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Dsrna As Insect Control Agent Dsrna Als Mittel Zur Insektenbekämpfung L’Arn À Double Brin Pour La Lutte Contre Les Insectes

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Dsrna As Insect Control Agent Dsrna Als Mittel Zur Insektenbekämpfung L’Arn À Double Brin Pour La Lutte Contre Les Insectes (19) TZZ___T (11) EP 1 971 688 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C12N 15/82 (2006.01) A01N 63/00 (2006.01) 14.03.2012 Bulletin 2012/11 (86) International application number: (21) Application number: 07700223.6 PCT/EP2007/000287 (22) Date of filing: 12.01.2007 (87) International publication number: WO 2007/080127 (19.07.2007 Gazette 2007/29) (54) DSRNA AS INSECT CONTROL AGENT DSRNA ALS MITTEL ZUR INSEKTENBEKÄMPFUNG L’ARN À DOUBLE BRIN POUR LA LUTTE CONTRE LES INSECTES (84) Designated Contracting States: (74) Representative: De Clercq, Ann G. Y. et al AT BE BG CH CY CZ DE DK EE ES FI FR GB GR De Clercq & Partners cvba HU IE IS IT LI LT LU LV MC NL PL PT RO SE SI Edgard Gevaertdreef 10 a SK TR 9830 Sint-Martens-Latem (BE) (30) Priority: 12.01.2006 EP 06447008 (56) References cited: 12.01.2006 US 758191 P WO-A-01/34815 WO-A-01/37654 07.02.2006 US 771160 P WO-A-02/46432 WO-A-03/004644 16.08.2006 US 837910 P WO-A-2005/019408 WO-A-2005/049841 18.12.2006 US 875362 P WO-A-2005/110068 (43) Date of publication of application: • DATABASE UniProt [Online] 30 August 2005 24.09.2008 Bulletin 2008/39 (2005-08-30), "Ribosomal protein S4e." XP002432593 retrieved from EBI accession no. (60) Divisional application: UNIPROT:Q4GXU7 Database accession no. 11157036.2 / 2 347 759 Q4GXU7 & DATABASE EMBL SEQUENCE 11157045.3 / 2 374 462 LIBRARY [Online] Ebi. hinxton; ribosomal protein S4e; rpS4e gene 16 July 2005 (73) Proprietor: Devgen NV (2005-07-16), LONGHORN,S.J.: "Biphyllus 9052 Gent-Zwijnaarde (BE) lunatus mRNA for ribosomal protein S4e" retrieved from EBI. HINXTON accession no. (72) Inventors: www.ebi.co.uk Database accession no. • RAEMAEKERS, Romaan AM048926 B-9840 De Pinte (BE) • WHYARD S ET AL: "Ingested double-stranded • KUBLER, Laurent RNAs can act as species-specific insecticides", F-59126 Linselles (FR) INSECT BIOCHEMISTRY AND MOLECULAR • PLAETINCK, Geert BIOLOGY, ELSEVIER SCIENCE LTD, GB, vol. 39, B-9820 Bottelare (BE) no. 11, 1 November 2009 (2009-11-01), pages • VANBLEU, Els 824-832, XP026777046, ISSN: 0965-1748, DOI: B-8900 Ieper (BE) DOI:10.1016/J.IBMB.2009.09.007 [retrieved on 2009-10-06] Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 1 971 688 B1 Printed by Jouve, 75001 PARIS (FR) EP 1 971 688 B1 Description Field of the Invention 5 [0001] The present invention relates to the field of double-stranded RNA (dsRNA)-mediated gene silencing in insect species. More particularly, the present invention relates to genetic constructs designed for the expression of dsRNA corresponding to novel target genes. These constructs are particularly useful in RNAi-mediated insect pest control. The invention further relates to methods for controlling insects, methods for preventing insect infestation and methods for down-regulating gene expression in insects using RNAi. 10 Background to the Invention [0002] Insect and other pests can cause injury and even death by their bites or stings. Additionally, many pests transmit bacteria and other pathogens that cause diseases. For example, mosquitoes transmit pathogens that cause malaria, 15 yellow fever, encephalitis, and other diseases. The bubonic plague, or black death, is caused by bacteria that infect rats and other rodents. Compositions for controlling microscopic pest infestations have been provided in the form of antibiotic, antiviral, and antifungal compositions. Methods for controlling infestations by pests, such as nematodes and insects, have typically been in the form of chemical compositions that are applied to surfaces on which pests reside, or admin- istered to infested animals in the form of pellets, powders, tablets, pastes, or capsules. 20 [0003] Control of insect pests on agronomically important crops is an important field, for instance insect pests which damage plants belonging to the Solanaceae family, especially potato (Solanum tuberosum), but also tomato (Solanum lycopersicum), eggplant (Solanum melongena), capsicums (Solanum capsicum), and nightshade (for example, Solanum aculeastrum, S. bulbocastanum, S. cardiophyllum, S. douglasii, S. dulcamara, S. lanceolatum, S. robustum, and S. triquetrum), particularly the control of coleopteran pests. 25 [0004] Substantial progress has been made in the last few decades towards developing more efficient methods and compositions for controlling insect infestations in plants. Chemical pesticides have been very effective in eradicating pest infestations. [0005] Biological control using extract from neem seed has been shown to work against coleopteran pests of vege- tables. Commercially available neem-based insecticides have azadirachtin as the primary active ingredient. These 30 insecticides are applicable to a broad spectrum of insects. They act as insect growth regulator; azadirachtin prevents insects from molting by inhibiting production of an insect hormone, ecdysone. [0006] Biological control using protein Cry3A from Bacillus thuringiensis varieties tenebrionis and san diego, and derived insecticidal proteins are alternatives to chemical control. The Bt toxin protein is effective in controlling Colorado potato beetle larvae either as formulations sprayed onto the foliage or expressed in the leaves of potatoes. 35 [0007] An alternative biological agent is dsRNA. Over the last few years, down-regulation of genes (also referred to as "gene silencing") in multicellular organisms by means of RNA interference or "RNAi" has become a well-established technique. [0008] RNA interference or "RNAi" is a process of sequence-specific down-regulation of gene expression (also referred to as "gene silencing" or "RNA-mediated gene silencing") initiated by double-stranded RNA (dsRNA) that is comple- 40 mentary in sequence to a region of the target gene to be down-regulated (Fire, A. Trends Genet. Vol. 15, 358-363, 1999; Sharp, P.A. Genes Dev. Vol. 15, 485-490, 2001). [0009] Over the last few years, down-regulation of target genes in multicellular organisms by means of RNA interference (RNAi) has become a well established technique. Reference may be made to International Applications WO 99/32619 (Carnegie Institution) and WO 00/01846 (by Applicant). 45 [0010] DsRNA gene silencing finds application in many different areas, such as for example dsRNA mediated gene silencing in clinical applications (WO2004/001013) and in plants. In plants, dsRNA constructs useful for gene silencing have also been designed to be cleaved and to be processed into short interfering RNAs (siRNAs). [0011] Although the technique of RNAi has been generally known in the art in plants, C. elegans and mammalian cells for some years, to date little is known about the use of RNAi to down-regulate gene expression in insects. Since the 50 filing and publication of the WO 00/01846 and WO 99/32619 applications, only few other applications have been published that relate to the use of RNAi to protect plants against insects. These include the International Applications WO 01/37654 (DNA Plant Technologies), WO 2005/019408 (Bar Ilan University), WO 2005/049841 (CSIRO, Bayer Cropscience), WO 05/047300 (University of Utah Research foundation), and the US application 2003/00150017 (Mesa et al.). The present disclosure provides target genes and constructs useful in the RNAi-mediated insect pest control. Accordingly, the present 55 disclosure provides methods and compositions for controlling pest infestation by repressing, delaying, or otherwise reducing gene expression within a particular pest. [0012] The invention provides a composition comprising at least one double-stranded RNA produced from the ex- pression of a polynucleotide sequence selected from the group consisting of: 2 EP 1 971 688 B1 (i) a polynucleotide sequence comprising the nucleic add sequence set forth in any of SEQ ID NOs: 1, 25, 49 to 55, 163 or 166, or the complement thereof, (ii) a polynucleotide sequence having at least 85% sequence identity as determined using the BLASTN alignment tool to the nucleic add sequence set forth In SEQ ID NO 1, or the complement thereof, 5 (iii) a polynucleotide sequence comprising a fragment of at least 21 contiguous nucleotides of any of SEQ ID NOs 1, 25, 49 to 55, 163 or 166, or the complement thereof, wherein said double stranded RNA comprises annealed complementary strands, one of which has a nucleotide sequence which is complementary to any one of the se- quences selected from the group consisting of: 10 (i) a polynucleotide sequence consisting of the nucleic add sequence set forth in any of SEQ ID NOs: 1, 25, 49 to 55, 163 or 166, or the complement thereof, (ii) a polynucleotide sequence having at least 85% sequence identity as determined using the BLASTN alignment tool to the nucleic add sequence set forth in SEQ ID NO 1, or the complement thereof, (iii) a polynucleotide sequence consisting of a fragment of at least 21 contiguous nucleotides of any of SEQ ID 15 NOs 1; 25, 49 to 55, 163 or 166, or the complement thereof, said composition further comprising at least one pesticidal agent selected from the group consisting of a chemical insecticide, a patatin, a Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal
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