US 20140348893A1 (19) United States (12) Patent Application Publication (10) Pub. N0.: US 2014/0348893 A1 KALETTA (43) Pub. Date: NOV. 27, 2014 (54) RNAI FOR THE CONTROL OF INSECTS AND A01N57/16 (2006.01) ARACHNIDS C12P 19/34 (2006.01) (52) US. Cl. (71) Applicant Devgen NV, Ghent-Zwijnaarde (BE) CPC ............ .. C12N15/113 (2013.01); A01N 57/16 _ _ (2013.01); C12P 19/34 (2013.01);A01N25/34 (73) Assignee:Inventor: DEVGENTltllS Jan NV, Ghent-ZWijnaardeNieder-Olm (BE) (201301) CNN 2310/1421 USPC 424/417; 536/245; 435/320.1; 435/252.3; (21) Appl' NO" 14/287’614 514/44 A; 435/913; 424/84; 424/93.2; 43/121 (22) Filed: May 27, 2014 (57) ABSTRACT Related US. Application Data _ _ _ _ _ The present invention describes a neW non-compound based (62) DWISIOn Of apphcanon NO' 11/921,370, ?led 911 Q“ approach for insect and/or arachnid control. The present 1’ 2009’ HOW Pat' NO' 83759306, ?led as apphcanon inventors have identi?ed for the ?rst time novel targets for NO' PCT/ 1132006/ 002360 on May 31’ 2006' RNAi, Which can effectively control insect and/or arachnid (60) Provisional application No. 60/685,765, ?led on May P€St POPUIations- Accordingly, the invention PTOVid?S bOth 31, 2005 nucleotide and amino acid sequences for the novel targets. Also provided are RNA constructs including double stranded Publication Classi?cation RNA regions for mediating RNAi in insects, DNA constructs, expression vectors, host cells and compositions for control (51) Int. Cl. ling insects and/ or arachnids using RNAi. Finally, the inven C12N 15/113 (2006.01) tion also provides for the use of the constructs, vectors, host A01M 1/20 (2006.01) cells and compositions in control of insects and/or arachnids A01N 25/34 (2006.01) populations and suitable kits for use in an RNAi based A01M 1/10 (2006.01) method of controlling insect and/ or arachnid pests. Patent Application Publication Nov. 27, 2014 Sheet 1 0f 13 US 2014/0348893 A1 Effect of 1% dsRNA on cockroach mortality 100— —&— 59033 80_ + 89001 + Bg003 60- + 89004 Mortality(%) —I—— 89005 40— —l— lmidacloprid 20_ —9— Solvent 0 I l I I l I I I I 0 5 10 15 20 25 30 3 4O 4 Days 120 100 : : : . I 80 /1/] it> Mortality(%) 60 0 3 610131720242731343841454852 Days + BgOO1 + 89001 concatemer2 —-l— Hexaflumuron FIG. 2 —6— Solvent Patent Application Publication Nov. 27, 2014 Sheet 2 0f 13 US 2014/0348893 A1 Month1 Month2 23 a? 89032 in LB 89032 in MO FIG. 3 100 90 80 7O 60 Mortality(%) 50 40 3O 20 1O 0 O 3 610131720242731343841 Days + 59001 —+— -ve control (miscellaneous dsRNA) -9- -ve control (solvent) FIG. 4 Patent Application Publication Nov. 27, 2014 Sheet 3 0f 13 US 2014/0348893 A1 SEQ ID NO 1; DNA; Blattella germanica taaggcatggatgttggacaagctcggtggagtgtatgctccaagaccaagcacaggacctcacaagttacgagagagtctgc cccttgtaatatttcttcgtaataggctgaaatatgcattaaccaactgtgaggttaagaaaattgttatgcagcgccttattaaggttg atggaaaagtcagaacagaccccaactatccagctggttttatggatgttgttacaattgaaaaaactggagaatttttccgtctgat ttatgacgtgaaaggacgtttcaccattcacagaataactgctgaagaagccaagtataaactgtgcaaggtaaagagagtgca gactgggcccaagggtattccattcttggtgacccatgatggtagaactcttagatatcctgatcctgtcatcaaagttaatgataca gttcaacttgacatcgctacttccaagattatggatagcatcaaatttgataatggtaatctctgtatgattactggaggccgtaacttg ggtcgtgttggaactgtagttaatcgagaacgtcatcctggttcctttgacattgtgcatgttaaagattcacaaggacacacatttgc taccagattgaa SEQ ID NO 2; PRT; Blattella germanica Lys Ala Trp Met Leu Asp Lys Leu Gly Gly Val Tyr Ala Pro Arg Pro Ser Thr Gly Pro His Lys Leu Arg Glu Ser Leu Pro Leu Val lle Phe Leu Arg Asn Arg Leu Lys Tyr Ala Leu Thr Asn Cys Glu Val Lys Lys lle Val Met Gln Arg Leu lle Lys Val Asp Gly Lys Val Arg Thr Asp Pro Asn Tyr Pro Ala Gly Phe Met Asp Val Val Thr lle Glu Lys Thr Gly Glu Phe Phe Arg Leu lle Tyr Asp Val Lys Gly Arg Phe Thr lle His Arg lle Thr Ala Glu Glu Ala Lys Tyr Lys Leu Cys Lys Val Lys Arg Val Gln Thr Gly Pro Lys Gly lle Pro Phe Leu Val Thr His Asp Gly Arg Thr Leu Arg Tyr Pro Asp Pro Val lle Lys Val Asn Asp Thr Val Gln Leu Asp lle Ala Thr Ser Lys lle Met Asp Ser lle Lys Phe Asp Asn Gly Asn Leu Cys Met lle Thr Gly Gly Arg Asn Leu Gly Arg Val Gly Thr Val Val Asn Arg Glu Arg His Pro Gly Ser Phe Asp lle Val His Val Lys Asp Ser Gln Gly His Thr Phe Ala Thr Arg Leu Asn SEQ ID NO 3; DNA; Blattella germanica; degenerate primer; w is a or t; r is a or g; m is a or c; y is c ort catttgaagc gtttwrmygc ycc SEQ ID NO 4; DNA; Blattella germanica; speci?c primer gtgcccttgc caatgatgaa cacgttg SEQ 10 NO 5; DNA; speci?c T7 primer cgctaatacg actcactata ggggagtgta tgctccaaga ccaag SEQ ID NO 6; DNA; Blattella germanica; speci?c primer caatctggta gcaaatgtgt gtcc SEQ ID NO 7; DNA; Blattella germanica; speci?c primer ggagtgtatg ctccaagacc aag SEQ ID NO 8; DNA; synthetic T7 primer cgctaatacg actcactata ggcaatctgg tagcaaatgt gtgtcc SEQ ID NO 9; RNA; Blattella germanica; u can be t (for DNA complement) ggaguguaug cuccaagacc aagcacagga ccucacaagu uacgagagag ucugccccuu FIG. 5 Patent Application Publication Nov. 27, 2014 Sheet 7 0f 13 US 2014/0348893 A1 SEQ ID NO 31; DNA; Blattella gemianica; ggcttgatcccaatgaaataaacgaaatlgcaaataccaattccagacaaaatattcgtaaactgattaaagatggtcttatcatc aagaagcccgtagctgtacactcaagggcccgtgttcgcaagaacaccgaagcaagaagaaaaggacgtcactgcggttttg gcaaaaggaagggtacggcaaatgcccgtatgccacagaaggtcttgtggattaatcgcatgcgtgttctgagaaggcttctca agaagtacagggaagcaaagaagatcgacagacatctataccaccagctglacatgaaggccaagggtaacgtgttcaaga acaagcgtglcctgatggagtlcatccacaagaagaaggctgagaaggccaggacaaagatgcttaacgac SEQ ID NO 32; PRT; BIatleIIa germanica; Leu Asp Pro Asn Glu IIe Asn Glu IIe Ala Asn ThrAsn Ser Arg Gln Asn Ile Arg Lys Leu Ile Lys Asp Gly Leu He He Lys Lys Pro Val Ala Val His Ser Arg Ala Arg Val Arg Lys Asn Thr Glu Ala Arg Arg Lys Gly Arg His Cys Gly Phe Gly Lys Arg Lys Gly Thr Ala Asn Ala Arg Met Pro Gln Lys Val Leu Trp Ile Asn Arg Mat Arg Val Leu Arg Arg Leu Leu Lys Lys Tyr Arg Glu Ala Lys Lys Ile Asp Arg His Leu Tyr His Gln Leu Tyr Met Lys Ala Lys Gly Asn Val Phe Lys Asn Lys Arg Val Leu Met Glu Phe IIe His Lys Lys Lys Ala Glu Lys Ala Arg Thr Lys Met Leu Asn Asp SEQ ID NO 33; DNA; Blatlella germanica; degenerative primer forward; r is a org; b is c org ort tgcgatgcgg caaraaraag gtbtgg SEQ ID NO 34; DNA; Blattella germanica; degenerative primer reverse; y is c or I; r is a or g gtcggcgagc ytcrgcytg SEQ ID NO 35; DNA; speci?c T7 primer fonlvard cgctaalacg actcactata ggggcttgat cccaatgaaa taaacg SEQ ID NO 36; DNA; Blattella germanica; speci?c primer reverse gtcgttaagc atctltgtcc tggc SEQ ID NO 37; DNA; Blattella germanica; speci?c primer forward ggcttgatcc caatgaaata aacg SEQ ID NO 38; DNA; speci?c T7 primer reverse cgctaatacg actcactata gggtcgttaa gcalctltgt cctggc SEQ ID NO 39; RNA; Blattella germanica; u can be t (for DNA complement) ggcuugaucc caaugaaaua aacgaaauug caaauaccaa uuccagacaa aauauucgua aacugauuaa agauggucuu aucaucaaga agcccguagc uguacacuca agggcccgug FIG. 5CONT’D Patent Application Publication Nov. 27, 2014 Sheet 13 0f 13 US 2014/0348893 A1 SEQ ID NO 66; DNA; synthetic BgOO1 fragment cttgggtcgtgttggaactgtagttaatcgagaacgtcatcctggttcct SEQ ID NO 67; DNA; synthetic BgOO1 concatemer 1 tatgctccaagaccaagcacaggacctcacaagttacgagagagtctgcctatgctccaagaccaagcacaggacctcacaa gttacgagagagtctgcctatgctccaagaccaagcacaggacctcacaagttacgagagagtctgcctatgctccaagacca agcacaggacctcacaagttacgagagagtctgcctatgctccaagaccaagcacaggacctcacaagttacgagagagtct gcc SEQ ID NO 68; DNA; synthetic BgOO1 concatemer 2 cttgggtcgtgttggaactgtagttaatcgagaacgtcatcctggttcctcttgggtcgtgttggaactgtagttaatcgagaacgtcat cctggttcctcttgggtcgtgttggaactgtagttaatcgagaacgtcatcctggttcctcttgggtcgtgttggaactgtagttaatcga gaacgtcatcctggttcctcttgggtcgtgttggaactgtagttaatcgagaacgtcatcctggttcct SEQ ID NO 69; DNA; synthetic BgOO1 fragment gctgaaatatgcattaaccaactgtgaggttaagaaaattgttatgcagcgccttattaaggttgatggaaaagtcagaacagacc ccaactatccagct SEQ ID NO 70; DNA; synthetic BgOO1 concatemer 3 gctgaaatatgcattaaccaactgtgaggttaagaaaattgttatgcagcgccttattaaggttgatggaaaagtcagaacagacc ccaactatccagctgctgaaatatgcattaaccaactgtgaggttaagaaaattgttatgcagcgccttattaaggttgatggaaaa gtcagaacagaccccaactatccagctgctgaaatatgcattaaccaactgtgaggttaagaaaattgttatgcagcgccttatta aggttgatggaaaagtcagaacagaccccaactatccagct FIG. 5CONT'D US 2014/0348893 A1 Nov. 27, 2014 RNAI FOR THE CONTROL OF INSECTS AND less toxic insecticides, there is an increased probability that ARACHNIDS the insect may become resistant over time. Insecticides act by binding to a certain insect protein, such as RELATED APPLICATION INFORMATION an acethylcholine receptor for example, and cause death of [0001] This is a divisional application ofU.S. patent appli the pest species by either deactivating or over-activating the cation Ser. No. 11/921,370 ?led on Oct. 1, 2009, which is a protein. Insecticides have been developed to be safe at certain 371 of International Application No. PCT/IB2006/002360, concentration, but can and do impact on human health when ?led May 31, 2006, which claims bene?t to US. Provisional incorporated at higher dosages or over long periods. In con Application No. 60/685,765, ?led May 31, 2005, the contents trary to agrochemicals, household insecticides are applied in of which are incorporated herein by reference herein. places where food is stored or prepared and food contamina tion and contact to humans cannot be avoided. STATEMENT REGARDING ELECTRONIC [0008] One alternative to chemical pesticides is to utilise SUBMISSION OF A SEQUENCE LISTING biological agents. Over the last few years, downregulation of genes (also referred to as “gene silencing”) in multicellular [0002] A Sequence Listing in ASCII text format, submitted organisms by means of RNA interference or “RNAi” has under 37 CPR. §1.821, entitled 803 84-US-REG-ORG-P-1_