Source: https://patents.google.com/patent/US9662348B2/en
Timestamp: 2019-06-17 00:58:52
Document Index: 253077029

Matched Legal Cases: ['§119', 'Application No. 61', 'Application No. 08847971', 'Application No. 08847971', 'Application No. 13156183', 'Application No. 10719620', 'Application No. 08847971', 'Application No. 13156185', 'Application No. 13156180', 'Application No. 10719620', 'Application No. 10779855', 'Application No. 13156183', 'Application No. 08847971', 'Application No. 201080056585', 'Application No. 13156180', 'Application No. 13156185', 'Application No. 13156183', 'Application No. 700791', '§ 3', '§ 2', 'art 12', 'Application No. 205594', 'Application No. 205594', 'Application No. 219193', 'Application No. 205594', 'Application No. 201080056585', 'Application No. 2010244122', 'Application No. 2008325989', 'Application No. 2', 'Application No. 219193', 'Application No. 216154', 'Application No. 205594', 'Application No. 201080056585', 'Application No. 201080056585']

US9662348B2 - Compositions for controlling Varroa mites in bees - Google Patents
US9662348B2
US9662348B2 US14/606,328 US201514606328A US9662348B2 US 9662348 B2 US9662348 B2 US 9662348B2 US 201514606328 A US201514606328 A US 201514606328A US 9662348 B2 US9662348 B2 US 9662348B2
US14/606,328
US20150133532A1 (en
Beeologics Inc
2009-10-14 Priority to US25133909P priority Critical
2010-10-14 Priority to PCT/IL2010/000844 priority patent/WO2011045796A1/en
2015-01-27 Application filed by Yissum Research Development Co of Hebrew University, Beeologics Inc filed Critical Yissum Research Development Co of Hebrew University
2015-01-27 Priority to US14/606,328 priority patent/US9662348B2/en
2015-05-14 Publication of US20150133532A1 publication Critical patent/US20150133532A1/en
2015-05-21 Assigned to BEEOLOGICS INC. reassignment BEEOLOGICS INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: BEN-CHANOCH, EYAL, YARDEN, GAL
2015-05-21 Assigned to YISSUM RESEARCH DEVELOPMENT COMPANY OF THE HEBREW UNIVERSITY OF JERUSALEM LTD. reassignment YISSUM RESEARCH DEVELOPMENT COMPANY OF THE HEBREW UNIVERSITY OF JERUSALEM LTD. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: MAORI, EYAL, SHAFIR, SHARONI, KALEV, HAIM, GARBIAN, YAEL, SELA, ILAN
2017-05-30 Publication of US9662348B2 publication Critical patent/US9662348B2/en
This application is continuation of U.S. patent application Ser. No. 13/446,557 filed on Apr. 13, 2012, which is a continuation-in-part (CIP) of PCT Patent Application No. PCT/IL2010/000844 filed Oct. 14, 2010, which claims the benefit of priority under 35 USC §119(e) of U.S. Provisional Patent Application No. 61/251,339 filed Oct. 14, 2009. The contents of the above applications are incorporated herein by reference in their entirety.
According to some embodiments of the invention, the gene product is an mRNA encoding a polypeptide selected from the group consisting of ATPase subunit A, RNA polymerase I, RNA polymerase III, Inhibitor of apoptosis (IAP), FAS apoptotic inhibitor and α-Tubulin.
According to some embodiments of the invention, the at least one nucleic acid agent comprises at least five nucleic acid agents, for down-regulating ATPase subunit A, RNA polymerase III, Inhibitor of apoptosis (IAP), FAS apoptotic inhibitor and α-Tubulin, each of the at least five nucleic acid agent targeting a different gene.
According to some embodiments of the invention, the at least one nucleic acid agent comprises at least six nucleic acid agents, for down-regulating ATPase subunit A, RNA polymerase I, RNA polymerase III, Inhibitor of apoptosis (IAP), FAS apoptotic inhibitor and α-Tubulin, each of the at least six nucleic acid agents for targeting a different gene.
FIG. 6 is a photograph illustrating dsRNA transmission from adult bees to Varroa mites. RT-PCR was performed on RNA from bees fed with GFP-specific dsRNA and untreated control bees (lanes B+, B−, respectively) and RNA from Varroa mites parasitizing the treated or untreated control bees (lanes V+ and V−, respectively). Lane C: Positive control (GFP-bearing plasmid). M=size markers;
FIG. 7 is a photograph illustrating dsRNA transmission from bees to Varroa and Varroa back to bees. Bees were infested with either Varroa mites carrying the GFP dsRNA or siRNA(V+) or control mites (V) devoid of GFP-specific dsRNA or siRNA. B+ is RNA amplified from bees infested with GFP-dsRNA or siRNA-fed mites, B− is RNA amplified from bees infested with control mites devoid of GFP-specific dsRNA or siRNA. Lane C: Positive control (GFP-bearing plasmid). M=size markers;
FIGS. 9A-9F illustrate silencing of Varroa gene expression following horizontal transfer of Varroa-specific dsRNA from bee to Varroa mite. FIGS. 9A-9C are graphs representing the means (±SE) of results of real-time RT-PCR of Varroa RNA with probes for Varroa gene mRNA: RNA polymerase III (9A, probes SEQ ID NOs. 137 and 138), IAP1 and IAP2 (9B, probes SEQ ID NOs. 141 and 142) and vacuolar proton ATPase (9C, probes SEQ ID NOs. 139 and 140), respectively. The Varroa RNA was extracted from mites infesting bees fed a mixture of 5 Varroa-specific dsRNAs (Mixture I), or from mites infesting bees fed a mixture of 14 Varroa-specific dsRNAs (Mixture II). Controls represent Varroa RNA extracted from mites infesting untreated bees or mites infesting bees fed irrelevant (GFP) dsRNA. FIGS. 9D-9F are photographs showing semi-quantitative RT-PCR of Varroa RNA illustrating specific silencing of Varroa apoptosis inhibitor FAS gene expression in mites infesting bees fed on Varroa-specific dsRNA. Apoptosis inhibitor FAS RNA was amplified (using primers SEQ ID NOs. 145 and 146) in Varroa RNA extracted from mites infesting bees fed a mixture of 5 Varroa-specific dsRNAs (9D, Mixture I), or from mites infesting bees fed a mixture of 14 Varroa-specific dsRNAs (9D, Mixture II). Controls represent amplification of Apoptosis inhibitor FAS RNA in Varroa RNA extracted from mites infesting untreated bees (9E, Untreated) or mites infesting bees fed irrelevant (9E, dsRNA-GFP) dsRNA. 9F is a control showing amplification of the housekeeping gene actin (using primers SEQ ID NOs. 147 and 148). Numbers indicate the number of cycles of amplification. −RT reactions serve as controls for DNA contamination. Note strong silencing of Apoptosis inhibitor FAS expression in mites infesting bees fed Mixture I or Mixture II (FIG. 9D);
FIG. 10 is a graph showing the mean (±SE) total number of bees (capped brood and adults) in bees fed a mixture of 5 Varroa-specific dsRNAs (Mixture I) or a mixture of 14 Varroa-specific dsRNAs (Mixture II), or control bees fed irrelevant (dsGFP) dsRNA or untreated (Untreated). No significant differences were detected;
The present inventors have shown that dsRNA can successfully be transferred to Vorroa mites (FIGS. 2A-E, 6 and 7), that the dsRNA can serve to down-regulate expression of a particular gene in the Varroa mite (FIGS. 4 and 9A-9E) and further that targeting of particular genes for down-regulation can result in a reduction in the number of Varroa mites (FIGS. 5 and 11). Yet further, the present inventors have shown that RNA sequences transferred to mites from bees fed dsRNA can be transferred back to untreated, “naïve” bees via Varroa infestation (FIG. 7).
As used herein, the term “bee” refers to both an adult bee and pupal cells thereof. According to one embodiment, the bee is in a hive.
The term “colony” refers to a population of bees comprising dozens to typically several tens of thousand bees that cooperate in nest building, food collection, and brood rearing. A colony normally has a single queen, the remainder of the bees being either “workers” (females) or “drones” (males). The social structure of the colony is maintained by the queen and workers and depends on an effective system of communication. Division of labor within the worker caste primarily depends on the age of the bee but varies with the needs of the colony. Reproduction and colony strength depend on the queen, the quantity of food stores, and the size of the worker force. Honeybees can also be subdivided into the categories of “hive bees”, usually for the first part of a workers lifetime, during which the “hive bee” performs tasks within the hive, and “forager bee”, during the latter part of the bee's lifetime, during which the “forager” locates and collects pollen and nectar from outside the hive, and brings the nectar or pollen into the hive for consumption and storage.
The phrase “Varroa destructor mite” refers to the external parasitic mite that attacks honey bees Apis cerana and Apis mellifera. The mite may be at an adult stage, feeding off the bee, or at a larval stage, inside the honey bee brood cell.
As used herein, the phrase “gene product” refers to an RNA molecule or a protein.
Exemplary gene products that may be down-regulated according to this aspect of the present invention include, but are not limited to NADH dehydrogenase; subunit 2—Genbank accession NC_004454; ATP synthetase; subunit 8—NC_004454; ATP synthetase; subunit 6—NC_004454; sodium channel gene—Genbank accession No. FJ216963; Cytochrome oxydase subunit I—Genbank accession No. EF025469.
It will be appreciated that whilst the agents of the present invention are capable of downregulating expression of a gene product of a Varroa destructor mite, it is preferable that they downregulate to a lesser extent expression of the gene product in other animals, such as the bee. Accordingly, the agents of the present invention must be able to distinguish between the mite gene and the bee gene, down-regulating the former to a greater extent than the latter. According to another embodiment the agents of the present invention do not down-regulate the bee gene whatsoever. This may be effected by targeting a gene that is expressed differentially in the mite and not in the bee e.g. the mite sodium channel gene—FJ216963. Alternatively, the agents of the present invention may be targeted to mite-specific sequences of a gene that is expressed both in the mite and in the bee.
SEQ ID NO: 1. Varroa gene homologous to ATPase subunit A (segment 1); SEQ ID NO: 2. Varroa gene homologous to ATPase subunit A (segment 2); SEQ ID NO: 3. Varroa gene homologous to ATPase subunit A (segment 3); SEQ ID NO: 4. Varroa gene homologous to ATPase subunit A (segment 4); SEQ ID NO: 5. Varroa gene homologous to ATPase subunit A (segment 5); SEQ ID NO: 6. Varroa gene homologous to ATPase subunit A (segment 6); SEQ ID NO: 7. Varroa gene homologous to ATPase subunit A (segment 7); SEQ ID NO: 8. Varroa gene homologous to ATPase subunit A (segment 8); SEQ ID NO: 9. Varroa gene homologous to ATPase subunit A (segment 9); SEQ ID NO: 10. Varroa gene homologous to RNA polymerase I (segment 1); SEQ ID NO: 11. Varroa gene homologous to RNA polymerase I (segment 2); SEQ ID NO: 12. Varroa gene homologous to RNA polymerase I (segment 3); SEQ ID NO: 13. Varroa gene homologous to RNA polymerase III (segment 1); SEQ ID NO: 14. Varroa gene homologous to RNA polymerase III (segment 2); SEQ ID NO: 15. Varroa gene homologous to RNA polymerase III (segment 3); SEQ ID NO: 16. Varroa gene homologous to RNA polymerase III (segment 4); SEQ ID NO: 17. Varroa gene homologous to RNA polymerase III (segment 5); SEQ ID NO: 18. Varroa gene homologous to RNA polymerase III (segment 6); SEQ ID NO: 19. Varroa gene homologous to RNA polymerase III (segment 7) SEQ ID NO: 20. Varroa gene homologous to RNA polymerase III (segment 8); SEQ ID NO: 21. Varroa gene homologous to RNA polymerase III (segment 9); SEQ ID NO: 22. Varroa gene homologous to Inhibitor of apoptosis (IAP; segment 1); SEQ ID NO: 23. Varroa gene homologous to Inhibitor of apoptosis (IAP; segment 2); SEQ ID NO: 24. Varroa gene homologous to Inhibitor of apoptosis (IAP; segment 3); SEQ ID NO: 25. Varroa gene homologous to Inhibitor of apoptosis (IAP; segment 4); SEQ ID NO: 26. Varroa gene homologous to Inhibitor of apoptosis (IAP; segment 5); SEQ ID NO: 27. Varroa gene homologous to Inhibitor of apoptosis (IAP; segment 6); SEQ ID NO: 28. Varroa gene homologous to Inhibitor of apoptosis (IAP; segment 7); SEQ ID NO: 29. Varroa gene homologous to Inhibitor of apoptosis (IAP; segment 8); SEQ ID NO: 30. Varroa gene homologous to FAS apoptotic inhibitor (segment 1); SEQ ID NO: 31. Varroa gene homologous to FAS apoptotic inhibitor (segment 2); SEQ ID NO: 32. Varroa gene homologous to FAS apoptotic inhibitor (segment 3); SEQ ID NO: 33. Varoa gene homologous to α-Tubulin (segment 1); SEQ ID NO: 34. Varoa gene homologous to α-Tubulin (segment 2); SEQ ID NO: 35. Varoa gene homologous to α-Tubulin (segment 3); SEQ ID NO: 36. Varoa gene homologous to α-Tubulin (segment 4); SEQ ID NO: 37. Varoa gene homologous to α-Tubulin (segment 5); SEQ ID NO: 38. Varoa gene homologous to α-Tubulin (segment 6); SEQ ID NO: 39. Varoa gene homologous to α-Tubulin (segment 7); SEQ ID NO: 40. Varoa gene homologous to α-Tubulin (segment 8); SEQ ID NO: 41. Varoa gene homologous to α-Tubulin (segment 9); SEQ ID NO: 42.NADH dehydrogenase; subunit 2 (NC_004454): bases 709 to 974; SEQ ID NO: 43. ATP synthetase; subunit 8 (NC_004454): bases 3545 to 3643; SEQ ID NO: 44. Sodium channel protein (AY259834): bases 3336-3836.
SEQ ID NO: 93-Varroa gene homologous to α-tubulin (411 bases); SEQ ID NO: 94-Varroa gene homologous to α-tubulin (277 bases); SEQ ID NO: 95-Varroa gene homologous to α-tubulin (329 bases); SEQ ID NO: 96-Varroa gene homologous to RNA polymerase III (380 bases); SEQ ID NO: 97-Varroa gene homologous to RNA polymerase III (426 bases); SEQ ID NO: 98-Varroa gene homologous to RNA polymerase II (366 bases); SEQ ID NO: 99-Varroa gene homologous to RNA polymerase I (324 bases); SEQ ID NO: 100-Varroa gene homologous to vacuolar translocating ATPase (311 bases); SEQ ID NO: 101-Varroa gene homologous to vacuolar proton ATPase (210 bases); SEQ ID NO: 102-Varroa gene homologous to Na+/K+ ATPase (307 bases); SEQ ID NO: 103-Varroa gene homologous to apoptosis inhibitor IAP (263 bases); SEQ ID NO: 104-Varroa gene homologous to apoptosis inhibitor FAS (277 bases); SEQ ID NO: 105-Varroa gene homologous to apoptosis inhibitor IAP 1 and IAP2 (263 bases); SEQ ID NO: 106-Varroa gene homologous to apoptosis inhibitor IAP 1 and IAP2, reverse orientation (282 bases).
Thus, according to one embodiment, the following group of genes are targeted—ATPase subunit A, RNA polymerase III, Inhibitor of apoptosis (IAP), FAS apoptotic inhibitor and α-Tubulin (e.g. using nucleic acid agents having the sequence as set forth in 1, 13, 27, 30 and 39, or nucleic acid agents having the sequence as set forth in SEQ ID Nos. 93, 96, 100, 104 and 106).
According to another embodiment, the following group of genes are targeted—ATPase subunit A, RNA polymerase I, RNA polymerase III, Inhibitor of apoptosis (IAP), FAS apoptotic inhibitor and α-Tubulin.
Tools which are capable of identifying species-specific sequences may be used for this purpose—e.g. BLASTN and other such computer programs
As used herein, the term “downregulating expression” refers to causing, directly or indirectly, reduction in the transcription of a desired gene, reduction in the amount, stability or translatability of transcription products (e.g. RNA) of the gene, and/or reduction in translation of the polypeptide(s) encoded by the desired gene.
As used herein, the phrase “RNA silencing” refers to a group of regulatory mechanisms [e.g. RNA interference (RNAi), transcriptional gene silencing (TGS), post-transcriptional gene silencing (PTGS), quelling, co-suppression, and translational repression] mediated by RNA molecules which result in the inhibition or “silencing” of the expression of a corresponding protein-coding gene or bee pathogen RNA sequence. RNA silencing has been observed in many types of organisms, including plants, animals, and fungi.
As used herein, the term “RNA silencing agent” refers to an RNA which is capable of inhibiting or “silencing” the expression of a target gene. In certain embodiments, the RNA silencing agent is capable of preventing complete processing (e.g, the full translation and/or expression) of an mRNA molecule through a post-transcriptional silencing mechanism. RNA silencing agents include noncoding RNA molecules, for example RNA duplexes comprising paired strands, as well as precursor RNAs from which such small non-coding RNAs can be generated. Exemplary RNA silencing agents include dsRNAs such as siRNAs, miRNAs and shRNAs. In one embodiment, the RNA silencing agent is capable of inducing RNA interference. In another embodiment, the RNA silencing agent is capable of mediating translational repression.
Various studies demonstrate that long dsRNAs can be used to silence gene expression without inducing the stress response or causing significant off-target effects—see for example [Strat et al., Nucleic Acids Research, 2006, Vol. 34, No. 13 3803-3810; Bhargava A et al. Brain Res. Protoc. 2004; 13:115-125; Diallo M., et al., Oligonucleotides. 2003; 13:381-392; Paddison P. J., et al., Proc. Natl Acad. Sci. USA. 2002; 99:1443-1448; Tran N., et al., FEBS Lett. 2004; 573:127-134].
The term “siRNA” refers to small inhibitory RNA duplexes (generally between 18-30 basepairs, between 19 and 25 basepairs) that induce the RNA interference (RNAi) pathway. Typically, siRNAs are chemically synthesized as 21 mers with a central 19 bp duplex region and symmetric 2-base 3′-overhangs on the termini, although it has been recently described that chemically synthesized RNA duplexes of 25-30 base length can have as much as a 100-fold increase in potency compared with 21 mers at the same location. The observed increased potency obtained using longer RNAs in triggering RNAi is theorized to result from providing Dicer with a substrate (27 mer) instead of a product (21 mer) and that this improves the rate or efficiency of entry of the siRNA duplex into RISC.
It has been found that position of the 3′-overhang influences potency of an siRNA and asymmetric duplexes having a 3′-overhang on the antisense strand are generally more potent than those with the 3′-overhang on the sense strand (Rose et al., 2005). This can be attributed to asymmetrical strand loading into RISC, as the opposite efficacy patterns are observed when targeting the antisense transcript.
The term “shRNA”, as used herein, refers to an RNA agent having a stem-loop structure, comprising a first and second region of complementary sequence, the degree of complementarity and orientation of the regions being sufficient such that base pairing occurs between the regions, the first and second regions being joined by a loop region, the loop resulting from a lack of base pairing between nucleotides (or nucleotide analogs) within the loop region. The number of nucleotides in the loop is a number between and including 3 to 23, or 5 to 15, or 7 to 13, or 4 to 9, or 9 to 11. Some of the nucleotides in the loop can be involved in base-pair interactions with other nucleotides in the loop. Examples of oligonucleotide sequences that can be used to form the loop include 5′-UUCAAGAGA-3′ (SEQ ID NO: 4; Brummelkamp, T. R. et al. (2002) Science 296: 550) and 5′-UUUGUGUAG-3′ (SEQ ID NO: 5; Castanotto, D. et al. (2002) RNA 8:1454). It will be recognized by one of skill in the art that the resulting single chain oligonucleotide forms a stem-loop or hairpin structure comprising a double-stranded region capable of interacting with the RNAi machinery.
According to another embodiment the RNA silencing agent may be a miRNA. miRNAs are small RNAs made from genes encoding primary transcripts of various sizes. They have been identified in both animals and plants. The primary transcript (termed the “pri-miRNA”) is processed through various nucleolytic steps to a shorter precursor miRNA, or “pre-miRNA.” The pre-miRNA is present in a folded form so that the final (mature) miRNA is present in a duplex, the two strands being referred to as the miRNA (the strand that will eventually basepair with the target) The pre-miRNA is a substrate for a form of dicer that removes the miRNA duplex from the precursor, after which, similarly to siRNAs, the duplex can be taken into the RISC complex. It has been demonstrated that miRNAs can be transgenically expressed and be effective through expression of a precursor form, rather than the entire primary form (Parizotto et al. (2004) Genes & Development 18:2237-2242 and Guo et al. (2005) Plant Cell 17:1376-1386).
In one embodiment of the present invention, synthesis of RNA silencing agents suitable for use with the present invention can be effected as follows. First, the Varroa mite target mRNA is scanned downstream of the AUG start codon for AA dinucleotide sequences. Occurrence of each AA and the 3′ adjacent 19 nucleotides is recorded as potential siRNA target sites. Preferably, siRNA target sites are selected from the open reading frame, as untranslated regions (UTRs) are richer in regulatory protein binding sites. UTR-binding proteins and/or translation initiation complexes may interfere with binding of the siRNA endonuclease complex [Tuschl ChemBiochem. 2:239-245]. It will be appreciated though, that siRNAs directed at untranslated regions may also be effective, as demonstrated for GAPDH wherein siRNA directed at the 5′ UTR mediated about 90% decrease in cellular GAPDH mRNA and completely abolished protein level (wwwdotambiondotcom/techlib/tn/91/912dothtml).
In some embodiments, the RNA silencing agent provided herein can be functionally associated with a cell-penetrating peptide. As used herein, a “cell-penetrating peptide” is a peptide that comprises a short (about 12-30 residues) amino acid sequence or functional motif that confers the energy-independent (i.e., non-endocytotic) translocation properties associated with transport of the membrane-permeable complex across the plasma and/or nuclear membranes of a cell. The cell-penetrating peptide used in the membrane-permeable complex of the present invention preferably comprises at least one non-functional cystein residue, which is either free or derivatized to form a disulfide link with a double-stranded ribonucleic acid that has been modified for such linkage. Representative amino acid motifs conferring such properties are listed in U.S. Pat. No. 6,348,185, the contents of which are expressly incorporated herein by reference. The cell-penetrating peptides of the present invention preferably include, but are not limited to, penetratin, transportan, pIsl, TAT (48-60), pVEC, MTS, and MAP.
Another agent capable of downregulating a Varroa mite gene product is a DNAzyme molecule capable of specifically cleaving an mRNA transcript or DNA sequence of the bee pathogen polypeptide. DNAzymes are single-stranded polynucleotides which are capable of cleaving both single and double stranded target sequences (Breaker, R. R. and Joyce, G. Chemistry and Biology 1995; 2:655; Santoro, S. W. & Joyce, G. F. Proc. Natl, Acad. Sci. USA 1997; 943:4262) A general model (the “10-23” model) for the DNAzyme has been proposed. “10-23” DNAzymes have a catalytic domain of 15 deoxyribonucleotides, flanked by two substrate-recognition domains of seven to nine deoxyribonucleotides each. This type of DNAzyme can effectively cleave its substrate RNA at purine:pyrimidine junctions (Santoro, S. W. & Joyce, G. F. Proc. Natl, Acad. Sci. USA 199; for rev of DNAzymes see Khachigian, L M [Curr Opin Mol Ther 4:119-21 (2002)].
duplex 3′-T C G A
The polynucleotide down-regulating agents of the present invention may be generated according to any polynucleotide synthesis method known in the art such as enzymatic synthesis or solid phase synthesis. Equipment and reagents for executing solid-phase synthesis are commercially available from, for example, Applied Biosystems. Any other means for such synthesis may also be employed; the actual synthesis of the polynucleotides is well within the capabilities of one skilled in the art and can be accomplished via established methodologies as detailed in, for example, “Molecular Cloning: A laboratory Manual” Sambrook et al., (1989); “Current Protocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., “Current Protocols in Molecular Biology”, John Wiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide to Molecular Cloning”, John Wiley & Sons, New York (1988) and “Oligonucleotide Synthesis” Gait, M. J., ed. (1984) utilizing solid phase chemistry, e.g. cyanoethyl phosphoramidite followed by deprotection, desalting and purification by for example, an automated trityl-on method or HPLC.
The polynucleotide agents of the present invention may comprise heterocylic nucleosides consisting of purines and the pyrimidines bases, bonded in a 3′ to 5′ phosphodiester linkage.
Preferred modified polynucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free acid forms can also be used.
Polynucleotide agents of the present invention may also include base modifications or substitutions. As used herein, “unmodified” or “natural” bases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified bases include but are not limited to other synthetic and natural bases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further bases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993. Such bases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. [Sanghvi Y S et al. (1993) Antisense Research and Applications, CRC Press, Boca Raton 276-278] and are presently preferred base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.
The polynucleotide sequences of the present invention, under the control of an operably linked promoter sequence, may further be flanked by additional sequences that advantageously affect its transcription and/or the stability of a resulting transcript. Such sequences are generally located upstream of the promoter and/or downstream of the 3′ end of the expression construct.
The term “operably linked”, as used in reference to a regulatory sequence and a structural nucleotide sequence, means that the regulatory sequence causes regulated expression of the linked structural nucleotide sequence. “Regulatory sequences” or “control elements” refer to nucleotide sequences located upstream, within, or downstream of a structural nucleotide sequence, and which influence the timing and level or amount of transcription, RNA processing or stability, or translation of the associated structural nucleotide sequence. Regulatory sequences may include promoters, translation leader sequences, introns, enhancers, stem-loop structures, repressor binding sequences, termination sequences, pausing sequences, polyadenylation recognition sequences, and the like.
Thus the polynucleotides of the present invention may be synthesized in vitro and added to the food. For example double stranded RNA may be synthesized by adding two opposing promoters (e.g. T7 promoters; SEQ ID NOs: 48 and 49) to the ends of the gene segments, wherein SEQ ID NO: 48 is placed immediately 5′ to the gene and SEQ ID NO: 49 is placed immediately 3′ to the gene segment. The dsRNA may then be transcribed in vitro with the T7 RNA polymerase.
Liquid feed can be supplied to bees inside the hive by, for example, any of the following methods: friction-top pail, combs within the brood chamber, division board feeder, boardman feeder, etc. Dry sugar may be fed by placing a pound or two on the inverted inner cover. A supply of water must be available to bees at all times. In one embodiment, pan or trays in which floating supports—such as wood chips, cork, or plastic sponge—are present are envisaged. Detailed descriptions of supplemental feeds for bees can be found in, for example, USDA publication by Standifer, et al 1977, entitled “Supplemental Feeding of Honey Bee Colonies” (USDA, Agriculture Information Bulletin No. 413).
It is expected that during the life of a patent maturing from this application many relevant methods for downregulating expression of gene products will be developed and the scope of the term “downregulating expression of a gene product of a Varroa destructor mite” is intended to include all such new technologies a priori.
Example 1 Feeding Varroa-Specific dsRNA Prevents Varroa Mite Infestation
Young, approximately 2-month-old queens, together with approximately 200 worker bees are collected from hives in a local apiary. The bees are transferred into mini-hives fitted with one mini comb that was previously built by a regular hive. All of the mini-hives are closed and placed in a temperature-controlled room (30° C.).
Varroa mite sequences are cloned into a plasmid between two opposing T7 promoters. Following propagation of plasmid DNA, the viral fragments, including the T7 promoters, are excised and gel-purified. These serve as templates for T7-directed in-vitro transcription (MEGAscript™, Ambion, Austin Tex.). The reaction product is submitted to DNase digestion followed by phenol extraction and ethanol precipitation. The final preparation is dissolved in nuclease-free water.
7 days after feeding in active hives, some of the colonies are placed in contact with a population of Varroa mites. Thereafter, dsRNA treatment is continued for a further 2 days. Samples of live and dead bees (larvae and adults) are collected daily from each mini-hive post introduction of the Varroa mite population for 32 consecutive days. Every bee collected is frozen in liquid nitrogen and preserved at −70° C. pending molecular analysis. Vitality of the colonies are monitored by opening the hives (without smoke), withdrawing the mini-comb and photographing the mini-comb from both sides. The hive-combs are photographed daily, and the numbers of remaining live bees are monitored. The photographs are downloaded onto a computer and the total number of bees is counted for every mini-hive.
Total RNA is extracted from treated and control bees. Formaldehyde is added to the RNA to 1.8% and warmed to 65° C. The RNA, 15 μg per lane is electrophoresed on a 1.2% agarose gel at 70 V, 4° C. with stirring. The previously described amplified Varroa mite-RNA product is digoxigenin labeled and serves as a probe for hybridization. Detection is performed with the DIG luminescent detection kit (Roche Diagnostics GmbH, Mannheim, Germany). RNA sizes are estimated by comparison to electrophoresed RNA Molecular Weight Markers I (Roche). Hybridization is carried out at high stringency (0.1×SSC; 65° C.).
Example 3 Large-Scale Field Trials of Varroa-Specific dsRNA for Prevention of Varroa Mite-Associated Disease of Honeybees
10 μg of freshly prepared RNA is measured using the nanodrop spectrophotometer and loaded on 12% Acrylamide gel (1:19 acrylamide:Bis acrylamide ratio) in denturation environment (gel contains 7M Urea). After electrophoresis samples are transferred to positively charged nylon membrane (Roch, USA) using electroblotting method.
Membrane is hybridized with freshly prepared DNA probe of Varroa mite segment, taken from a region that does not correspond to the dsRNA of the Varroa mite-specific dsRNA itself. This is made using DIG PCR probe preparation Kit (Roch, USA) o/n 42° C. in DIG easyhyb solution (Roch, USA) according to manufacturer protocol. The membrane is washed twice with 2×SSC/0.1 SDS, than washed for stringency with 0.1×SSC/0.1% SDS in 65° C. Membranes are further washed using DIG Wash and Block Kit (Roch, USA) according to manufacturer protocol. Detection is preformed using CSPD-star substrate (Roch, USA). Positive control is 21nt DNA primers corresponding to the hybridized sequence.
Example 4 Bi-Directional Transfer of Bee-Ingested dsRNA from Bee to Varroa Mite and Back to Bee Via Varroa Infestation
In Examples 1 and 2 it was shown that dsRNA can be transferred from bees to Varroa directly into mites infesting bees ingesting the dsRNA, or indirectly into mites infesting larva fed by bees which ingested the dsRNA. In order to uncover whether the mites can further serve as an additional vector, transferring the dsRNA or siRNA from the mite back to a “naïve” bee via parasitisation, “naïve” bees were infested with Varroa following infestation of dsRNA-fed bees.
Primers for dsRNA preparation
specific dsRNA
SEQUENCE Amplicon
(SEQ ID NO:) Primers (F = Forward; R = Reverse) /SEQ ID NO: (bp)
SEQ ID NO: 93 F: 5′ CTAATACGACTCACTATAGGGCGAATGGAGAACATCGCACAG3′/SEQ ID NO: 107 411 bp
R: 5′ CTAATACGACTCACTATAGGGCGATTCCAGTACGTTATGTTGCTC3′/SEQ ID NO: 108
SEQ ID NO: 94 F: 5′ CTAATACGACTCACTATAGGGCGAGGTCTTGACAACACATGCTAC 3′/SEQ ID NO: 109 277 bp
R: 5′ CTAATACGACTCACTATAGGGCGACTCAGCAGAAATGATCGG3′/SEQ ID NO: 110
SEQ ID NO: 95 F: 5′ CTAATACGACTCACTATAGGGCGAAACGCTGTGCTTCACGTA 3′/SEQ ID NO: 111 329 bp
R: 5′ CTAATACGACTCACTATAGGGCGATCACGAGTAATCTCCACGA 3′/SEQ ID NO: 112
SEQ ID NO: 96 F: 5′ CTAATACGACTCACTATAGGGCGATCAGATGATTGGAACGGA 3′/SEQ ID NO: 113 380 bp
R: 5′ CTAATACGACTCACTATAGGGCGAAACAGGTCTTCAAACAGCAG 3′/SEQ ID NO: 114
SEQ ID NO: 97 F: 5′ CTAATACGACTCACTATAGGGCGATCAATTCGTCTGCAGATCTC 3′/SEQ ID NO: 115 426 bp
R: 5′ CTAATACGACTCACTATAGGGCGACATAAATGGCGATAAGCG 3′/SEQ ID NO: 116
SEQ ID NO: 98 F: 5′ CTAATACGACTCACTATAGGGCGAAATGAGTGTTGAGCGCGG 3′/SEQ ID NO: 117 366 bp
R: 5′ CTAATACGACTCACTATAGGGCGACTCCGATCATTTGGCGTT 3′/SEQ ID NO: 118
SEQ ID NO: 99 F: 5′ CTAATACGACTCACTATAGGGCGAAGGTGACATCCGTGTTCG 3′/SEQ ID NO: 119 324 bp
R: 5′ CTAATACGACTCACTATAGGGCGAATGAAGACATATAGGGTCGCT 3′/SEQ ID NO: 120
SEQ ID NO: 100 F: 5′ CTAATACGACTCACTATAGGGCGACTGTACAGGGTCCGAATATAAA 3′/SEQ ID NO: 121 311 bp
R: 5′ CTAATACGACTCACTATAGGGCGATTCGAGTTICTCAAAGGITG 3′/SEQ ID NO: 122
SEQ ID NO: 101 F: 5′ CTAATACGACTCACTATAGGGCGACAATTGAATATGGACGTCACTC 3′/SEQ ID NO: 123 201 bp
R: 5′ CTAATACGACTCACTATAGGGCGATTGAAAGCCAGCAGTAAACG 3′/SEQ ID NO: 124
SEQ ID NO: 102 F: 5′ CTAATACGACTCACTATAGGGCGACATCATCTTCTTCATCTGCTTG 3′/SEQ ID NO: 125 290 bp
R: 5′ CTAATACGACTCACTATAGGGCGAGGTTCCCACGGTTGGTAT 3′/SEQ ID NO: 126
SEQ ID NO: 103 F: 5′ CTAATACGACTCACTATAGGGCGAAATGGTTTCTGCTACCTGTG 3′/SEQ ID NO: 127 263 bp
R: 5′ CTAATACGACTCACTATAGGGCGAATTGGAAGCTGATACATTGG 3′/SEQ ID NO: 128
SEQ ID NO: 104 F: 5′ CTAATACGACTCACTATAGGGCGATGGCTAATTAATAGTAGGCCG 3′/SEQ ID NO: 129 277 bp
R: 5′ CTAATACGACTCACTATAGGGCGATGGAGTTTGCTACCAACCT 3′/SEQ ID NO: 130
SEQ ID NO: 105 F: 5′ CTAATACGACTCACTATAGGGCGAAGCCGGCTTCTTCTTCCT 3′/SEQ ID NO: 131 263 bp
R: 5′ CTAATACGACTCACTATAGGGCGAAGTCACTGCCTGTTCCTCC 3′/SEQ ID NO: 132
SEQ ID NO: 106 F: 5′ CTAATACGACTCACTATAGGGCGATTCCGCTTCATTTGAGAAC 3′/SEQ ID NO: 133 282 bp
R: 5′ CTAATACGACTCACTATAGGGCGATCTGAATCAACCTCATCGG 3′/SEQ ID NO: 134
SEQ ID NO: 92  F: 5′ TAATACGACTCACTATAGGGCGAGCCAACACTTGTCACTACTAGAAAGAGAA 3′/SEQ ID NO: 135 431 bp
R: 5′ TAATACGACTCACTATAGGGCGAAGGTAATGGITGTCTGGTAAAGGAC 3′/SEQ ID NO: 136
Total RNA for dsRNA-GFP detection experiments was isolated from a single honeybee or from 10 Varroa mites, using phenol-chloroform extraction (peqGOLD Trifast™, Peqlab). Total RNA for Varroa dsRNA experiments was isolated from 5 Varroa mites by tissue homogenization binding to a mini-column, DNA-removal and RNA elution (ZR Tissue & Insect RNA MicroPrep, Zymo Research, Irvine Calif.). DNA was digested in the eluted RNA by nucleases (TURBO DNA-free kit, Ambion, Austin, Tex., USA) and the RNA was tested for DNA contamination. Varroa RNA was then co-precipitated with glycogen and 3 M sodium acetate in 70% ethanol and resuspended in 20 μl of RNAse-free water. The amount and quality of the RNA were determined spectrophotometrically using the nanodrop method (NanoDrop Technologies, Wilmington, Del., USA).
List of primers and probes used for real-time and semi-quantitative
RT-PCR assays.
(SEQ ID NO) Primers/SEQ ID NO: Amplicon (bp)
Varroa RNA F: 5′ AAAGGGCAGGTGCTTATCAA 3′/137 65
Polymerase III R: 5′ TGTCCAGGGTCGAGAGTAGC 3′/138
Varroa vacuolar F: 5′ ACCTTTTTCAAAGACCGAACC 3′/139 62
proton ATPase R: 5′ CGAAGACTCCGTTCGAAAAC 3′/140
Varroa IAP1 and F: 5′ CTAGTTAATGGCGCGGTAGC 3′/141 63
IAP2, reverse R: 5′ TCCTCCCGGTTCTACTTCAC 3′/142
Varroa 18S RNA F: 5′ AATGCCATCATTACCATCCTG 3′/143 60
R: 5′ CAAAAACCAATCGGCAATCT 3′/144
Varroa Apoptosis F: 5′ ATCTGCCCACGTCAGCGTTT 3′/145 317
Inhibitor FAS R: 5′ GTCCGTCATTTCGGCTTTGG 3′/146
Varroa Actin F: 5′ AAGTCGTACGAGCTTCCCGAC 3′/147 336
R: 5′ ACAGGGAGGCAAGGATGGAAC 3′/148
The real-time PCR program was as follows: 95° C. for 10 min, followed by 45 cycles of 95° C. for 10 seconds and 60° C. for 30 seconds, and finally 40° C. for 30 seconds. 18S rRNA was used as an internal control for the standardization of RNA levels.
The semi-quantitative PCR program was as follows: 95° C. for 10 min, followed by 40 cycles, each consisting of 95° C. for 10 seconds and 65° C. and 55° C. for 30 seconds for the apoptosis inhibitor (FAS, primers were SEQ ID Nos. 145 and 146) and its internal standardization control (actin, primers were SEQ ID Nos. 147 and 148), respectively, followed by 72° C. for 30 seconds. Reaction products were sampled every three cycles starting from cycle 31 for FAS and from cycle 29 for actin, the sample incubated for 5 min at 72° C. and stored at −20° C. Samples were analyzed on a 1.2% agarose gel. Each semi-quantitative PCR experiment was repeated three times.
1-day-old bees were placed in four plastic containers (30 bees per container). Two containers were fed with 30 μg dsRNA-GFP in 200 μl of 50% sucrose solution for 8 days, and the other two control containers fed 50% sucrose solution without dsRNA. Mite infestation was initiated by introduction of adult female Varroa (n=30) into each container on day 5. After 3 days, Varroa that were attached to bees were removed and collected, and their RNA isolated for dsRNA-GFP analysis. To test for bidirectional transfer of dsRNA-GFP from bee to mite and on to another bee, newly emerged, untreated bees were infested by some of the Varroa that had been detached from the dsRNA-fed bees for 4 days and the bee's RNA isolated for dsRNA-GFP analysis. Each day, bees in all containers were given an additional 1 ml sucrose solution after finishing their treatment. In addition, bees had free access to a pollen patty consisting of 70% pollen mixed with sugar powder.
The experiment with Varroa dsRNA was conducted in mini-hives, 12 mini-hives per repetition, for three repetitions. In each repetition, a cup of bees and a laying queen were placed in each mini-hive. Three mini-hives were randomly assigned to one of four netted enclosures, each representing a different feeding treatment. Bees were fed 5 ml of 50% sucrose solution in troughs placed in each mini-hive. The four treatments were: 1) sucrose solution only (untreated control), 2) Mixture I (200 μg each of five dsRNAs added to the sugar solution), 3) Mixture II (200 μg each of 14 dsRNAs added to the sugar solution), and 4) dsRNA-GFP (200 μg dsRNA) serving as a dsRNA-positive control. Bees that fully consumed the treatment solutions were supplemented with candy (67% sugar powder and 33% honey). In addition, the bees were routinely fed pollen patties (70% pollen and 30% sugar powder). Each repetition of the experiment lasted for 60 days (FIG. 8). Bees in each treatment were fed the respective solution daily for the first 10 days and for the last 14 days, and twice a week in the interim. Infestation with Varroa mites was initiated by introducing mites into each mini-hive from day 7 until day 14. In the first repetition, 30 mites were introduced into each mini-hive; in the latter two repetitions, 100 mites were introduced into each mini-hive. On day 60, all mature bees were collected, counted and shaken with 70% ethanol overnight in order to collect and count Varroa mites falling off the bees. All capped brood cells were opened to collect and count Varroa mites. Number of mites per bee included mature and developing (capped brood) bees. Varroa mites, adult bees, emerging bees and pupae were stored for molecular analyses.
To test for bidirectional horizontal transfer, mites feeding on bees ingesting GFP-specific dsRNA were removed from the bees after 3 days and introduced into a container with untreated, “naïve” bees for 4 days. RT-PCR of Varroa and bee RNA reveals that GFP-specific RNA sequences were detectable in RNA extracts of “naïve” bees which had been parasitized by Varroa mites previously infesting bees carrying GFP-dsRNA (see FIG. 7, lanes B− and B+). The presence of GFP-specific sequences in the parasitized “naïve” bees indicates reciprocal, bi-directional transfer of the GFP-specific sequences derived from dsRNA, from bee to Varroa and then to another bee by mite infestation.
These results clearly point to a surprising additional means for transmission, from dsRNA-fed bees to mites and back to “naïve” bees, of RNAi sequences derived from the dsRNA. Such bi-directional transmission can be effective in further disseminating the silencing effect of ectoparasite (e.g. mite)-specific dsRNA fed to bees.
Example 5 Silencing of Varroa Gene Expression Mediated by Bees Ingesting dsRNA
In order to determine whether feeding the dsRNA mixtures affected bee survival, all mature bees and sealed brood in the mini-hives at completion of the protocol (see FIG. 8) were counted. Bee population size did not differ between control and dsRNA-treated mini-hives (F3,29=0.62, P=0.608; FIG. 10). The results were similar when brood and adult bees were analyzed separately (not shown). Thus, feeding the dsRNA mixtures is not deleterious to bees, indicating no off-target effect of the feeding.
Varroa infestation was reduced in bees of mini-hives fed with Varroa dsRNA compared to the controls (F3,29=5.65, P=0.0035; FIG. 11). The effect was even more significant in bees of hives fed Mixture II, which targeted more genes than Mixture I, reducing Varroa infestation by an average 53% compared to control hives fed the dsRNA-GFP control, and by 61% compared to hives receiving no dsRNA control.
Taken together, these results indicate that feeding bees Varroa-specific dsRNA results in both direct and indirect transmission of mite-specific dsRNA and siRNA to mites feeding off the bees and larval/pupae in the hives, as well as bi-directional transmission of the Varroa-specific RNA sequences from parasitizing mites back to “naïve” bees, and that feeding the Varroa-specific dsRNA is an effective and safe method for reducing mite infestation in the hives.
10. The method of claim 1, wherein the Varroa destructor mite mRNA encodes a polypeptide selected from the group consisting of ATPase subunit A, RNA polymerase I, RNA polymerase III, Inhibitor of apoptosis (IAP), FAS apoptotic inhibitor and α-Tubulin.
18. The method of claim 11, wherein the Varroa destructor mite mRNA encodes a polypeptide selected from the group consisting of ATPase subunit A, RNA polymerase I, RNA polymerase III, Inhibitor of apoptosis (IAP), FAS apoptotic inhibitor and α-Tubulin.
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