(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date ;n n /n 19 May 2011 (19.05.2011) 2U11/U57825 Al (51) International Patent Classification: (74) Agent: LAHRTZ, Fritz; Isenbruck Bosl Horschler LLP, A01K 67/033 (2006.01) C12N 15/63 (2006.01) Prinzregentenstrasse 68, 81675 Munchen (DE). (21) International Application Number: (81) Designated States (unless otherwise indicated, for every PCT/EP20 10/006982 kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, (22) International Filing Date: CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO, 16 November 2010 (16.1 1.2010) DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, (25) Filing Language: English HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, (26) Publication Langi English ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, (30) Priority Data: NO, NZ, OM, PE, PG, PH, PL, PT, RO, RS, RU, SC, SD, 10 2009 053 469.5 SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, 16 November 2009 (16.1 1.2009) DE TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. 10 2009 054 265.5 (84) Designated States (unless otherwise indicated, for every 23 November 2009 (23.1 1.2009) DE kind of regional protection available): ARIPO (BW, GH, 10001476.0 12 February 2010 (12.02.2010) EP GM, KE, LR, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, 61/308,143 25 February 2010 (25.02.2010) US ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, (71) Applicant (for all designated States except US): TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, FRAUNHOFER-GESELLSCHAFT ZUR EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, FORDERUNG DER ANGEWANDTEN LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, FORSCHUNG E.V. [DE/DE]; Hansastrasse 27c, 80686 SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, Munchen (DE). GW, ML, MR, NE, SN, TD, TG). (72) Inventors; and Published: (75) Inventors/ Applicants (for US only): RODRIGUES — with international search report (Art. 21(3)) MARKERT DOS SANTOS, Gustavo [DE/DE]; Giebel- — before the expiration of the time limit for amending the str. 29, 70499 Stuttgart (DE). DELAROQUE, Nicolas claims and to be republished in the event of receipt of [FR/DE]; Kobisstr. 2, 043 17 Leipzig (DE). SZARDEN- amendments (Rule 48.2(h)) INGS, Michael [DE/DE]; Oselweg 20, 38302 Wolfen- buttel (DE). ULBERT, Sebastian [DE/DE]; Kochstr. 35, 04275 Leipzig (DE). GIESE, Matthias [DE/DE]; Im Schaffher 24, 69123 Heidelberg (DE). 00 o (54) Title: INDUCTION OF GENE EXPRESSION IN ARTHROPODS (57) Abstract: The present invention relates to a method for the production of a gene encoded molecule in an arthropod in vivo, o wherein a nucleic acid comprising a gene encoding said molecule is fed to the arthropod. Furthermore, the present invention re lates to a method for the delivery of a purified nucleic acid to arthropod cells in vivo, wherein the purified nucleic acid is fed to the arthropod. The present invention relates to methods, uses and solutions to introduce heterologous nucleic acids into insects, hi particular, the present invention discloses that DNA may be introduced into insects via an oral route, may be transcribed there and may also be retrieved in the sanguivorous (ecto- and endo-)parasites of the insects. Induction of Gene Expression in Arthropods The present invention relates to the induction of gene expression in arthropods in vivo, especially to the induction of protein expression in insects in vivo. Further, the present invention relates to methods, uses and solutions to introduce heterologous nucleic acids into insects. In particular, the present invention discloses that DNA may be introduced into insects via an oral route, may be transcribed there and may also be retrieved in the sanguivorous (ecto- and endo-)parasites of the insects. Arthropods are a highly diverse group of animals comprising about 80% of all described living animal species. The most important classes of arthropods are hexapoda (which include insects), chelicerata (including spiders, mites and scorpions), Crustacea and myriapoda (Brusca and Brusca 2003). An arthropod may be a pest in agriculture, an ectoparasite on animals, including human, an endoparasite in animals, including human, an epidemiologic vector, a pest destroying property by feeding or contamination or a pest endangering ecological balance. On the other hand, an arthropod may serve as native or agricultural animal feed, as nutrition for humans, as factor to balance an ecosystem or as a source of material susceptible of the industrial utilisation. It is well-known in the art that insect cell cultures e.g. SF-9 or SF-21 cell cultures, are widely used in the art and are capable of producing considerable amounts of a gene encoded molecule, in particular a protein. In contrast, the production of a gene encoded molecule in an arthropod in vivo is still hampered by the problem to incorporate the required nucleic acid in the cells of said arthropod. There is still a considerable lack of capable techniques to enable a nucleic acid to enter into arthropod cells, which is different to the laborious and time consuming manipulation of germ cell lines. Arthropods bear an impermeable barrier, in form of a chitin shield, all over their body surface and are therefore extensively protected from exogenous influences and extensively isolated from the molecular environment. Therefore, the application of a nucleic acid by methods regularly used in the art, such as the use of gene guns (Tang et al 1992), may fail or may be complicated. Furthermore, it is well-known in the art that the use of a nucleic acid, in particular the use of DNA and/or RNA, in vivo does mostly not lead to the desired result. It is well-known that an aforementioned nucleic acid is highly ineffective in in vivo applications in nearly all animal species (Wahren and Liu 2005). It is a common prejudice that this is also true for the use of nucleic acids in living arthropods. There is a need for an efficient and simple method for inducing gene expression in arthropods in vivo. Said efficient and simple method for inducing gene expression in arthropods in vivo may be used to treat arthropodes suffering from a disease or disorder. Or, said efficient and simple method for inducing gene expression in arthropods in vivo may be used to prevent a disorder or disease in arthropodes. There are numerous disorders that may influence the live of insects, particularly bees, in particular bacterial and viral infectious diseases and various parasites. It is also discussed whether monocultures limit the habitat of the insects and, in this context, reduce diversity of species. Or whether the use of pesticides impairs the immune system of bees so that the animals die. Also, the disputed cultivation of genetically modified plants fell into the focus of criticism again. In spring 2007, it was reported from the U.S.A. that approximately 70 percent of the bees from the east to the west coast disappeared tracelessly, designated as "Colony Collapse Disorder" by the experts. Reason unknown. However, the infestation by the Varroa mite (Varroa destructor) remains the major verifiable pest of the honey bee. The mite pest is accompanied by the infection of various pathogenic agents. Some of the strains of this mite have recently developed some resistance against certain active agents that were still effective until now. For this reason, the alarming situation must be approached in an alternative way. Most of the higher organisms have developed a specific defense mechanism that protects them from bacteria, viruses but also parasites. Arthropodes do not have this adaptive immune system of the vertebrates. However, they possess an innate unspecific though highly effective immune system that is based on a cascade of differently inducible components. Just recently, for insects, a humoral as well as a cellular immune system has been described that is regulated by various genes. In this context, a background for the following project description is given. From an economical point of view, the bee is, beside pig and cattle, the third-most important production animal of humans and their pollination activity in agriculture is irreplaceable. In this respect, research and development of biologically safe and highly effective products against the Varroa mite is urgently demanded. As presented in the following, an immunization of the bees against Varroa mites and, in this context, also against Varroa-associated viral pathogenic agents is possible. Until now, it was found that it is possible to downregulate viruses in bees by orally administered dsRNA constructs (Maori et al., Insect Molecular Biology (2009), 18 (1), 55- 60). The introduction of the dsRNA constructs are attributed to a particular transmembrane protein in the bee that is known to enable to translocate dsRNA to the interior of a cell (Aronstein et al., Insect Biochem Mol Biol (2006); 36, 683-693). However, the manufacture and the handling of dsRNA is difficult as it can be degraded easily. Further, there is a need for novel insecticides that are easier to handle and that are associated with lesser side effects for humans and the environment. Previous biological insecticides require a viral or animal vector to introduce toxic gene products in certain insects. Therefore, it was the object of the present invention to provide a method that enables to introduce nucleic acids into insects which method avoids the disadvantages of the state of the art and optionally enables a gene-therapeutic method for insects easily applicable in practice.