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WO2007092181A2 - Compositions et méthodes permettant de moduler la répression traductionnelle - Google Patents

Compositions et méthodes permettant de moduler la répression traductionnelle Download PDF

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WO2007092181A2
WO2007092181A2 PCT/US2007/002203 US2007002203W WO2007092181A2 WO 2007092181 A2 WO2007092181 A2 WO 2007092181A2 US 2007002203 W US2007002203 W US 2007002203W WO 2007092181 A2 WO2007092181 A2 WO 2007092181A2
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rck
polypeptide
rna
construct
protein
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WO2007092181A3 (fr
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Tariq M. Rana
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Unversity Of Massachusetts
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—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
    • C12N15/1137—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 against enzymes
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/14—Type of nucleic acid interfering N.A.
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    • C12N2330/10—Production naturally occurring

Definitions

  • RNA interference e.g. RNA interference (RNAi), transcriptional gene silencing (TGS), post-transriptional gene silencing (PTGS), quelling, co-suppression, and translational repression
  • RNA silencing has been observed in many types of eukaryotes, including humans, and utility of RNA silencing agents as both therapeutics and research tools is the subject of intense interest.
  • RNA silencing in eukaryotes, including small interfering RNAs (siRNAs) and endogenously expressed microRNAs (miRNAs, also known as small temporal RNAs (stRNAs)).
  • siRNAs small interfering RNAs
  • miRNAs endogenously expressed microRNAs
  • stRNAs small temporal RNAs
  • RNA silencing agents are produced by the cleavage of double-stranded RNA (dsRNA) precursors by Dicer, a nuclease of the RNase III family of dsRNA-specific endonucleases (Bernstein et al.,2001; Billy et al., 2001; Grishok et al., 2001; Hutvagner et al., 2001; Ketting et al., 2001; Knight and Bass, 2001; Paddison et al., " 2002; Park et al., 2002; Provost et al., 2002; Reinhart et al., 2002; Zhang et al., 2002; Doi et al., 2003; Myers et al., 2003).
  • dsRNA double-stranded RNA
  • siRNAs result when transposons, viruses or endogenous genes express long dsRNA or when dsRNA is introduced experimentally into plant or animal cells to associate with and guide a protein complex ca lied RNA-induced silencing complex (RISC) to direct the sequence-specific destruction of a complementary target mRNA by endonucleo lytic cleavage, a process known as RNA interference (RNAi) (Fire et al., 1998; Hamilton and Baulcombe, 1999; Zamore et al., 2000; Elbashir et al., 2001a; Hammond et al., 2001; Sijen et al., 2001; Catalanotto et al., 2002).
  • RISC RNA-induced silencing complex
  • miRNAs are the products of endogenous, non-coding genes whose transcripts form long, largely single-stranded RNA transcripts termed pri-rniRNAs.
  • Pri-miRNAs are sequentially processed, first in the nucleus by Drosha to form a ⁇ 65nt stem-loop RNA precursor termed a pre-miRNA, then in the cytoplasm by Dicer to form mature miRNAs of 21-23 nucleotides (Lagos-Quintana et al., 2001; Lau et al., 2001; Lee and Ambros, 2001; Lagos-Quintana et al., 2002; Mourelatos et al., 2002; Reinhart et al., 2002; Ambros et al., 2003; Brennecke et al., 2003; Lagos-Quintana et al., 2003; Lim et al., 2003a; Lim et al., 2003b).
  • miRNAs exist transiently in the cell as double-stranded molecules, one strand (usually the antisense strand) is incorporated into RISC while the other strand (usually the sense strand) is rapidly degraded. In recent years, hundreds of miRNAs have been identified in animals and plants
  • miRNAs have been shown to play a key role in human disease, including cancer (He et al, 2005; Lu et al, 2005; Poy et al, 2004; Vella et al, 2004).
  • cancer He et al, 2005; Lu et al, 2005; Poy et al, 2004; Vella et al, 2004.
  • let-7 miRNA up-regulate RAS protein in lung cancer cells, demonstrating a possible role of miRNA in tumorigenesis.
  • miRNAs may mediate RNA silencing by distinct but interchangeable mechanisms which are largely determined by the degree of complementarity between the miRNA and its target mRNA (Schwarz and Zatnore, 2002; Hutvagner and Zamore, 2002; Zeng et al., 2003; Doench et al., 2003; Ambros, 2004; He and Hannon, 2004).
  • miRNAs with a perfect complementarity to a corresponding target mRNA behave like siRNAs in that they direct target mRNA cleavage by the RNAi mechanism (Zamore et al., 2000; Elbashir et al., 2001a; Rhoades et al., 2002; Reinhart et al., 2002; Llave et al., 2002a; Llave et al., 2002b; Xie et al., 2003; Kasschau et al., 2003; Tang et al., 2003; Chen, 2003; Zeng et al., 2003; Yekta et al., 2004).
  • miRNAs are generally not fully complementary to their mRNA target. Rather, most animal miRNAs have imperfect complementarity with sequences in the 3' untranslated region (3'-UTR) of the target mRNA and mediate RNA silencing by facilitating the recruitment of the miRNA-loaded RISC complex (tniRISC) to the target mRNA, thereby blocking its translation (Mourelatos et al., 2002; Hutvagner and Zamore, 2002; Caudy et al., 2002; Martinez et al., 2002; Abrahante et al, 2003; Brennecke et al., 2003; Lin et al, 2003; Xu et al., 2003).
  • tniRISC miRNA-loaded RISC complex
  • the present invention is based, at least in part, on the discovery that certain protein components of P-bodies (cytoplasmic foci of translational repression) are also found within miRNA-loaded RISC ("miRISC ”) complexes and are key mediators of miRNA-mediated translational repression in vitro and in vivo.
  • miRISC miRNA-loaded RISC
  • the invention is based, at least in part, on the identification of sequestration proteins (e.g. RCK polypeptides and orthologs thereof) which facilitate the recruitment of miRISC complexes and formation of P-bodies where translational repression of a complementary target mRNA by the miRNA takes place.
  • the invention demonstrates that the identified sequestration proteins are capable of modulating miRNA-mediated translational repression of endogenous miRNAs. Further, the invention provides compositions and methods of modulating said sequestration proteins, thereby modulating said miRNA-mediated translation repression. In particular, the invention provides methods for regulating miRNA-mediated RNA silencing by modulating the sequestration of miRISC to the P-body both in vivo and in vitro. Still further, the compositions and methods of the invention have application in the development of human diagnostic and therapeutic agents.
  • the invention provides methods for enhancing the potency or specificity of miRNA-mediated RNA silencing of one or more miRNA target genes in a cell by increasing the activity or concentration of a sequestration protein in a cell such that number or concentration of miRISC complexes in the P-bodies of a cell are increased.
  • the invention provides methods for decreasing rniRNA- mediated RNA silencing of one or more miRNA target genes by decreasing the activity or concentration of a sequestration protein such that the number or concentration of m ⁇ RISC complexes in a P-body is decreased.
  • the methods of the invention employ modulators of endogenous sequestration proteins (e.g. inhibitors or enhancers of sequestration proteins).
  • th e methods of the invention employ exogenous sequestration proteins which are added to a cell to increase sequestration activity.
  • the methods of the invention are suitable for modulating miRNA silencing both in vitro and in vivo. In vivo methodologies are useful for both general miRNA silencing modulatory purposes as well as in therapeutic applications in which miRNA silencing modulation (e.g., inhibition) is desirable. Modulation of miRNA silencing is of use in investigation of disease states (e.g., oncogenesis and infectious disease) where miRNA silencing is aberrant or altered. Insulin secretion has recently been shown to be regulated by at least one miRNA (Poy et al. 2004), and a role for miRNAs has also been implicated in spinal muscular atrophy (SMA; Mourelatos et al.
  • SMA spinal muscular atrophy
  • the invention is directed to a method for enhancing miRNA- mediated translational repression in an extract, cell, cell lysate, or organism, the method comprising introducing an effective amount of a sequestration polypeptide or an ortholog or bioactive fragment thereof, to the extract, cell, cell lysate, or organism.
  • the invention is directed to a method for modulating miRNA-mediated translational repression in an extract, cell, cell lysate, or organism, the method comprising introducing an effective amount of a modulator of (i) a sequestration polypeptide, (ii) an ortholog of a sequestration polypeptide, or (iii) a bioactive fragment of a sequestration polypeptide, to the extract, cell, cell lysate, or organism.
  • the sequestration polypeptide is an RCK-polypeptide.
  • the ortholog is a RCK ortholog.
  • the bioactive fragment is a RCK bioactive fragment.
  • the invention is directed to the method of reducing miRNA-mediated translation repression.
  • the modulator is a siKNA or shRNA capable of silencing: (i) the sequestration polypeptide, (ii) the ortholog, or (iii) the sequestration polypeptide fragment.
  • the invention is directed to a method for treating a miRNA- associated disease or disorder in a subject, the method comprising administering to the subject an effective amount of a sequestration polypeptide or an ortholog or bioactive fragment thereof, thereby treating the disease or disorder.
  • the invention is directed to a method for treating a miRNA- associated disease or disorder in a subject, the method comprising introducing an effective amount of a modulator of: (i) a sequestration polypeptide, (ii) an ortholog of a sequestration polypeptide, or (iii) a bioactive fragment of a sequestration polypeptide, thereby treating the disease or disorder.
  • the sequestration polypeptide is an RCK-polypeptide.
  • the ortholog is a RCK ortholog.
  • the bioactive fragment is a RCK bioactive fragment.
  • the modulator is a siRNA or shRNA capable of silencing: (i) the sequestration polypeptide, (ii) the ortholog, or (iii) the sequestration polypeptide fragment.
  • the sequestration polypeptide is expressed by an exogenously added vector encoding said polypeptide.
  • the cell is a mammalian cell.
  • the extract is selected from the group consisting of a cellular extract, nuclear extract, cytoplasmic extract, protein extract, partially purified protein extract, and purified protein extract.
  • the bioactive fragment is a DEAD box domain.
  • the present invention provides compositions for both general and sequence-specific miRISC modulation.
  • the invention provides miRISC sequestration agents (e.g., small RNA oligonucleotides (e.g. 15-30mer RNA oligonucleotides) as well as chemically-modified variants thereof, e.g., locked nucleic acid (LNA) and phosphorothioate-modif ⁇ ed small RNA oligonucleotides) for modulating miRNA silencing in vitro and in vivo.
  • LNA locked nucleic acid
  • the present invention also contemplates compositions and methods by which to selectively inhibit miRNA silencing in a targeted (e.g-, sequence-specific) manner.
  • the invention therefore also features miRISC tethering constructs which recruit a miRISC complex to a particular target RNA in a cell.
  • the invention features a miRISC tethering construct having the formula, S L T, wherein S is a sequestration moiety, L is a Unking moiety, and T is a targeting moiety.
  • the invention is directed to miRISC tethering construct suitable for use in repressing translation of a target RNA, comprising (a) a sequestration moiety which is capable of recruiting a miRISC complex; (b) a targeting moiety capable of binding to the target RNA; and (c) a linker moiety that links the targeting moiety and the sequestration moiety, wherein the tethering construct tethers the miRISC complex to the target mRNA.
  • the sequestration moiety of the construct is a RCK polypeptide or an ortholog or bioactive fragment thereof. In another embodiment, the sequestration moiety of the construct is a RCK interacting polypeptide or an ortholog or bioactive fragment thereof. In some embodiments, the RCK interacting polypeptide of the construct is a component of the miRISC complex. In further embodiments, the component of the miRISC complex of the construct is Argonaute-1 or Arognaute-2. In still further embodiments, the sequestration moiety of the construct is a RCK modulatory agent. In some embodiments, the RCK modulatory agent of the construct is a RCK interacting nucleic acid.
  • the RCK interacting nucleic acid is a small RNA having high binding affinity to a RCK polypeptide or an ortholog or bioactive fragment thereof.
  • the small RNA is chemically- modified.
  • the chemically-modified RNA comprises a modified nucleotide selected from the group consisting of a 2-fluoro nucleotide, a 2'-O-methyl nucleotide, and a backbone-modified nucleotide.
  • the targeting moiety of the construct is an oligonucleotide targeting moiety that is complementary to the target RNA.
  • the oligonucleotide targeting moiety is a small RNA oligonucleotide of about 10 to about 30 nucleotides in length. In further embodiments, the oligonucleotide targeting moiety is about 15 nucleotides in length. In still further embodiments, the oligonucleotide targeting moiety is chemically-modified.
  • the chemically-modified targeting moiety comprises a modified nucleotide selected from the group consisting of a 2-fluoro nucleotide, a 2'-O-methyl nucleotide, and a backbone- modified nucleotide.
  • the oligonucleotide targeting moiety of the construct is complementary to a 3'UTR of a target mRNA.
  • the targeting moiety is a polypeptide targeting moiety.
  • the polypeptide targeting moiety of the construct is a RNA binding polypeptide, or an ortholog or bioactive fragment thereof.
  • the RNA binding polypeptide of the construct is a mammalian RNA binding polypeptide. In further embodiments, the RNA binding polypeptide of the construct is a viral RNA binding polypeptide. In another embodiment, the viral RNA binding polypeptide of the construct is selected from the group consisting of a TAT polypeptide, a REV polypeptide, or a TAR binding polypeptide.
  • the targeting moiety of the construct targets an mRNA encoding a protein involved in a disease or disorder.
  • the disease or disorder is ALS.
  • the mRNA of the construct is a mutant SODl mRNA.
  • the linking moiety of the construct comprises a phosphodiester or peptide bond. In other embodiments, the linking of the construct moiety comprises an oligonucleotide or polypeptide linker.
  • the invention is directed to a pharmaceutical composition
  • a pharmaceutical composition comprising a tethering construct a pharmaceutically acceptable carrier.
  • the pharmaceutically-acceptable carrier is a nanotransporter.
  • the invention is directed to a method of repressing translation of a target RNA in a cell, comprising introducing a tether construct, under conditions such that the agent represses translation of the target RNA within the cell.
  • the gene encodes a protein associated with a disease or disorder.
  • the gene encodes a mutant protein.
  • the gene encodes a mutant SODl protein.
  • the invention is directed to a method for treating a subject having or at risk for a disease or disorder characterized or caused by the overexpression or overactivity of a cellular protein, comprising administering to the subject an effective amount of a tethering construct.
  • Figure IA depicts results indicating that depletion of RCK/p54 has no significant effect on RNAi activity in vivo.
  • Total cell extracts from HeLa cells co-expressing Myc- Ago2 and YFP-Agol, YFP-Dcp2, YFP-RCK/p54, YFP-eBF4E, YFP-Lsml or YFP were treated with +/- RNase A followed by Myc-Ago2 immunoprecipitation (IP).
  • TCE Total cell extract
  • anti-Myc IPs were analyzed by immunoblot using anti-GFP and anti- Myc antibodies.
  • Figure IB depicts in vivo localization of RCK/p54 and Ago2 to P-bodies.
  • HeLa cells expressing YFP-Lsml and CFP-Ago2 (panels a, b, and c), YFP-RCK/p54 and CFP- Ago2 (panels d, e, and f) were visualized by confocal microscopy at 24 h post- transfection.
  • FIG. 1C depicts interactions between RCK/p54 and Ago2 in P-bodies by fluorescence resonance energy transfer (FRET).
  • FRET fluorescence resonance energy transfer
  • Figure ID depicts FRET efficiencies between different P-body protein donor: acceptor pairs.
  • HeLa cells co-expressing YFP-RCK/p54 and CFP-Ago2, YFP-Lsml and CFP-Ago2, YFP-RCK/p54 and CFP, YFP-Agol and CFP-Ago2, YFP-Ago2 and CFP- Agol, YFP-RCK/p54 and CFP-Ago-1, as well as YFP-Agol and CFP were fixed and FRET efficiencies were measured.
  • Figure IE depicts results further indicating that depletion of RCK/p54 has no significant effect on RNAi activity in vivo.
  • HeLa cells were transfected with siRNAs against CDK9 mismatch (control), RCK/p54, and Ago2. 24 h later cells were transfected again with EGFP and RFP reporter plasmids and varying amounts (2, 10, and 5OnM) of siRNA against EGFP. 24 h after the second transfection, RNAi efficiencies were analyzed (see Experimental Procedures). To quantify the effect of depleting RCK/p54 and Ago2 on RNAi, the ratio of GFP/RFP signals was normalized to that observed in the absence of GFP siRNA (0 nM).
  • Figure IF depicts quantification of siRISC cleavage activity in vitro after depletion of RCK/ ⁇ 54. Cleavage activity of siRISC targeting EGFP mRNA was quantified as a function of protein content in extracts of HeLa cells depleted of RCK/p54.
  • Figure IG depicts subcellular localization of endogenous Ago2 in HeLa cells.
  • HeLa cells were analyzed by immunofluorescence using antibodies against endogenous Ago2 (panel a) and Lsml (panel b), and stained with Hoechest 33258 to visualize the nucleus. The images were digitally merged to indicate co-localization of Ago2 and Lsml (panel c). Arrows point to P-bodies.
  • Figure 2A depicts the experimental outline to purify active human RISC.
  • the guide strands of siRNA complexes targeting GFP (si-GFP) were conjugated with 3 ' biotin (si-GFP-Bi; blue strands) and transfected into HeLa cells.
  • RISCs were captured by incubating cell extracts with streptavidin-magnetic beads.
  • Figure 2B depicts results indicating that target mRNA is cleaved by biotin- captured RISC.
  • Bead (B) and supernatant (S) phases of captured RISC were incubated with 124-nt 32 P-cap-labeled GFP target mRNA. The reactions were stopped after 120 min, and products were resolved on 6% denaturing polyacrylamide gels.
  • Figure 2C depicts results indicating biotin-captured RISC contains proteins associated with mRNA processing.
  • Active human RISC from HeLa cells expressing Flag- Ago 1 was captured by biotin-siRNA and its protein composition was analyzed by immunoblot using anti-Flag, anti-Ago2, anti-RCK/p54, anti-Lsml, and anti-eIF4E antibodies.
  • FIG. 2D depicts results indicating that RCK/p54 is a translational repressor in human cells.
  • Incorporation of [ 35 S]methionine into HeLa cells was used to measure general translational activity.
  • Cells were transfected with siRNAs targeting mismatched CDK9 (control) or RCK/p54. Mock control cells were treated with the transfection reagent only.
  • At 24 h post-transfection cells were incubated for 1 h in medium lacking Met and Cys, and metabolically labeled with [ ⁇ S]methionine (see Experimental Procedures).
  • As a control for passive uptake of [ ⁇ ⁇ S] mock cells were treated with the translation inhibitor, cycloheximide (CHX). Cell incorporation Of[ 3 ⁇ S] is shown as cpm versus time after adding [35S].
  • CHX cycloheximide
  • Figure 2E depicts specific knockdown of RCK/p54 in HeLa cells.
  • HeLa cells were transfected with 50 nM siRNA against RCK/p54, harvested at 24, 48, and 72 h post-transfection, and total cell extracts were prepared. Analysis by immunoblot shows the specific knockdown of RCK/p54 protein without changing the protein levels of Lsml or Ago2.
  • Figure 2F depicts specific depletion of RCK/p54 mRNA after siRNA treatment.
  • Total RNA samples (3 ⁇ g) from HeLa cells transfected with siRNA against RCK/p54 were reverse-transcribed and analyzed by quantitative PCR to quantify mRNA levels.
  • RCK/p54 mRNA levels were normalized to GAPDH mRNA and are presented relative to mock treatment. Data are from two representative, independent experiments.
  • Figure SA depicts affinity-purified miRISCs associated with PCK/p54 retain cleavage activity.
  • To purify miRISC associated with RCK/p54 magnetic protein A beads coupled with rabbit IgG, rabbit anti-Ago2 or rabbit anti-RCK/p54 antibodies were incubated with HeLa cytoplasmic extracts. After immunoprecipitation, RISC activities were analyzed by incubating the supernatant (S) or bead (B) phases with 182-nt 32 P-cap- labeled let-1 substrate mRNAs having a perfectly complementary or mismatched sequence to the let-1 miRNA. Cleavage products were resolved on 6% denaturing polyacrylamide gels (CE, cytoplasmic extract; PM, perfect match; MM, mismatch).
  • Figure 3B depicts affinity-purified miRISCs retain cleavage activity, let-7 miRISC cleavage of a perfectly matched RNA target was inhibited by 2'-O-Me oligonucleotides complementary to let-7 miRNA (let-7-2'-O-Me or let-7-2'-O-Me-biotin [Bi]).
  • a 182-nt 32 P-cap-labeled let-7 substrate mRNA was incubated with the supernatant (S) or bead (B) phases of captured miRISC. The reactions were stopped after 120 min, and products were resolved on 6% denaturing polyacrylamide gels.
  • FIG. 3C depicts miRISCs contain proteins associated with mRNA processing.
  • Cytoplasmic extracts of HeLa cells expressing Flag-Ago2 and Myc-Agol were incubated with 2'-0-Me oligonucleotides complementary to let-7 miRNA (let-7-2'-O- Me or /e/-7-2'-O-Me-biotin [Bi]), affinity purified by streptavidin-magnetic beads to capture let-7 miRISC.
  • Supernatant (S) and beads (B) after biotin capture were analyzed by immunoblot using anti-Myc, anti-Flag, anti-RCK/p54, and anti-eIF4E antibodies.
  • Figure 4 depicts results indicating that depletion of RCK/p54 disrupts P-bodies and disperses the localization of human Ago2.
  • HeLa cells were co-transfected with Myc-Ago2 and siRNA against human RCK/p54 (lower panels) or CDK9 mismatch (control; upper panels).
  • cells were analyzed by immunofluorescence using antibodies against Myc-Ago2 (panels a and e) and against the P-body proteins Lsml (panels b and f) and RCK/p54 (panels c and g). Cells were stained with Hoechst 33258 to visualize nuclei and images were digitally merged (panels d and h).
  • Figure 5A depicts specific knockdown of Lsml in HeLa cells by siRNA.
  • HeLa cells were transfected with siRNA against Lsml and harvested at 24, 48, and 72 h post- transfection, and total cell extracts were analyzed by immunoblot with antibodies against Lsml or GAPDH.
  • Figure 5B depicts depletion of Lsml disrupts P-bodies.
  • HeLa cells were transfected with siRNA against Lsml, Ago2, RCK7p54, or GAPDH.
  • cells were analyzed by immunofluorescence using antibodies against Lsml and Myc tag for Ago2.
  • Cells were stained with Hoechst33258 to visualize nuclei, and images were digitally merged.
  • FIG. 5C depicts results indicating that RCK/p54 interacts with Myc-Ago2 in Lsml-depleted cells.
  • HeLa cells were transfected for 48 h with Myc-Ago2 and control siRNA or siRNA against Lsml, total cell extracts (TCE) were prepared, and Myc-Ago2 was immunoprecipitated from an aliquot of TCE (EP).
  • TCE and anti-Myc IPs were analyzed by immunoblot using anti-Myc, anti-RCK/p54 and anti-Lsml antibodies.
  • Figure 5D depicts affinity-purified RCK/p54 and Ago2 from Lsml-depleted cell extracts retain miRISC activity.
  • HeLa cells were transfected for 48 h with siRNA against Lsml, and cytoplasmic extracts were prepared. These extracts were incubated with magnetic protein A beads coupled with rabbit IgG, rabbit anti-Ago2 or rabbit anti- RCK/p54 antibodies to purify miRISC associated with RCK/p54. After immunoprecipitation, RISC activities were analyzed by incubating the supernatant (S) or bead (B) phases with 182-nt 32 P-cap-labeled let-1 substrate mRNAs having a perfectly matched or a mismatched sequence to the let-1 miRNA. Cleavage products were resolved on 6% denaturing polyacrylamide gels (MM, mismatch).
  • Figure 6A depicts the in vivo effect of depleting RCK/p54, Lsml, and Ago2 on siRNA dose-dependent RNAi activity.
  • HeLa cells were transfected with siRNAs against CDK9 mismatch (control), RCK/p54, Lsml, and Ago2. 24 h later cells were transfected again with EGFP and RFP reporter plasmids and varying amounts (2, 10, and 5OnM) of siRNA against EGFP. 24 h after the second transfection, RNAi efficiencies were analyzed (see Materials and Methods).
  • Figure 6B depicts results indicating that depletion of RCK/p54 has no effect on in vitro siRISC cleavage activity.
  • HeLa cells were transfected with siRNAs targeting RCK/p54, Lsml, Ago2, or CDK9 mismatch (control).
  • 24 h after the first transfection cells were again transfected with 50 nM siRNA targeting EGFP.
  • 24 h later cytoplasmic extracts (CE) were made (see Materials and Methods), and varying amounts (20, 50, 100 ⁇ g) of total CE protein were incubated with a 124-nt, 32 P-cap-labeled GFP target mRNA. The reactions were stopped after 60 min, and products were resolved on 6% denaturing polyacrylamide gels.
  • Figure 6C depicts quantification of siRISC cleavage activity in vitro after depletion of RCK/p54, Lsml, or Ago2.
  • Cleavage activity of siRISC targeting EGFP mRNA was quantified as a function of protein content in extracts of HeLa cells depleted ofRCK/p54, Lsml, or Ago2.
  • Figure 7 A depicts release of general translational repression by silencing
  • Figure 7B depicts results indicating that depletion of RCK/p54 releases translational repression in a luciferase reporter system.
  • HeLa cells were transfected with siRNAs against CDK9 mm (control), RCK/p54, GWl 82, Lsml, or Ago2. 24 h later, cells were co-transfected with siRNA (1 x perfectly matched [PM] site) or miRNA (4 x bulged sites) luciferase reporters in the presence of CXCR4 siRNA.
  • R. reniformis luciferase (RL) activities were measured 24 h after the second transfection and normalized to firefly luciferase (FL) activity as a control.
  • Figure 7C depicts results indicating that depletion of RCK/p54 releases repression of RAS protein translation.
  • HeLa cells were transfected with 100 nM of 2'- O-Me oligonucleotide (let-12'-0-Me inhibitor or 2'-0-Me control), and siRNA against RCK/p54 or CDK9 mm control. 24 h after transfection, the cells were counted and harvested (see Materials and Methods). The protein contents of total cell extracts were resolved by SDS-PAGE and analyzed by immunoblot using anti-RAS and anti-actin antibodies.
  • Figure 7D depicts results indicating that depletion of RCK/p54 releases repression of luciferase protein translation by 3'UTRs of NRAS and KRAS.
  • HeLa cells transfected with an ifr-luc-expressing vector, pRL-TK, and a Pp- luc-expressing vector, pGL3-controL, pGL3-NRAS, or pGL3-KRAS were co-transfected with 100 nM of 2'-O- Me oligonucleotide (let-1 2' -O-Me inhibitor or 2'-O-Me control) and siRNA against RCK/p54 or CDK9 mm control.
  • Figure 8 depicts quantitative PCR (qPCR) results indicating that RCK/p54 is a general translation repressor.
  • Total RNA samples from HeLa cells transfected with let-1 2'0Me inhibitor or siRNA against RCK/p54 were reverse-transcribed and analyzed by qPCR to quantify mRNA levels.
  • RAS mRNA levels were normalized to GAPDH mRNA and are presented relative to mock treatment.
  • FIG. 9 depicts a model for human RISC function involving miRNA and siRNA.
  • RISC contains Ago2, Agol, RCK/p54 (labeled p54), and other known (e.g., dicer and TRBP) and unidentified proteins and is distributed throughout the cytoplasm.
  • RISC programmed with the guide strand of siRNA binds to its target mRNA by perfectly matching, base pairs, cleaves the target RNA for degradation, recycles the complex, and does not require Pbody structures for its function.
  • RISC programmed with miRNA
  • P-bodies Multiple numbers (n) of RISC programmed with miRNA (miRISC) bind to target mRNA by forming a bulge sequence in the middle that is not suitable for RNA cleavage, accumulate in P-bodies, and repress translation by exploiting global translational suppressors such as RCK/p54.
  • the translationally repressed mRNA is either stored in P-bodies or enters the mRNA decay- pathway for destruction.
  • the present invention is based, at least in part, on the discovery of previously unrecognized activity of several P-body proteins in miRNA-mediated translational repression.
  • the invention features the defining of a role for several sequestration polypeptides (e.g. RCK polypeptides and orthologs thereof) in the formation of P-bodies, the recruitment of miRISC to P-bodies, and the translational repression of target mRNAs by miRISC complexes.
  • the invention provides modulators of sequestration polypeptides and orthologs thereof.
  • the invention features the activity of the RCK polypeptides or orthologs thereof in modulating miRNA translational repression.
  • RISC refers to the proteins and single-stranded polynucleotides that interact to recognize target RNA molecules. Demonstrated components of RISC include Dicer, R2D2 and the Argonaute family of proteins, as well as the guide strands of siRNAs and miRNAs.
  • miRISC complex refers to a complex of RISC proteins loaded with miRNA.
  • siRISC complex refers to a complex of RISC proteins loaded with siRNA.
  • miRISC sequestration agent refers to an agent (e.g. a sequestration polypeptide or sequestration oligonucleotide) which modulates (e.g. promotes) translation repression in a cell.
  • a sequestration agent of the invention promotes miRNA-mediated translation repression.
  • the sequestration agents of the invention can promote miRNA-mediated translation repression by recruiting miRISC to the P bodies of a cell or promoting the formation of P bodies which are capable of miRNA-mediated translational repression (e.g., P bodies containing miRISC).
  • exemplary sequestering agents include the sequestering polypeptides of the invention.
  • the term "sequestration polypeptide" includes polypeptides having the amino acid sequences set forth in subsections II, infra, (e.g. RCK polypeptides) as well as variants having sufficient sequence identity to function in the same manner as the described polypeptides.
  • a certain amount of amino acid substitution e.g., conservative amino acid substitution
  • some deletion or insertion can be tolerated in a sequestration polypeptide of the invention (or homolog or bioactive fragment thereof) while retaining the same function of the polypeptide.
  • tethering construct refers to a synthetic or non-natural construct of the invention which is capable of promoting the recruitment of miRISC to P-bodies in a sequence-specific manner.
  • a tethering construct of the invention is designed to facilitate the recruitment of a sequestering polypeptide to a target RNA (e.g. target mRNA), i.e., the agent recruits a miRISC complex containing a particular miRNA having a sequence complementary (i.e., sufficiently complementary) to the sequence of a target mRNA.
  • the sequestration agent in a manner that is independent of the sequence of the miRNA component of the miRISC complex.
  • P body refers to a site or foci of mRNA turnover within the cytoplasm of eukaryotic cells.
  • P-bodies contain repressor proteins and lack translation initiation factors and ribosomal components, thereby repressing the translation of associated mRNAs unl til they are eventually degraded (see Liu et al., 2005; Pillai et al., 2005; Rossi et aL, 2005; Send and Blau, 2005).
  • modulator or “modulating agent” as used herein shall refer to any compound or molecule, for example, a small molecule, nucleic acid (e.g., RNAi agent, siRNA, miRNA, shRNA, antisense molecule, ribozyme), peptide, or polypeptide (e.g. antibody) capable of affecting a change in the expression, activity, function, or structure of a protein.
  • nucleic acid e.g., siRNA, miRNA, shRNA, antisense molecule, ribozyme
  • peptide e.g. antibody
  • sequestration modulating agent includes agents or modulators capable of affecting a change in the activity (e.g., dsRNA binding) or function of a sequestration protein either directly or indirectly.
  • exemplary modulating agents include, but are not limited to, small molecules, nucleic acids (e.g., RNAi agents, siRNAs, miRNAs, shRNAs, antisense molecules, ribozymes ), peptides, and polypeptides (e.g. antibodies).
  • homolog of a given gene product is one of functional similarity as well as sequence similarity. If the homolog is derived from a different organism, the homolog may be referred to as the ortholog. If several homo logs exist in a given organism, the homolog may be referred to as a paralog. Typically, the sequence similarity/identity between homologs is at least about 40%, 50%, 60%, 70%, 80%, 90%, or more (or a percentage falling within any interval or range of the foregoing). Methods for determining such similarity/identity are described, infra. Motifs (e.g.
  • dsRNA- binding domains conserved between homologs can have a sequence similarity/identity of at least about 70%, 80%, 90%, or more. It is understood that when comparing gene product sequence between diverse organisms, for example, flies and humans, sequence similarity between given homologs across the entire protein sequence may be low. However, if functional complementarity exists, and in addition, if conserved motifs exist, e.g., dsRNA binding motifs, then the gene products being compared can be considered homologs and thus selected as compositions for use in the methods of the invention, as described herein.
  • bioactive fragment includes any portion (e.g., a segment of contiguous amino acids) of a polypeptide, e.g., a sequestration polypeptide or otholog thereof, sufficient to exhibit or exert at least activity of the polypeptide, e.g., the ability to recruit miRISC to P-bodies, the ability to facilitate formation of P-bodies, or the ability to facilitate miRNA-mediated translational repression.
  • target gene includes a gene intended for downregulation via RNA silencing (e.g. miRNA-mediated translational repression).
  • target gene product or “target protein” refers to a gene product, e.g., a nucleic acid or protein, intended for downregulation via RNA silencing (e.g. miRNA-mediated translational repression).
  • target RNA refers to an RNA molecule intended for RNA silencing, e.g., by miRNA-mediated translational repression.
  • An exemplary "target RNA” is a coding RNA molecule (e.g., an RNA molecule encoding a gene product, e.g., an mRNA and protein so encoded therefrom).
  • oligonucleotide sequestration agent refers to oligonucleotides having a length of about 15 to 40 nucleotides (or nucleotide analogs), e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 nucleotides (or nucleotide analogs).
  • oligonucleotide sequestration agents are modified RNA oligonucleotides having a length of about 15 to 35 nucleotides (or nucleotide analogs).
  • sequestration agents are modified oligonucleotides having a length of about 5 to 60 nucleotides (or nucleotide analogs), or for example, about 5-10, 10-15, 15-20, 20-25, 25- 30, 30-35, 35-40, 40-45, 45-50, 50-55, 55-60, 60 or more nucleotides (or nucleotide analogs).
  • nuclease-resistant oligonucleotide refers to any oligonucleotide that has been modified to inhibit degradation by enzymes such as, for example, the exonucleases known to be present in the cytoplasm of a eukaryotic cell.
  • RNA molecules e.g., RNA oligonucleotides
  • ribonuclease-resistant sequestration oligonucleotide is thus defined as a miRISC sequestration agent that is relatively resistant to ribonuclease enzymes (e.g.
  • Preferred miRISC sequestration agents of the invention include those that have been modifi ed to render the oligonucleotide relatively nuclease-resistant or ribonuclease-resistant.
  • the oligonucleotide sequestration agent of the invention has been modified with a 2'-O-methyl group.
  • RNA silencing refers to all forms of RISC-mediated small RNA-directed silencing and includes both RNAi (siRNA-mediated or miRNA- mediated cleavage of target mRNA) and miRNA-mediated translational repression.
  • RNAi RNA interference
  • target mRNA silencing refers to a selective intracellular degradation of RNA.
  • RNAi occurs in cells naturally to remove foreign RNAs (e.g., viral RNAs). Natural RNAi proceeds via fragments cleaved from free dsRNA which direct the degradative mechanism to other similar RNA sequences.
  • translational repression refers to a selective inhibition of mRNA translation. Natural translational repression proceeds via miRNAs cleaved from shRNA precursors. Both RNAi and translational repression are mediated by RISC. Both RNAi and translational repression occur naturally or can be initiated by the hand of man, for example, to silence the expression of target genes.
  • RNA silencing agent refers to an RNA (or analog thereof), having sufficient sequence complementarity to a target RNA (i.e., the RNA being degraded) to direct RNA silencing (e.g., RNAi or translational repression) .
  • RNA silencing agent having a " sequence sufficiently complementary to a target RNA sequence to direct RNAi” means that the RNA silencing agent (e.g. miRNA or siRNA) has a sequence sufficient to trigger RNAi by RISC.
  • RNA silencing agent having a "sequence sufficiently complementary to a target RNA sequence to direct translational repression" is intended to mean that the RNA silencing agent (e.g.
  • miRNA has a sequence sufficient to trigger the translational repression of the target RNA by RISC.
  • small interfering RNA (“ siRNA”) (also referred to in the art as “short interfering RNAs”) refers to an RNA (or RNA analog) comprising between about 10-50 nucleotides (or nucleotide analogs) which is capable of directing or mediating RNA interference.
  • microRNA refers to a small (10-50 nucleotide, e.g. a 21-23 nucleotide) RNA comprising the product of an endogenous, non-coding gene whose precursor RNA transcripts can form small stem-loops from which mature miRNAs are cleaved by Dicer (Lagos- Quintana et al, 2001; Lau et ah, 2001; Lee and Ambros, 2001; Lagos-Quintana et al, 2002; Mourelatos et al, 2002; Reinhart et al, 2002; Ambros et al, 2003; Brennecke et al, 2003b; Lagos-Quintana et al, 2003; Lim et al, 2003a; Lim et al, 2003b).
  • miRNAs are encoded in genes distinct from the mRNAs whose expression they control. Mature miRNAs represent the single stranded product of Dicer cleavage that then function as guide RNA fragments in mediating RNA silencing when incorporated into RISC. A miRNA can be identified from publically-available and searchable databases (see Griffiths-Jones S. "The microRNA Registry", NAR (2004) 32, Database Issue, D 109- Dl 11 or through online searching at the Sanger Institute website, both of which are hereby incorporated herein by reference).
  • miRNAs are clustered together in the introns of pre-mRNAs and can be identified in silico using homo logy-based searches (Pasquinelli et al., 2000; Lagos-Quintana et al., 2001; Lau et al., 2001; Lee and Ambros, 2001) or computer algorithms (e.g. MiRScan, MiRSeeker) that predict the capability of a candidate miRNA gene to form the stem loop structure of a pri-mRNA (Grad et al., MoI. Cell, 2003; Lim et al., Genes Dev., 2003; Lim et al., Science, 2003; Lai EC et al., Genome Bio., 2003).
  • homo logy-based searches Pasquinelli et al., 2000; Lagos-Quintana et al., 2001; Lau et al., 2001; Lee and Ambros, 2001
  • computer algorithms e.g. MiRScan, MiRSeeker
  • miRNA may be cloned from a cell using methods that are known in the art, for example as described in International PCT Publication No. WO 03/029459; Elbasbir et al., Genes & Dev., (2001), 15: 188). Briefly, these methods may comprise isolating total RNA, size-fractionating the total RNA (e.g.
  • RNA molecules by gel electrophoresis or gel filtration) to obtain a population of small RNAs, ligating 5'- and 3 '-adapter molecules to the ends of the fractionated small RNA molecules, reverse- transcribing said adapter-ligated RNA molecules, and characterizing said reverse transcribed RNA molecules, for example, by amplification (e.g., RT-PCR), concatamerization, cloning, and sequencing.
  • amplification e.g., RT-PCR
  • concatamerization e.g., cloning, and sequencing.
  • the term "antisense strand" or "first strand” of an RNA silencing agent refers to a strand that is substantially complementary to a section of about 10-50 nucleotides, e.g., about 15-30, 16-25, 18-23 or 19-22 nucleotides of the mRNA of the gene targeted for silencing.
  • the antisense strand has sequence sufficiently complementary to the desired target mRNA sequence to direct target-specific RNA silencing, (e.g., for RNAi, complementarity sufficient to trigger the destruction of the desired target mRNA by the RNAi machinery or process).
  • the term “sense strand” or “second strand” of an siRNA or RNAi agent refers to a strand that is complementary to the antisense strand or first strand.
  • guide strand refers to a stran d of an RNA silencing agent, e.g., an antisense strand of an siRNA duplex, that enters into RISC and directs cleavage or translational silencing of the target mRNA.
  • an RNA silencing agent e.g., an antisense strand of an siRNA duplex
  • the "5' end”, as in the 5' end of an antisense strand, refers to the 5' terminal nucleotides, e.g., between one and about 5 nucleotides at the 5' terminus of the antisense strand.
  • the "3' end”, as in the 3' end of a sense strand refers to the region, e.g., a region of between one and about 5 nucleotides, that is complementary to the nucleotides of the 5' end of the complementary antisense strand.
  • a “target gene” is a gene whose expression is to be selectively inhibited or “silenced.” This silencing is achieved by cleaving or translationally silencing the mRNA of the target gene (also referred to herein as the "target mRNA") by an siRNA or miRNA, e.g., an siRNA or miRNA that is created from an engineered RNA precursor by a cell's RNA silencing system.
  • siRNA or miRNA e.g., an siRNA or miRNA that is created from an engineered RNA precursor by a cell's RNA silencing system.
  • One portion or segment of a duplex stem of the RNA precursor is an anti-sense strand that is complementary, e.g. , sufficiently complementary to trigger the destruction of the desired target mRNA by the RNAi machinery or process, to a section of about 18 to about 40 or more nucleotides of the mRNA of the target gene.
  • nucleoside refers to a molecule having a purine or pyrimidine base covalently linked to a ribose or deoxyribose sugar.
  • exemplary nucleosides include adenosine, guanosine, cytidine, uridine and thymidine.
  • nucleotide refers to a nucleoside having one or more phosphate groups joined in ester linkages to the sugar moiety.
  • Exemplary nucleotides include nucleoside monophosphates, diphosphates and triphosphates.
  • RNA and RNA are used interchangeably herein and refer to a polymer of nucleotides joined together by a phosphodiester linkage between 5' and 3 ' carbon atoms.
  • RNA or "RNA molecule” or “ribonucleic acid molecule” refers to a polymer of ribonucleotides.
  • DNA or “DNA molecule” or “deoxyribonucleic acid molecule” refers to a polymer of deoxyribonucleotides.
  • DNA and RNA can be synthesized naturally (e.g., by DNA replication or transcription of DNA, respectively). RNA can be post-transcriptionally modified.
  • DNA and RNA can also be chemically synthesized.
  • DNA and RNA can be single-stranded (i.e., ssRNA and ssDNA, respectively) or multi-stranded (e.g., double stranded, i.e., dsRNA and dsDNA, respectively).
  • mRNA or “messenger RNA” is single-stranded RNA that specifies the amino acid sequence of one or more polypeptide chains. This information is translated during protein synthesis when ribosomes bind to the mRNA.
  • nucleotide analog or altered nucleotide or “modified nucleotide” refers to a non-standard nucleotide, including non-naturally occurring ribonucleotides or deoxyribonucleotides.
  • Preferred nucleotide analogs are modified at any position so as to alter certain chemical properties of the nucleotide yet retain the ability of the nucleotide analog to perform its intended function. Examples of preferred modified nucleotides include, but are not limited to, 2-amino-guanosine, 2-amino-adenosine, 2,6-diamino- guanosine and 2,6-diamino-adenosine.
  • positions of the nucleotide which may be derivitized include the 5 position, e.g, 5-(2-amino)propyl uridine, 5-bromo uridine, 5-propyne uridine, 5-propenyl uridine, etc.; the 6 position, e.g., 6-(2- amino)propyl uridine; the 8-position for adenosine and/or guanosines, e.g., 8-bromo guanosine, 8-chloro guanosine, 8-fluoroguanosine, etc.
  • 5 position e.g, 5-(2-amino)propyl uridine, 5-bromo uridine, 5-propyne uridine, 5-propenyl uridine, etc.
  • the 6 position e.g., 6-(2- amino)propyl uridine
  • the 8-position for adenosine and/or guanosines e.g., 8-brom
  • Nucleotide analogs also include deaza nucleotides, e.g., 7-deaza-adenosine; O- and N-modified (e.g., alkylated, e.g., N6- methyl adenosine, or as otherwise known in the art) nucleotides; and other heterocyclically modified nucleotide analogs such as those described in Herdewijn, Antisense Nucleic Acid Drug Dev., 2000 Aug. 10(4):297-310.
  • Nucleotide analogs may also comprise modifications to the sugar portion of the nucleotides.
  • the 2' OH-group may be replaced by a group selected from H, OR, R 7 F, Cl, Br 7 1, SH, SR, NH2, NHR, NI ⁇ COOR, or OR, wherein R is substituted or unsubstituted Cj -C $ alkyl, alkenyl, alkynyl, aryl, etc.
  • Other possible modifications include those described in U.S. Patent Nos. 5,858,988, and 6,291,438.
  • the phosphate group of the nucleotide may also be modified, e.g., by substituting one or more of the oxygens of the phosphate group with sulfur (e.g., phosphorothioates), or by making other substitutions which allow the nucleotide to perform its intended function such as described in, for example, Eckstein, Antisense
  • oligonucleotide refers to a short polymer of nucleotides and/or nucleotide analogs.
  • RNA analog refers to a polynucleotide (e.g., a chemically synthesized polynucleotide) having at least one altered or modified nucleotide as compared to a corresponding unaltered or unmodified RNA but retaining the same or similar nature or function as the corresponding unaltered or unmodified RNA.
  • the oligonucleotides may be linked with linkages which result in a lower rate of hydrolysis of the RNA analog as compared to an RNA molecule with phosphodiester linkages.
  • the nucleotides of the analog may comprise methylenediol, ethylene diol, oxymethylthio, oxyethylthio, oxycarbonyloxy, phosphorodiamidate, and/or phosphorothioate linkages.
  • exemplary RNA analogues include sugar- and/or backbone-modified ribonucleotides and/or deoxyribonucleotides. Such alterations or modifications can further include addition of non-nucleotide material, such as to the end(s) of the RNA or internally (at one or more nucleotides of the RNA).
  • An RNA analog need only be sufficiently similar to natural RNA that it has the ability to mediate (mediates) RNA silencing.
  • oligonucleotides comprise Locked Nucleic Acids (LNAs) or Peptide Nucleic Acids (PNAs).
  • LNAs Locked Nucleic Acids
  • PNAs Peptide Nucleic Acids
  • the term "compound” includes any reagent which is tested using the assays of the invention to determine whether it modulates RNA silencing (e.g. miRNA-mediated translational repression) activity. More than one compound, e.g., a plurality of compounds, can be tested at the same time for their ability to modulate RNAi activity in a screening assay.
  • test compounds comprise any selection of the group consisting of a small molecule, a peptide, a polynucleotide, an antibody or biologically active portion thereof, a peptidomimetic, and a non-pepdide oligomer.
  • in vitro has its art recognized meaning, e.g., involving purified reagents or extracts, e.g., cell extracts.
  • in vivo also has its art recognized meaning, e.g., involving living cells, e.g., immortalized cells, primary cells, cell lines, and/or cells in an organism.
  • a gene "involved" in a disorder includes a gene, the normal or aberrant expression or function of which effects or causes a disease or disorder or at least one symptom of said disease or disorder
  • Various methodologies of the invention include a step that involves comparing a value, level, feature, characteristic, property, etc. to a "suitable control", referred to interchangeably herein as an "appropriate control”.
  • a "suitable control” or “appropriate control” is any control or standard familiar to one of ordinary skill in the art useful for comparison purposes.
  • a "suitable control” or “appropriate control” is a value, level, feature, characteristic, property, etc. determined prior to performing an RNAi methodology, as described herein.
  • a transcription rate, mRNA level, translation rate, protein level, biological activity, cellular characteristic or property, genotype, phenotype, etc. can be determined prior to introducing an RNAi- modulatory agent (e.g., an oligonucleotide, compound, etc., that alters sequence-specific RNAi activity) of the invention into a cell or organism.
  • an RNAi- modulatory agent e.g., an oligonucleotide, compound, etc., that alters sequence-specific RNAi activity
  • a "suitable control” or “appropriate control” is a value, level, feature, characteristic, property, etc. determined in a cell or organism, e.g., a control or normal cell or organism, exhibiting, for example, normal traits.
  • a "suitable control” or “appropriate control” is a predefined value, level, feature, characteristic, property, etc.
  • IL Sequestration Polypeptides Preferred aspects of the invention feature sequestration polypeptides, homologs thereof, as well a bio active fragments thereof. Exemplary sequestration polypeptides, are set forth in the following subsections.
  • RCK Polypeptides, homologs and fragments Preferred aspects of the invention feature RCK (p54) polypeptides, RCK homologs (e.g., RCK orthologs) and/or biologically active portions (i.e., bioactive fragments) of RCK polypeptides or RCK orthologs, including polypeptide fragments suitable for use in making RCK fusion proteins.
  • RCK polypeptides or RCK orthologs can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques.
  • RCK polypeptide or RCK orthologs can be further derived from said isolated polypeptides using standard enzymatic techniques.
  • RCK orthologs, RCK polypeptides or bioactive fragments thereof are produced by recombinant DNA techniques.
  • RCK orthologs, RCK polypeptides or bioactive fragments thereof can be synthesized chemically using standard peptide synthesis techniques.
  • Polypeptides of the invention are preferably “isolated” or “purified”.
  • isolated and purified are used interchangeably herein.
  • Isolated or purified means that the protein or polypeptide is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the polypeptide is derived, substantially free of other protein fragments, for example, non-desired fragments in a digestion mixture, or substantially free from chemical precursors or other chemicals when chemically synthesized.
  • substantially free of cellular material includes preparations in which the polypeptide is separated from other components of the cells from which it is isolated or recombinantly produced.
  • the language "substantially free of cellular material” includes preparation s of polypeptide having less than about 30% (by dry weight) of non-RCK ortholog polypeptide or non-RCK polypeptide (also referred to herein as a "contaminating protein"), more preferably less than about 20% of non-RCK ortholog polypeptide or non-RCK polypeptide, still more preferably less than about 10% of non-RCK ortholog polypeptide or non-RCK polypeptide, and most preferably less than about 5% non-RCK ortholog polypeptide or non-RCK polypeptide.
  • non-RCK ortholog polypeptide or non-RCK polypeptide also referred to herein as a "contaminating protein”
  • culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the polypeptide preparation.
  • the preparation is preferably free of enzyme reaction components or chemical reaction components and is free of non-desired RCK orthologs or RCK fragments, i.e., the desired polypeptide represents at least 75% (by dry weight) of the preparation, preferably at least 80%, more preferably at least 85%, and even more preferably at least 90%, 95%, 99% or more or the preparation.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of polypeptide in which the polypeptide is separated from chemical precursors or other chemicals which are involved in the synthesis of the polypeptide.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations having less than about 30% (by dry weight) of chemical precursors or reagents, more preferably less than about 20% chemical precursors or reagents, still more preferably less than about 10% chemical precursors or reagents, and most preferably less than about 5% chemical precursors or reagents.
  • Bioactive fragments of RCK orthologs or RCK polypeptides include polypeptides comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence of the RCK ortholog or the RCK protein, respectively, which include less amino acids than the foil length protein, and exhibit at least one biological activity of the full-length protein.
  • biologically active portions comprise a domain or motif with at least one activity of the full-length protein.
  • a biologically active portion of a RCK ortholog or RCK polypeptide can. be a polypeptide which is, for example, 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350 or more amino acids in length.
  • a bioactive portion of a RCK protein comprises a RNA-binding domain (e.g.
  • a bioactive portion of a RCK polypeptide comprises a portion which is capable of interacting with a protein subunit of the miRISC complex (e.g. Dicer, transactivation-responsive RNA-binding protein (TRBP), Argonaute-1 or Argonaute-2).
  • a bioactive portion of a RCK polypeptide comprises a portion which is capable of interacting with a protein subunit of aP-body (e.g. Lsm-1, Dcp2, eIF4E, Xrnl).
  • RCK and/or RCK ortholog can also be utilized as assay reagents, for example, mutants having reduced, enhanced or otherwise altered biological properties identified according to one of the activity assays described herein.
  • a RCK polypeptide or RCK ortholog polypeptide of the invention includes polypeptides having the amino acid sequences set infra, as well as homologs an/or orthologs of said polypeptides, i.e. polypeptides having sufficient sequence identity to function in the same manner as the described polypeptides.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be in troduced in the first sequence or second sequence for optimal alignment).
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In one embodiment, the alignment generated over a certain portion of the sequence aligned having sufficient identity but not over portions having low degree of identity (i.e., a local alignment).
  • the alignment is optimized by introducing appropriate gaps and percent identity is determined over the length of the aligned sequences (i.e., a gapped alignment).
  • Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Research 25(17):3389-3402.
  • the alignment is optimized by introducing appropriate gaps and percent identity is determined over the entire length of the sequences aligned (i.e., a global alignment).
  • a preferred, non-limiting example of a mathematical algorithm utilized for the global comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package.
  • a PAMl 20 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.
  • RCK polypeptides and homo logs are as f follows.
  • the mRNA (coding) sequences and amino acid sequences for RCK/p54, the yeast homo log Dhhlp, and the Xenopus homolog Xp54 are set forth in Table 1, 2, and 3, respectively.
  • a RCK family member is identified based on the presence of a "DEA(DZH) box helicase domain" and one "helicase C-terminal domain".
  • DEA(DZH) box helicase domain refers to a protein domain having a conserved motif Asp-Glu- Ala- AspZHis (DEAD/H).
  • DEAD/H conserved motif Asp-Glu- Ala- AspZHis
  • members of the DEA(DZH) box helicase family are involved in various aspects of RNA metabolism, including nuclear transcription, pre-mRNA splicing, ribosome biogenesis, nucleocytoplasmic transport, translation, RNA decay and organellar gene expression.
  • DEAD box protein family bind ATP and nucleic acid.
  • a protein that "binds nucleic acid” is defined a protein that forms complexes with nucleic acid.
  • the DEAD region additionally couples RNA helicase activity to ATPase activity. The hydrolysis of ATP provides the energy needed for RNA unwinding during helicase activity.
  • a RCK protein contains a OBA(DfH) box helicase domain containing about amino acids 80-290.
  • a RCK protein contains a DEAD(D/H) box helicase domain containing about amino acids 80-250.
  • a RCK protein contains a DEAD(D/H) box helicase domain containing about amino acids 120-290.
  • a RCK protein contains a DEAD(DZH) box helicase domain containing about amino acids 109-280.
  • a "helicase C-terminal domain" refers to a protein domain containing about 76 amino acids having nucleic acid binding, helicase activity, and ATP-binding. The domain, which defines this group of proteins, is found in a wide variety of helicases and helicase related proteins.
  • Helicases are nucleotide thriphosphate (NTP)-dependent enzymes responsible for unwinding duplex DNA and RNA during replication, repair, recombination, transcription, transalation and processing of nucleic acids.
  • NTP nucleotide thriphosphate
  • a RCK protein contains a helicase C-terminal domain containing about amino acids 314-430.
  • a RCK protein contains a helicase C-terminal domain containing about amino acids 340-420. In another embodiment, a RCK protein contains a helicase C-terminal domain containing about amino acids 314-390. In yet another embodiment, a RCK protein contains a helicase C-terminal domain containing about amino acids 350-430.
  • the amino acid sequence of the polypeptide can be searched against a database of conserved protein domains (e.g., the CD database at the NCBI) using the default parameters (see Marchler-Bauer et al. (2005) Nucleic Acids Res. 33: D192-6). NCBI conserved Domains Database
  • a RCK bioactive fragment is any fragment of RCK having sufficient size and structure to carry out at least one activity (e.g., biological activity) of the corresponding full-length RCK protein.
  • exemplary bioactive fragments include, but are not limited to, enzymatic domains, protein binding and/or interaction domains, and nucleic acid binding domains.
  • Biologically active portions of a RCK protein include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequence of the RCK protein, e.g., the amino acid sequence shown in SEQ ID NO:1 or the full length RCK proteins, and exhibit at least one activity of a RCK protein.
  • biologically' active portions comprise a domain or motif with at least one activity of the RCK protein.
  • a biologically active portion of a RCK protein can be a polypeptide which is, for example, 10, 25, 50, 100 or more amino acids in length.
  • a preferred biologically active portion of a RCK protein of the present invention may contain at least one of the above-identified structural domains.
  • other biologically active portions, in which other regions ' of the protein are deleted can be prepared by recombinant techniques and evaluated for one or more of the functional activities of a native RCK protein.
  • RCK Interactor Polypeptides e.g., RCK binding polypeptides
  • RCK ortholog interactor proteins e.g., RCK binding polypeptides
  • biologically active portions ⁇ i.e., bioactive fragments
  • RCK interactor polypeptides include components of the miRISC complex listed below.
  • a preferred RCK interactor polypeptide (or RCK ortholog interactor polypeptide) is Argonaute-2 (or fragments thereof).
  • RCK polypeptides, RCK orthologs, and fragments thereof are similarly applicable to RCK interactor polypeptides, RCK ortholog interactor polypeptides, and fragments thereof.
  • a "chimeric protein” or “fusion protein” comprises a polypeptide sequence derived from RCK or RCK orthologs operatively linked to a non-RCK polypeptide, non-ortholog polypeptide or non-interactor polypeptide, respectively.
  • RCK polypeptide refers to a polypeptide having an amino acid sequence corresponding to the RCK protein or RCK ortholog, respectively
  • a or “non- RCK polypeptide”, “non-RCK ortholog polypeptide” or “non-RCK interactor polypeptide” refers to a polypeptide having an amino acid sequence corresponding to a protein which is not substantially identical to the RCK protein, RCK ortholog or RCK interactor protein.
  • the RCK protein, RCK ortholog, or RCK interactor polypeptide can correspond to all or a portion of a RCK protein, RCK ortholog or RCK interactor protein.
  • a RCK protein, RCK ortholog or RCK interactor fusion protein comprises at least one biologically active portion of a RCK protein, RCK ortholog or RCK interactor protein, respectively.
  • a RCK protein, RCK ortholog or RCK interactor fusion protein comprises at least two biologically active portions of a RCK protein, RCK ortholog or RCK interactor protein, respectively.
  • a fusion protein can comprise RCK protein, or a bioactive portion thereof, operatively linked to RCK interactor, or a bioactive portion thereof, such that RCK and RCK interactor, or their respective bioactive portions are brought into close proximity.
  • the term "operatively linked" is intended to indicate that the RCK polypeptide, RCK ortholog or RCK interactor polypeptide and the non-RCK > polypeptide, non-RCK ortholog polypeptide or non-RCK interactor polypeptide are fused in-frame to each other.
  • the non-RCK polypeptide, non-RCK ortholog polypeptide or non-RCK interactor polypeptide can be fused to the N-terminus or C- te ⁇ ninus of the RCK polypeptide, RCK ortholog polypeptide or RCK interactor polypeptide, respectively.
  • the fusion protein is a GST-fusion protein in which the RCK protein, RCK ortholog or RCK interactor sequences are fused to the C- terminus of the GST sequences.
  • the fusion protein is a chitin binding domain (CBD) fusion protein in which the RCK protein, RCK ortholog or RCK interactor sequences are fused to the N-terminus of chitin binding domain (CBD) sequences.
  • CBD chitin binding domain
  • Such fusion proteins can facilitate the purification of recombinant RCK • protein, RCK ortholog or RCK interactor.
  • a chimeric or fusion protein of the invention is produced by standard recombinant DNA techniques.
  • DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, for example by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-i ⁇ of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and . enzymatic ligation.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can , subsequently be annealed and reamplifled to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).
  • anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can , subsequently be annealed and reamplifled to generate a chimeric gene sequence
  • many expression vectors are commercially available that already encode a fusion moiety.
  • a RCK protein- or RCK - ' encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the RCK polypeptide, RCK ortholog or RCK polypeptide interactor.
  • the invention relates to methods and compositions for modulating RCBC, or a RCK ortholog, for example, to modulate general miRNA mediated translational repression.
  • the invention relates to methods and compositions (e.g., RCK polypeptide compositions) for modulating (e.g., upregulating) miRISC , sequestration to P-bodies.
  • the invention relates to methods and compositions (e.g., RCK polypeptide compositions) for modulating (e.g., upregulating) , miRISC sequestration in a subject (e.g., a human subject).
  • a subject e.g., a human subject.
  • exemplary modulators are described in detail below.
  • small molecule modulators of RCK may be identified, for example, from a library of test compounds or a collection of compounds produced by rationale drug design. Generally, small molecules are those of a molecular weigh of 1000 daltons or less.
  • Suitable test compounds can include, for example, compounds suspected of having the ability to disrupt protein:protein interactions (e.g., RCK:Argonaute-2 interactions, RCK ortholog:Argonaute-2 interactions), modulate (e.g., enhance or miRNA activity), modulate RNAi, etc.
  • the following assays may be used to identify compounds that modulate the activity of RCK or orthologs or bioactive fragments thereof (or RCK interactor polypeptides or bioactive fragments thereof).
  • modulatory compounds are identified in a cell-free assay in which a composition comprising assay reagents (e.g., a RCK polypeptide, RCK interactor polypeptide, or biologically active portions thereof), is contacted with a test compound and the ability of the test compound to modulate binding of the RCK polypeptide (or RCK ortholog) (or biologically active portions thereof) to the RCK interactor polypeptide (or bioactive fragments thereof) is determined.
  • assay reagents e.g., a RCK polypeptide, RCK interactor polypeptide, or biologically active portions thereof
  • Binding of the RCK or RCK interactor can be accomplished, for example, by coupling the polypeptide or fragment with a radioisotope or enzymatic label such that binding of polypeptide reagents can be determined by detecting the labeled compound or polypeptide in a complex.
  • test compounds or polypeptides can be labeled with 125 I, 35 S, 33 P 5 32 P 5 1 ⁇ C, or 3 H, either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting.
  • polypeptides can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate protein to product.
  • Biomolecular Interaction Analysis Sjolander, S. and Urbaniczky, C. (1991) Anal Chem. 63 :2338-2345 and Szabo et al. (1995) Curr. Opin. Struct. Biol. 5:699-705.
  • BIOA Biomolecular Interaction Analysis
  • BIA is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore). Changes in the optical phenomenon of surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules.
  • SPR surface plasmon resonance
  • the assay includes contacting RCK polypeptide or biologically active portion thereof with a RCK target molecule, e.g., a RCK interactor or a bioactive fragment thereof to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with the RCK polypeptide, wherein determining the ability of the test compound to interact with • the RCK polypeptide comprises determining the ability of the test compound to preferentially bind to RCK or the bioactive portion thereof as compared to the RCK target molecule (e.g., a RCK interactor protein).
  • a RCK target molecule e.g., a RCK interactor or a bioactive fragment thereof
  • the assay i includes contacting the RCK polypeptide or biologically active portion thereof with a RCK interactor target molecule, e.g., RCK interactor protein or a bioactive fragment thereof to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to modulate binding between the RCK polypeptide and the RCK interactor polypeptide.
  • a RCK interactor target molecule e.g., RCK interactor protein or a bioactive fragment thereof
  • the assay is a cell-free assay in which a composition comprising a RCK polypeptide and a RCK interactor polypeptide (or bioactive portions thereof) is contacted with a test compound and the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the RCK polypeptide or RCK interactor polypeptide (or biologically active portions thereof) is determined.
  • Determining the ability of the test compound to modulate the activity of a RCK or a RCK interactor polypeptide can be accomplished, for example, by determining the ability of the RCK interactor polypeptide to modulate the activity of a downstream binding partner or target molecule by one of the methods described herein for cell or organism-based assays. For example, the catalytic/enzymatic activity of the target molecule on an appropriate downstream substrate (e.g., dsRNA) can be determined as previously described. '
  • the cell-free assay involves contacting a RCK polypeptide or biologically active portion thereof with a RCK target molecule that binds the RCK polypeptide to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to preferentially modulate ⁇ the activity of the RCK protein, as compared to the RCK interactor binding partner or target molecule.
  • RCK protein or RCK or target ' molecules
  • the ability of a test compound to modulate RCK polypeptide activity, RCK interactor polypeptide activity, interaction of a RCK polypeptide with a RCK interactor polypeptide (or target interaction or activity) in the presence and absence of a candidate compound can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtitre plates, test tubes, and micro-centrifuge tubes.
  • a fusion protein can be provided so as to add a domain that allows one or both of the proteins to be bound to a matrix.
  • a fusion protein can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtitre plates, which are ' then combined with the test compound or the test compound and either the non-adsorbed RCK polypeptide or RCK interactor polypeptide (or target polypeptide), and the mixture incubated under conditions conducive to complex formation (e.g.
  • the beads or microtitre plate wells are • washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described above.
  • the complexes can be dissociated from the matrix, and the level of RCK interactor binding or activity or RCK binding or activity (or target binding or activity) determined using standard techniques.
  • RCK and/or RCK interactor fusion proteins include, but are not limited to, chitin binding domain (CBD) fusion proteins, hemagglutinin epitope tagged (HA)-fiision proteins, His fusion proteins (e.g., Hisg tagged proteins), FLAG tagged fusion proteins, AUl tagged proteins, and the like.
  • CBD chitin binding domain
  • HA hemagglutinin epitope tagged
  • His fusion proteins e.g., Hisg tagged proteins
  • FLAG tagged fusion proteins e.g., FLAG tagged fusion proteins
  • AUl tagged proteins e.g., AUl tagged proteins
  • RCK polypeptide, a RCK interactor polypeptide or target polypeptide can be immobilized utilizing conjugation of biotin and streptavidin.
  • Biotinylated RCK polypeptide, RCK interactor polypeptide or target polypeptide can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, IL), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).
  • antibodies reactive with RCK polypeptide, RCK interactor polypeptide or target polypeptide but which do not interfere with binding of the RCK interactor polypeptide to RCK polypeptide (or protein to target binding) can be derivatized to the wells of the plate, and unbound RCK or RCK interactor polypeptide (or target) trapped in the wells by antibody conjugation.
  • Methods for detecting such complexes include immunodetection of complexes using antibodies reactive with the RCK interactor polypeptide, RCK polypeptide or target polypeptide, as well as enzyme- linked assays which rely on detecting an enzymatic activity associated with the RCK interactor polypeptide, RCK polypeptide or target polypeptide.
  • the RCK or RCK interactor polypeptides can be used as "bait proteins" in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Patent No. 5,283,317; Zervos et al.
  • At least one exemplary two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains.
  • the assay utilizes two different DNA constructs.
  • the gene that codes for a first polypeptide e.g., RCK or RCK protein
  • a gene encoding the DNA binding domain of a known transcription factor e.g., GAL-4
  • a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein (“prey” or "sample” is fused to a gene that codes for the activation domain of the known transcription factor.
  • the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) which is operably linked to a transcriptional , regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene that encodes the protein that interacts with the bait polypeptide.
  • a reporter gene e.g., LacZ
  • CytoTrapTM Another exemplary two-hybrid system, referred to in the art as the CytoTrapTM system, is based in the modular nature of molecules of the Ras signal transduction • cascade.
  • the assay features a fusion protein comprising the "bait” protein and Son-of-Sevenless (SOS) and the cDNAs for unidentified proteins (the "prey") in a vector that encodes myristylated target proteins .
  • SOS Son-of-Sevenless
  • prey unidentified proteins
  • Expression of an appropriate bait-prey combination results in translocation of SOS to the cell membrane where it activates Ras.
  • Cytoplasmic reconstitution of the Ras signaling pathway allows identification of proteins that interact with the bait protein of interest, for example, a RCK or RCK interactor protein.
  • Additional mammalian two hybrid systems are also known in the art . and can be utilized to identify RCK or RCK interactor interacting proteins. Moreover, at , least one of the above-described assays can be utilized to identify RCK -interacting domains or regions of the RCK interactor protein or alternatively, to identify RCK protein-interacting domain or regions of the RCK protein.
  • a modulator of a RCK protein is identified in a. cell or organism-based assay in which a cell or organism , capable of expressing the RCK protein (or RCK ortholog or RCK interactor), or biologically active portion thereof, is contacted with a test compound and the ability of the test compound to modulate the expression of the RCK protein (or RCK ortholog or • RCK interactor), or biologically active portion thereof, determined.
  • an assay is a cell or organism-based assay in which a cell or organism which expresses a RCK protein (or a RCK ortholog or RCK interactor) (or biologically active portions thereof) is contacted with a test compound and the ability of the test compound to modulate the activity of the RCK protein (or RCK ortholog or RCK interactor (or biologically active portions thereof) determined.
  • the cell for example, can be of mammalian origin or a yeast cell.
  • the organism can be a nematode, for example, C. elegans or C. briggsae or D. melanogaster.
  • the polypeptides for example, can be expressed heterologously or native to the cell or organism.
  • Determining the ability of the test compound to modulate the activity of the RCK protein (or RCK ortholog or RCK interactor (or biologically active portions thereof) can be accomplished by assaying for any of the activities of a RCK protein (or RCK ortholog or RCK interactor) described herein. Determining the ability of the test compound to modulate the activity of a RCK protein (or RCK ortholog or RCK interactor) (or biologically . active portions thereof) can also be accomplished by assaying for the activity of a RCK downstream molecule.
  • determining the ability of the test compound to modulate the activity of the RCK protein (or RCK ortholog or RCK interactor), or biologically active portion thereof is accomplished by assaying for the ability to bind the RCK protein (or RCK ortholog or RCK interactor) or a bioactive portion thereof. In another embodiment, determining the ability of the test compound to modulate the activity of the RCK protein (or RCK ortholog or RCK interactor), or biologically active portion thereof, is accomplished by assaying for the activity of the RCK protein (or RCK ortholog or RCK interactor).
  • the cell or organism overexpresses the RCK protein (or RCK ortholog), or biologically active portion thereof, and optionally, overexpresses RCK interactor, or biologically active portion thereof.
  • the cell or organism expresses RCK, or biologically active portion thereof.
  • the cell or organism is contacted with a compound that stimulates a RCK protein-associated activity or RCK - associated activity and the ability of a test compound to modulate the RCK protein- associated activity is determined.
  • determining the ability of the test compound to modulate the activity of the RCK protein (or RCK ortholog or RCK interactor) or biologically active portion thereof can be determined by assaying for any of the native activities of a RCK protein (or RCK ortholog or RCK interactor) as described herein.
  • the activity of a RCK protein (or RCK ortholog or RCK interactor) can be determined by assaying for an indirect activity which is coincident to the activity of the RCK protein (or RCK ortholog or RCK interactor).
  • determining the ability of the test compound to modulate the activity of RCK protein (or RCK ortholog or RCK interactor) or biologically active portion thereof can be determined by assaying for an activity which is not native to the RCK protein (or RCK ortholog or RCK interactor), but for which the cell or organism has been recombinantly engineered. It is also intended that in certain embodiments, the cell or organism-based assays of the present invention comprise a • final step of identifying the compound as a modulator of RCK activity (or RCK ortholog activity or RCK interactor activity).
  • test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: , biological libraries; spatially addressable parallel solid phase or solution phase libraries; . synthetic library methods requiring deconvolution; the One-bead one-compound' library method; and synthetic library methods using affinity chromatography selection.
  • the biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, K.S. (1997) Anticancer Drug Des. 12:145).
  • the library is a natural product library.
  • the invention relates to nucleic acid-based compositions for modulating RCK, or a RCK ortholog, for example, to modulate RNA interference.
  • RNAi agents e.g., siRNAs, rniRNAs, shRNAs
  • antisense molecules e.g., ribozymes
  • a modulatory compound of the invention is an antisense • nucleic acid molecule that is complementary to a mRNA encoding RCK (or a RCK ortholog or RCK interactor), or to a portion of said mRNA, or a recombinant expression vector encoding said antisense nucleic acid molecule.
  • RCK Ribozymes
  • a modulatory compound of the invention is an antisense • nucleic acid molecule that is complementary to a mRNA encoding RCK (or a RCK ortholog or RCK interactor), or to a portion of said mRNA, or a recombinant expression vector encoding said antisense nucleic acid molecule.
  • the use of antisense nucleic acids to downregulate the expression of a particular protein in a cell is well known in the art (see e.g., Weintraub, H. et al, Antisense RNA as a molecular tool for genetic analysis, Reviews - Trends in Genetic
  • An antisense nucleic acid molecule comprises a nucleotide sequence that is complementary to the mRNA sequence of RCK (or a RCK ortholog or RCK interactor) , and accordingly is capable of hydrogen bonding to the mRNA.
  • Antisense sequences complementary to a sequence of an mRNA can be complementary to a sequence found in the coding region of the mRNA, the 5' or 3' untranslated region of the mRNA or a region bridging the coding region and an untranslated region (e.g., at the junction of the 5' untranslated region and the coding region).
  • an antisense nucleic acid is designed so as to be complementary to a region preceding or spanning the initiation co don in the 3 1 untranslated region of an mRNA.
  • antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick base pairing.
  • the antisense nucleic acid molecule can be complementary to the entire coding region of an mRNA, but more preferably is antisense to only a portion of the coding or noncoding region of an mRNA.
  • the antisense oligonucleotide can be complementary to the region surrounding the translation start site of a RCK (or a RCK ortholog or RCK interactor)mRNA.
  • An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.
  • An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art.
  • an antisense nucleic acid e.g., an antisense oligonucleotide
  • an antisense nucleic acid e.g., an antisense oligonucleotide
  • modified nucleotides which can be used to generate the antisense nucleic acid include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthi ⁇ e, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5- carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1- methylguanine, 1-methylinosi ⁇ e, 2,2-dimethylguanine, 2-methyladenine, 2- methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5- methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D- mannosylqueosine, 5'
  • an antisense nucleic acid can be produced biologically using an expression vector into which all or a portion of a cDNA has been subcloned in an antisense orientation (i.e., nucleic acid transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest).
  • Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the expression of the antisense RNA molecule in a cell of interest, for instance promoters and/or enhancers or other regulatory sequences can be chosen which direct constitutive, tissue specific or inducible expression of antisense RNA.
  • the antisense expression vector is prepared according to standard recombinant DNA methods for constructing recombinant expression vectors, except that the cDNA (or portion thereof) is cloned into the vector in the antisense orientation.
  • the antisense expression vector can be in the form of, for example, a recombinant plasmid, phagemid or attenuated virus.
  • the antisense expression vector can be introduced into cells using a standard transfection technique.
  • the antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a protein to thereby inhibit expression of the protein, e.g. , by inhibiting transcription and/or translation.
  • the hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix.
  • An example of a route of administration of an antisense nucleic acid molecule of the invention includes direct injection at a tissue site.
  • an antisense nucleic acid molecule can be modified to target selected cells and then administered systemically.
  • an antisense molecule can be modified such that it specifically binds to a receptor or an antigen expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecule to a peptide or an antibody which binds to a cell surface receptor or antigen.
  • the antisense nucleic acid molecule can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of ( antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol in promoter are preferred.
  • an antisense nucleic acid molecule of the invention is an -anomeric nucleic acid molecule.
  • An -anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual - units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641).
  • the antisense nucleic acid molecule can also comprise a 2'-o- methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBS Lett. 215:327-330).
  • an antisense nucleic acid molecule of the invention is a ribozyme.
  • Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region.
  • ribozymes ⁇ e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used to catalytically cleave mRNA transcripts to thereby inhibit translation mRNAs.
  • a ribozyme having specificity e.g., for a RCK (or a RCK ortholog or RCK interactor)- encoding nucleic acid can be designed based upon the nucleotide sequence of the cDNA.
  • a derivative of a Tetrahymena L- 19 IVS RNA can be constructed in which' the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in, e.g., a RCK (or a RCK ortholog or RCK interactor)-encoding mRNA. See, e.g., Cech et al. U.S. Patent No. 4,987,071 and Cech et al U.S. Patent No.
  • RCK or a RCK ortholog or RCK interactor
  • mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel, D. and Szostak, J.W. (1993) Science 261:1411-1418.
  • gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of a gene (e.g., a RCK (or a RCK ortholog or , RCK interactor) promoter and/or enhancer) to form triple helical structures that prevent t ranscription of a gene in target cells. See generally, Helene, C.
  • RNA silencing agents e.g. siRNAs, miRNAs, shRNAs
  • a modulatory compound of the invention is used to effect RNA silencing induced by an RNA silencing agent.
  • an RNA silencing agent is used as a modulatory agent of the invention, for example, to silence RCK, RCK orthologs or RCK interactors (e.g., Dicer proteins).
  • the present invention features "small interfering RNA molecules" (“siRNA molecules” or “siRNA”), “miRNA” molecules, methods of making said siRNA and miRNA molecules, and methods (e.g., research and/or therapeutic methods) for using said siRNA and miRNA molecules.
  • An RNA silencing agent (e.g-.siRNA or miRNA molecule) of the invention can be a duplex consisting of a sense strand and complementary antisense strand, the antisense strand having sufficient complementarity to a target mRNA to mediate RNAi or translational repression.
  • the strands are aligned such that there are at least 1, 2, or 3 bases at the end of the strands which do not align (i.e., for which no complementary bases occur in the opposing strand) such that an overhang of 1, 2 or 3 residues occurs at one or both ends of the duplex when strands are annealed.
  • the siRNA or miRNA molecule has a length from about 10-50 or more nucleotides, i.e., each strand comprises 10-50 nucleotides (or nucleotide analogs). More preferably, the siKNA or miRNA molecule has a length from about 15-45 or 15-30 nucleotides. Even more preferably, the siRNA or miRNA molecule has a length from about 16-25 or 18-23 nucleotides.
  • siRNA-like duplex molecules as well as . methods, for making and using such molecules in RNA silencing.
  • siRNA-like duplexes include a first or miRNA strand and a second or miRNA* strand and are structurally similar to siRNA duplexes. Modifications within the duplex are permitted. Modifications that do not significantly affect RNA-silencing activity are particularly tolerated.
  • siRNA-like duplexes mediate translational repression or RNAi depending on the degree of complementarity with target mRNA, as described supra.
  • RNA silencing agents e.g. siRNAs or miRNAs
  • the ⁇ base pair strength between the antisense strand 5' end (AS 5') and the sense strand 3' end (S 3') of the siRNA, or miRNAs molecule is less than the bond strength or base pair strength between the antisense strand 3' end (AS 3' ) and the sense strand 5' end (S '5), such that the antisense strand preferentially guides cleavage or translational repression of a target mRNA.
  • the bond strength or base-pair strength is less due to fewer G:C base pairs between the 5' end of the first or antisense strand and the 3' end of the second or sense strand than between the 3 ' end of the first or antisense strand and the 5' end of the second or sense strand.
  • the bond strength or base pair strength is less due to at least one mismatched base pair between the 5' end of the first or antisense strand and the 3' end of the second or sense strand.
  • the mismatched base pair is selected from the group consisting of G:A, C:A, C:U. G:G, A: A, C:C and U:U.
  • the bond strength or base pair strength is less due to at least one wobble base pair, e.g., G:U, between the 5' end of the first or antisense strand and the 3' end of the second or sense strand.
  • the bond strength or base pair strength is less due to at least, one base pair comprising a rare nucleotide, e.g., inosine (I).
  • the base pair is selected from the group consisting of an I: A, I:U and I:C.
  • the bond strength or base pair strength is less due to at least one base pair comprising a modified nucleotide.
  • the modified nucleotide is selected from the group consisting of 2-amino-G, 2-amino-A, 2,6-diamino-G, and 2,6-diamino-A.
  • siRNA molecules, or siRNA-like molecules, of the invention further have a sequence that is "sufficiently complementary" to a target mRNA sequence to direct target-specific RNA interference (ECSfAi), as defined herein, i.e., the siRNA has a sequence sufficient to trigger the destruction of the target mRNA by the RNAi machinery or process.
  • ECSfAi target-specific RNA interference
  • nucleotide sequences sufficiently identical to a portion of the target gene to effect RISC-mediated cleavage of the target gene are preferred. 100% sequence identity between the siRNA and the target gene is not required to practice the present invention.
  • the invention has the advantage of being able to tolerate certain sequence variations to enhance efficiency and specificity of RNAi.
  • sequences with insertions, deletions, and single point mutations relative to the target sequence can also be effective for inhibition.
  • sequences with nucleotide analog substitutions or insertions can be effective for inhibition.
  • siRNA-like molecules of the invention have a sequence (i.e., have a strand having a sequence) that is "sufficiently complementary" to a target mRNA sequence to direct gene silencing either by RNAi or translational repression.
  • a sequence i.e., have a strand having a sequence
  • the degree of sequence identity between a miRNA sequence and the corresponding target gene sequence is decreased, the tendency to mediate post-transcriptional gene silencing by translational repression rather than RNAi is increased. Therefore, in an alternative embodiment, where post-transcriptional gene silencing by translational repression of the target gene is desired, the miRNA sequence has partial complementarity with the target gene sequence.
  • the miRNA sequence has partial complementarity with one or more short sequences (complementarity sites) dispersed within the target mRNA (e.g. within the 3'-UTR of the target mRNA) (Hutvagner and Zamore, Science, 2002; Zeng et al., MoI. Cell, 2002; Zeng et al., RNA, 2003; Doench et al., Genes & Dev., 2003). Since the mechanism of translational repression is cooperative, multiple complementarity sites (e.g., 2, 3, 4, 5, or 6) may be targeted in certain embodiments.
  • multiple complementarity sites e.g., 2, 3, 4, 5, or 6
  • the instant invention provides shRNAs having enhanced specificity or efficacy in mediating gene silencing (e.g. RNAi or translational repression).
  • gene silencing e.g. RNAi or translational repression
  • short hairpin RNAs e.g. siRNA or siRNA-like duplexes
  • shRNAs mimic the natural precursors of miRNAs and enter at the top of the gene silencing pathway. For this reason, shRNAs are believed to mediate gene silencing more efficiently by being fed through the entire natural gene silencing pathway.
  • a preferred shRNA of the invention is one that has been redesigned for increased specificity or enhancement relative to a previous shRNA.
  • the new shRNA differs from a previous shRNA in that a silencing duplex produced from the new shRNA has less base pair strength between the 5' end of the antisense strand or first strand and the 3' end of the sense strand or second strand than the base pair strength between the 3' end of the ' antisense strand or first strand and the 5' end of the sense strand or second strand.
  • Naturally-occurring miRNA precursors have a single strand that forms a duplex stem including two portions that are generally complementary, and a ⁇ loop, that connects the two portions of the stem.
  • the stem includes one or more bulges, e.g., extra nucleotides that create a single nucleotide "loop" in one portion of the stem, and/or one or more unpaired nucleotides that create a gap in the hybridization of the two portions of the stem to each other.
  • Short hairpin RNAs or engineered RNA precursors, of the invention are artificial constructs based on these naturally occurring pre-miRNAs, but which are engineered to deliver desired RNA silencing agents (e.g., siRNAs or siRNA-like duplexes).
  • desired RNA silencing agents e.g., siRNAs or siRNA-like duplexes.
  • one portion of the duplex stem is a nucleic acid sequence that is complementary (or anti-sense) to the target mRNA.
  • engineered RNA precursors include a duplex stem with two portions and a loop connecting the two stem portions. The two stem portions are about 18 or 19 to about 25, 30, 35, 37, 38, 39, or 40 or more nucleotides in length.
  • the length of the stem portions should be less than about 30 nucleotides to avoid provoking non-specific responses like the interferon pathway.
  • the stem can be longer than 30 nucleotides.
  • the stem can include much larger sections complementary to the target mRNA (up to, and including the entire mRNA).
  • the two portions of the duplex stem must be sufficiently complementary to hybridize to form the duplex stem.
  • the two portions can. be, but need not be, fully or perfectly complementary.
  • the two stem portions can be the same length, or one portion can include an overhang of 1, 2, 3, or 4 nucleotides.
  • the overhanging nucleotides can include, for example, uracils (Us), e.g., all Us.
  • the loop in , the shRNAs or engineered RNA precursors may differ from natural pre-miRNA i sequences by m odifying the loop sequence to increase or decrease the number of paired nucleotides, or replacing all or part of the loop sequence with a tetraloop or other loop sequences.
  • the loop in the shRNAs or engineered RNA precursors can be 2, 3, 4, ' 5, 6, 7, 8, 9, or more, e.g., 15 or 20, or more nucleotides in length.
  • shRNAs of the invention include the sequences of a desired RNA silencing agent (e.g. siRNA or siRNA-like duplex).
  • a desired RNA silencing agent e.g. siRNA or siRNA-like duplex.
  • the desired RNA , silencing duplex (e.g. siRNA or siRNA-like duplex), and thus both of the two stem portions in the engineered RNA precursor, are selected by methods known in the art. These include, but are not limited to, selecting an 18, 19, 20, 21 nucleotide, or longer, sequence from the target gene rriRNA s equence from a region 100 to 200 or 300 ' nucleotides on the 3' side of the start of translation.
  • the sequence can be selected from any portion of the mRNA from the target gene, such as the 5 r UTR (untranslated region), coding sequence, or 3' UTR.
  • This sequence can optionally follow • immediately after a region of the target gene containing two adjacent AA nucleotides.
  • the last two nucleotides of the 21 or so nucleotide sequence can be selected td be UU (so that the anti-sense strand of the siRNA begins with UU).
  • This 21 or so nucleotide sequence is used to create one portion of a duplex stem in the engineered RNA precursor.
  • This sequence can replace a stem portion of a wild-type pre-stRNA sequence, e.g., enzymatically, or is included in a complete sequence that is synthesized.
  • a stem portion of a wild-type pre-stRNA sequence e.g., enzymatically, or is included in a complete sequence that is synthesized.
  • Engineered RNA precursors include in the duplex stem the 21-22 or so nucleotide sequences of the siRNA or siRNA-like duplex desired to be produced in vivo.
  • the stem portion of the engineered RNA precursor includes at least 18 or 19 • nucleotide pairs corresponding to the sequence of an exonic portion of the gene whose expression is to be reduced or inhibited.
  • the two 3 1 nucleotides flanking this region of the stem are chosen so as to maximize the production of the siRNA from the engineered RNA precursor, and to maximize the efficacy of the resulting siRNA in targeting the corresponding mRNA for translational repression or destruction by RNAi in vivo and in vitro.
  • shRNAs of the invention include miRNA sequences, optionally end-modified miRNA sequences, to enhance entry into RISC.
  • the miRNA sequence can be similar or identical to that of any naturally occurring miRNA (see e.g. The miRNA Registry; Griffiths- Jones S. Nuc. Acids Res., 2004). Over one thousand natural miRNAs have been identified to date and together they are thought to comprise , ⁇ 1% of all predicted genes in the genome.
  • miRNAs are clustered together in the introns of pre-mRNAs and can be identified in silico using homology-based searches (Pasquinelli et al, 2000; Lagos-Quintana et al., 2001; Lau et al, 2001; Lee and Ambros, 2001) or computer algorithms (e.g. MiRScan, MiRSeeker) that predict the capability of a candidate miRNA gene to form the stem loop structure of a pri-mRNA (Grad et al., MoI. Cell., 2003; Lim et al, Genes Dev., 2003; Lim et al, Science, 2003; Lai EC et al., Genome Bio., 2003).
  • homology-based searches Pasquinelli et al, 2000; Lagos-Quintana et al., 2001; Lau et al, 2001; Lee and Ambros, 2001
  • computer algorithms e.g. MiRScan, MiRSeeker
  • RNA Registry provides a searchable database of all published miRNA sequences (The miRNA Registry at the Sanger Institute website; Griffiths- Jones S. Nuc. Acids Res.. 2004).
  • natural miRNAs include lin-4, let-7, miR-10, mirR-15, miR-16, miR-168, miR-175, miR-196 and their homologs, as well as other natural miRNAs from humans and certain model organisms including Drosopbila melanogaster, Caenorhabditis elegans, zebraf ⁇ sh, Arabidopsis thalania, mouse, and rat as described in International PCT Publication No. WO 03/029459.
  • Naturally-occurring miRNAs are expressed by endogenous genes in vivo and are processed from a hairpin or stem-loop precursor (pre-miRNA or pri-miRNAs) by Dicer , or other RNAses (Lagos-Quintana et al., Science, 2001; Lau et al., Science, 2001; Lee and Ambros, Science, 2001; Lagos-Quintana et al.,Curr. Biol, 2002; Mourelatos et al., Genes Dev., 2002; Reinhart et al., Science, 2002; Ambros et al., Curr.
  • pre-miRNA or pri-miRNAs hairpin or stem-loop precursor
  • Dicer or other RNAses
  • miRNAs can exist transiently in vivo as a double-stranded duplex b ut only one strand is taken up by the RISC complex to direct gene silencing.
  • Certain miRNAs e.g. plant miRNAs, have perfect or near-perfect complementarity to their target mRNAs and, hence, direct cleavage of the target mRNAs.
  • Other miRNAs have less than perfect complementarity to their target mRNAs and, hence, direct translational repression of the target mRNAs.
  • the degree of complementarity between > an miRNA and its target mRNA is believed to determine its mechanism of action. For example, perfect or near-perfect complementarity between a miRNA and its target mRNA is predictive of a cleavage mechanism (Yekta et al, Science, 2004), whereas less than perfect complementarity is predictive of a translational repression mechanism.
  • the miRNA sequence is that of a naturally-occurring miRNA sequence, the aberrant expression or activity of which is correlated with a miRNA disorder.
  • RNA precursors As a consequence of their length, sequence, and/or structure, they do not induce sequence non-specific responses, such as induction of the interferon response or apoptosis, or that they induce a lower level of such sequence non-specific responses than long, double- ' stranded RNA (>150bp) that has been used to induce post-transcriptional gene silencing (e.g. RNAi).
  • sequence non-specific responses such as induction of the interferon response or apoptosis
  • RNAi post-transcriptional gene silencing
  • the interferon response is triggered by dsRNA longer than
  • a modulatory compound of the invention is a nucleic acid molecule that is capable of interacting with (e.g. binding to) a sequestration polypeptide.
  • the invention is directed to nucleic acid molecules that are capable of binding with high affinity (e.g. at least InM) to an RCK polypeptide, a RCK ortholog, or a RCK interact ing polypeptide (herein, "RCK interacting nucleic acids”) as well as recombinant expression vectors encoding said nucleic acid molecule.
  • Nucleic acids that , bind with high affinity to the target proteins in a cell are well known in the art.
  • RCK interacting nucleic acid for example, can be an RNA oligonucleotide of about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.
  • the RCK ' interacting nucleic acid comprises a small RNA or aptamer (see e.g., Ellington and Szostak, Nature 346:818 (1990), Tuerk and Gold, Science 249:505 (1990), U.S. Pat. No. 5,582,981, PCT Publication No. WO 00/20040, U.S. Pat. No.
  • the RCK interacting nucleic acids of the invention are capable of binding to a RCK polypeptide (or a RCK ortholog or RCK interactor) without substantially interfering with a desired activity of the RCK polypeptide (or RCK ortholog or RCK interacting polypeptide).
  • the RCK interacting nucleic acids, of the invention a capable of binding (e.g. hydrogen bonding ) to a region of the polypeptide sequence outside a domain that confers a biological activity to the RCK polypeptide.
  • the RCK interacting nucleic acids of the invention bind to a polypeptide sequence adjacent to a DEAD/H box domain of a RCK polypeptide.
  • the RCK interacting nucleic acids of the invention may be identified using methods that are well known in the art. Said methods generally involve using the RCK polypeptide (or RCK ortholog or RCK interacting polypeptide) to screen (most , typically, in vitro) a library of nucleic acids of variant sequence and selecting those nucleic acids which form high affinity binding interactions with the RCK polypeptide.
  • an in vitro selection techniques for identifying RNA aptamers involve first preparing a large pool of DNA molecules of the desired length that contain at least some region that is randomized or mutagenized.
  • a common oligonucleotide pool for aptamer selection might contain a region of 20-100 randomized nucleotides flanked on both ends by an about 15-25 nucleotide long region of defined sequence useful for the binding of PCR primers.
  • the oligonucleotide pool is amplified using standard PCR techniques.
  • the DNA pool is then transcribed in vitro.
  • the RNA transcripts are then subjected to affinity chromatography. The transcripts are most typically passed through a column or contacted with magnetic beads or the like on which the target ligand has been immobilized. RNA molecules in the pool which bind to the ligand are retained on the column or bead, while nonbinding sequences are washed away.
  • RNA molecules which bind the ligand are then reverse transcribed and amplified again by PCR (usually after elution).
  • the selected pool sequences are then put through another round of the same type of selection. Typically, the pool sequences are put through a total of about three to ten iterative rounds of the selection procedure.
  • the cDNA is then amplified, cloned, and sequenced using standard procedures to identify the sequence of the RNA molecules which are capable of acting as aptamers for the target ligand.
  • An exemplary screening method well known in the art is the SELEX method (Systematic Evolution of Ligands by Exponential Enrichment, see, for example, U.S. Pat. Nos. 5,270,163 and 5,567,588; herein incorporated by reference).
  • RCK interacting nucleic acid identified by art-recognized methods may then be constructed using chemical synthesis and enzymatic ligation reactions using any of the procedures described supra for the synthesis of antisense olignucleotides (e.g., an RCK interacting oligonucleotide).
  • RCK interacting nucleic acids may be modified with any of the modified nucleotides described supra. Examples of preferred modified nucleotides 5-fluoro nucleotides, 2'-O-methyl nucleotides, and backbone modified nucleotides.
  • an RCK interacting nucleic acid can be produced biologically and delivered to a cell using an y of the methods described supra for antisense nucleotides.
  • a RCK polypeptide (or a RCK ortholog polypeptide or RCK interactor polypeptide), or fragment thereof, is used in a method of the invention, for example, to modulate RNAi.
  • Methods for introducing polypeptides into cells and/or administering polypeptides to organisms are well known in the art. Any of the RCK polypeptides, RCK ortholog polypeptides, RCK interactor polypeptides, or bioactive fragments thereof, described supra are suitable for use in such methodologies.
  • the invention provides an antibody that specifically binds to
  • RCK a RCK orthologue, a RCK -interacting protein, or fragment thereof
  • the antibody is capable of identifying, altering, or interfering with a RCK:RCK interactor interaction.
  • the invention provides an antibody capable of binding an epitope within amino acid residue positions 1145 to 1347 of RCK (DCR-I), or corresponding residues of a homolog thereof.
  • the invention also provides polypeptides comprising RCK epitopes suitable for raising such antibodies, e.g., for use as immunogens or screening polypeptides.
  • the epitope is within amino acid residue positions 1145 to 1347 of RCK (DCR-I), or corresponding residues of a homolog thereof.
  • a RCK polypeptide, RCK ortholog polypeptide or RCK interactor polypeptide, or a portion or fragment of the RCK polypeptide, RCK ortholog polypeptide or RCK interactor polypeptide, can be used as an immunogen to generate antibodies that bind RCK, the RCK ortholog or the RCK interactor or that block RCK protein/RCK interactor binding using Standard techniques for polyclonal and monoclonal antibody . preparation.
  • a full-length polypeptide can be used or, alternatively, the invention provides antigenic peptide fragments for use as immunogens.
  • an antigenic fragment comprises at least 8 amino acid residues of the amino acid sequence of a RCK polypeptide, RCK ortholog polypeptide or RCK interactor polypeptide and encompasses an epitope of the RCK polypeptide, RCK ortholog polypeptide or RCK interactor polypeptide such that an antibody raised against the peptide forms a specific immune complex with RCK polypeptide, RCK ortholog polypeptide or RCK interactor polypeptide, respectively.
  • the antigenic peptide comprises at least 10 amino acid residues, more preferably at least 15 amino acid residues, even more preferably at least 20 amino acid residues, and most preferably at least 30 amino acid residues of the RCK polypeptide, RCK ortholog polypeptide or RCK interactor polypeptide.
  • Preferred epitopes encompassed by the antigenic peptide are regions of RCK polypeptide, RCK ortholog polypeptide or RCK interactor polypeptide that are located on the surface of the protein, e.g., hydrophilic regions.
  • Antigenic determinants at the termini of RCK polypeptide, RCK ortholog polypeptide or RCK interactor polypeptide are preferred for the development of antibodies that do not interfere with the RCK protein: RCK interactor protein interaction.
  • interfering antibodies can be generated towards ⁇ antigenic determinants located within the RCK interacting domain of RCK protein. The latter are preferred for inhibitory purposes.
  • a RCK polypeptide, RCK ortholog polypeptide or RCK interactor immunogen typically is used to prepare antibodies by immunizing a suitable subject, (e.g., rabbit, goat, mouse or other mammal) with the immunogen.
  • An appropriate immunogenic preparation can contain, for example, recombinantly polypeptide or a chemically synthesized polypeptide.
  • the preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or similar immunostimulatory agent.
  • Immunization of a suitable subject with an immunogenic RCK polypeptide, RCK ortholog polypeptide or RCK interactor preparation induces a polyclonal anti-RCK , anti-RCK ortholog or anti-RCK interactor antibody response, respectively.
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site which specifically binds (immunoreacts with) an antigen, such as RCK, RCK ortholog or RCK interactor.
  • immunologically active portions of immunoglobulin molecules include F(ab) and F(ab')2 fragments which can be generated by treating the antibody with an enzyme such as pepsin.
  • the invention provides polyclonal and monoclonal antibodies that bind RCK protein, RCK ortholog, or RCK interactor protein.
  • monoclonal antibody or “monoclonal antibody composition”, as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of RCK protein, RCK ortholog or RCK interactor protein.
  • a monoclonal antibody composition thus typically displays a single binding affinity for a particular RCK polypeptide, RCK ortholog polypeptide or RCK interactor polypeptide with which it immunoreacts.
  • Polyclonal anti-RCK, anti-RCK ortholog or anti-RCK interactor antibodies can be prepared as described above by immunizing a suitable subject with a RCK, RCK ortholog or RCK interactor immunogen, respectively.
  • the antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized RCK 3 RCK ortholog or RCK interactor.
  • ELISA enzyme linked immunosorbent assay
  • the antibody molecules can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as protein A chromatography to obtain the IgG fraction.
  • antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein (1975) Nature 256:495-497) (see also, Brown et al. (1981) J. Immunol. 127:539-46; Brown et al. (1980) J. Biol. Chem
  • an immortal cell line typically a myeloma
  • lymphocytes typically splenocytes
  • the culture supernatants ' of the resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that binds RCK, RCK ortholog or RCK interactor, respectively.
  • any of the many well known protocols used for fusing lymphocytes and irnmortalized cell lines can be applied for the purpose of generating an anti-RCK monoclonal antibody, anti-RCK ortholog antibody or anti-RCK interactor antibody (see, ' e.g., G. Galfre et al. (1977) Nature 266:550-52; Gefter et al. Somatic Cell Genet, cited supra; Lerner, Yale J. Biol. Med., cited supra; Kenneth, Monoclonal Antibodies, cited supra). Moreover, the ordinarily skilled worker will appreciate that there are many variations of such methods which also would be useful.
  • the immortal cell line (e.g., a myeloma cell line) is derived from the same mammalian species as the lymphocytes.
  • murine hybridomas can be made by fusing lymphocytes from a mouse immunized with an immunogenic preparation of the present invention with an immortalized mouse cell line.
  • Preferred • immortal cell lines are mouse myeloma cell lines that are sensitive to culture medium containing hypoxanthine, aminopterin and thymidine ("HAT medium").
  • myeloma cell lines can be used as a fusion partner according to standard ' techniques, e.g., the P3-NS 1/1-Ag4-1 , P3-x63-Ag8.653 or Sp2/O-Agl4 myeloma lines.
  • These myeloma lines are available from ATCC.
  • HAT ⁇ sensitive mouse myeloma cells are fused to mouse splenocytes using polyethylene glycol ("PEG").
  • PEG polyethylene glycol
  • Hybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfused and unproductively fused myeloma cells (unfused splenocytes die after several days because they are not transformed).
  • Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma culture supernatants for antibodies that bind RCK, RCK ortholog or RCK interactor, e.g., using a standard ELISA assay.
  • a monoclonal antibody can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with RCK, RCK ortholog or RCK interactor to thereby isolate immunoglobulin library members that bind RCK, RCK ortholog or RCK interactor, respectively.
  • Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the Stratagene SurfZAPTM Phage Display Kit, Catalog No. 240612).
  • examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, Ladner et al. U.S. Patent No. 5,223,409; Kang et al. PCT International Publication No. WO 92/18619; Dower et al. PCT International Publication No. WO 91/17271; Winter et al. PCT International Publication WO 92/20791; Markland et al. PCT International Publication No. WO 92/15679; Breitling et al. PCT International Publication WO 93/01288; McCafferty et ⁇ al. PCT International Publication No.
  • An anti-RCK, anti-RCK ortholog or anti-RCK interactor antibody (e.g., , monoclonal antibody) can be used to isolate RCK, RCK ortholog or RCK interactor, bioactive portions thereof, or fusion proteins by standard techniques, such as affinity chromatography or immunoprecipitation.
  • Anti-RCK antibodies, anti-RCK ortholog ' antibodies or anti-RCK interactor antibodies made according to any of the above- described techniques can be used to detect protein levels in donor or acceptor fractions as part of certain assay methodologies described herein. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance.
  • detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bio luminescent materials, and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, -galactosidase, or acetylcholinesterase
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythri ⁇
  • an example of a luminescent material includes luminol
  • examples of bio luminescent materials include luciferase, luciferin, and aequorin
  • suitable radioactive material include 1251, 1311, 35S or 3H.
  • Antibodies that are specific for the RCK protein, RCK ortholog or RCK interactor protein, and oprtionally interfere with its activity may also be used to modulate (e.g., inhibit) at least one activity or function of the RCK protein, RCK ortholog or RCK interactor protein.
  • Such antibodies may be generated using standard techniques described herein, against the RCK protein, RCK ortholog or RCK interactor protein itself or against peptides corresponding to portions of said proteins.
  • Such antibodies include but are not limited to polyclonal, monoclonal, Fab fragments, single chain antibodies, or chimeric antibodies.
  • Lipofectin liposomes may be used to deliver the antibody or a fragment of the Fab region which binds to the target epitope into cells. Where fragments of the antibody are used, the smallest inhibitory fragment which binds to the target protein's binding domain is preferred.
  • peptides ' having an amino acid sequence corresponding to the domain of the variable region of the antibody that binds to the target gene protein may be used. Such peptides may be synthesized chemically or produced via recombinant DNA technology using methods well known in the art (described in, for example, Creighton (1983), supra; and '
  • Single chain neutralizing antibodies which bind to intracellular target gene epitopes may also be administered.
  • Such single chain antibodies may be administered, for example, by expressing nucleotide sequences encoding single-chain antibodies within the target cell population by utilizing, for example, techniques such as those described in Marasco et al. (1993) Proc. Natl. Acad. Sci. USA 90:7889-7893).
  • the present invention also features mJRISC tethering constructs, which are suitable for use in modulating (e.g., enhancing) miRNA-mediated translational repression of a particular target RNA both in vitro and in vivo.
  • the miRISC tethering constructs of the invention are designed to facilitate the recruitment of (e.g. tethering) sequestration agents (e.g. exogenous or endogenous RCK polypeptides, RCK interacting polypeptides, or modulators thereof) to a target RNA bound within a P-body complex so as to modulate (e.g., enhance) the translational repression of the target RNA.
  • sequestration agents e.g. exogenous or endogenous RCK polypeptides, RCK interacting polypeptides, or modulators thereof
  • the tethering constructs are useful both for sequence-specific translation repression purposes as well as for therapeutic applications in which modulation (e.g., enhancement) of translational repression is desirable.
  • the tethering constructs of the invention comprise a targeting moiety (T) which is capable of binding to a target RNA and facilitating the recruitment of a miRISC complex to the target RNA.
  • T targeting moiety
  • Exemplary targeting moieties are described, infra .
  • the tethering constructs have the formula T-L-S, wherein T is the , targeting moiety, S is a sequestration moiety and L is a linking moiety described, infra ⁇ which operably links the T and S moieties.
  • a tether employed by the methods of the invention is a dual-functional oligonucleotide (see, e.g. International Patent Publication No. WO 2005/078096 of Zamore et al., which is incorporated by reference herein).
  • a tethering construct of the invention is any tethering construct capable of binding to a target RNA and facilitating the recruitment of a sequestration polypeptide to the target RNA, with the proviso that said tethering constructs do not bind to miRNA within the miRISC complex.
  • Moieties within the tethering constructs can be arranged or linked as depicted in the formula T-L-S,or alternatively, the moieties can be arranged or linked in the tethering construct in the opposite configuration (i.e. S-L-T).
  • Multimeric configurations of the tethering constructs are also contemplated, e.g., tethering constructs having the formula T-L-S-L-T or S-L-T-L-S.
  • a tethering construct of the invention comprises a RCK polypeptide (or an ortholog or bioactive fragment thereof) as a sequestration moiety.
  • a tethering construct of the invention comprises a RCK polypeptide (or an ortholog or bioactive fragment thereof) as a sequestration moiety and an oligonucleotide as a targeting moiety, (e.g. an RNA oligonucleotide targeting moiety described infra).
  • a tethering construct of the invention comprises a RCK polypeptide (or an ortholog or bioactive fragment thereof) as sequestration moiety and a RNA binding polypeptide (e.g. TAT, REV, or TAR binding protein) as a targeting moiety.
  • a RCK polypeptide or an ortholog or bioactive fragment thereof
  • a RNA binding polypeptide e.g. TAT, REV, or TAR binding protein
  • an RCK interacting polypeptide e.g. Ago-1, Ago-2, or another polypeptide of the miRISC complex
  • a tethering construct of the invention comprises a RCK interacting polypeptide (or an ortholog or bioactive fragment thereof) as a sequestration moiety and an oligonucleotide (e.g. an RNA oligonucleotide targeting moiety described infra) as a targeting moiety.
  • a tethering construct of the invention comprises a RCK interacting polypeptide (or an ortholog or bioactive fragment thereof) as sequestration moiety and a RNA binding polypeptide (e.g. TAT, REV, or TAR binding protein) as a targeting moiety.
  • a RCK interacting polypeptide or an ortholog or bioactive fragment thereof
  • a RNA binding polypeptide e.g. TAT, REV, or TAR binding protein
  • the tethering constructs of the invention may also feature certain RCK ⁇ modulatory agents as sequestration moieties.
  • RCK modulatory agents which are capable of forming stable binding interactions with an RCK polypeptide (or an ortholog or bioactive fragment thereof) are employed as sequestration moieties (e.g., a compound or an anti-RCK antibody or binding fragment thereof).
  • a tethering construct of the invention may be a hybrid oligonucleotide wherein a tethering moiety of the construct is RCK- interacting nucleic acid (e.g.
  • a targeting moiety of the construct is a oligonucleotide targeting moiety (e.g. a small RNA oligonucleotide targeting moiety).
  • exemplary tethering constructs feature an RCK-interacting nucleic acid (e.g. a small, RCK-interacting RNA) as a sequestration moiety and a RNA binding polypeptide (e.g. a TAT, REV, or TRBP) as a targeting moiety.
  • the tethering constructs are formed within a cell by separately introducing the targeting moiety (T) and the linker moiety (L) to the cell such that T and L associate to form a partial tethering construct T-L (or L-T) which in turn associates with a sequestration polypeptide (S) to form a tethering construct of the invention.
  • T targeting moiety
  • L linker moiety
  • S sequestration polypeptide
  • a partial tethering construct having the formula T-L (L-T) is introduced to the cell where it associates with an endogenous sequestration polypeptide to form a tethering construct of the invention having the formula T-L-S (or S-L-T).
  • T is introduced to the cell where it associates with an endogenous L moiety (e.g. a sequestration polypeptide binding protein) and an endogenous sequestration polypeptide to from a tethering construct of the invention having the formula T-L-S (or S-L-T).
  • an endogenous L moiety e.g. a sequestration polypeptide binding protein
  • an endogenous sequestration polypeptide binding protein e.g. a sequestration polypeptide binding protein
  • an endogenous sequestration polypeptide binding protein e.g. a sequestration polypeptide binding protein
  • Targeting moieties (T) The targeting moiety, as described above, is capable of capturing a specific target
  • RNA e.g. a target mRNA
  • expression of the target RNA is undesirable, and, thus, translational repression of the target RNA is desired.
  • the target RNA is a mRNA that encodes a protein involved in a disease or a disorder.
  • the mRNA may encode for huntingtin protein (e.g. mutant huntingtin protein), which is associated with Huntington's disease (a genetic ⁇ neurodegenerative disease).
  • the RNA is a viral RNA.
  • the targeting moieties of the tethering constructs of the ' invention comprise an oligonucleotide sequence (e.g. a DNA or RNA oligonucleotide J sequences) having complementarity to the target RNA (herein, "oligonucleotide targeting moieties").
  • the oligonucleotide targeting moiety is a DNA oligonucleotide.
  • the oligonucleotide is a RNA oligonucleotide.
  • the oligonucleotide is an RNA/DNA hybrid t molecule comprising both ribonucleotides and deoxyribonucleotides.
  • the complementarity of the oligonucleotide targeting moiety and the target RNA sequence should be designed to promote binding of the oligonucleotide targeting moiety with the target RNA.
  • the oligonucleotide targeting moiety should include a sequence of sufficient size and of sufficient degree of complementarity to the target RNA so as to effectively and selectively bind the target RNA.
  • the oligonucleotide targeting moiety has sufficient complementarity to one or more sequences within the 3 ' Untranslated Region (3 '-UTR) of a target mRNA.
  • the oligonucleotide targeting moiety has sufficient complementarity to one or more sequences in the 5 '-untranslated region (5'-UTR) of a target mKNA. In another embodiment, the oligonucleotide targeting moiety has sufficient complementarity to one or more sequences in the coding region of a target mRNA. In another embodiment, the olignucleotide targeting moiety has sufficient complementarity to a plurality of sites on a target RNA sequence (e.g., about 10, 5, 4, 3, or 2 sites). In another embodiment, the tethering construct contains a plurality of oligonucleotide targeting moieties, each with sufficient complementarity to one or more sites on the target RNA sequence.
  • oligonucleotide targeting moieties may have sufficient complementarity to the same site on the target RNA sequence.
  • a construct of the invention comprises an oligonucleotide RNA targeting moiety with complementarity to one site on a target RNA sequence.
  • the length of the oligonucleotide targeting moiety will vary greatly depending, in part, on the length of the target RNA and the degree of complementarity between the '. target RNA and the oligonucleotide targeting moiety. In various embodiments, the oligonucleotide targeting moiety is less than about 200, 100, 50, or 25 nucleotides in • length.
  • the oligonucleotide targeting moieties have a length of between about 10-100 nucleotides (or modified nucleotides), preferably between about 10-40 nucleotides (or modified nucleotides), for example, between about 15-35, e.g., about 15-20, 15-25, 20-25, 25-30, 30-35 (31, 32, 33, 34, 35), or 35-40 nucleotides , (or modified nucleotides).
  • the oligonucleotide targeting moiety is about 15 to about 25 nucleotides in length.
  • the oligonucleotide targeting moiety is about 15 nucleotides in length, e.g., 15, 16, 17 or 18 nucleotides in length.
  • Oligonucleotide targeting moieties should have sufficient complementarity with a target RNA to mediate binding to the target RNA. However, 100% sequence complementarity between the oligonucleotide targeting moiety and the target RNA is not required to practice the present invention. Oligonucleotide targeting moieties are able to tolerate sequence variations that might be expected due to genetic mutation, strain polymorphism, or evolutionary divergence. Insertions, deletions, and single point mutations relative to the target sequence may be tolerated in the oligonucleotide targeting moiety such that remains effective in binding the target RNA.
  • Sequence complementarity may be determined by sequence comparison and alignment algorithms known in the art. To determine the percent complementarity of oligonucleotide targeting moiety and the target RNA, the reverse complement sequence of either the oligonucleotide targeting moiety sequence or the target RNA sequence is aligned with the target RNA sequence or the oligonucleotide targeting moiety sequence, respectively, for optimal comparison purposes (e.g., gaps can be introduced in the either ' sequence for optimal alignment). The nucleotides (or amino acid residues) at corresponding nucleotide (or amino acid) positions are then compared. When a position ' in the first sequence is occupied by the same residue as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the comparison of sequences and determination of percent identity between two ' sequences can be accomplished using a mathematical algorithm.
  • the alignment generated over a certain portion of the sequence aligned having sufficient identity but not over portions having low degree of identity i.e., a local alignment.
  • a ' preferred, non-limiting example of a local alignment algorithm utilized for the comparison of sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. ScI USA 87:2264-68, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77. Such an algorithm is incorporated into the BLAST programs
  • the alignment is optimized by introducing appropriate gaps and percent identity is determined over the length of the aligned sequences (i.e., a gapped alignment).
  • a gapped alignment i.e., Gapped BLAST can be utilized as described in Altschul et al., ( ⁇ 997) Nucleic Acids Res.
  • the alignment is optimized by introducing appropriate gaps and percent identity is determined over the entire length of the sequences aligned (i.e., a global alignment).
  • a global alignment i.e., a mathematical algorithm utilized for the global comparison of sequences.
  • a mathematical algorithm utilized for the global comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the
  • ALIGN program version 2.0 which is part of the GCG sequence alignment software package.
  • a PAM120 weight residue table a gap length penalty of 12, and a gap penalty of 4 can be used.
  • 70% sequence complementarity e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100% sequence complementarity, between the oligonucleotide targeting moiety and the target RNA sequence is preferred.
  • the oligonucleotide targeting moiety sequence may be defined functionally as a nucleotide sequence (or oligonucleotide sequence) a portion of which is capable of hybridizing with the target RNA sequence ⁇ e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 5OyC or 7OyC hybridization for 12-16 hours; followed by washing).
  • Additional preferred hybridization conditions include ⁇ hybridization at 70 0 C in IxSSC or 50 0 C in IxSSC, 50% fo ⁇ namide followed by washing at 70 0 C in 0.3xSSC or hybridization at 70 0 C in 4xSSC or 5O 0 C in 4xSSC, 50% formamide followed by washing at 67°C in IxSSC.
  • the length of the complementary between nucleotide sequences may be at least about 10, 12, 15, 17, 20, 22, 25, 27, 30, 32, 35, 37, 40, 42, 45, 47 or 50 bases.
  • oligonucleotide targeting moieties comprise modified nucleotides, e.g. 2'-O-methyl ribonucleotides.
  • modified nucleotides e.g. 2'-O-methyl ribonucleotides.
  • Many other forms of oligonucleotide modification can be incorporated into the oligonucleotide targeting ⁇ moiety, for example, locked nucleic acids (oligonucleotides comprising at least one 2'- C,4'-C-oxy-methylene-linked bicyclic ribonucleotide monomer) and phosphorotbioate modifications.
  • the oligonucleotide targeting moieties are modified to improve stability in serum or in growth medium for cell cultures.
  • the 3 '-residues may be stabilized against degradation, e.g., they may be selected such that they consist of purine nucleotides, particularly adenosine or guanosine nucleotides.
  • substitution of pyrimidine nucleotides by modified analogues e.g., substitution of uridine by 2'-deoxythymidine.
  • the 5'-te ⁇ ninus is, most ' preferably, phosphorylated (i.e., comprises a phosphate, diphosphate, or triphosphate group).
  • Preferred nucleotide analogues include sugar- and/or backbone-modified ribonucleotides (i.e., include modifications to the phosphate-sugar backbone).
  • the phosphodiester linkages of an RNA oligonucleotide targeting moiety may be modified to include at least one of a nitrogen or sulfur heteroatom.
  • the phosphoester group connecting to adjacent ribonucleotides is replaced by a modified group, e.g., of phosphothioate group.
  • the 2' OH-group is replaced by a group selected from H, OR, R, halo, SH, SR, NH2, NHR, NR2 or ON, wherein R is C1-C6 alkyl, alkenyl or alkynyl and halo is F, Cl, Br or I.
  • the modifications are 2'-fluoro, 2'-amino and/or 2'-thio modifications.
  • Particularly preferred modifications include 2'-fluoro-cytidine, 2'-fluoro-uridine, 2'-fluoro-adenosine, 2'-fluoro-guanosine, 2'-amino-cytidine, 2'-amino-uridine, 2'-amino-adenosine, 2'-amino- guanosine, 2,6-diaminopurine, 4-thio-uridine, and/or 5-amino-allyl-uridine.
  • the 2'-fluoro ribonucleotides are every uridine and cytidine.
  • Additional exemplary modifications include 5-bromo-uridine, 5-iodo-uridine, 5-methyl- cytidine, ribo-thymidine, 2-aminopurine, 2'-amino-butyryl-pyrene-uridine, 5-fluoro- cytidine, and 5-fluoro-uridine.
  • 2'-deoxy-nucleotides and 2'-0me nucleotides can also be used within modified oligonucleotide targeting moieties of the instant invention.
  • Additional modified residues include, deoxy-abasic, inosine, N3-methyl-uridine, N6, N6-dimethyl-adenosine, pseudouridine, purine ribonucleoside and ribavirin.
  • nucleobase-modified nucleotides containing at least one non- naturally occurring nucleobase instead of a naturally occurring nucleobase.
  • Bases may be modified to block the activity of adenosine deaminase.
  • modified nucleobases include, but are not limited to, uridine and/or cytidine modified at the 5- position, e.g., 5-(2-amino)propyl uridine, 5-bromo uridine; adenosine and/or guanosines modified at the 8 position, e.g., 8-bromo guanosine; deaza nucleotides, e.g.
  • Oligonucleotide targeting moieties may also be modified with chemical moieties (e.g., cholesterol) that improve their in vivo pharmacological properties.
  • the oligonucleotide targeting moiety comprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleobase-modified nucleotides (i.e., the nucleotides contain at least one non-naturally occurring nucleobase instead of a naturally occurring nucleobase).
  • the internal residues of the oligonucleotide targeting ' moiety are modified.
  • an "internal" nucleotide is one occurring at any . position other than the 5' end or 3' end of the oligonucleotide.
  • the oligonucleotide targeting moiety is modified by the substitution of at least one internal ' nucleotide.
  • the oligonucleotide targeting moiety is modified by ' the substitution of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more internal nucleotides.
  • the oligonucleotide targeting moiety is modified by the substitution of at least 5%, 10%, . 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of the internal nucleotides.
  • the modified oligonucleotide , targeting moiety can contain mismatches or bulges.
  • the oligonucleotide targeting moiety comprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, ( 9, 10, or more) backbone-modified nucleotides (i.e., modifications to the phosphate sugar backbone).
  • the phosphodiester linkages of an RNA oligonucleotide targeting moiety may be modified to include at least one of a nitrogen or sulfur heteroatom.
  • the phosphoester group connecting to adjacent ribonucleotides is replaced by a modified group, e.g., of phosphothioate group.
  • the olignucleotide targeting moiety comprises a sequence wherein at least a portion contains one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) mismatches with the respective target RNA ⁇ e.g., mRNA).
  • the oligmicleotide targeting moiety comprises any combination of two or i more (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) modifications as described herein.
  • the olignucleotide targeting moiety can comprise a combination of two sugar-modified nucleotides, wherein the sugar-modified nucleotides are 2'-fluoro ' modified ribonucleotides (e.g.
  • 2'-fluoro uridine or 2'-fluoro cytidine 2'-fluoro uridine or 2'-fluoro cytidine
  • 2'-deoxy ribonucleotides e.g., 2'-deoxy adenosine or 2'-deoxy guanosine
  • Oligonucleotide targeting moieties may be produced enzymatically or by partial/total organic synthesis, any modified nucleotide can be introduced by in vitro enzymatic or organic synthesis.
  • an oligonucleotide targeting moiety is prepared chemically. Methods of synthesizing RNA and DNA molecules are known t in the art, in particular, the chemical synthesis methods as de scribed in Verma and Eckstein (1998) Annul Rev. Biochem. 67:99-134.
  • an oligonucleotide targeting moiety is prepared enzymatically, for example by enzymatic transcription from synthetic DNA templates or from DNA plasmids isolated from recombinant bacteria.
  • polypeptides are attached to either end of the oligonucleotide, or, if necessary, to both ends of the molecule.
  • the targeting moieties of the tethering constructs of the invention comprise a polypeptide sequence which is capable of selectively binding a particular target RNA or class of target RNAs (herein, "polypeptide targeting moieties").
  • the polypeptide targeting moiety is an RNA binding protein. or an active portion thereof.
  • the binding affinity of the polypeptide targeting moiety and the target RNA sequence should be sufficient to promote binding of the tethering construct with the target RNA.
  • the specific binding affinity of an RNA binding polypeptide for an RNA binding site refers to a dissociation constant of at least 10 ⁇ M, preferably at least 100 nM and most preferably at least 10 nM, and the capacity to bind one (or more) RNA binding sites more strongly (Le., at least 5-fold, 10-fold, 100-fold or 1000-fold) than others. Dissociation constants as low as 1 nM, 1 pM or 1 fM are possible for polypeptide-RNA binding. '
  • the polypeptide targeting moiety should include a minimal portion of the RNA binding domain of the RNA binding protein which is sufficient to effectively and selectively bind the target RNA.
  • the polypeptide targeting moiety has sufficient binding affinity for the 5' Untranslated Region (UTR) of , the target mRNA.
  • the polypeptide targeting moiety has sufficient binding affinity for the 3' Untranslated Region (UTR) of a target mRNA.
  • the oligonucleotide targeting moiety has sufficient binding affinity to a plurality stem loops on a target RNA sequence (e.g., about 10, 5, 4, 3, or 2 stem loops).
  • the tethering construct contains a plurality of polypeptide targeting moieties, each with sufficient binding affinity for one or more stem-loops on the target RNA sequence.
  • at least two of the polypeptide targeting moieties may have sufficient binding affinity to the same site on the target RNA sequence.
  • the targeting polypeptide moiety may be a recombinant polypeptide, natural polypeptide, or synthetic polypeptide, preferably a recombinant polypeptide.
  • the polypeptide of the invention may be a purified natural product or a chemically synthetic , product. Alternatively, it may be produced from prokaryotic or eukaryotic hosts, such as ' , bacteria, yeast, higher plant, insect, and mammal cells, using recombinant techniques. Depending on the host used in the protocol of recombinant production, the polypeptide of the invention may be glycosylated or non-glycosylated.
  • the polypeptide targeting moiety is a sequence-specific RNA binding protein or an active portion thereof which is capable of interacting specifically with a target RNA.
  • Sequence specific RNA binding molecules comprise RNA binding protein motifs which are capable of recognizing binding sites found within the untranslated regions of a particular target RNA molecule or a subset of target RNA molecules. This is a specific interaction.
  • some RNA binding proteins bind RNA molecules in general (i.e. non-specifically) based on the general chemical characteristics of all RNA molecules.
  • Common sequence-specific RNA binding protein motifs include the RNP motif, Arg-rich motif (ARM) , RGG box, and double-stranded RNA-binding motif (see, e.g.
  • RNA binding proteins useful as polypeptide targeting moieties include members of the RRM superfamily of RNA binding proteins.
  • the RRM superfamily of RNA binding proteins contain an RNP consensus octamer and an 80 amino acid motif implicated in RNA recognition (see, e.g. Query et aL, Cell, 57: 89-101 (1989); Kenan et al, Trends Biochem. ScL, 16: 214-220 (1991)).
  • Many members of the RRM superfamily have been reported to date, the majority of which reside in all tissues and are ubiquitously conserved in phylogeny.
  • Tissue-specific members of the RRM family are less common, but include IMP, Bruno, AZP-RRMI, and Xl 6 which are expressed in pre-B cells and ELAV (embryonic lethal abnormal vision) proteins which are neuronal-specific RNA binding proteins involved in the development of the nervous system. Good et al, Proc. Natl. Acad. ScL USA, 92, 4557-4561 (1995). Human ELAV proteins (Hu proteins, e.g.
  • HuA HuB
  • HuC and HuD HuA
  • HuB HuB
  • HuC and HuD HuA
  • HuD HuA
  • HuB HuB
  • HuC and HuD and their respective alternatively-spliced isofbrms
  • AU Hu proteins contain three RNA-recognition motifs (RRMs), which confer their binding specificity for AU-rich RNA elements (AREs) in target mRNAs.
  • RRMs RNA-recognition motifs
  • the tethering constructs of the invention comprise polypeptide targeting moieties derived from the RRM protein termed HeI-Nl (Human elav-like Neuronal protein- 1), or related proteins (see US Patent No. 5,144,449).
  • HeI-Nl proteins are able to specifically bind to 3'-UTR sequences that are uniquely present in the mRNAs that encode oncoproteins (e.g. c-src, c-myc and c-fos) and lymphokines (e.g., GM-CSF).
  • Tethering constructs capable of translationally repressing oncoproteins and lymphokines mRNAs are useful regulation of tumor cell growth and proliferation in vitro and in vivo, and the regulation of immune cell expression.
  • Other RNA binding proteins which are useful as polypeptide targeting moieties include members the PUF family of RNA binding proteins (see, e.g. Wickens et al., Trends Genet., 18(3): 150-7, (2002)). PUF proteins bind 3* UTR elements and reduce expression, either by repressing translation or causing mRNA instability. PUF proteins contain, eight repeats of ⁇ 40 amino acids, called Puf repeats. All RNA targets of PUF proteins contain a UGU trinucleotide that is critical for binding. Exemplary PUFG proteins include CPEB, Pumilio- 1 , Pumilio-2, FBF- 1 , and FBF-2.
  • the polypeptide targeting moiety is an RNA binding protein capable of targeting a viral RNA 3 e.g., picornavirus RNA, retroviral, and flaviviral RNA (herein a "viral RNA binding protein").
  • viral RNAs often form highly structured motifs at their 3y or 5y UTRs or other regions. These motifs play pivotal roles in viral replication and translation through their interaction with virus encoded proteins as well as essential host factors. Examples of such RNA motifs include the internal ribosome entry sites (IRES), the 3y polypyrimidine tract, and the TAR element of the human immunodeficiency virus (HIV).
  • IRS internal ribosome entry sites
  • HAV human immunodeficiency virus
  • IRES sites have been identified , in, for example, HCV, HIV, rhinovirus, poliovirus, encephalomyocarditis virus, foot and i mouth disease virus, friend murine leukemia virus, Moloney murine leukemia virus, cricket paralysis virus, Kaposi's sarcoma-associated virus and rous sarcoma virus (see, e.g., Hellen e* ⁇ /., Genes and Dev. 15:1593-1612 (2001)).
  • TAT examples of viral RNA-binding proteins useful in the practice of the present invention are the TAT and REV proteins encoded by HIV-I, and the human TAR binding protein, (TRBP).
  • TAT consists of 86 amino acids and is a potent transactivator of long terminal repeat (LTR)-directed viral gene expression and is essential for HIV replication.
  • LTR long terminal repeat
  • the TAT protein is capable of binding specifically to the TAR
  • RNA element located within the 5' UTR of all viral mRNAs transcribed from the HTV-I genome.
  • the TAR element is capable of forming a stable stem-loop structure in the native viral RNA.
  • a 3 nucleotide (nt) bulge on the stem of TAR has been demonstrated to be essential for specific and high-affinity binding of the TAT protein to the TAR element.
  • a full- length TAT protein or a TAT polypeptide comprising the portion of the TAT protein involved in binding to TAR element (e.g.
  • the N-te ⁇ ninal 72 amino acids or a fragment comprising amino acid residues 49-72, 49-86, or 48-61 of TAT protein may be employed as a targeting polypeptide in a tethering construct.
  • the tethering of sequestration peptides to TAR using the tethering constructs of the invention is expected to enhance the translational repression of HIV transcripts whose translation is required for the replication of the HIV virus, thereby serving as an effective AID S therapy.
  • a second HTV protein that is useful in the present invention is the REV protein.
  • REV protein is a regulatory factor essential for viral replication; it is required for the production of viral structural proteins.
  • the REV protein consists of 116 amino acids, encoded by two exons.
  • the REV protein is capable of specifically binding a target RNA sequence termed the REV response element (RRE), located in the transcript of the HIV ' envelope gene.
  • RRE has been mapped to a 234-nucleotide region capable of forming four stem-loop structures and one branched stem-loop structure.
  • REV offers ⁇ another potential means of tethering a sequestration peptide specifically to HIV RNA.
  • Tethering constructs which employ the REV polypeptide, or an RNA binding portion thereof, are expected to enhance the translational repression of HIV transcripts (e.g. env transcripts) required for the replication of the HIV virus, thereby serving as an effective AIDS therapy.
  • TRBP human TAR binding protein
  • ' TRBP is similar to TAT in that it is capable of binding the TAR element in HIV-I transcripts, however it is encoded by the genome of the human host cell (see Gatignol et ah. Science, 251: 1597— 1600(1991)).
  • An exemplary sequence of human TAR binding ' protein is set forth in Table 5 below. ' ⁇
  • Polypeptide targeting moieties may be fused by well-known recombinant methods to a sequestration polypeptide moiety to generate a tethering construct of the invention. Such hybrid proteins can be then be expressed using any number of standard expression systems known in the art. Typically, DNA sequences encoding the polypeptide targeting moieties (e.g. TAT, REV, or RNA binding portions thereof) are cloned adjacent to and in-frame with a DNA sequence encoding a sequestration polypeptide or an active portion thereof. Alternatively, the polypeptide targeting moieties may be fused to the sequestration polypeptide or active portion thereof via an intervening peptide linker as described below. b) Linking Moieties (L)
  • the linking moiety refers to a domain, portion or region of the tethering construct which covalently or non-covalently joins or links a targeting moiety with a sequestration polypeptide or portion thereof. Accordingly, the linking moiety merely tethers the sequestration polypeptide with the targeting moiety. Accordingly, the linking moiety may be a discrete entity as known in the art, including, . but not limited to, a carbon chain, a polypeptide sequence, a nucleotide sequence, polyethylene glycol (PEG) or a cholesterol.
  • PEG polyethylene glycol
  • the linking moiety may be a simple phosphorus-containing moiety, such as a phosphodiester linkage, a phosphorothioate, methylphosphonates.
  • the linking moiety is a phosphodiester bond or a peptide bond.
  • the linking moiety may be a linker peptide that tethers a polypeptide targeting moiety to a sequestration polypeptide or active fragment thereof.
  • the linker peptide may be of any length suitable to allow the respective moieties to retain their normal functions such that the tethering construct is capable of modulating the translational repression of the corresponding target RNA.
  • the linking moiety is less than about 50, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 amino acids in length.
  • the linker peptide is about 10 to about 20 amino acids in length.
  • the linker peptide is absent such that the polypeptide targeting moiety is directly linked to the sequestration polypeptide via a peptide bond.
  • the linking peptide may comprise a polypeptide or portion thereof which is capable of binding (i.e. non-covalently interacting) to a sequestration polypeptide.
  • the binding affinity of the linking polypeptide should be sufficient such that the sequestration polypeptide is tethered to a target RNA corresponding to the targeting moiety of the tethering construct.
  • Exemplary linking peptides of this kind include the RCK interacting polypeptides described supra, for example, Ago-2 polypeptides or portions thereof.
  • the linking moiety may comprise a bifunctional agent (e.g. an RNA-protein crosslinking agent) that tethers an oligonucleotide targeting moiety to a sequestration polypeptide or active fragment thereof.
  • the bifunctional agent is capable of linking an amino acid of the sequestering polypeptide or active portion thereof (e.g. preferably an N-terminal or C-terminal amino acid of the sequestering polypeptide) with a nucleotide (e.g. a modified nucleotide, preferably a modified nucleotide at the 5' or 3' terminus of the oligonucleotide) of the oligonucleotide targeting moiety.
  • a nucleotide e.g. a modified nucleotide, preferably a modified nucleotide at the 5' or 3' terminus of the oligonucleotide
  • Exemplary bifunctional agents and methods for conjugating oligonucleotides to polypeptides are well-known in the art (see e.g., Tamilarasu et al, Bioorg. &Mea ⁇ Chem. Lett. 11:505-507 (2001)).
  • the linkage of a primary amine in the polypeptide to amino-allyl uridine or amino groups in a modified oligonucleotide can be accomplished using amino-allyl coupling methods (e.g., isothiocyanate, N-hydroxysuccinimide (NHS) esters).
  • photocrosslinkers e.g., thiouracil, thioguanosine, psoralens, benzophenones
  • photocrosslinkers are attached at the 3' or 5' terminus of an oligonucleotide derivative to facilitate crosslinking to a polypeptide.
  • one end of the pair to be linked can be made amine reactive and the other thiol reactive (see e.g., Wang et al, Biochemistry, 40:6458-6464, (2001)).
  • the oligonucleotide targeting moiety may comprise a modified nucleotide having one or more 2'-aldehyde groups capable of being coupled to a synthetic sequestering polypeptide , an active portion thereof, or a linker peptide comprising an N-terminal cysteine, aminooxy, or hydrazide group (see, e.g. Zatsepin et at, Bioconjugate Chem., 13(4):822-30, (2002)).
  • an oligonucleotide comprising an amine-containing modified nucleotide e.g. 3'N3
  • a maleimide crosslinker e.g.
  • RNA-peptide crosslinking methods employ platinum crosslinking reagents (e.g. trans-diamine-dichloroplatinum( ⁇ ); see, e.g. Tukalo et ah, Biochemistry; 26(16):5200-8 (1987)).
  • the target RNA of the invention is a target mRNA that specifies the amino acid sequence of a cellular protein (e.g., a nuclear, cytoplasmic, transmembrane, or membrane-associated protein).
  • the target RNA of the invention is a target mRNA that specifies the amino acid sequence of an extracellular protein (e.g., an extracellular matrix protein or secreted protein).
  • the phrase "specifies the amino acid sequence" of a protein means that the mRNA sequence is translated into the amino acid sequence according to the rules of the genetic code.
  • developmental proteins e.g., adhesion molecules, cyclin kinase inhibitors, Wnt family members, Pax ⁇ family members, Winged helix family members, Hox family members, cytokines/lymphokines and their receptors, growth/differentiation factors and their receptors, neurotransmitters and their receptors
  • oncogene-encoded proteins e.g., ABLI, BCLI, BCL2, BCL6, CBFA2, CBL, CSFlR, ERBA, ERBB, EBRB2, ETSI, ETSI, ETV6, FGR, FOS, FYN, HCR, HElAS, JUN, KRAS, LCK, LYN, MDM2, MLL, ' MYB, MYC, MYCLI, MYCN, NRAS, PIM I, PML, RET, SRC, TALI, TCL3, and YES); tumor suppressor proteins (e.g., tumor suppressor proteins (e.g., tumor suppress
  • the target RNA of the invention is an ! mRNA that specifies the amino acid sequence of a protein associated with a pathological condition.
  • the protein may be a pathogen-associated protein (e.g., a viral ' protein involved in immunosuppression of the host, replication of the pathogen, transmission of the pathogen, or maintenance of the infection), or a host protein which facilitates entry of the pathogen into the host, drug metabolism by the pathogen or host, replication or integration of the pathogen's genome, establishment or spread of infection in the host, or assembly of the next generation of pathogen.
  • the protein may be a tumor-associated protein or an autoimmune disease-associated protein.
  • the target RNA of the invention is an mRNA that specifies the amino acid sequence of an endogenous protein (i.e. , a protein present in the genome of a cell or organism).
  • the target mRNA specifies the amino acid sequence of a heterologous protein expressed in a recombinant cell or a genetically
  • the target mRNA molecule of the invention specifies the amino acid sequence of a protein encoded by a transgene (i.e., a gene construct inserted at an ectopic site in the genome of the cell).
  • the target mKNA specifies the amino acid sequence of a protein encoded by a pathogen genome which is capable of infecting a cell or an organism from which the cell is derived.
  • the target RNA of the invention is an mKNA that specifies the amino acid sequence of a protein associated with cholesterol production, including, but not limited to, apolipoprotein B (ApoB).
  • ApoB is the main apolipoprotein of ( chylomicrons and low density lipoproteins (LDL).
  • ApoB is found in the plasma in two main isoforms, apoB-48 and apoB-100. The first is synthesized by the gut, the second by the liver. The intestinal (apoB-48) and hepatic (apoB-100) forms of apoB are coded by a single gene and by a single mRNA transcript.
  • the nucleotide and amino acid sequence of human ApoB can be found in GenBank record GI 4502152, the entire , contents of which are incorporated by reference herein.
  • the target RNA of a tethering construct of the invention can include the RNA sequence of the apoB gene, including apoB-100, apoB-48, or both apoB-100 and apoB-48.
  • a target RNA of the invention is a mRNA that specifies the amino acid sequence of endogenous SODl. In another embodiment, a target RNA of the invention is mRNA that specifies the amino acid sequence of mutant SODl.
  • SODl mutations More than 100 SODl mutations have been identified. Most of these mutations produce a single amino acid replacement in the superoxide dismutase enzyme's chain of amino acids. The most common substitution, which occurs in 50 percent of American patients with type 1 amyotrophic lateral sclerosis, is the replacement of arginine with valine at position 4 in the amino acid chain (also written as Arg4Val). Exemplary mutations to SODl can be found, for example, in International Patent Publication No. WO 2004/042027, published May 21, 2004, which is incorporated by reference herein.
  • VJ Recombinant Expression Vectors and Assay Cells or Organisms
  • vectors preferably expression vectors, for producing the proteins reagents of the instant invention.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • a preferred vector is a "plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • plasmid and “vector” can be used interchangeably as the plasmid is the most commonly used form of vector.
  • the recombinant expression vectors of the invention comprise a nucleic acid that encodes, for example RCK protein (or RCK orthologue protein) or a bioactive fragment ⁇ thereof, or RCK interactor protein or bioactive fragment, in a form suitable for ' expression of the nucleic acid in a host cell or organism, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells or organisms to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed.
  • RCK protein or RCK orthologue protein
  • bioactive fragment ⁇ thereof or RCK interactor protein or bioactive fragment
  • operably linked is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell • or organism when the vector is introduced into the host cell or organism).
  • regulatory sequence is intended to includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals).
  • the expression vectors can be introduced into host cell or organisms to thereby produce proteins, including fusion proteins or peptides.
  • retroviral expression vectors and/or adenoviral expression vectors can be utilized to express the proteins of the present invention.
  • the recombinant expression vectors of the invention can be designed for expression of RCK polypeptides, RCK otholog polypeptide or RCK interactor polypeptides in prokaryotic or eukaryotic cells.
  • RCK polypeptides, RCK otholog polypeptides or RCK interactor polypeptides can be expressed in bacterial cells such as E. coli, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells.
  • Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein.
  • Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification.
  • a proteolytic cleavage site is introduced at the junction of the fusion ' moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein.
  • Purified fusion proteins are particularly useful in the cell-free assay methodologies of the present invention.
  • a RCK-encodin g, RCK ortholog-encoding or RCK interactor-encoding nucleic acid is expressed in mammalian cells, for example, for use in the cell or organism-based assays described herein.
  • the expression vector's control functions are often provided by viral regulatory elements.
  • the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid).
  • An assay cell can be prokaryotic or eukaryotic, but preferably is eukaryotic.
  • Cell lines are cultured according to art-recognized techniques.
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nd, ecL, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), and other laboratory manuals.
  • An assay cell of the invention can be contacted with a test compound and assayed for ( any RCK, RCK ortholog and/or RCK interactor biological activity in order to identify the compound as a modulator.
  • Biological activities that can further be assayed as part of the methodologies of the present invention include, but are not limited to, (1) recruiting miRISC to target RNAs; (2) mediating P-body formation, (3) mediating assembly of miRISC; and (4) directing translation repression (e.g., via miRNAs).
  • modulators described supra For pharmaceutical suitability, modulators can be tested in an appropriate animal model.
  • a RCK modulator, translational repression modulator and/or gene silencing modulator identified as described herein can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such a modulator.
  • a modulator identified as described herein can be used in an animal model to determine the mechanism of action of such an agent.
  • this invention pertains to uses of modulators identified by the above-described screening assays for therapeutic treatments as described infra. Accordingly, the modulators of the present invention can be incorporated into pharmaceutical compositions suitable for administration.
  • Such compositions typically comprise the nucleic acid molecule, protein, antibody, or modulatory compound and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as , any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • a pharmaceutical composition of the invention is formulated to be compatible ⁇ with its intended route of administration.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosaL, and rectal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following ⁇ components: a sterile diluent such as water for injection, saline solution, fixed oils, ' polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic ' acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • a sterile diluent such as water for injection, saline solution, fixed oils, ' polyethylene glycols, glycerine, propylene glycol or other synthetic solvents
  • antibacterial agents such as benzyl alcohol or methyl
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the . maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, and sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as > micro crystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as > micro crystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide
  • the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and j fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • the compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
  • the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.
  • the materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
  • Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.
  • the present invention relates to delivery of the compositions of the invention via nanotransporters.
  • Nanotransporters are comprised of a central core with at least one functional surface group attached.
  • Various molecules can be associated with the nanotransporter for delivery to a desired target, e.g., a cell or tissue.
  • Molecules capable of associating with the nanotransporter include, but are not limited to, nucleic . acid molecules and/or pharmaceutical agents.
  • the > miRISC tethering constructs of the invention are delivered to a desired target cell or tissue (e.g. a tumor cell or other diseased cell) via a nanotransporter.
  • a desired target cell or tissue e.g. a tumor cell or other diseased cell
  • the sequestration or sequestration interactor polypeptides of the invention are delivered to a desired target cell or tissue via a nanotransporter.
  • the core of the nanotransporter is comprised of a nanotube, e.g., a carbon nanotube.
  • Nanotubes for use in the present invention may be either single walled ("SWNTs") or multi-walled (“MWNTs").
  • SWNT is a single tube that is about 1 nanometer in diameter and about 1 to about 100 microns in length.
  • MWNTs are tubes ( with at least one other tube embedded within it.
  • the core of the nanotransporter is comprised of a nanoparticle.
  • Nanoparticles of the present invention include, but are not limited to, dendrimers. Dendrimers are highly branched polymers with a well-defined architecture. Many dendrimers are commercially available.
  • the dendrimers of the invention include but are not limited to: polylysine dendrimers, Polyamidoamine (PAMAM): Amine terminated and /or PAMAM: Carboxylic acid terminated (available, e.g., from Dendritech, Inc., Midland, MI); Diaminobutane (DAB) - DAB: Amine terminated and/or DAB: Carboxylic acid terminated; and PEGs: OH terminated (Frechet et al., JACS 123:5908 (2001)), among others.
  • PAMAM Polyamidoamine
  • DAB Diaminobutane
  • PEGs OH terminated
  • ! polylysine dendrimers or a variant thereof are used.
  • various functional surface groups are conjugated to the core of the nanotransporter.
  • the term "functional surface group” refers to molecules that upon binding to ' the core increase the functionality of the nanotransporter, e.g., to increase cell targeting specificity, to increase delivery of the nanotransporter to the target cell, and/or to impart ' a precise biological function.
  • functional surface groups of the invention include, but are not limited to, lipids, fatty acids and derivatives, fluorescent and charge , controlling molecules, and cell type specific targeting moieties.
  • a single type of functional surface group or multiple types of functional surface groups may be present on the surface of the core of the nanotransporter.
  • Exemplary ' nanotransporters can be found, for example, in USSN 60/762,956, filed January 26, [ 2006, and U.S. application number XX/XXX,XXX, filed on January 26, 2007, each of which is incorporated by reference herein.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
  • Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD5O/ED5O.
  • Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture.
  • IC50 i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms
  • levels in plasma may be measured, for example, by high performance liquid chromatography.
  • the pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration. ,
  • VJJI Methods of Treatment
  • the present invention also features methods of treatment or therapeutic methods.
  • the invention features a method of treating a subject (e.g., a human subject in need thereof) with a pharmaceutical composition according to the present » invention, such that a desired therapeutic effect is achieved.
  • the method involves administering to an isolated tissue or cell line from the subject a modulatory compound identified according to the methodology described herein, such , that a desired therapeutic effect is achieved.
  • the invention features a method of treating a subject having a disease or disorder characterized by . overexpression or aberrant expression of a particular protein. For example, positive , modulators of RCK and/or translational repression can be used to enhance translational repression of deleterious proteins.
  • negative modulators of RCK and/or translational repression can be used to alleviate symptoms resulting from the ' translational repression pathway.
  • a tethering construct of the invention may be used to treat or prevent a disease or disorder characterized by overexpression or aberrant expression of a particular protein. Desired therapeutic effects include a modulation of a miRNA disease or disorder, as described herein. Desired therapeutic i effects also include, but are not limited to curing or healing the subject, alleviating, relieving, altering or ameliorating a disease or disorder in the subject or at least one symptom of said disease or disorder in the subject, or otherwise improving or affecting the health of the subject.
  • the effectiveness of treatment of a subject with a RCK modulator, translational repression modulator and/or gene silencing modulator can be accomplished by (i) detecting the level of activity in the subject prior to treating with an appropriate modulator; ( ⁇ ) detecting the level of activity in the subject post treatment with the modulator; (iii) comparing the levels pre-administration and post administration; and (iv) altering the administration of the modulator to the subject accordingly. For example, increased administration of the modulator may be desirable if the subject continues to demonstrate undesirable symptoms of the disease or disorder being treated. ' The present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant or unwanted gene expression or activity.
  • Treatment is defined as the application or administration of a therapeutic agent (e.g., a sequestration polypeptide or a miKNA tethering construct) to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient, who has a disease or disorder, a symptom of disease or disorder or a predisposition toward a > disease or disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease or disorder, the symptoms of the disease or disorder, or the predisposition toward disease.
  • a therapeutic agent e.g., a sequestration polypeptide or a miKNA tethering construct
  • prophylactic and therapeutic methods of treatment such treatments may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics.
  • “Pharmacogenomics” refers to the application of genomics technologies such as gene sequencing, statistical genetics, and gene expression analysis to drugs in clinical development and on the market. More specifically, the term refers the study of how a patient's genes determine his or her response to a drug (e.g., a patient's "drug response phenotype", or “drug response genotype”).
  • another aspect of the invention provides methods for tailoring an individual's prophylactic or therapeutic treatment with either the target gene molecules of the present invention or target gene modulators according to that individual's drug response genotype.
  • Pharmacogenomics allows a clinician or physician to target prophylactic or therapeutic treatments to patients who will most benefit from the treatment and to avoid treatment of patients who will experience toxic drug-related side effects.
  • the practice of the present invention employs, unless otherwise indicated, conventional techniques of nucleic acid chemistry, recombinant DNA technology, molecular biology, biochemistry, cell biology and transgenic animal biology. See, e.g., DNA Cloning, VoIs. 1 and 2, (D.N. Glover, Ed. 1985); Oligonucleotide Synthesis (MJ. Gait, Ed. 1984); Oxford Handbook of Nucleic Acid
  • RNA Interference The Nuts & Bolts of siRNA Technology, by D. Engelke, DNA Press, (2003); Gene Silencing by RNA Interference: Technology and Application, by M. Sohail, CRC Press (2004); Sambrook, Fritsch and Maniatis, Molecular Cloning: Cold Spring Harbor Laboratory Press (1989); and Current Protocols in Molecular Biology, eds. Ausubel et al., John Wiley & Sons (1992), which are incorporated in their entireties by reference herein.
  • Agol and Ago2 expression vectors with a N-terminal YEP- or CFP-epitope tags were generated by PCR amplification of Agol and Ago2 coding sequences from pMyc- Ago 1 and pMyc-Ago2 followed by cloning into the Xbal and EcoRl sites of pEYFP-Cl and pECFP-Cl (BD Biosciences Palo Alto, CA).
  • Vectors for expressing YFP-tagged Lsml, p54, eIF4E, eIF4E-T and Dcp2 were generated through PCR amplification of their coding sequences from 293T cDNA followed by cloning into the BgIII and Sail sites of pEYFP-Cl.
  • siRNAs Small interfering RNAs (siRNAs) siRNAs against GFP, human Ago2, RCK/p54, and CDK9 mismatch were synthesized by Dharmacon (Dharmacon, Lafayette, CO), 2'-OH deprotected according to the manufacturer's protocols.
  • the sequences of siRNAs (passenger strand) for our e xperiments are:
  • GFP 5'-GCAGCACGACUUCUUCAAGdTdT-3'
  • hAgo2 5'-GCACGGAAGUCCA UCUGAAdTdT-3'
  • RCK/p54 5'-GCAGAAACCCUAUGAGAUUUU-S'
  • CDK9mm 5'-CCAAAGCUC
  • HeLa cells were cultured in Dulbecco's minimal essential medium (DMEM) with 10% fetal bovine serum (FBS) at 37°C with 5% CO2. Cells were transfected using ' Lipofectami ⁇ e (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. HeLa cell lines stably expressing EGFP-Cl (GFP-HeLa) were generated as described (Malik and Roeder, 2003) and cultured at 37°C in DMEM with 10% FBS and 400 yg/mL G418 (Invitrogen). Cells were cloned and selected for expression of GFP. Immunoprecipitation and imntunoblotting '
  • Total cell extracts were prepared by incubating in lysis buffer (20 mM HEPES, pH 7.9, 10 mM NaCl, 1 mM MgC12, 0.2 mM EDTA, 0.35% (v/v) Triton X-100, 1/100 (v/v) dilution in protease inhibitor cocktail) and centrifuging at 14000 rpm for 10 min at 4°C. Protein concentration was determined by Dc protein assay (Bio-Rad, Hercules, CA). To examine the RNA dependence of protein-protein interactions, total cell extracts (250 yg) were treated with 0.2 yg/ul of RNase A for 20 min at room temperature before i immunoprecipitation.
  • Myc-tagged proteins were precipitated by incubating overnight with anti-myc rabbit polyclonal antibodies directly conjugated to agarose beads (Santa Cruz Biotech, CA). Samples were washed 4 times in lysis buffer and eluted by boiling for 5 min at 100 0 C in SDS-PAGE sample loading buffer, separated by SDS-PAGE, and analyzed by immunoblot.
  • antibodies included monoclonal mouse anti-GFP (BD Biosciences), anti-eIF4E and anti-myc (Santa Cruz Biotech, CA), anti- Flag (Sigma); polyclonal rabbit anti-myc (Santa Cruz Biotech, CA), anti-DDX6 (rck/p54; Bethyl Laboratories, Montgomery, TX); and polyclonal chicken anti-Lsml (GenWay Biotech, Inc., San Diego, CA).
  • HeLa cells were cultured in 35 mm dishes with glass coverslip bottoms (MatTek Corporation, Ashland, MA). Expression vectors for CFP or YFP-tagged proteins were transfected into cells using Lipofectamine as described above. 24 h later, the live cells were monitored for CFP and YFP signals of the transiently expressed proteins. The signals were detected by a Leica confocal imaging spectrophotometer system (TCS-SP2) attached to a Leica DMERE inverted fluorescence microscope equipped with an argon laser, two HeNe lasers, an acousto-optic tunable filter (AOTF) to attenuate individual visible laser lines, and a tunable acousto-optical beam splitter (AOBS).
  • TCS-SP2 Leica confocal imaging spectrophotometer system
  • AOTF acousto-optic tunable filter
  • AOBS tunable acousto-optical beam splitter
  • Anti-sense strands of the GFP siRNA duplex were chemically synthesized and ' biotinconjugated at the 3 '-end (Dharmacon, Lafayette, CO). Synthetic oligonucleotides were deprotected and annealed with the unmodified sense strand RNA to form duplex siRNA.
  • HeLa cells which had been plated at 70% confluency in 100 mm plates, were sequentially transfected with siRNA duplexes with or without biotin (50 pmole) as described above. Cells were harvested after 24 h with trypsin and centrifuged at 1000 x G for 5 min at 4°C.
  • the pellets were washed 3x with ice-cold PBS pH 7.2 and lysed by adding three packed cell volumes of lysis buffer (20 mM HEPES pH 7.9, 10 mM NaCl, ImM MgC12, 0.5 M sucrose, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF, and 0.35% Triton X-100).
  • Biotinylated siRNA with RISC was captured by incubating 50 yl cytoplasmic extract overnight with streptavidin-magnetic beads (30 yl) in lysis buffer (500 yl) at 4°C.
  • HeLa cells sequentially transfected with siRNAs against P-body components and EGFP were harvested 24 h after the second transfection.
  • Cytoplasmic extracts of HeLa cells were prepared as described above and the protein concentration was determined by Dc protein assay (Bio-Rad).
  • Target mRNAs were prepared and the in vitro cleavage assay for GFP-siRISC and /e*-7-miRISC was performed as. described (Hutvagner et aL, 2004; Meister et at, 2004). Dual fluorescence assay
  • a dual fluorescence assay was used to quantify the RNAi activity of siRNAs against GFP.
  • cell lysates were prepared from siRNA-treated • cells 24 h post-transfection.
  • Total cell lysate 150 yg in 200 yl of reporter lysis buffer) ' was measured using a Safire plate reader (TECAN).
  • GFP fluorescence was detected in cell lysates by exciting at 488 nm and recording emissions from 504 to 514 nm.
  • the spectrum peak at 509 nm represents the fluorescence intensity of GFP.
  • RFP fluorescence- was detected in the same cell lysates by exciting at 558 nm and recording emissions from 578 to 588 nm.
  • the spectrum peak at 583 nm represents the fluorescence intensity of RFP.
  • the fluorescence intensity ratio of target (GFP) to control (RFP) fiuorophores was determined in the presence of siRNA duplexes and normalized to the emissions measured in mock-treated cells. Normalized ratios ⁇ 1.0 indicated specific RNA interference.
  • HeLa cells cultured in 6-well plates were transfected with siRNAs against RCK/p54 or CDK9 mismatch as a scramble control. 24 h after transfection, cells were incubated for 1 h in culture medium lacking methionine and cysteine, and metabolically • labeled by incubating in culture medium containing 100 yCi/ml Tran35S-label (MP Biomedicals, Irvine CA). At 0, 15, 30, 60, and 120 min after metabolic labeling, cells were washed twice and harvested in 300yl M-PER buffer (Pierce) with protease inhibitors (Sigma). For each experiment, two extra sets of siRNA-treated cells were trypsinized and counted for total cell numbers.
  • Example I Human argonaute proteins interact with RCK/p54, a component of mRNA processing P-bodies
  • RCK/p54 a component of mRNA processing P-bodies
  • YFP-tagged P-body proteins Lsml, RCK/p54, Dc ⁇ 2, and elF4E
  • TCE total cell extracts
  • P-bodies contain RNA and proteins, many protein components of P-bodies are likely to be assembled on a common RNA scaffold without forming functional protein-protein interactions.
  • HeLa cells were transfected with vectors to co-express myc-Ago2 and the YFP-tagged P-body proteins, Lsml, RCK/p54, Dcp2, and elF4E, subjected to RNase A digestion, and immunopurified. Analysis of immunopurified complexes showed that Agol and RCK/p54 interactions with myc- Ago2 were not affected by RNase treatment, whereas the amounts of Dcp2 and elF4E protein that co-purified with myc-Ago2 decreased significantly (see Figure IA).
  • RCK/p54 is a protein found in cytoplasmic P-bodies with Lsml, Dcp2, and elF4E (Andrei et ah, 2005; Coller and Parker, 2004; Cougot et ah, 2004).
  • cytoplasmic structures also contain Ago2 as recently reported (Liu et ah, , 2005; Sen and Blau, 2005)
  • HeLa cells were transfected with expression vectors containing YFP-Agol, CFP-Agol, YFP-Ago2, and CFP-Ago2. Transiently expressed YFP- and CFP-tagged Agol and Ago2 were co-localized at specific foci in cytoplasm.
  • HeLa cells were transfected with expression vectors for YFP-Lsml and CFP- Ago2, or YFP-RCK/ ⁇ 54 and CFP- Ago2 and visualized 24 hours later by confocal microscopy (see Figure IB).
  • CFP-Ago2 co- localized with YFP-Lsml and YFP-RCK/p54, two bonaflde P-body proteins (Andrei et ah, 2005; Coller and Parker, 2004; Cougot et ah, 2004).
  • overexpressing ⁇ YFP-RCK/p54 increased the average number of P-bodies in every cell.
  • RCK/p54 is a component of RISC containing the guide strand of siRNA
  • active RISCs were affinity purified, analyzed for protein composition, and assayed for RISC function (see Figure 2A).
  • siRNA duplexes against EGFP si-GFP
  • 3'-bitoin moieties were conjugated to the 3 '-end of the guide strand (si-GFP-Bi).
  • RISCs were captured by incubating cell extracts with straptavidin-conjugated magnetic beads (Meister et ah, 2004). Beads , and supernatants were analyzed for RISC function, i.e., the ability to cleave target mRNA in vitro (Hutvagner et ah; 2004; Meister eial, 2004).
  • RISCs primed with siGFP or siGFP-Bi guide strands efficiently cleaved their target mRNA, but only biotin-containing RISC purified on streptavidin-magnetic beads showed cleavage activity (see Figure 2B).
  • RCK/p54 is a component of RISC containing miRNA
  • RISCs containing miRNA were isolated from HeLa cells by affinity capture.
  • 2 ' -O-methyl (2'-0-Me) inhibitors of let-7 miRNA were employed (Hutvagner et ah, 2004; Meister et ah, 2004).
  • HeLa cytoplasmic extracts expressing myc-Agol and Flag-Ago2 were incubated with 3'-biotinylated ⁇ /e_ * -7-2'-C>-methyl inhibitor, which is complementary to the let-7 miRNA, for 20 min, then incubated with streptavidin-conjugated magnetic beads overnight.
  • cell extracts were treated with the /e*-7-2'-O-niethyl inhibitor without 3'-biotinylation.
  • beads containing miRISCs were washed with lysis buffer and split into two aliquots to determine miRISC function and protein composition.
  • RCK/p54 was depleted in P-bodies of HeLa cells by siRNA-mediated RNAi. 24 hours after transfecting cells with siRNA, real-time quantitative PCR (qPCR) showed that mRNA levels decreased by more than 90% and immunoblot analysis showed that protein levels ' decreased significantly.
  • qPCR real-time quantitative PCR
  • the effect of depleting RCK/p54 on localization of Ago2 was next examined by immunofluorescence analysis of HeLa cells expressing myc-Ago2 and siRNAs against RCK/p54. Depleting RCK/p54 disrupted the cellular P-body structures.
  • Ago2 proteins were diffused throughout the cytoplasm and no longer accumulated at specific foci. This cytoplasmic redistribution of Ago2 suggests that its localization to P-bodies is driven in mammalian cells by factors such as RCK/p54.
  • Example V Depletion of RCK/p54 does not affect RNAi activity in vivo
  • RNAi-mediated gene silencing was quantified in RCK/p54-depleted HeLa cells using a dual fluorescence reporter assay. Briefly, GFP and RFP were constitutively expressed in cells transfected with reporter plasmids for EGFP and RFP, respectively. GFP expression was silenced by treating cells with a 21-nt siRNA targeting nt 238-258 of the EGFP mRNA. The fluorescence intensity ratio of target (GFP) to control (RFP) fluorophore was determined in the presence of siRNA duplexes and normalized to that observed in control-treated cells.
  • GFP target
  • RFP control fluorophore
  • Example VI Depletion of RCK/p54 does not affect RISC-mediated mRNA cleavage in vitro
  • RCK7p54 might be a general translation repressor used by the miRNA machinery to ' dictate translation suppression of target mRNAs.
  • RCK/p54 was a general translation repressor in human cells.
  • RCK/p54 expression was silenced in HeLa cells and general translational activity was analyzed by [ 3 ⁇ S]methionine incorporation (Fig. 2).
  • Cells were transfected with 5OnM siRNAs targeting mismatched CDK9 (control) or RCK/p54. Mock control cells were treated with the transfection reagent only. At 24 h post-transfection, cells were incubated for 1 h in medium lacking Met and Cys, and metabolically labeled with [ 35 S

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Abstract

L'invention concerne des compositions permettant de moduler la répression traductionnelle ainsi que leurs méthodes d'utilisation. L'invention concerne notamment, des constructions d'attache capables de médier la répression traductionnelle spécifique d'une séquence d'ARN cibles. L'invention concerne également un procédé permettant d'améliorer la répression traductionnelle médiée par l'ARNmi dans un extrait, une cellule, un lysat cellulaire ou un organisme en introduisant une quantité efficace d'un polypeptide de séquestration, de son orthologue ou de son fragment dans l'extrait, la cellule, le lysat cellulaire ou l'organisme. Des polypeptides de séquestration préférés sont RCK, son orthologue ou son fragment bioactif. L'invention concerne enfin des méthodes thérapeutiques.
PCT/US2007/002203 2006-01-26 2007-01-26 Compositions et méthodes permettant de moduler la répression traductionnelle WO2007092181A2 (fr)

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Publication number Priority date Publication date Assignee Title
US8513209B2 (en) 2007-11-09 2013-08-20 The Board Of Regents, The University Of Texas System Micro-RNAS of the MIR-15 family modulate cardiomyocyte survival and cardiac repair
US9078919B2 (en) 2007-11-09 2015-07-14 The Board Of Regents, The University Of Texas System Micro-RNAs of the miR-15 family modulate cardiomyocyte survival and cardiac repair
US20120053223A1 (en) * 2008-11-26 2012-03-01 Centre National De La Recherche Scientifique (C.N.R.S) Compositions and methods for treating retrovirus infections
US9439923B2 (en) * 2008-11-26 2016-09-13 Centre National De La Recherche Scientifique (C.N.R.S) Compositions and methods for treating retrovirus infections
US9163235B2 (en) 2012-06-21 2015-10-20 MiRagen Therapeutics, Inc. Inhibitors of the miR-15 family of micro-RNAs
WO2015157534A1 (fr) * 2014-04-10 2015-10-15 The Regents Of The University Of California Procédés et compositions pour l'utilisation d'un argonaute pour modifier un acide nucléique simple brin cible
US10253311B2 (en) 2014-04-10 2019-04-09 The Regents Of The University Of California Methods and compositions for using argonaute to modify a single stranded target nucleic acid
EP4035659A1 (fr) 2016-11-29 2022-08-03 PureTech LYT, Inc. Exosomes destinés à l'administration d'agents thérapeutiques

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