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Methods for Controlling Pests Using Rnai

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Methods for Controlling Pests Using Rnai (19) & (11) EP 2 347 759 A2 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: (51) Int Cl.: 27.07.2011 Bulletin 2011/30 A61K 31/711 (2006.01) A61K 31/7105 (2006.01) C12N 5/10 (2006.01) A01N 57/16 (2006.01) (2006.01) (21) Application number: 11157036.2 A01N 61/00 (22) Date of filing: 12.01.2007 (84) Designated Contracting States: • Raemaekers, Romaan AT BE BG CH CY CZ DE DK EE ES FI FR GB GR 9840 De Pinte (BE) HU IE IS IT LI LT LU LV MC NL PL PT RO SE SI • Bogaert, Thierry SK TR 8500 Kortrijk (BE) • Plaetinck, Geert (30) Priority: 12.01.2006 US 758191 P 9820 Bottelare (BE) 07.02.2006 US 771160 P 16.08.2006 US 837910 P (74) Representative: De Clercq, Ann G. Y. et al 18.12.2006 US 875362 P De Clercq & Partners 12.01.2006 EP 06447008 Edgard Gevaertdreef 10a 9830 Sint-Martens-Latem (BE) (62) Document number(s) of the earlier application(s) in accordance with Art. 76 EPC: Remarks: 07700223.6 / 1 971 688 •Thecomplete document including Reference Tables and the Sequence Listing can be downloaded from (71) Applicant: deVGen N.V. the EPO website 9052 Zwijnaarde (BE) •This application was filed on 04-03-2011 as a divisional application to the application mentioned (72) Inventors: under INID code 62. • van Bleu, Els •Claims filed after the date of filing of the application 9290 Berlare (BE) / after the date of receipt of the divisional application • Kubler, Laurent (Rule 68(4) EPC). 01700 Beynost (FR) (54) Methods for controlling pests using RNAi (57) The present invention concerns methods for from insect pests. Alternatively, the methods are used controlling insect infestation via RNAi- mediated gene si- for treating and/or preventing insect infestation on a sub- lencing, whereby the intact insect cell(s) are contacted strate or a subject in need of such treatment and/or pre- with a double-stranded RNA from outside the insect cell vention. Suitable insect target genes and fragments (s) and whereby the double-stranded RNA is taken up thereof, dsRNA constructs, recombinant constructs and by the intact insect cell (s). In one particular embodiment, compositions are disclosed. the methods of the invention are used to alleviate plants EP 2 347 759 A2 Printed by Jouve, 75001 PARIS (FR) EP 2 347 759 A2 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, havetypically been in the form of chemical compositions that are applied to surfaces on which pests reside, or administered 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 fromBacillus 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 (W02004/001013) 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 invention provides target genes and constructs useful in the RNAi-mediated insect pest control. Accordingly, the present 55 invention provides methods and compositions for controlling pest infestation by repressing, delaying, or otherwise re- ducing gene expression within a particular pest. 2 EP 2 347 759 A2 Description of the invention [0012] The present invention describes a novel non-compound, non-protein based approach for the control of insect crop pests. The active ingredient is a nucleic acid, a double- stranded RNA (dsRNA), which can be used as an insecticidal 5 formulation, for example, as a foliar spray. The sequence of the dsRNA corresponds to part or whole of an essential insect gene and causes downregulation of the insect target via RNA interference (RNAi). As a result of the downregulation of mRNA, the dsRNA prevents expression of the target insect protein and hence causes death, growth arrest or sterility of the insect. [0013] The methods of the invention can find practical application in any area of technology where it is desirable to 10 inhibit viability, growth, development or reproduction of the insect, or to decrease pathogenicity or infectivity of the insect. The methods of the invention further find practical application where it is desirable to specifically down-regulate expression of one or more target genes in an insect. Particularly useful practical applications include, but are not limited to, (1) protecting plants against insect pest infestation; (2) pharmaceutical or veterinary use in humans and animals (for example to control, treat or prevent insect infections in humans and animals); (3) protecting materials against damage caused 15 by insects; (4) protecting perishable materials (such as foodstuffs, seed, etc.) against damage caused by insects; and generally any application wherein insects need to be controlled and/or wherein damage caused by insects needs to be prevented. [0014] In accordance with one embodiment the invention relates to a method for controlling insect growth on a cell or an organism, or for preventing insect infestation of a cell or an organism susceptible to insect infection, comprising 20 contacting insects with a double- stranded RNA, wherein the double- stranded RNA comprises annealed complementary strands, one of which has a nucleotide sequence which is complementary to at least part of the nucleotide sequence of an insect target gene, whereby
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