Down-regulating gene expression in insect pests

• US 9,206,438 B2
• Filed: 05/02/2012
• Issued: 12/08/2015
• Est. Priority Date: 04/20/2011
• Status: Expired due to Fees

• Current Assignee: deVGen NV (China National Chemical Corporation)
• Original Assignee: deVGen NV (China National Chemical Corporation)
• Inventors: Beghyn, Myriam, Bogaert, Thierry Andre Olivier Eddy, Feldmann, Pascale, Raemaekers, Romaan
• Primary Examiner(s): Ibrahim, Medina A
• Assistant Examiner(s): DEVEAU ROSEN, JASON
• Application Number:
  US13/462,636
• Publication Number:
  US 20120322660A1
• Time in Patent Office: 1,315 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: A01N 57/16 containing heterocyclic radicals
• CPC Class Codes: A01N 63/60 Isolated nucleic acids
• CPC Class Codes: C07K 14/43563 from insects
• CPC Class Codes: C12N 15/113 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 when used in plants C12N15/8218
• 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: C12N 2310/14 interfering N.A.
• CPC Class Codes: Y02A 40/146 Genetically Modified [GMO] plants, e.g. transgenic plants

Abstract

The present invention relates to genetic control of infestation by insect pest species, particularly prevention and/or control of pest infestation of plants, using interfering ribonucleic acid (RNA) molecules. Compositions and combinations containing the interfering RNA molecules of the invention for use in topical applications, for example in the form of insecticides.

Claims

1. An interfering ribonucleic acid (RNA) wherein the RNA comprises at least one silencing element wherein the silencing element is a region of double-stranded RNA comprising annealed complementary strands, one strand of which comprises a sequence of at least 21 contiguous nucleotides which is at least partially complementary to a target nucleotide sequence within an insect troponin I target gene, and wherein the interfering RNA (i) is at least 85% identical to at a 21 contiguous nucleotide fragment of SEQ ID NO:

1, or the complement thereof;

or (ii) comprises at least a 21 contiguous nucleotide fragment of SEQ ID NO;

1, or the complement thereof;

or (iii) comprises at least a 21 contiguous fragment of a nucleotide sequence encoding an amino acid sequence encoded by SEQ ID NO;

1, or the complement thereof, wherein the inferring RNA down regulates the troponin I target gene in a target insect pest species.

2. The interfering RNA of claim 1 wherein the RNA comprises at least two silencing elements, wherein each silencing element comprises a sequence of nucleotides which is at least partially complementary to a target nucleotide sequence within the troponin I target gene.

3. The interfering RNA of claim 2 wherein each of the silencing elements comprises a different sequence of nucleotides which is complementary to a different target nucleotide sequence within the troponin I target gene.

4. The interfering RNA of claim 3 wherein the different target nucleotides sequences originate from a single troponin I target gene or from different target troponin I genes selected from Lygus herperus, Leptinotarsa decemlineata, Nilaparvata lugens, and Acyrthosiphon pisum.

5. The interfering RNA of claim 4 wherein the different target nucleotide sequences originate from the same insect pest species or different insect pest species.

6. The interfering RNA of claim 1 wherein the insect pest species is a plant pest.

7. The interfering RNA of claim 6 wherein the plant pest is selected from the orders Coleoptera, Hemiptera, Lepidoptera, Diptera, Dichyoptera, Orthoptera, and Siphonaptera.

8. The interfering RNA of claim 7 wherein the insect plant pest is selected from the group consisting of Leptinotarsa spp., optionally L. decemlineata (Colorado potato beetle), L. juncta (false potato beetle), and L. texana (Texan false potato beetle);

Nilaparvata spp., optionally N. lugens (brown planthopper);

Lygus spp., optionally L. lineolaris (tarnished plant bug) and L. hesperus (western tarnished plant bug);

Myzus spp., optionally M. persicae (green peach aphid);

Diabrotica spp., optionally D. virgifera virgifera (western corn rootworm), D. barberi (northern corn rootworm), D. undecimpunctata howardi (southern corn rootworm), and D. virgifera zeae (Mexican corn rootworm).

9. The interfering RNA of claim 1 wherein down-regulating expression of the troponin I target gene causes decreased growth, development, reproduction, or survival of the pest as compared with the pest species that is not exposed to said interfering RNA [ribonucleic acid targeting a non-essential gene or an interfering ribonucleic acidnthaqt does not down-regulate any genes within the pest species].

10. A polynucleotide comprising a sequence of nucleotides encoding the interfering RNA of claim 1.

11. A DNA construct comprising the polynucleotide of claim 10.

12. The DNA construct of claim 11 which is an expression construct, wherein the polynucleotide sequence encoding the interfering RNA is operably linked to at least one regulatory sequence capable of driving expression of the polynucleotide sequence.

13. A host cell comprising the RNA of claim 1.

14. The host cell of claim 13 wherein the host cell is a prokaryotic or a eukaryotic cell.

15. The host cell of claim 14 wherein the host cell is a bacterial cell.

16. A composition for preventing and/or controlling insect pest infestation comprising the interfering ribonucleic acid (RNA) according to claim 1 and at least one suitable carrier, excipient or diluent.

17. The composition of claim 16 wherein the interfering RNA is encoded by a polynucleotide comprising a sequence of nucleotides encoding the interfering RNA which is comprised in a DNA construct, which optionally is an expression construct wherein the polynucleotide sequence encoding the interfering RNA is operably linked to at least one regulatory sequence capable of driving expression of the polynucleotide sequence.

18. The composition of claim 16 comprising at least one host cell capable of expressing said interfering RNA [according to claim1].

19. The composition of claim 18 wherein the host cell is a bacterial cell.

20. The composition of claim 16 wherein the composition is in a form suitable for ingestion by an insect.

21. The composition of claim 16 wherein the composition is in solid, liquid or gel form.

22. The composition of claim 16 wherein the composition is formulated as an insecticidal spray.

23. The composition of claim 22 wherein the spray is a pressurized/aerosolized spray or a pump spray.

24. The composition of claim 16 wherein the composition further comprises at least one pesticidal agent selected from the group consisting of a Bacillus thuringiensis insecticidal protein.

25. The composition of claim 24 wherein said Bacillus thuringiensis insecticidal protein is a Cry3.

26. A combination for preventing and/or controlling pest infestation comprising the composition of claim 16 and at least one other active agent.

27. The combination of claim 26 wherein the combination is for preventing and/or controlling pest infestation of a plant and the other active agent is an agronomical agent.

28. The combination of claim 27 wherein the agronomical agent comprises a herbicide.

29. The combination of claim 27 wherein the agronomical agent comprises a pesticide.

30. The combination of claim 29 wherein the pesticide is a Bacillus thuringiensis insecticidal protein.

31. The combination of claim 30 wherein said Bacillus thuringiensis insecticidal protein is a Cry3.

32. A method for down-regulating expression of a troponin I target gene in an insect pest species in order to prevent and/or control pest infestation, comprising contacting said pest species with the interfering ribonucleic acid (RNA) according to claim 1.

33. The method of claim 32 wherein down-regulation of expression of the target gene in an insect pest species is used to obtain at least 20% pest control or at least 20% pest mortality as compared to said insect pests that are not contacted with said interfering ribonucleic acid (RNA) [targeting a non-essential pest gene or a target gene not expressed in said pest].

34. The method of claim 32 wherein the method is used to prevent or control pest infestation of a plant.

35. The method of claim 34 wherein the plant is chosen from the group comprising cotton, potato, rice, canola, sunflower, sorghum, pearl millet, corn, strawberries, soy, alfalfa, tomato, eggplant, pepper and tobacco.