3b). A structure-neutral but energy- increasing change from structure 0-0 to structure 0-0-ΔG (3 exchanges) and the change to unstable unfavorable structure 8-2-US (5 exchanges) did not improve the parental molecule indicating that ΔG is not a determinant of RNAi. Changes from structure 2-9 to higher energy structure 2-4 (1 exchange) and internal loop structures IL1 and IL2 (2 exchanges) did not reduce silencing. Structure 2-4 was even more active compared to the parental molecule 2-9. Changes to unstable but unfavorable (only 3 free 3'nts) structures 7-3-US1 and 7-3-US2 resulted in loss of activity. Hence, target-neutral but guide-structure-relevant exchanges can improve active siRNAs or transform inactive species into active ones. The low activity of favorable structure 3-8, the decrease in activity from structure 2-4 to IL1 , and possibly the loss of function of unstable structures 7-3-US1 and 7-3-US2 indicate that wobble pairing with the target impairs silencing at 5' terminal regions of guide strands but is tolerated in a central position of structure 2-4. This finding is consistent with recent observations with miRNA²². According to equations in Fig. 4c, A>G and C>U exchanges increase the numbers of complementary guide-siRNA by > 3 Iog10 for target sequences with G/U base contents of 50% allowing accessing new active and more powerful siRNA (see Supplementary Discussion A online). Analogue degenerations are observed among sequences of mature miRNAs²¹ implying that miRNA- activity is modulated by mature miRNA structures.

The observed dependency on free ends of guide-structures has implications for gene silencing. The 5'region was described to determine specificity and binding strength of RISC^* whereas central positions and 3' ends seem to participate in catalysis²³²⁴. The Ago PIWI domain of A. fulgidus and recombinant human Ago-2 anchor guide-RNA 5'-ends which were suggested to initiate nucleation and to determine the distance to mRNA cleavage sites²⁵²⁶. Free dangling ends of guide-RNA structures appear more flexible and accessible than base-paired ends and more suitable for nucleation or interaction with proteins. The length of free ends of antisense RNA structures was reported to directly correlate with the kinetics of mRNA targeting and with activity²⁷. Guide-strands can be regarded as RISC- associated antisense RNA and we assume that terminal free nts determine the efficiency of mRNA targeting which might be rate-limiting in RNAi. We cannot decide whether mRNA targeting by RISC initiates via 5' or 3' ends of guide-siRNA. Empirically, cooperative base pairing after nucleation requires > 2 or 3 unpaired nts and our finding that 2 free 5' nts but > 3 free 3' nts are required for guide-siRNA function favors the idea that mRNA targeting initiates via 3' ends.

The decision which Argonaute protein is chosen for RISC and which subsequent pathway is initiated must be made before the mRNA is encountered and base matching can be monitored, possibly at the stage of the effector duplex representing a common precursor of the primarily Ago-1 - dependent miRNA pathway and Ago-2-dependent siRNA-mediated target cleavage. Silencing activities of duplexes described in Fig. 4 were monitored in Ago-1 , Ago-2, and Ago-1+2 knock-down cells (see methods). Silencing induced by mismatched duplexes, i.e. those with a mismatch at antisense strand position 15, was found to be Ago-1 -dependent whereas silencing induced by all other duplexes distinguished with Ago-2 knock-down indicating that the structure of the effector duplex determines the choice of the Argonaute protein and, hence, the silencing pathway. The moderate activity of structure 3-8, which gives rise to a single wobble base pair with the target but not within the siRNA duplex, did neither depend on Ago-1 nor on Ago-2. This can be explained if wobble pairing between targets and guide-RNA 5'regions induces reprogramming or resolving of RISC* leading to Ago-1 /2-independent silencing or antisense effects (see Fig. 9). Comparisons of duplexes IL2 with IL3 and 2-9 with 2-9-1 and 2-9-2 indicate that duplex-intrinsic base-wobbling and mismatches only marginally impair silencing (Fig. 4).

For computation, guide-RNA is treated like free molecules although they exhibit cellular function only in association with RISC. Such simplification can lead to misinterpretations. In this study, the profound correlations between parameters calculated for isolated guide-RNA and RNAi provide compelling evidence that guide-RNA structures play a crucial role in RNA silencing and can serve as basis for predicting siRNA activity with a resolution at the single-nt-level.

Targets of functional siRNA can coincide with targets of effective antisense oligodeoxyribonucleotides (asODN)^{13 15} and target structure predictions can improve the prediction of site efficacy^16"18. In these studies however, the impact of duplex or as-siRNA-related features on RNAi was not considered. AsODN activity depends on the accessibility of the target structure, which can be predicted by in silico methods^{33 36}. We investigated the impact of target accessibilities on RNAi independent of as-siRNA structures. Using our program TARGETscot/f we selected a highly accessible target site T-a and an inaccessible site T-i within the jagged-1 mRNA. Target T-a meets the requirements of an accessible site for asODN, i.e. containing a contiguous stretch of >10 bases (loop L1) likely of being unpaired. Average accessibility scores SC_acc of the targets T {SC_a∞ = 21.1%), T-a {SC_a∞ - 76.2%), and T-i (SCacc = 13.0%), as well as individual scores for each nt within these targets were calculated. Predicted mfe structures and calculated accessibility profiles are shown in Figure 7). Accessibility scores describe probabilities of local target sites or nts of being unpaired for the complete Boltzmann ensemble of mRNA secondary structures²⁰. High scores reflect targets accessible for hybridization with complementary nucleic acids. To verify target accessibility predictions, antisense oligodeoxyribonucleotides (asODN) corresponding in sequence to selected as-siRNA were tested for gene silencing. Only the sequence of asODN t-a directed against accessible target T-a showed detectable inhibition of target gene expression (see Fig. 7d). The strong differences in siRNA activities which were reflected by the predicted as-siRNA secondary structures were not related to the accessibility scores of the corresponding target sites in T (Fig. 1b and Fig. 7). Favorable as-siRNA structures 6-5, 4-7, and 2-9 each targeting inaccessible local sites in T successfully mediated gene suppression. Conversely, the unfavorable as-siRNA structure t-a (type 0-10) directed against an accessible mRNA target T-a as well as the unfavorable siRNA structures 10-1 , 0-0, 0-11 , and t-i targeting the inaccessible targets T and T- i, failed to induce RNAi. Thus, as-siRNA structures rather than target structures determine RNAi and accessible target structures are neither necessary nor sufficient for RNAi.

Thermodynamic structure predictions are based on the assumptions that the lowest free energy structure, the minimum free energy (mfe) structure, is the most likely one. Nevertheless, suboptimal foldings can exist and can be relevant as well. Mfe structures and suboptimal folds can be predicted by mfold and other related algorithms. The so called partition function considers all possible folds for a given RNA sequence including the mfe structure and suboptimal foldings as generated by mfold. In many cases partition structures can be drawn from the partition function. If no suboptimal folds occur, the partition structure is equivalent to the mfe structure. For highest congruity between predictions and expected real RNA structures we applied both the partition function and mfold and exclusively selected sequences for which partition structures were equivalent to mfe structures. These structures are depicted figures. That is, for selected RNA sequences no relevant suboptimal foldings exist and it is likely that the mfe structures comply with the real RNA structures. For sequences 2-4, IL1 , and IL2, however, suboptimal foldings occurred. In these cases we show partition structures which consider both mfe and suboptimal structures. In our examples, partition structures are highly compatible with observed siRNA activities.

G and U bases can form Watson-Crick and Wobble-base pairs. Consequently, sequences generated by A->G and C->U base exchanges are more competent in forming secondary structures compared to parental sequences and contain a smaller fraction of most active unstructured RNA. Furthermore, not all base exchanges may be tolerated during RNAi. Thus, as-siRNA sequences generated by base exchanges are expected to contain less than 0.14% of highly active species as calculated for random sequences. Nonetheless, the base-exchange technique dramatically increases the numbers of complementary guide strands allowing to access new active and more powerful siRNA.

It has been speculated that stable internal fold-back structures of guide- strands may exist in equilibrium with the duplex form, reducing the effective concentration and activity of siRNA¹¹. Thus, observed structure-function relationships could be crucial for siRNA duplex formation in vitro and/or in vivo. On that level, structures of sense- and as-siRNA would be on a par and one would assume equivalent relations between structures of sense-siRNA and RNAi. Such correlations were not observed. SiRNA activities do not correlate with Δ (r = 0.15), free 3' (r = 0.26) or free 5¹ nts (r = 0.17) of sense- siRNA structures, indicating that structures of as-siRNA play no role in the formation of effector duplexes in vivo. The quality of siRNA duplexes was monitored using a bioanalyzer and did not provide any evidence that guide- RNA fold-back structures impair duplex formation in vitro.

3' dT overhangs are standard in siRNA synthesis but difficult to consider by RNA folding algorithms which are based on the ribo-alphabet. Uracil but not Thymin can pair with Guanin and the decision of using dT or T instead of dU or U overhangs can alter guide structures if terminal dU/U was predicted to pair with G. In this study, 3'terminal dU/U was only substituted by dT/T if no impact on guide-structures was to be expected. The comparison of structures IL1 with IL2 (Fig. 4) and of structure 7-3-US1 with 7-3-US2 (Fig. 3) did not indicate any difference between 3' as dT and dU overhangs in our set of structures.

Further data show that programming of RNA silencing pathways may be effected by the structures of siRNA double strands (see Fig. 10). Wobble- base pairs within central regions of the guide strands mediate Ago-2 dependent gene silencing, wherein wobble base pairing between the target and the 5'-terminus of the antisense strands prevents both Ago-1 and Ago-2 dependency. Mismatches within siRNA duplexes result in Ago-1 -dependent silencing. A knockdown of the Ago-1 -dependent silencing pathway by co-delivery of Ago-1 -specific siRNA and jagged-1 specific siRNA enhances total RNA silencing (see Fig. 11). Ago-1 -dependent siRNA directed against Ago-1 is more effective than Ago-2-dependent siRNA directed against Ago-1. Ago-2- dependent siRNA directed against Ago-2 decreases silencing activity (see Fig. 12).

Over-expression of proteins of the silencing machinery, e.g. Ago-1 , enhances specific siRNA mediated gene silencing in human tissue culture cells (Fig. 13).

Knocking down expression of proteins involved in the RNA silencing machinery such as for example Ago-1 , Ago-2, and/or Diceri using gene- specific siRNAs disables RNAi and results in increased susceptibility of human tissue culture cells to microbial (bacterial) pathogens. Conversely, over-expression of proteins of the silencing machinery protects human tissue culture cells from microbial (bacterial) infection (Figure 14). Thus, RNAi defends human tissue culture cells from microbial (bacterial) invasion or mediates defence.

Susceptibility of human tissue culture cells to S. thyphimurium increases with knock-down of Ago-1 , Ago-2, and Diceri . Susceptibility of human tissue culture cells to L. monocytogenes increases strongly with siRNA-mediated knock-down of Diceri and slightly with Ago-1 knock-down (Fig. 13).

Susceptibility of human tissue culture cells to S. thyphimurium decreases with over-expression of Argonaute-2 protein (pAgo). A control plasmid carrying the same promoter has no effect (pControl). Concomitant siRNA- mediated knock-down of Diceri (siDcri ) annuls this effect resulting even in increased susceptibility. Control siRNAs directed against EGFP (siGFP) and Luciferase (siLuci) have no effect. Susceptibility of human tissue culture cells to L. monocytogenes increases strongly with knock-down of Diceri and slightly with Ago-1 knock-down (Fig. 16). REFERENCES

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Claims

1. A method for preparing a double stranded RNA molecule with target gene specific silencing activity which selectively interacts with an RNA- induced silencing complex (RISC) containing a predetermined species of Argonaute protein, comprising the steps

(a) identifying a double stranded RNA molecule directed to the mRNA of a target gene, wherein said RNA molecule comprises: (i) a double stranded portion of 9-35 nucleotides and optionally at least one 3¹ overhang,

(ii) an antisense strand which has a sufficient degree of complementarity to the mRNA of the target gene for RISC formation, and (iii) a sense strand which has a predetermined degree of complementarity to the antisense strand to provide a double stranded RNA molecule for specific interaction with an RISC containing a predetermined species of Argonaute protein, and

2. The method according to claim 1 wherein the antisense strand has a degree of complementarity of at least 50%, preferably of at least 70%, more preferably of at least 90% to said mRNA, wherein complementarity comprises Watson-Crick base pairs and wobble (G-U, U-G) base pairs.

3. The method according to any one claims 1 or 2 wherein the target gene specific silencing activity is a transcriptional gene silencing (TGS) activity or a post-transcriptional gene silencing (PTGS) activity preferably selected from RNA interference, and/or translational attenuation.

4. The method according to any one of claims 1-3 wherein a double stranded RNA molecule which selectively interacts with an Ago-2- containing RISC is prepared.

5. The method according to claim 4 wherein the sense strand has a degree of complementarity of 100% to the antisense strand, wherein complementarity comprises Watson-Crick base pairs and wobble (G-U, U-G) base pairs.

6. The method according to any one of claims 1-3 wherein a double stranded RNA molecule which selectively interacts with an Ago-1- containing RISC is prepared.

7. The method according to claim 6 wherein the sense strand has a degree of complementarity of less than 100% to the antisense strand, wherein complementarity comprises Watson-Crick base pairs and wobble (G-U, U-G) base pairs.

8. The method according to claim 7 wherein the double stranded portion of sense and antisense strand comprises at least one mismatch.

9. A method according to claim 8 wherein at least one mismatch is located between position 13-17 of an antisense strand having an length of 19-23 nucleotides.

10. The method according to any one of claims 1-3, wherein a double- stranded RNA molecule which has Argonaute-1 or/and Argonaute-2 independent cellular gene silencing activity is prepared.

11. The method according to claim 10, wherein a double stranded RNA molecule which selectively interacts with a RISC different from a RISC selected from Ago-1 containing RISC and Ago-2 containing RISC is prepared.

12. The method according to any one of claims 10 or 11 , wherein a double stranded RNA molecule which selectively interacts with a RISC selected from Ago-3 containing RISC and Ago-4 containing RISC is prepared.

13. The method according to any one of claims 10 to 12, wherein the antisense strand of the double stranded RNA is capable of forming at least one Wobble base pair (U-G, G-U) with the mRNA of the target gene.

14. The method according to any one of claims 1-13 wherein the double stranded RNA is a siRNA, shRNA or a miRNA.

15. The method according to any one of claims 1-14 wherein the length of the antisense strand and the sense strand is between 15 and 30 nucleotides, preferably between 19 and 25 nucleotides.

16. The method according to any one of claims 1-15 wherein the double stranded RNA molecule comprises a double stranded portion and at least one 3' overhang of from 1-10, preferably from 1-5 nucleotides.

17. The method of any one of the claims 1 to 16, wherein said antisense and/or said sense strand of the double stranded RNA molecule comprises at least one nucleotide analogue.

18. The method of claim 17 wherein said modified nucleotide analogue is selected from sugar-, backbone- and nucleobase-modified nucleotides and combinations thereof.

19. The method according to any one of claims 1-18 wherein the double stranded RNA molecule further comprises 5'- and/or 3'-modifications preferably selected from lipid groups, e.g. cholesterol groups and vitamins.

20. The method of any one of the claims 1 to 19, wherein said sense and said antisense strands are chemically and/or enzymatically synthesized.

21. Use of a double stranded RNA molecule obtainable by a method according to any one of the claims 1 to 20 for the manufacture of a reagent, a diagnostic or a medicament.

22. A method for regulating the expression of a target gene in a cell, an organism or a cell-free system, comprising the steps of

(a) preparing a double stranded RNA molecule according to any one of claims 1 to 20 or a precursor thereof, or a DNA molecule encoding

^■ said RNA molecule or precursor, and

23. A method for regulating the expression of a target gene in a cell, an organism or a cell-free system, comprising the steps of:

(i) a double stranded portion of 9-35 nucleotides and optionally at least one 3¹ overhang, (ii) an antisense strand which has a sufficient degree of complementarity to the mRNA of the target gene for RISC formation, and

24. A method for producing a cell, organism or cell-free system comprising a 5 double stranded RNA molecule which selectively interacts with a RISC containing a predetermined species of Argonaute protein, comprising the steps:

(a) preparing a double stranded RNA molecule according to any one of claims 1 to 16 or a precursor thereof, or a DNA molecule encoding o said RNA molecule or precursor, and

(b) introducing the molecule of (a) into said cell, organism or cell-free system under conditions under which target-specific nucleic acid silencing selectively occurs with a RISC containing a predetermined species of Argonaute protein. 5

25. A knockdown cell, non-human organism or cell-free system obtainable by the method of claim 24.

26. A method for examining the function of a target gene in a cell, an 0 organism or a cell-free system comprising:

(a) preparing a double stranded RNA molecule according to any one of claims 1 to 17 or a precursor thereof, or a DNA molecule encoding said RNA molecule or precursor,

(b) introducing the molecule of (a) into said cell, organism or cell-free 5 system under conditions under which target-specific nucleic acid silencing selectively occurs with a RISC containing a predetermined species of Argonaute protein, and

(c) observing the phenotype of the cell, organism or system of (b) and optionally comparing said phenotype to that of an appropriate o control cell, organism or system.

27. A reagent, diagnostic or medicament comprising a double stranded RNA molecule obtainable by a method according to any one of the claims 1 to

20.

28. A method for modulating target gene specific silencing activity in a cell, an organism or a cell-free system, comprising selectively increasing and/or suppressing the activity of at least one polypeptide of the gene silencing machinery selected from Argonaute proteins such as Ago-1 (elF2C1 ), Ago-2 (elF2C2), Ago-3 (elF2C3), Ago-4 (elF2C4), PIWIL 1 (HIWI)₁ PIWIL 2 (HILI), PIWIL 3 and PIWIL 4 (HIW! 2), preferably Ago-1 and/or Ago-2, and other proteins of the gene silencing machinery such as Dicer proteins, e.g. Dcr1 , or Dcr2; DGCR8 (Drosha, Pasha), R2D2

(dsRBD), NR, Fmri/Fxr, Vig, Tsn, Dmp68, Gemin3, Gemin4, Exportin-5 and Loquacious, and providing a double stranded RNA molecule directed to the mRNA of a target gene in said cell, organism or cell-free system.

29. The method according to claim 28 wherein the activity of said at least one polypeptide is selectively increased.

30. The method according to claim 29 wherein the activity of Ago-2 is selectively increased.

31. The method according to any one of claims 29 or 30 wherein the activity is selectively increased by overexpression of said at least one polypeptide and/or by adding an excess of said at least one polypeptide.

32. The method according to claim 28 wherein the activity of said at least one polypeptide is selectively suppressed.

33. The method according to claim 32 wherein the activity is selectively suppressed by gene-specific silencing of said polypeptide.

34. The method according to claim 33 wherein gene-specific silencing comprises administering double stranded RNA molecules, precursors thereof or DNA molecules encoding said RNA molecules or precursors thereof directed against the mRNA encoding said polypeptide of the gene silencing machinery.

35. The method according to any one of claims 32-34 wherein the activity of Ago-1 is suppressed.

36. The method according to claim 35 wherein Ago-1 suppression comprises administering double stranded RNA molecules, precursors thereof or DNA molecules encoding said RNA molecules or precursors thereof directed against Ago-1 mRNA.

37. The method according to claim 36 wherein the RNA molecules directed against Ago-1 mRNA specifically interact with an Ago-1 -containing RISC.

38. A Composition for target gene specific silencing comprising:

(a) a double stranded RNA molecule directed to the mRNA of a target gene, a precursor thereof or a DNA molecule encoding the double stranded RNA molecule or the precursor thereof, and

(b) (i) at least one polypeptide of the gene silencing machinery selected from Argonaute proteins, preferably Ago-1 and/or Ago

2, and other proteins of the gene silencing machinery or (ii) a nucleic acid encoding the polypeptide of (i), wherein component (b) is present in an amount or form to provide a selective activity increase of the polypeptide (i) or nucleic acid (ii).

39. The composition of claim 35 which is an expression system comprising (a) a DNA molecule encoding the double stranded RNA molecule directed to the mRNA of the target gene or a precursor thereof and (b) a DNA molecule encoding said at least one polypeptide of the gene silencing machinery wherein (a) and (b) are operatively linked to expression control sequences.

40. The composition of claim 38 or 39 wherein said polypeptide of the gene silencing machinery is Ago-2.

41. A composition for target gene specific silencing comprising:

(b) a double stranded RNA molecule directed to the mRNA encoding at least one polypeptide of the gene silencing machinery selected from Argonaute proteins, preferably Ago-1 and/or Ago-2, and other proteins of the gene silencing machinery, a precursor of the RNA molecule or a DNA molecule encoding the double stranded RNA molecule or the precursor thereof.

42. The composition according to claim 41 comprising

(a) a double stranded RNA molecule directed to the mRNA of the target gene and

(b) a double stranded RNA molecule directed to the mRNA of a polypeptide of the gene silencing machinery.

43. The composition of any one of claims 41 to 42 wherein said polypeptide is Ago-1.

44. The composition of any one of claims 38 to 41 which is a reagent, a diagnostic or a medicament.

45. A cell or non-human organism transformed or transfected with a composition of any one of claims 38-41.

46. An expression system which comprises at least one expression vehicle comprising the composition of claim 39.

47. A double stranded RNA molecule with gene silencing activity directed against an mRNA of a polypeptide of the gene silencing machinery, or a precursor thereof or a DNA molecule encoding said RNA molecule or precursor.

48. The compound of claim 47 directed against Ago-1 or Ago-2.

49. The compound of claim 48 directed against Ago-1 wherein the antisense strand and the sense strand comprise at least one mismatch.

50. A reagent, a diagnostic or a medicament comprising the compound of any one of claims 47-49.

51. Use of a polypeptide of the gene silencing machinery or a nucleic acid coding therefor for the manufacture of a medicament for the prophylaxis or treatment of disorders associated with dysfunctional gene expression including infectious diseases, particularly viral, bacterial or protozoal diseases.

52. The use of claim 47 wherein the polypeptide is Ago-2 and/or Diceri .

53. Use of a double stranded RNA with directed against Ago-1 or a precursor thereof or a DNA molecule encoding the RNA molecule or the precursor thereof for the manufacture of a medicament for the prophylaxis or treatment of disorders associated with dysfunctional gene expression including infection diseases, particularly viral, bacterial or protozoal diseases.