Methods for controlling pests using RNAi

• US 8,906,876 B2
• Filed: 08/23/2012
• Issued: 12/09/2014
• Est. Priority Date: 01/12/2006
• Status: Expired due to Fees

• Current Assignee: deVGen NV (China National Chemical Corporation)
• Original Assignee: deVGen NV (China National Chemical Corporation)
• Inventors: Raemaekers, Romaan, Kubler, Laurent, Plaetinck, Geert Karel Maria, Vanbleu, Els, Bogaert, Thierry Andre Olivier Eddy
• Primary Examiner(s): Bowman, Amy
• Application Number:
  US13/592,513
• Publication Number:
  US 20130058890A1
• Time in Patent Office: 838 Days
• Field of Search: None
• US Class Current: 514/44
• CPC Class Codes: A01N 2300/00 Combinations or mixtures of active ingredients covered by classes A01N27/00 - A01N65/48 with other active or formulation relevant ingredients, e.g. specific carrier materials or surfactants, covered by classes A01N25/00 - A01N65/48
• CPC Class Codes: A01N 25/00 Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application , e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
• CPC Class Codes: A01N 57/16 containing heterocyclic radicals
• CPC Class Codes: A01N 63/14 Insects
• CPC Class Codes: A01N 63/20 Bacteria; Substances produced thereby or obtained therefrom
• CPC Class Codes: A01N 63/22 Bacillus
• CPC Class Codes: A01N 63/23 B. thuringiensis
• CPC Class Codes: A01N 63/50 Isolated enzymes; Isolated proteins peptides A01N37/46
• CPC Class Codes: A01N 63/60 Isolated nucleic acids
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: C07K 14/43563 from insects
• CPC Class Codes: C12N 15/8218 Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
• CPC Class Codes: C12N 15/8286 for insect resistance
• CPC Class Codes: Y02A 40/146 Genetically Modified [GMO] plants, e.g. transgenic plants

Abstract

The present invention concerns methods for controlling insect infestation via RNAi-mediated gene silencing, whereby the intact insect cell(s) are contacted with a double-stranded RNA from outside the insect cell(s) and whereby the double-stranded RNA is taken up by the intact insect cell(s). In one particular embodiment, the methods of the invention are used to alleviate plants from insect pests. Alternatively, the methods are used for treating and/or preventing insect infestation on a substrate or a subject in need of such treatment and/or prevention. Suitable insect target genes and fragments thereof, dsRNA constructs, recombinant constructs and compositions are disclosed.

Claims

1. An isolated double stranded RNA molecule comprising annealed complementary strands, wherein at least one of said strands comprises a polyribonucleotide selected from the group consisting of:

(i) polyribonucleotides complementary to at least 21 contiguous nucleotides of a target gene represented by SEQ ID NO;

2493 or SEQ ID NO;

11, or the complement thereof,

(ii) polyribonucleotides complementary to at least 21 contiguous nucleotides of a target gene encoding the amino acid sequence represented by SEQ ID NO;

2494, and

(iii) polyribonucleotides having at least 85% sequence identity with the polyribonucleotides of (i) or (ii),

wherein ingestion of said double stranded RNA molecule inhibits expression of said target gene.

2. An isolated polynucleotide or set of polynucleotides encoding the double stranded RNA molecule of claim 1.

3. A composition comprising a double stranded RNA molecule according to claim 1 and further comprising at least one adjuvant and optionally at least one surfactant.

4. A composition comprising at least one double-stranded RNA molecule according to claim 1, and further comprising at least one adjuvant and optionally at least one suitable carrier, excipient or diluent.

5. A cell transformed with a polynucleotide or set of polynucleotides according to claim 2, optionally operably linked to a regulatory sequence.

6. The cell of claim 5, wherein said cell is a prokaryotic cell or wherein said cell is an eukaryotic cell.

7. The cell of claim 6 wherein said cell is a bacterial cell.

8. The cell of claim 6 wherein said cell is a yeast cell.

9. A composition comprising at least one bacterial cell or yeast cell comprising at least one double stranded RNA molecule as defined in claim 1.

10. The composition of claim 9 wherein said bacterial or yeast cell(s) is (are) inactivated or killed.

11. The composition of claim 10, wherein said bacterial or yeast cell is inactivated or killed by heat treatment.

12. A composition comprising at least one bacterial or yeast cell expressing at least one double-stranded RNA molecule according to claim 1, and further comprising at least one adjuvant and optionally at least one surfactant.

13. The composition of claim 12 wherein said bacterial cell or cells are inactivated or killed by heat treatment.

14. The composition of claim 4, 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 protein, a Bacillus laterosporous insecticidal protein, and a Bacillus sphearicus insecticidal protein.

15. The composition of claim 9, wherein said at least one bacterial or yeast cell further comprises at least one pesticidal agent selected from the group consisting of a Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, and a Bacillus sphearicus insecticidal protein.

16. The composition of claim 12, wherein said at least one bacterial or yeast cell further comprises or further expresses at least one pesticidal agent selected from the group consisting of a Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, and a Bacillus sphearicus insecticidal protein.

17. A composition of claim 9, further comprising at least one further bacterial or yeast cell comprising or expressing at least one pesticidal agent selected from the group consisting of a Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, and a Bacillus sphearicus insecticidal protein.

18. The composition of any of claims 14-17 wherein said Bacillus thuringiensis insecticidal protein is selected from the group consisting of a Cry1, a Cry3, a TIC851, a CryET170, a Cry22, a binary insecticidal protein CryET33 and CryET34, a binary insecticidal protein CryET80 and CryET76, a binary insecticidal protein TIC100 and TIC101, and a binary insecticidal protein PS149B1.

19. The composition according to claim 9 formulated as a spray and optionally further comprising at least one adjuvant and at least one surfactant.

20. The composition according to claim 13 formulated as a spray and optionally further comprising at least one adjuvant and at least one surfactant.

21. A housing or trap or bait for a pest comprising a double stranded polyribonucleotide as defined in claim 1.

22. A method for killing or inhibiting growth of an insect chosen from the group comprising Leptinotarsa spp., optionally L. decemlineata (Colorado potato beetle), L. juncta (false potato beetle), and L. texana (Texan false potato beetle), comprising contacting the insect with a composition according to claim 9.

23. A method for reducing insect growth on a plant or for reducing insect infestation of a plant comprising applying to the plant an effective amount of a composition of claim 9.

24. A method for improving yield, comprising applying to a plant an effective amount of a composition of claim 9.

25. The method of claim 23 or claim 24 wherein said plant is chosen from the group comprising alfalfa, apple, apricot, artichoke, asparagus, avocado, banana, barley, beans, beet, blackberry, blueberry, broccoli, Brussels sprouts, cabbage, canola, carrot, cassava, cauliflower, a cereal, celery, cherry, citrus, clementine, coffee, corn, cotton, cucumber, eggplant, endive, eucalyptus, figs, grape, grapefruit, groundnuts, ground cherry, kiwifruit, lettuce, leek, lemon, lime, pine, maize, mango, melon, millet, mushroom, nut, oat, okra, onion, orange, an ornamental plant or flower or tree, papaya, parsley, pea, peach, peanut, peat, pepper, persimmon, pineapple, plantain, plum, pomegranate, potato, pumpkin, radicchio, radish, rapeseed, raspberry, rice, rye, sorghum, soy, soybean, spinach, strawberry, sugarbeet, sugarcane, sunflower, sweet potato, tangerine, tea, tobacco, tomato, a vine, watermelon, wheat, yams and zucchini.

26. The method of claim 23 or claim 24 wherein said insect is selected from the group comprising Leptinotarsa spp., optionally L. decemlineata (Colorado potato beetle), L. juncta (false potato beetle), or L. texana (Texan false potato beetle).

27. The method of claim 23 or claim 24, wherein said composition is topically applied to said plant, optionally wherein said composition is sprayed on said plant.

28. A method for reducing insect growth on a substrate comprising applying a composition of claim 9 to said substrate.

29. The composition of any of claim 3, 19 or 20, wherein the adjuvant comprises alkylphenyl Ω

hydroxy-polyoxyethylene, polymerized resins and fatty acids.

30. The composition of claim 9 or claim 12 comprising between about 10⁸ and 10¹¹ bacteria per milliliter.