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Patent US8101343 - protecting against parasites and predators; transgenic organisims - Google PatentsSearch Images Maps Play YouTube News Gmail Drive More »Sign in<nobr>Advanced Patent Search</nobr>PatentsThe invention is to methods of gene silencing in arthropods using dsRNA. The method is include contacting the arthropod with, and/or directly feeding the arthropod, the dsRNA to the arthropods to deliver the dsRNA to arthropod tissues. It is envisaged that the methods of the invention will have use in...http://www.google.com/patents/US8101343?utm_source=gb-gplus-sharePatent US8101343 - protecting against parasites and predators; transgenic organisimsAdvanced Patent SearchPublication numberUS8101343 B2Publication typeGrantApplication numberUS 10/482,888PCT numberPCT/AU2002/000897Publication dateJan 24, 2012Filing dateJul 5, 2002Priority dateJul 6, 2001Also published asCA2455490A1, CA2455490C, EP1414959A1, EP1414959A4, EP2333061A1, US8263573, US8415320, US20050095199, US20120041053, US20120309813, US20130237586, WO2003004644A1Publication number10482888, 482888, PCT/2002/897, PCT/AU/2/000897, PCT/AU/2/00897, PCT/AU/2002/000897, PCT/AU/2002/00897, PCT/AU2/000897, PCT/AU2/00897, PCT/AU2000897, PCT/AU2002/000897, PCT/AU2002/00897, PCT/AU2002000897, PCT/AU200200897, PCT/AU200897, US 8101343 B2, US 8101343B2, US-B2-8101343, US8101343 B2, US8101343B2InventorsSteven Whyard, Fiona Helen Cameron, Minoo Moghaddam, Trevor J. LockettOriginal AssigneeCommonwealth Scientific And Industrial Research OrganisationExport CitationBiBTeX, EndNote, RefManPatent Citations (69), Non-Patent Citations (521), Referenced by (4), Classifications (31), Legal Events (1) External Links: USPTO, USPTO Assignment, Espacenetprotecting against parasites and predators; transgenic organisimsUS 8101343 B2Abstract The invention is to methods of gene silencing in arthropods using dsRNA. The method is include contacting the arthropod with, and/or directly feeding the arthropod, the dsRNA to the arthropods to deliver the dsRNA to arthropod tissues. It is envisaged that the methods of the invention will have use in determining the biological function of genes in arthropods. Methods of pest control of arthropods, and of protecting arthropods against parasites and predators are provided. Transgenic arthropods expressing dsRNA molecules are also provided by the present invention.
CROSS REFERENCE TO RELATED APPLICATIONS This application is the National Stage of International Application No. PCT/AU02/00897, filed 5 Jul. 2002, which claims the benefit under the Paris Convention and under 35 U.S.C. �119 of Australian Application No. PR 6215, filed 6 Jul. 2001.
FIELD OF THE INVENTION The present invention relates generally to dsRNA and its use in gene silencing. Furthermore, the present invention relates to methods of delivering dsRNA to an arthropod.
BACKGROUND OF THE INVENTION RNA interference (RNAi) is considered as a naturally occurring adaptive defence in at least some organisms against viruses and the production of aberrant transcripts, such as those produced by transposon mobility (Bosher and Labouesse, 2000; Waterhouse et al., 2001).
The actual process by which dsRNA mediates target RNA degradation is not fully understood, but the cellular machinery involved is gradually being identified. Full-length dsRNAs have been observed to be progressively degraded into �21-nucleotide dsRNAs, by an enzyme called Dicer-1 (Elbashir et al., 2001). It is believed that the Dicer-1 proteins, along with their associated 21-mer dsRNA, seek single stranded RNAs with sequence identity, and promote the cleavage of single stranded RNA targets (Waterhouse et al., 2001).
SUMMARY OF THE INVENTION The present invention provides methods that utilize dsRNA to determine the biological function of an RNA in an arthropod. In particular, the invention provides efficient mechanisms of delivering dsRNA to an arthropod with the aid of transfection promoting agents. Furthermore, the present invention provides methods for controlling pest arthropod populations, methods for controlling pathogens carried by arthropods, as well as methods for protecting an arthropod from a pathogen, parasite or predatory organism. In addition, the present invention provides transgenic organisms, in particular arthropods, expressing small dsRNA molecules.
BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1. PCR detection of phspGUS[i/r] plasmid in insects injected with the plasmid as late embryos. In the leftmost 7 lanes, the presence of the GUS transgene in the flies is evident by the production of the 1 kb PCR product in all developmental stages. There was no PCR product in wild type, non-transgenic flies (wt). In the right hand side of the gel, only L1 (1st instar larvae) show evidence of the injected plasmid, as indicated by the single 500 bp PCR product.
DETAILED DESCRIPTION OF THE INVENTION General Techniques
Standard methods for the production of transgenic insects are outlined in �Insect Transgenesis�Methods and Applications� (Ed. A. M. Handler and A. A. James, CRC Press, London, 2000).
As used herein, �dsRNA� or �RNAi� refers to a polyribonucleotide structure formed either by a single self-complementary RNA strand or at least by two complementary RNA strands. The degree of complementary, in other words the % identity, need not necessarily be 100%. Rather, it must be sufficient to allow the formation of a double-stranded structure under the conditions employed.
As used herein, the term �specifically reduce the level of a target RNA and/or the production of a target protein encoded by the RNA�, and variations thereof, refers to the sequence of a portion of one strand of the dsRNA being sufficiently identical to the target RNA such that the presence of the dsRNA in a cell reduces the steady state level and/or the production of said RNA. In many instances, the target RNA will be mRNA, and the presence of the dsRNA in a cell producing the mRNA will result in a reduction in the production of said protein. Preferably, this accumulation or production is reduced at least 10%, more preferably at least 50%, even more preferably at least 75%, yet even more preferably at least 95% and most preferably 100%, when compared to a wild-type cell.
The formulation of polynucleotides encapsulated in lipid-containing compounds in known in the art and described in, for example, �Liposomes: from physical structure to therapeutic applications� (Ed. C. G. Knight. Elsevier Press, 1981).
As used in the present invention, the terms �micelle� and �liposome� mean vesicles composed of amphiphilic lipids self-assembled in aqueous solution to form tertiary structures.
Liposomes also include �sterically stabilized� liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) comprises one or more glycolipids, or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen and Chonn, 1987; Wu et al., 1993).
Transfection promoting agents useful for the methods and compositions of the present invention include �Tris cationic lipids� which are disclosed in WO 96/05218, U.S. Pat. No. 5,854,224, U.S. Pat. Nos. 5,583,198 and 5,869,606, the contents of which are incorporated by reference. These agents include compounds having a formula selected from the group consisting of:
R4 is H or CH2O�R3; and R1, R2 and R3 are the same or different and are either hydrogen, methyl, ethyl, hydroxyl or an acyl group derived from a fatty acid having a carbon chain of 3 to 24 carbon atoms saturated or unsaturated, with the proviso that at least one of R1, R2 and R3 is an acyl group derived from a fatty acid;
w . . . x-y-NH�CH2�CH2O�R5 ii)
w . . . x-y-NH�CH2�CH2O�R5 iv)
R4 is H or halogen or CH2O�R3; and R1, R2 and R3 are the same or different and are either hydrogen, methyl, ethyl, alkyl, alkenyl, hydroxylated alkyl, hydroxylated alkenyl groups or ether containing alkyl, alkenyl, hydroxylated alkyl or hydroxylated alkenyl groups, optionally being an acyl group derived from a fatty acid having a carbon chain length equivalent to 3-24 carbon atoms saturated or unsaturated, with the proviso that at least one of R1, R2 and R3 includes a group having a carbon chain of 3-24 carbon atoms saturated or unsaturated.
H2N�(�(CH)m�NH�)n�H
Agriculturally acceptable carriers are also referred to herein as an �excipient�. An excipient can be any material that the animal, plant or environment to be treated can tolerate. Furthermore, the excipient must be such that the composition of the present invention is still capable of causing gene silencing. Examples of such excipients include water, saline, Ringer's solution, dextrose or other sugar solutions, Hank's solution, and other aqueous physiologically balanced salt solutions, phosphate buffer, bicarbonate buffer and Tris buffer. In addition, the composition may include compounds that increase the half-life of a composition. Such compounds are be known to the skilled person in the art.
A particularly preferred expression vector is a baculovirus. By �baculovirus� it is meant any virus of the family Baculoviridae, such as a nuclear polyhedrosis virus (NPV). Baculoviruses are a large group of evolutionarily related viruses, which infect only arthropods; indeed, some baculoviruses only infect insects that are pests of commercially important agricultural and forestry crops, while others are known that specifically infect other insect pests. Because baculoviruses infect only arthropods, they pose little or no risk to humans, plants, or the environment.
Suitable baculoviruses for practicing this invention may be occluded or non-occluded. The nuclear polyhedrosis viruses (�NPV�) are one baculovirus sub-group, which are �occluded.� That is, a characteristic feature of the NPV group is that many virions are embedded in a crystalline protein matrix referred to as an �occlusion body.� Examples of NPVs include Lymantria dispar NPV (gypsy moth NPV), Autographa californica MNPV, Anagrapha falcifera NPV (celery looper NPV), Spodoptera litturalis NPV, Spodoptera frugiperda NPV, Heliothis armigera NPV, Mamestra brassicae NPV, Choristoneura fumiferana NPV, Trichoplusia ni NPV, Helicoverpa zea NPV, and Rachiplusia ou NPV. For field use occluded viruses often are preferable due to their greater stability since the viral polyhedrin coat provides protection for the enclosed infectious nucleocapsids.
Among illustrative, useful baculoviruses in practicing this invention are those isolated from Anagrapha falcifera, Anticarsia gemmatalis, Buzura suppressuria, Cydia pomonella, Helicoverpa zea, Heliothis armigera, Manestia brassicae, Plutella xylostella, Spodoptera exigua, Spodoptera littoralis, and Spodoptera litura. A particularly useful �NPV� baculovirus for practicing this invention is AcNPV, which is a nuclear polyhedrosis virus from Autographa californica. Autographa californica is of particular interest because various major pest species within the genera Spodoptera, Trichoplusia, and Heliothis are susceptible to this virus.
The term �plant� refers to whole plants, plant organs (e.g. leaves, stems roots, etc), seeds, plant cells and the like. Plants contemplated for use in the practice of the present invention include both monocotyledonous and dicotyledonous plants. Exemplary dicotyledonous plants include cotton, oilseeds and other brassicas, tomato, tobacco, potato, bean, and soybean. Exemplary monocotyledonous plants include wheat, maize, barley, rice, and sorghum. The choice of the plant species is determined by the intended use of the plant or parts thereof and the amenability of the plant species to transformation.
EXAMPLES Methods GUS RNA In Vitro Transcription Plasmids
A selection of transfection promoting agents was kindly provided by Trevor Lockett and colleagues (CSIRO Molecular Science). These transfection promoting agents are thoroughly described in the patent �Delivery of Nucleic Acids� (PCT/AU95/00505, U.S. Pat. No. 5,906,922). A comparison of 11 of these CSIRO reagents with the 5 commercially available reagents was conducted, and many of the CSIRO liposomes were more effective at producing an RNAi effect in Drosophila (Table 5). In particular, liposomes CS096, CS102, and CS129 performed better than the best-performing commercially available liposome, DMRIE-C. All of the CSIRO liposomes tested produced a greater number of individuals affected by RNAI than the poorest commercially available liposome, DOTAP. These results confirm that optimised delivery of dsRNA to insects can be achieved by selecting appropriate transfection promoting agents.
*Complete names of the transfection promoting agents are provided in the �Transfection Promoting Agent� section of the Detailed Description. Lipofectin, Lipofectamine, Cellfectin, and DMRIE-C were obtained from Life Technologies, whereas DOTAP was obtained from Boehringer Mannheim.
REFERENCES Allen, T. M. and Chonn, A. (1987) FEBS Lett. 223:4246.
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(2000) "RNAi: Double-Stranded RNA Directs the ATP-Dependent Cleavege of mRNA at 21 to 23 Nucleotide Intervals," Cell 101:25-33.* Cited by examinerReferenced byCiting PatentFiling datePublication dateApplicantTitleUS8263573Sep 23, 2011Sep 11, 2012Commonwealth Scientific And Industrial Research OrganisationDelivery of dsRNA to arthropodsUS8415320Jul 10, 2012Apr 9, 2013Commonwealth Scientific And Industrial Research OrganisationDelivery of dsRNA to arthropodsUS20080226551 *Dec 12, 2007Sep 18, 2008Revance Therapeutics, Inc.Binding to support; transdermal penetrationUS20090012021 *Apr 17, 2006Jan 8, 2009Sood Anil KDelivery of Sirna by Neutral Lipid Compositions* Cited by examinerClassifications U.S. Classification435/6.1, 435/6.13, 536/23.1, 514/44.00A, 536/24.5International ClassificationC12N15/09, C07H21/02, C12N15/113, C12Q1/68, A01K67/033, A61K38/00, A61K48/00, A01H5/00, C07H21/04, A01K67/027Cooperative ClassificationC12Y302/01031, A01K2217/05, A01N57/16, A61K31/713, C12N15/1137, C12N2310/14, A01K67/0337, C12N15/111, A61K38/00, C12N2320/32, C12N15/113European ClassificationC12N15/11M, C12N15/113D, C12N15/113, A01K67/033M30, C12Y302/01031Legal EventsDateCodeEventDescriptionApr 2, 2004ASAssignmentOwner name: COMMONWEALTH SCIENTIFIC AND INDUSTRIAL RESEARCH ORFree format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:WHYARD, STEVEN;CAMERON, FIONA HELEN;MOGHADDAM, MINOO;ANDOTHERS;REEL/FRAME:014489/0635;SIGNING DATES FROM 20040311 TO 20040322Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:WHYARD, STEVEN;CAMERON, FIONA HELEN;MOGHADDAM, MINOO;ANDOTHERS;SIGNING DATES FROM 20040311 TO 20040322;REEL/FRAME:014489/0635RotateOriginal ImageGoogle Home - Sitemap - USPTO Bulk Downloads - Privacy Policy - Terms of Service - About Google Patents - Send FeedbackData provided by IFI CLAIMS Patent Services©2012 Google