U.S. patent application number 14/483182 was filed with the patent office on 2015-01-01 for methods and compositions for increasing rna interference.
This patent application is currently assigned to DEVGEN N.V.. The applicant listed for this patent is DEVGEN N.V.. Invention is credited to Annelies Philips, Romaan Raemaekers.
Application Number | 20150004148 14/483182 |
Document ID | / |
Family ID | 41089148 |
Filed Date | 2015-01-01 |
United States Patent
Application |
20150004148 |
Kind Code |
A1 |
Raemaekers; Romaan ; et
al. |
January 1, 2015 |
METHODS AND COMPOSITIONS FOR INCREASING RNA INTERFERENCE
Abstract
The present invention relates to compositions containing a
combination of a first active component comprising one or more
sulfated polysaccharides and/or glycosaminoglycans and a second
active component comprising one or more RNAi-inducing molecules,
and methods for using these compositions to enhance double-stranded
RNA (dsRNA)-mediated gene silencing in pest or pathogen species.
The invention further relates to methods for controlling pests or
pathogens, methods for preventing pest infestations or pathogen
infections and methods for knocking down gene expression in pests
or pathogens using the compositions and methods of the
invention.
Inventors: |
Raemaekers; Romaan; (De
Pinte, BE) ; Philips; Annelies; (Oostakker,
BE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
DEVGEN N.V. |
Zwijnnaarde |
|
BE |
|
|
Assignee: |
DEVGEN N.V.
Zwijnnaarde
BE
|
Family ID: |
41089148 |
Appl. No.: |
14/483182 |
Filed: |
September 11, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12389635 |
Feb 20, 2009 |
8865668 |
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14483182 |
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61030029 |
Feb 20, 2008 |
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61087315 |
Aug 8, 2008 |
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61118105 |
Nov 26, 2008 |
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Current U.S.
Class: |
424/93.45 ;
424/93.1; 424/93.4; 424/93.461; 424/93.51; 424/93.7; 514/44A |
Current CPC
Class: |
A01N 43/16 20130101;
A61K 31/7105 20130101; A61K 45/06 20130101; A01N 43/90 20130101;
A61K 31/727 20130101; C12N 15/8279 20130101; C12N 15/8218 20130101;
C12N 15/8286 20130101; C12N 2310/14 20130101; A61K 31/721 20130101;
C12N 15/113 20130101; A61K 31/74 20130101; Y02A 40/162 20180101;
Y02A 50/30 20180101; A61K 31/715 20130101; Y02A 50/473 20180101;
A61K 31/737 20130101; Y02A 40/146 20180101; C12N 15/8261 20130101;
A61K 31/726 20130101; A01N 43/16 20130101; A01N 57/16 20130101;
A01N 63/00 20130101; A01N 43/16 20130101; A01N 2300/00 20130101;
A61K 31/7105 20130101; A61K 2300/00 20130101; A61K 31/715 20130101;
A61K 2300/00 20130101; A61K 31/721 20130101; A61K 2300/00 20130101;
A61K 31/726 20130101; A61K 2300/00 20130101; A61K 31/727 20130101;
A61K 2300/00 20130101; A61K 31/74 20130101; A61K 2300/00
20130101 |
Class at
Publication: |
424/93.45 ;
514/44.A; 424/93.1; 424/93.4; 424/93.461; 424/93.7; 424/93.51 |
International
Class: |
A01N 43/90 20060101
A01N043/90; C12N 15/113 20060101 C12N015/113; A01N 43/16 20060101
A01N043/16; A61K 31/737 20060101 A61K031/737 |
Claims
1. A method for increasing a RNA interference (RNAi) effect of a
molecule or composition that reduces expression of a selected gene
or genes by RNA interference in cell(s) or an organism comprising
contacting the cell(s) or organism with (1) a first active
component comprising an amount of one or more sulfated
polysaccharides and/or glycosaminoglycans that is effective to
enhance the RNAi effect of a molecule(s) or a composition and (2) a
second active component comprising a molecule(s) or a composition
that reduces expression of a selected gene or genes by RNA
interference.
2. The method of claim 1, wherein the cell(s) or organism is
contacted with the first active component (i) before or after
contacting the cell(s) or organism with the second active
component, (ii) simultaneously with contacting the cell(s) or
organism with the second active component, or (iii) before,
simultaneously with and/or after contacting the cell(s) or organism
with the second active component.
3. The method of claim 1, wherein the contacting comprises applying
or spraying the second active component and the effective amount of
the first active component onto the target cell(s) or target
organism or on food for the target organism.
4. A method for protecting plants or foodstuffs or substances from
pest infestation or pathogen infection comprising contacting the
plant or the soil on which the plant is rooted, or foodstuff or
substance with (1) a first active component comprising an amount of
one or more sulfated polysaccharides and/or glycosaminoglycans that
is effective to enhance the RNAi effect of a molecule(s) or a
composition and (2) a second active component comprising a
molecule(s) or a composition that reduces expression of a selected
gene or genes by RNA interference.
5. The method of claim 4, wherein the plant or the soil on which
the plant is rooted, or foodstuff or substance is contacted with
the first active component (i) before or after contacting the plant
or the soil on which the plant is rooted, or foodstuff or substance
with the second active component, (ii) simultaneously with
contacting the plant or the soil on which the plant is rooted, or
foodstuff or substance with the second active component, or (iii)
before, simultaneously with and/or after contacting the plant or
the soil on which the plant is rooted, or foodstuff or substance
with the second active component.
6. The method of claim 4, wherein the contacting comprises applying
or spraying the second active component and the effective amount of
the first active component onto the plant or foodstuff or
substance.
7. A method for treating a plant infection by a pathogen or plant
infestation by a pest comprising contacting the plant or the soil
in which the plant is rooted with (1) a first active component
comprising an amount of one or more sulfated polysaccharides and/or
glycosaminoglycans that is effective to enhance the RNAi effect of
a molecule(s) or a composition and (2) a second active component
comprising a molecule(s) or a composition that reduces expression
of a selected gene or genes by RNA interference.
8. A method for increasing crop yield or reducing a decline in crop
yield that results from pest infestation and/or pathogen infection
comprising contacting the plant or the soil wherein the plant is
rooted with (1) a first active component comprising an amount of
one or more sulfated polysaccharides and/or glycosaminoglycans that
is effective to enhance the RNAi effect of a molecule(s) or a
composition and (2) a second active component comprising a
molecule(s) or a composition that reduces expression of a selected
gene or genes by RNA interference.
9. The method of claim 7, wherein the plant or the soil on which
the plant is rooted is contacted with the first active component
(i) before or after contacting the plant or the soil on which the
plant is rooted with the second active component, (ii)
simultaneously with contacting the plant or the soil on which the
plant is rooted with the second active component, or (iii) before,
simultaneously with and/or after contacting the plant or the soil
on which the plant is rooted with the second active component.
10. The method of claim 7, wherein the contacting comprises
applying or spraying the second active component and the effective
amount of the first active component onto the plant or onto the
soil wherein the plant is rooted.
11. The method of claim 1, wherein the first active component is as
recited in claim 20 and the second active component comprises at
least one RNA molecule of which at least a portion is a
double-stranded (dsRNA) molecule.
12. The method of claim 1, wherein the first active component is as
recited in claim 20 and the second active component is as recited
in claim 20.
13. The method of claim 1, wherein the first active component and
the second active component are combined in a composition as
recited in claim 20.
14. A method for protecting a human or an animal from pest
infestation or pathogen infection comprising administering to the
human or animal (1) a first active component comprising an amount
of one or more sulfated polysaccharides and/or glycosaminoglycans
that is effective to enhance the RNAi effect of a molecule(s) or a
composition and (2) a second active component comprising a
molecule(s) or a composition that reduces expression of a selected
gene or genes by RNA interference.
15. A method for treating a pest infestation or pathogen infection
of a human or an animal, comprising administering to the human or
animal an effective amount of (1) a first active component
comprising an amount of one or more sulfated polysaccharides and/or
glycosaminoglycans that is effective to enhance the RNAi effect of
a molecule(s) or a composition, and (2) a second active component
comprising a molecule(s) or a composition that reduces expression
of a selected gene or genes by RNA interference.
16. The method of claim 14, wherein the human or animal is
administered the first active component (i) before or after
administering the second active component to the human or animal,
(ii) simultaneously with administering the second active component
to the human or animal, or (iii) before, simultaneously with and/or
after administering the second active component to the human or
animal.
17. The method of claim 14, wherein the administering comprises
topical, parenteral, enteral, transdermal, cutaneous, subcutaneous,
intravenous, intraperitoneal, intramuscular or oral
administration.
18. The method of claim 14, wherein the first active component is
as recited in claim 20 and the second active component is as
recited in claim 20.
19. The method of claim 14, wherein the first active component and
the second active component are combined in a composition as
recited in claim 20.
20. A composition comprising a first active component comprising an
amount of one or more sulfated polysaccharides and/or
glycosaminoglycans, wherein the amount of one or more sulfated
polysaccharides and/or glycosaminoglycans is effective to increase
the RNAi effect of a molecule(s) or a composition on a selected
gene or genes and a second active component comprising one or more
prokaryotic cells or eukaryotic cells or host organisms that
express double-stranded RNA molecule(s) that reduce expression of a
selected gene or genes by RNA interference (RNAi) in a target cell
or target organism.
21. The composition of claim 20, wherein the first active component
is a composition of one or more sulfated polysaccharides and/or
glycosaminoglycans.
22. The composition of claim 20, wherein the first active component
is dextran sulfate.
23. The composition of claim 20, wherein the first active component
is fucoidan.
24. The composition of claim 20, wherein the first active component
is heparin.
25. The composition of claim 22, wherein the dextran sulfate has an
average molecular weight of between 8 and 40 kilodaltons.
26. The composition of claim 20, wherein the one or more
prokaryotic cells or eukaryotic cells or host organisms express at
least one RNA molecule of which at least a portion is a
double-stranded (dsRNA) molecule.
27. The composition of claim 20, wherein the one or more
prokaryotic cells or eukaryotic cells or host organisms comprise at
least one expression vector that expresses at least one RNA
molecule of which at least a portion is a double-stranded RNA
(dsRNA) molecule.
28. The composition of claim 26, wherein the dsRNA molecule is a
dsRNA molecule consisting of at least two separate strands, an
siRNA molecule or a hairpin molecule.
29. The composition of claim 27, wherein the expression vector
comprises at least one regulatory sequence operably linked to the
nucleotide sequence encoding the at least one RNA molecule.
30. The composition of claim 20, wherein the prokaryotic cell is a
bacterial cell chosen from the group comprising Gram positive and
Gram negative cells comprising Escherichia spp. (e.g. E. coli),
Bacillus spp. (e.g. B. thuringiensis), Rhizobium spp.,
Lactobacilllus spp., and Lactococcus spp.
31. The composition of claim 30, wherein the bacterial cell is
inactivated by heat or by chemical treatment.
32. The composition of claim 20, wherein the eukaryotic cell is
chosen from the group comprising mammalian cells and non-human
mammalian cells.
33. The composition of claim 20, wherein the eukaryotic cell is a
yeast cell.
34. The composition of claim 20, wherein at least one strand of the
dsRNA comprises a nucleotide sequence that is complementary to a
portion of the nucleotide sequence of a selected gene or genes from
the target cell or target organism.
35. The composition of claim 20, wherein said gene or genes is
essential for the viability, growth, development or reproduction of
the target cell or target organism.
36. The composition of claim 20, wherein said gene or genes in the
second active component comprises (a) the sequence of any of SEQ ID
NOs 1 to 6, (b) the RNA equivalent thereof, (3) the complement of
(a) or (b), or a fragment of any of (a) to (c) consisting of at
least 17 contiguous nucleotides of any of (a) to (c).
37. The composition of claim 20, wherein the target organism is a
pest species, a pathogen species, or a cell thereof.
38. The composition of claim 37, wherein said pest species or
pathogen species is a fungus, an insect or a nematode.
39. A composition comprising a first active component comprising an
amount of dextrane sulfate having a molecular weight between 5 and
2000 kDA, preferably between 8 and 40 kDa, wherein the amount of
dextrane sulfate is effective to increase the RNAi effect of a
molecule(s) or a composition on a selected gene or genes and a
second active component comprising at least one RNA molecule of
which at least a portion is a double-stranded RNA (dsRNA)
molecule.
40. The composition of claim 39 wherein the at least one RNA
molecule in the second active compound is as recited in claim
28.
41. The composition of claim 20, wherein the first active component
is present in an amount of 0.000001% to 99% by weight of the
composition (W/W), preferably 0.00001% to 99% by weight (W/W), more
preferably, 0.0001% to 99% by weight (W/W), still more preferably
0.0002% to 99% by weight (W/W).
42. The composition of claim 20, wherein the second active
component is present in an amount of 0.0000000001% to 99% by weight
(W/W) of the composition, preferably 0.000000001% to 99% by weight
(W/W), more preferably 0.00000001% to 99% by weight (W/W).
43. A kit comprising a first container containing an amount of a
first active component comprising one or more sulfated
polysaccharides and/or glycosaminoglycans as recited in claim 20,
wherein the amount of one or more sulfated polysaccharides and/or
glycosaminoglycans is effective to increase the RNAi effect of a
molecule(s) or a composition, and a second container containing a
second active component comprising a molecule(s) or a composition
that reduces expression of a selected gene or genes by RNA
interference (RNAi) in a target cell or target organism as recited
in claim 20.
42. A method for making a RNAi composition comprising combining a
first active component comprising an amount of the one or more
sulfated polysaccharides and/or glycosaminoglycans as recited in
claim 20, wherein the amount of one or more sulfated
polysaccharides and/or glycosaminoglycans is effective to increase
the RNAi effect of a molecule(s) or a composition on a selected
gene or genes, and a second active component comprising a
molecule(s) or a composition that reduces expression of a selected
gene or genes by RNA interference (RNAi) in a cell or organism as
recited in claim 20, to make a RNAi composition.
43. A medicament comprising an effective amount of: (1) a first
active component comprising an amount of one or more sulfated
polysaccharides and/or glycosaminoglycans that is effective to
enhance the RNAi effect of a molecule(s) or a composition, wherein
said first active compound is recited claim 20, and (2) a second
active component comprising a molecule(s) or a composition that
reduces expression of a selected gene or genes by RNA interference,
wherein said second active compound is recited in claim 20.
44. The medicament of claim 43 which is formulated for topical,
parenteral, enteral, transdermal, cutaneous, subcutaneous,
intravenous, intraperitoneal, intramuscular or oral administration.
Description
RELATED APPLICATIONS
[0001] This divisional application claims the benefit under 35
U.S.C. .sctn.119(e) of nonprovisional application Ser. No.
12/389,635, which claims the benefit of U.S. provisional
application 61/030,029, filed Feb. 20, 2008, U.S. provisional
application 61/087,315, filed Aug. 8, 2008, and U.S. provisional
application 61/118,105, filed Nov. 26, 2008, the entire disclosures
of which are incorporated herein by reference.
STATEMENT REGARDING ELECTRONIC SUBMISSION OF A SEQUENCE LISTING
[0002] A Sequence Listing in ASCII text format, submitted under 37
C.F.R. .sctn.1.821, entitled "80366USRNAi_ST25.txt", 11 KB in size,
generated on Feb. 20, 2009, and filed via EFS-Web is provided in
lieu of a paper copy. This Sequence Listing is hereby incorporated
by reference into the specification for its disclosures.
FIELD OF THE INVENTION
[0003] The present invention relates to various compositions
containing a combination of (1) one or more sulfated
polysaccharides and/or glycosaminoglycans and (2) one or more
RNAi-inducing molecules, and methods for using these compositions
to enhance double-stranded RNA (dsRNA)-mediated gene silencing in
pest and/or pathogen species.
BACKGROUND OF THE INVENTION
[0004] Over the last few years, reduction of gene expression (also
referred to as "knockdown" or "gene silencing") in multicellular
organisms by means of RNA interference or "RNAi" has become a
well-established technique. RNAi is a process of sequence-specific
knockdown of gene expression initiated by double-stranded RNA
(dsRNA) that is complementary in sequence to a region of the target
gene to be knocked down (Fire, A. Trends Genet. Vol. 15, 358-363,
1999; Sharp, P. A. Genes Dev. Vol. 15, 485-490, 2001). Reference
may also be made to International applications WO 99/32619
(Carnegie Institution) and WO 00/01846 (Devgen NV), and U.S. Pat.
No. 6,506,559.
[0005] Gene silencing by dsRNA finds application in many different
areas, such as dsRNA-mediated gene silencing in clinical
applications (WO 2004/001013). The technique of RNAi has also been
used to knock down gene expression in pests, including insects.
Other published applications that relate to the use of RNAi to
protect plants against insects include the International
applications WO 2006/046148A2 (Devgen NV); WO 2006/045591A2 (Devgen
NV); WO 2006/045590A2 (Devgen NV); WO 2006129204 (Devgen NV); WO
2001/37654 (DNA Plant Technologies), WO 2005/019408 (Bar Ilan
University), WO 2005/049841 (CSIRO, Bayer Cropscience), WO
2005/047300 (University of Utah Research foundation), WO
2005/110068 (Monsanto), WO2007/035650 (Monsanto), WO2007/083193
(Devgen NV), WO2007/074405 (Devgen NV), WO2007/080127 (Devgen NV),
WO2007/080126 (Devgen NV) and the US published application
2003/00150017 (Mesa et al.).
SUMMARY OF THE INVENTION
[0006] To date, no methods of pest and/or pathogen control have
been described which utilize compounds or compositions that further
increase the RNA interference (RNAi) effect of dsRNA, further
reduce expression of a selected gene or genes, and thereby further
increase the mortality of pests and/or pathogens. In addition, no
such methods have been described which utilize compounds or
compositions that further increase the RNA interference (RNAi)
effect of dsRNA that is expressed by microorganisms such as
bacteria. Such methods and compositions would be of benefit by
either reducing the amount of dsRNA that is necessary to control
pests and/or pathogens, or by increasing the lethality of any given
quantity of dsRNA, or by shortening the period to kill the pest
and/or pathogen after application of the dsRNA.
[0007] In the research leading to identification of RNAi receptors,
the present inventors found that certain molecules enhanced the
RNAi effect whilst they were searching for molecules that would
prevent RNAi. Scavenger receptors have been shown to be implicated
in target dsRNA uptake and initiation of RNA interference. Certain
polyanions and sulfated polysaccharides are antagonists of
scavenger receptors and have been reported to prevent RNA
interference when administered in the presence of target dsRNA
(Saleh et al., 2006). In addition, the complex branched sulfated
polysaccharide dextran sulfate specifically inhibited poly I:C
dsRNA activation of intracellular signaling required for cytokine
and chemokine secretion via scavenger receptor type SR-A (Limmon et
al., 2007). Based on these reports the inventors tested whether
scavenger receptor antagonists could prevent target dsRNA from
setting off the RNAi process in insecta and thereby demonstrate
that these receptors play a pivotal role in initiation of RNA
interference. Quite surprisingly, ingestion of target dsRNA in the
presence of the sulfated polysaccharide dextran sulfate led to
substantially increased lethality when compared to target dsRNA
alone. Contrary to the expectations, sulfated polysaccharides, such
as dextran sulfate, substantially increased the RNAi-induced
lethality of target dsRNA (i.e., insect mortality) when the two
components were coadministered in insect feeding bioassays.
Additionally, ingestion of target dsRNA in the presence of
glycosaminoglycans, such as heparin, also unexpectedly increased
insect mortality when compared to target dsRNA alone.
[0008] While pest- and/or pathogen-specific target double-stranded
RNA molecules (dsRNA), once ingested, have been shown to be lethal
by RNA interference (RNAi) to certain pests, the present inventors
have now developed methods by which the RNAi-induced lethality of
dsRNA and lethality in pests and/or pathogens can be substantially
increased by feeding target dsRNA to the pests and/or pathogens in
the presence of enhancing molecules or compounds. These methods
comprise the feeding to pests and/or pathogens of such enhancing
molecules or compounds either before, during, or after the feeding
to pests and/or pathogens of either target dsRNA or microorganisms,
such as bacteria, which express dsRNA.
[0009] This increase in the RNAi-induced mortality in pests and/or
pathogens was a surprisingly effective result of feeding both
enhancing molecules or compounds and dsRNA or microorganisms which
express dsRNA to pest and/or pathogens. Although sulfated compounds
have been used to mobilize polynucleotides into cells (e.g. for
transfection of cells with nucleic acids), the compositions of the
present invention (1) increased the effectiveness of RNAi in pests
and/or pathogens, (2) and/or increased the lethality of dsRNA
toward pests and/or pathogens, (3) and/or increased the yields of
crops that were susceptible to pests and/or pathogens. Furthermore,
the above-mentioned results were achieved, unexpectedly, despite
the simplicity of the methods of the invention, in which the
compositions comprising the enhancing compounds and either target
dsRNA or microorganisms expressing such dsRNA are merely sprayed on
or near the crops of interest. Moreover, the dsRNA in the
composition is not complexed with the enhancing molecules.
[0010] The present invention provides a method for substantially
increasing the efficiency of controlling pest infestation and/or
pathogen infection by RNA interference via contacting an organism
or cell, or administering to an organism (e.g. by ingestion),
target dsRNA in the presence of enhancing molecules or compounds.
The target dsRNA can be in the form of nucleic acid molecules or
prokaryotic or eukaryotic cells, such as bacteria or other
microorganisms, that express the target dsRNA.
[0011] One aspect of the invention includes methods for increasing
the RNA interference (RNAi) effect of a molecule or composition
that reduces expression of a selected gene or genes by RNA
interference in cell(s) or an organism. The methods include
contacting the cell(s) or organism with (1) a first active
component including an amount of one or more sulfated
polysaccharides and/or glycosaminoglycans that is effective to
enhance the RNAi effect of the molecules or composition and (2) a
second active component including molecules or a composition that
reduces expression of a selected gene or genes by RNA interference.
In some embodiments of the foregoing methods the cell(s) or
organism is contacted with the first active component before or
after contacting the cell(s) or organism with the second active
component. In further embodiments of the foregoing methods the cell
or organism is contacted with the first active component
simultaneously with contacting the cell(s) or organism with the
second active component. In yet other embodiments of the foregoing
methods the cell or organism is contacted with the first active
component before, simultaneously with and/or after contacting the
cell(s) or organism with the second active component. "Contacting"
includes applying or spraying the second active component and the
effective amount of the first active component onto the cell(s) or
organism or on food for the organism. In embodiments of the
foregoing methods the first active component and the second active
component are independently applied or the first active component
and the second active component are combined in a composition as
described herein.
[0012] In another aspect of the invention, methods for protecting
plants or foodstuffs or substances from pest infestation or
pathogen infection are provided. The methods include contacting the
plant or soil wherein the plant is rooted, or foodstuff or
substance with (1) a first active component including an amount of
one or more sulfated polysaccharides and/or glycosaminoglycans that
is effective to enhance the RNAi effect of the molecules or
composition and (2) a second active component comprising molecules
or a composition that reduces expression of a selected gene or
genes by RNA interference. In embodiments of the foregoing methods,
the plant or soil wherein the plant is rooted, or foodstuff or
substance is contacted with the first active component before or
after contacting the plant or soil wherein the plant is rooted, or
foodstuff or substance with the second active component. In further
embodiments of the foregoing methods, the plant or soil wherein the
plant is rooted, or foodstuff or substance is contacted with the
first active component simultaneously with contacting the plant or
soil wherein the plant is rooted, or foodstuff or substance with
the second active component. In yet other embodiments of the
foregoing methods, the plant or soil wherein the plant is rooted,
or foodstuff or substance is contacted with the first active
component before, simultaneously with and/or after contacting the
plant or soil wherein the plant is rooted, or foodstuff or
substance with the second active component. "Contacting" includes
applying or spraying the second active component and the effective
amount of the first active component onto the plant or foodstuff or
substance. In embodiments of the foregoing methods the first active
component and the second active component are independently applied
or the first active component and the second active component are
combined in a composition as described herein.
[0013] According to another aspect of the invention, methods for
treating a plant infection by a pathogen or plant infestation by a
pest are provided. The methods include contacting the plant or the
soil wherein the plant is rooted with (1) a first active component
including an amount of one or more sulfated polysaccharides and/or
glycosaminoglycans that is effective to enhance the RNAi effect of
the molecules or composition and (2) a second active component
including molecules or a composition that reduces expression of a
selected gene or genes by RNA interference. In embodiments of the
foregoing methods, the plant or the soil wherein the plant is
rooted is contacted with the first active component before or after
contacting the plant with the second active component. In other
embodiments of the foregoing methods, the plant or the soil wherein
the plant is rooted is contacted with the first active component
simultaneously with contacting the plant or soil wherein the plant
is rooted, with the second active component. In further embodiments
of the foregoing methods, the plant or the soil wherein the plant
is rooted is contacted with the first active component before,
simultaneously with and/or after contacting the plant or soil
wherein the plant is rooted, with the second active component.
"Contacting" includes applying or spraying the second active
component and the effective amount of the first active component
onto the plant. In embodiments of the foregoing methods, the first
active component and the second active component are independently
applied or the first active component and the second active
component are combined in a composition as described herein.
[0014] According to a further aspect of the invention, methods are
provided for increasing crop yield or reducing a decline in crop
yield that results from pest infestation and/or pathogen infection,
comprising contacting a plant or soil surrounding a plant with a
composition. Such compositions include a first active component
comprising an amount of one or more sulfated polysaccharides and/or
glycosaminoglycans, wherein the amount of one or more sulfated
polysaccharides and/or glycosaminoglycans is effective to increase
the RNAi effect of a molecule(s) or a composition on a selected
gene or genes and a second active component comprising one or more
prokaryotic cells or eukaryotic cells or host organisms that
express double-stranded RNA molecule(s) that reduce (the)
expression of a selected gene or genes by RNA interference (RNAi)
in a target cell or target organism. Both the increases in crop
yields and the reductions in the decline of crop yields are
established from comparisons of yields of crops which have been
treated with the compositions described herein relative to the
yields of crops which have not been treated with the compositions
and methods described herein. Both the increases in crop yields and
the reductions in the decline of crop yields are also established
from comparisons of yields of crops rooted in soils which have been
treated with the compositions and methods described herein relative
to the yields of crops rooted in soils which have not been treated
with the compositions and methods described herein. Increases in
crop yields can range between 1% and 100%, including 1%, 2%, 3%,
4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%,
18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%,
31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%,
44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%,
57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,
70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99%, 100%, 120%, 140%, 160%, 180%, 200%, 220%, 240%,
260%, 280%, 300%, 320%, 340%, 360%, 380%, 400%, 420%, 440%, 460%,
480%, 500%, 520%, 540%, 560%, 580%, 600%, 620%, 640%, 660%, 680%,
700%, 720%, 740%, 760%, 780%, 800%, 820%, 840%, 860%, 880%, 900%,
920%, 940%, 960%, 980%, and 1000%. Reductions in the decline of
crop yields can range between 1% and 100%, including 1%, 2%, 3%,
4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%,
18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%,
31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%,
44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%,
57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,
70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99%, and 100%.
[0015] Another aspect of the invention provides methods for
protecting a human or an animal from pest infestation or pathogen
infection or methods for treating a pest infestation or pathogen
infection of a human or an animal. The methods include
administering to the human or animal an effective amount of (1) a
first active component comprising an amount of one or more sulfated
polysaccharides and/or glycosaminoglycans that is effective to
enhance the RNAi effect of the molecules or composition and (2) a
second active component including molecules or a composition that
reduces expression of a selected gene or genes by RNA interference.
In embodiments of the foregoing methods, the human or animal is
administered the first active component before or after
administering the second active component to the human or animal.
In other embodiments of the foregoing methods, the human or animal
is administered the first active component simultaneously with
administering the second active component to the human or animal.
In further embodiments of the foregoing methods, the human or
animal is administered the first active component before,
simultaneously with and/or after administering the second active
component to the human or animal. In other embodiments of the
foregoing methods, the administering includes topical, parenteral,
enteral, transdermal, cutaneous, subcutaneous, intravenous,
intraperitoneal, intramuscular or oral administration. In
embodiments of the foregoing methods the first active component and
the second active component are independently applied or the first
active component and the second active component are combined in a
composition as described herein.
[0016] In another aspect of the invention, methods for the
preparation of a medicament for preventing or treating a pest
infestation or pathogen infection of a human or an animal are
provided, these medicaments comprising an effective amount of (1) a
first active component comprising an amount of one or more sulfated
polysaccharides and/or glycosaminoglycans that is effective to
enhance the RNAi effect of the molecules or composition and (2) a
second active component comprising molecules or a composition that
reduces expression of a selected gene or genes by RNA
interference.
[0017] According to another embodiment, medicaments are provided
comprising an effective amount of (1) a first active component
comprising an amount of one or more sulfated polysaccharides and/or
glycosaminoglycans that is effective to enhance the RNAi effect of
the molecules or composition, and (2) a second active component
comprising molecules or a composition that reduces expression of a
selected gene or genes by RNA interference. In embodiments of the
foregoing is the medicament formulated for topical, parenteral,
enteral, transdermal, cutaneous, subcutaneous, intravenous,
intraperitoneal, intramuscular or oral administration.
[0018] According to another aspect of the invention, compositions
are provided. The compositions include a first active component
including an amount of one or more sulfated polysaccharides and/or
glycosaminoglycans, wherein the amount of one or more sulfated
polysaccharides and/or glycosaminoglycans is effective to increase
the RNAi effect of a molecule(s) or a composition on a selected
gene or genes; and a second active component comprising (i) at
least one RNA molecule of which at least a portion is a double
stranded RNA (dsRNA) molecule; or (ii) one or more prokaryotic
cells or eukaryotic cells or host organisms that express
double-stranded RNA molecule(s) that reduce expression of a
selected gene or genes by RNA interference (RNAi) in a pest and/or
pathogen cell or organism.
[0019] In some embodiments, the first active component is one or
more sulfated polysaccharides or one or more glycosaminoglycans or
a combination thereof. In preferred embodiments, the first active
component is dextran sulfate, or fucoidan, or heparin. More
preferably, the dextran sulfate has an average molecular weight of
about between 8 and 40 kilodaltons.
[0020] In other embodiments of the foregoing compositions, the
second active component expresses at least one RNA molecule of
which at least a portion is double-stranded (dsRNA) or is a
double-stranded RNA molecule. Preferably, the dsRNA molecules are
dsRNA molecules consisting of two separate strands, siRNA molecules
or hairpin molecules. In still other embodiments of the foregoing
compositions, the one or more prokaryotic or eukaryotic cells of
the second active component comprise an expression vector that
expresses at least one RNA molecule of which at least a portion is
double-stranded. Preferably the expression vector includes at least
one regulatory sequence operably linked to the nucleotide sequence
encoding the at least one RNA molecule and which nucleotide
sequence is complementary to at least part of the nucleotide
sequence of a selected gene or genes to be knocked down.
[0021] In yet other embodiments of the foregoing compositions, the
second active component includes one or more prokaryotic cells or
eukaryotic cells or host organisms that express the double-stranded
RNA. Preferably the prokaryotic cell is a bacterial cell.
Preferably the bacterial cell is an Escherichia coli (E. coli)
cell. Preferably the eukaryotic cell is a yeast cell.
[0022] In yet other embodiments of the foregoing compositions, at
least one strand of the dsRNA includes a nucleotide sequence that
is complementary to a portion of the nucleotide sequence of a
selected gene or genes from the pest and/or pathogen cell or
organism.
[0023] In some embodiments of the foregoing compositions, the gene
or genes is/are essential for the viability, growth, development or
reproduction of the pest and/or pathogen cell or organism.
Preferably, the gene or genes in the second active component
comprise at least one nucleic acid comprising (i) the sequence of
any of SEQ ID NOs 1 to 6, (ii) the rNA equivalent thereof, (iii)
the complement of (i) or (ii), or (iv) a fragment of any of (i) to
(iii) consisting of at least 17 contiguous nucleotides of any of
(i) to (iii).
[0024] In some embodiments of the foregoing compositions, the pest
and/or pathogen cells or organisms are pest species, pathogen
species, or cells thereof. Preferably, the pest or pathogen is a
fungus, an insect or a nematode.
[0025] In some embodiments of the foregoing compositions, the first
active component is present in an amount of 0.000001%-99% by weight
of the composition (W/W), or preferably 0.00001%-99% by weight
(W/W), or more preferably, 0.0001%-99% by weight (W/W), or still
more preferably 0.0002%-99% by weight (W/W). In some embodiments of
the foregoing compositions, the second active component is present
in an amount of 0.0000000001%-99% by weight (W/W) of the
composition, or preferably 0.000000001%-99% by weight (W/W), or
more preferably 0.00000001%-99% by weight (W/W).
[0026] In additional embodiments, the foregoing compositions
consist essentially of the first and second active components as
described above.
[0027] According to another aspect of the invention, kits are
provided. The kits include a first container containing an amount
of a first active component including one or more sulfated
polysaccharides and/or glycosaminoglycans as provided in any of the
foregoing compositions, and a second container including a second
active component comprising a molecule(s) or a composition that
reduces expression of a selected gene or genes by RNA interference
(RNAi) in a pest and/or pathogen cell or organism as provided in
any of the foregoing compositions. The amount of one or more
sulfated polysaccharides and/or glycosaminoglycans is effective to
increase the RNAi effect of the molecule(s) or composition.
[0028] According to another aspect of the invention, methods for
making a RNAi composition are provided. The methods include
combining a first active component including an amount of the one
or more sulfated polysaccharides and/or glycosaminoglycans as
provided in any of the foregoing compositions, and a second active
component including a molecule(s) or a composition that reduces
expression of a selected gene or genes by RNA interference (RNAi)
in a pest and/or pathogen cell or organism as provided in any of
the foregoing compositions, to make a RNAi composition. The amount
of one or more sulfated polysaccharides and/or glycosaminoglycans
is effective to increase the RNAi effect of the molecule(s) or
composition on the selected gene or genes. These and other aspects
of the invention will be described in greater detail below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] FIG. 1 shows survival of L. decemlineata larvae on
artificial diet treated with target Ld105 double-stranded RNA (SEQ
ID NO:1) in the presence of varying amounts of dextran sulfate
(Mw.+-.8 kDa). On the artificial diet, each larva was exposed to
water only, 50 ng target Ld105 dsRNA only (designated as Ld105), 50
.mu.g, 5 .mu.g, 500 ng or 50 ng dextran sulfate in the absence
(designated as DS-Ld105) or presence (designated as DS+Ld105) of 50
ng target Ld105 dsRNA. Twenty-four larvae were tested per
condition. Number of survivors were assessed on 5, 7, 9, 12 and 14
days post infestation. Mortality versus treatment and survival
curves are shown.
[0030] FIG. 2 shows survival of L. decemlineata larvae on
artificial diet treated with target Ld105 double-stranded RNA (SEQ
ID NO:1) in the presence of varying amounts of fucoidan. On the
artificial diet, each larva was exposed to water only, 50 ng target
Ld105 dsRNA only (designated as Ld105), 50 .mu.g, 5 .mu.g, 500 ng
or 50 ng fucoidan in the absence (designated as Fucoidan-Ld105) or
presence (designated as Fucoidan+Ld105) of 50 ng target Ld105
dsRNA. Twenty-four larvae were tested per condition. Number of
survivors were assessed on 5, 6, 7, 8, 11 and 12 days post
infestation. Mortality versus treatment and survival curves are
shown.
[0031] FIG. 3 shows survival of L. decemlineata larvae on
artificial diet treated with target Ld105 double-stranded RNA (SEQ
ID NO:1) in the presence of varying amounts of the polyanion,
polyinosine. On the artificial diet, each larva was exposed to 0.9
M NaCl only (designated as NaCl), 50 ng target Ld105 dsRNA only
(designated as Ld105), 50 .mu.g, 5 .mu.g, 500 ng or 50 ng
polyinosine in the absence (designated as Poly(I)-Ld105) or
presence (designated as Poly(I)+Ld105) of 50 ng target Ld105 dsRNA.
Twenty-four larvae were tested per condition. Number of survivors
were assessed on 5, 6, 7, 8, 9 and 12 days post infestation.
Mortality versus treatment and survival curves are shown.
[0032] FIG. 4 shows survival of L. decemlineata larvae on
artificial diet treated with target Ld105 double-stranded RNA (SEQ
ID NO:1) at different amounts in the presence of dextran sulfate
(Mw.+-.8 kDa) at one quantity. On the artificial diet, each larva
was exposed to water only, 50 .mu.g of dextran sulfate only
(designated as DS), or 1 .mu.g to 1 pg in ten-fold serial dilutions
of target Ld105 dsRNA in the absence (designated as Ld105-DS) or
presence (designated as Ld105+DS) of 50 .mu.g dextran sulfate.
Twenty-four larvae were tested per condition. Number of survivors
were assessed on 5, 6, 7, 8, 11 and 12 days post infestation.
Mortality versus treatment and survival curves are shown (only
selected data represented in the latter figure).
[0033] FIG. 5 shows survival of L. decemlineata larvae on
artificial diet treated with target Ld105 double-stranded RNA (SEQ
ID NO:1) in the presence of varying amounts of dextran sulfate
(Mw.+-.1400 kDa). On the artificial diet, each larva was exposed to
water only, 50 ng target Ld105 dsRNA only (designated as Ld105), 50
.mu.g, 5 .mu.g, 500 ng or 50 ng dextran sulfate in the absence
(designated as DS-Ld105) or presence (designated as DS+Ld105) of 50
ng target Ld105 dsRNA. Twenty-four larvae were tested per
condition. Number of survivors were assessed on 5, 6, 7, 8, 11 and
12 days post infestation. Mortality versus treatment and survival
curves are shown.
[0034] FIG. 6 shows survival of L. decemlineata larvae on
artificial diet treated with target Ld105 double-stranded RNA (SEQ
ID NO:1) in the presence of varying amounts of heparin. On the
artificial diet, each larva was exposed to water only, 50 ng target
Ld105 dsRNA only (designated as Ld105), 50 .mu.g, 5 .mu.g, 500 ng
or 50 ng heparin in the absence (designated as heparin-Ld105) or
presence (designated as DS+Ld105) of 50 ng target Ld105 dsRNA.
Twenty-four larvae were tested per condition. Number of survivors
were assessed on 5, 6, 7, 10, 11 and 13 days post infestation.
Mortality versus treatment and survival curves are shown.
[0035] FIG. 7 shows survival curves of L. decemlineata larvae on
artificial diet with target Ld105 double-stranded RNA (SEQ ID NO:1)
in the presence of chondroitin sulfate or hyaluronic acid. On the
artificial diet, each larva was exposed to water only, 50 ng Ld105
dsRNA in the absence or presence of 50 .mu.g chondroitin sulfate
(CS) or hyaluronic acid (HA). Twenty-four larvae were tested per
condition. Numbers of survivors were assessed regularly for up to
13 days in the bioassay.
[0036] FIG. 8 shows survival curves of L. decemlineata larvae on
artificial diet treated with E. coli-expressed Ld105
double-stranded RNA (SEQ ID NO:1) at different amounts in the
presence of dextran sulfate (Mw.+-.8 kDa) at one quantity. On the
diet, each larva was exposed to 0.25 U pGN29 control, 0.25 U
pGBNJ003, 0.08 U pGBNJ003 or 0.027 U pGBNJ003 with or without 50
.mu.g dextran sulfate (DS). One unit (U) corresponds to the amount
of bacteria present in 1 mL culture with an OD600 nm value of 1
prior to heat-inactivation. Sixteen larvae were tested per
condition. Numbers of survivors were assessed regularly for up to
12 days in the bioassay.
[0037] FIG. 9 shows survival of L. decemlineata larvae on
artificial diet treated with different target double-stranded RNAs
(SEQ ID NO:1, 2, 3, 4; Ld105 dsRNA=SEQ ID NO:1, Ld013=SEQ ID NO:2,
Ld009=SEQ ID NO:3 and Ld248=SEQ ID NO:4) at one quantity (100 ng)
in the presence of dextran sulfate (DS; .+-.8 kDa) at once quantity
(50 .mu.g). On the artificial diet, each larva was exposed to water
only or water plus dextran sulfate, gfp dsRNA only or gfp dsRNA
plus dextran sulfate, Ld009 dsRNA only or Ld009 dsRNA plus dextran
sulfate, Ld013 dsRNA only or Ld013 dsRNA plus dextran sulfate,
Ld105 dsRNA only or Ld105 dsRNA plus dextran sulfate, Ld 248 dsRNA
only or Ld248 dsRNA plus dextran sulfate. Twenty-four larvae were
tested per condition. Number of survivors were assessed on 5, 6, 7,
11, 12, 13, 14, 15 and 18 days post infestation. Survival curves
for each target dsRNA shown.
[0038] FIG. 10 shows survival of L. decemlineata larvae on potato
leaf discs treated with target Ld105 double-stranded RNA (SEQ ID
NO:1) at different amounts in the presence of dextran sulfate
(Mw.+-.8 kDa) at one quantity. Each larva was first exposed to leaf
discs treated with water only, 50 .mu.g dextran sulfate only (DS),
10 ng Ld105 dsRNA only, 100 ng Ld105 dsRNA only, 10 ng Ld105 dsRNA
plus 50 .mu.g dextran sulfate, or 100 ng Ld105 dsRNA plus 50 .mu.g
dextran sulfate. Twenty-four larvae were tested per condition.
After 24 hours, each larva was transferred to untreated leaf discs.
This was repeated once before transferring them each to a well
containing artificial diet. Number of survivors were assessed on 2,
3, 6, 7, 8, 9 and 13 days post infestation (starting on treated
leaf discs). Survival curves are shown.
[0039] FIG. 11 shows survival of L. decemlineata larvae on
artificial diet in a sequential feeding assay with dextran sulfate
(DS; .+-.8 kDa) and target Ld105 double-stranded RNA (SEQ ID NO:1).
First, each larva was exposed to artificial diet topically applied
with either dextran sulfate or water only. After two full days of
feeding, each larva was then transferred to fresh artificial diet
topically applied with target Ld105 dsRNA, dextran sulfate or water
only. Thus the treatments were with first component/second
component: water/water, DS/DS, water/dsRNA, and DS/dsRNA. Amount of
dextran sulfate used was 50 .mu.g and target Ld105 dsRNA 1 .mu.g.
Twenty-four larvae were tested per condition. Number of survivors
were assessed on 5, 6, 7, 8, 9, 12, 14 and 15 days post
infestation. Survival curves are shown.
[0040] FIG. 12 shows survival of P. cochleariae larvae on oilseed
rape leaf discs treated with target Pc105 double-stranded RNA (SEQ
ID NO:5) at one quantity (50 ng) in the presence of dextran sulfate
(Mw.+-.8 kDa) at one quantity (50 ng). Each larva was first exposed
to leaf discs treated with 25 .mu.A of 0.05% Triton X-100 solution
with water only (designated water), dextran sulfate only
(designated DS), Pc105 dsRNA only or Pc105 dsRNA in combination
with dextran sulfate. Thirty larvae were tested per condition. Once
the treated leaf disc was consumed by each larva, the larvae were
transferred to untreated leaf discs. This was repeated until the
end of the bioassay. Number of survivors were assessed on 4, 5, 6
and 7 days post infestation. Survival curves are shown.
[0041] FIG. 13 shows survival and developmental stages of C.
elegans soaked in target rps-14 double-stranded RNA (SEQ ID NO:6)
at one concentration (50 ng/.mu.l) in the presence of different
concentrations of dextran sulfate (Mw.+-.8 kDa). Percentage of
survival numbers and developmental stages of the nematodes were
assessed. AD: adults; L4: fourth larval stage; L3: third larval
stage; dead: non-living and/or missing nematodes calculated by
subtracting total numbers of L3+L4+AD from the starting count.
DETAILED DESCRIPTION OF THE INVENTION
[0042] The invention provides compositions and methods based on the
discovery that certain sulfated polysaccharides and
glycosaminoglycans increase the RNAi effect of target dsRNA.
Therefore, the invention includes compositions containing two
"active components" including (1) one or more sulfated
polysaccharides and/or glycosaminoglycans and (2) one or more
RNAi-inducing double-stranded RNA (dsRNA) molecules. The present
invention also includes methods for increasing RNA interference by
the application of (1) one or more sulfated polysaccharides and/or
glycosaminoglycans and (2) one or more RNAi-inducing
double-stranded RNA (dsRNA) molecules. In certain embodiments, the
methods for increasing RNA interference-induced effects include
increasing dsRNA-mediated gene silencing in particular for the
control of pests and/or pathogens, including insects, nematodes,
and fungi, in particular for the protection of plants from
infestation and/or damage by such pests and/or pathogens.
[0043] As indicated, compositions of the invention contain two
"active components". The "first active component" is one or more
sulfated polysaccharides and/or glycosaminoglycans and the "second
active component" is one or more nucleic acid molecules that
induces RNA interference, as is well known in the art, and in
particular embodiments is one or more RNA molecules of which at
least a portion is double-stranded. The sequence of one of the
strands of the dsRNA corresponds to part or whole of an essential
pest and/or pathogen gene and causes knockdown of this pest and/or
pathogen gene expression via RNA interference. As a result of mRNA
knockdown the dsRNA reduces or prevents expression of the target
pest and/or pathogen protein and, hence, causes death, growth
arrest or sterility of the pest and/or pathogen.
[0044] The dsRNA-induced RNA interference is rendered more
effective when the dsRNA, is coadministered in the presence of one
or more various sulfated polysaccharides and/or glycosaminoglycans.
In which the presence of sulfated polysaccharides and/or
glycosaminoglycans, the effectiveness of dsRNA increases such that
a lesser amount can be used to knock down gene expression in a pest
and/or pathogen. For example, in the context of protecting plants
against insects and other plant pests and/or pathogens, the use of
sulfated polysaccharides and/or glycosaminoglycans in combination
with dsRNA increases the effectiveness of the dsRNA and/or
increases the speed of killing or disabling the plant pests and/or
pathogens.
[0045] The methods of the invention can find practical applications
in any area of technology where it is desirable to inhibit
viability, growth, development or reproduction of a pest, or to
decrease pathogenicity or infectivity of a pathogen. The methods of
the invention further find practical applications where it is
desirable to specifically knock down expression of one or more
genes in a pest and/or pathogen. Particularly useful practical
applications include, but are not limited to, (1) protecting plants
against pest infestation or pathogen infection; (2) pharmaceutical
or veterinary use in humans and animals (for example, to control,
treat or prevent pest or pathogen infections in humans and
animals); (3) protecting materials against damage caused by pests
or pathogens; (4) protecting perishable materials (such as
foodstuffs, seed, etc.) against damage caused by pests or
pathogens; (5) generally any application wherein pests or pathogens
need to be controlled and/or wherein damage caused by pests or
pathogens needs to be reduced or prevented; (6) reducing the amount
of dsRNA that is necessary to induce RNAi due to the added
effectiveness of compositions comprising both active components,
the target dsRNA and one or several sulfated polysaccharides and/or
glycosaminoglycans; (7) increasing the
speed-to-RNAi-induced-lethality of pest(s) or pathogen(s) utilizing
a composition comprising both active components, the target dsRNA
and one or more sulfated polysaccharides and/or glycosaminoglycans
(8) reducing the time-to-kill of pest(s) or pathogen(s) utilizing a
composition comprising both active components, the target dsRNA and
one or more sulfated polysaccharides and/or glycosaminoglycans.
[0046] Compositions of the invention include two "active
components": (1) one or more sulfated polysaccharides and/or
glycosaminoglycans, such as but not limited to dextran sulfate,
fucoidan or heparin, and (2) one or more molecules or a composition
that reduce expression of a selected gene or genes by RNA
interference in a cell or organism. The amount of one or more
sulfated polysaccharides and/or glycosaminoglycans is present in an
amount that is effective to increase the RNAi effect of the
molecule(s) or composition on the selected gene or genes.
[0047] The "first active component" of the invention comprises one
or more sulfated polysaccharides and/or glycosaminoglycans.
"Sulfated polysaccharides" as used in the application are
relatively complex carbohydrates containing up to 30% sulfur by
weight. Polysaccharides are polymers made up of many
monosaccharides joined together by glycosidic bonds. They are often
branched, and therefore large macromolecules. Polysaccharides have
a general formula of C.sub.n(H.sub.2O).sub.n-1 where n is usually a
large number between 200 and 2500. Considering that the repeating
units in the polymer backbone are often six-carbon monosaccharides,
the general formula can also be represented as
(C.sub.6H.sub.10O.sub.5).sub.n where n={40 . . . 3000}.
[0048] One example of sulfated polysaccharides are "dextran
sulfates", which are long-chain polymers of glucose containing
approximately 10-40% of sulfur. Preferably the dextran sulfates
contain approximately 17-20% of sulfur, many of which are
commercially available. Low molecular weight dextran sulfates have
a molecular weight ranging from about 5000 to about 10000 daltons
whereas high molecular weight dextran sulfates can go to 2000
kilodaltons (kDa). As illustrated later on, any of the molecular
weight variants of dextran sulfate can be used in the methods and
compositions of the invention. Another example of a sulfated
polysaccharide is fucoidan, which consists in two distinct forms:
F-fucoidan, which is >95% composed of sulfated esters of fucose,
and U-fucoidan, which is approximately 20% glucuronic acid.
[0049] "Glycosaminoglycans" (also known as GAGs or
mucopolysaccharides) belong to the family of polysaccharides and
are long unbranched polysaccharides consisting of a repeating
disaccharide unit. This unit consists of N-acetyl-hexosamine and a
hexose or hexuronic acid, either or both of which may be sulfated.
As used herein, the term "sulfated glycosaminoglycans" means
glycosaminoglycans that are sulfated. The family of sulfated
glucosaminoglycans, therefore, overlaps with the family of sulfated
polysaccharides. Heparin is a highly-sulfated glycosaminoglycan
consisting of a variably-sulfated repeating disaccharide unit
having a molecular weight ranging from about 3 kDa to about 40 kDa.
Hyaluronan is the only GAG that is exclusively not sulfated.
Average molecular weights of the sulfated glycosaminoglycans can be
any known in the art. Preferably the average molecular weight of
glycosaminoglycans is between about 500 daltons and about 3000
kilodaltons.
[0050] Some of these compounds, such as dextrane sulfate and
fucoidan are known as complexing agents for complexing nucleic
acids upon transfection of cells. Surprisingly, these compounds are
now demonstrated to enhance the RNA interference effect when they
are fed in combination to pest organisms such as insects and
nematodes. Moreover, the ingestion of dsRNA in the presence of
sulfated polysaccharides and/or glycosaminoglycans reaches the
appropriate target sites to initiate the systemic RNAi effect
resulting in increased and/or more efficient lethality of the pest
organism.
[0051] Sulfated polysaccharides and/or glycosaminoglycans may be
obtained from natural sources, such as from naturally occurring
organisms, recombinant organisms, synthetic methods, or any
combination thereof.
[0052] In preferred embodiments, the "second active component" of
the invention comprises RNA molecules of which at least a portion
is double-stranded. Examples of double-stranded RNA molecules are
small interfering RNA (siRNA) molecules, hairpin RNA (hpRNA)
molecules, etc. Other examples are well known in the art. The
expression "at least part" or "at least a portion" as used herein
means for instance over at least 15, 16, 17, 18, 19, 20, 21, 22,
23, 24 or more contiguous nucleotides, preferably at least 30, 50,
100, or at least 150 contiguous nucleotides.
[0053] The RNA molecule that reduces expression of a selected gene
or genes by RNA interference effect in a target cell or target
organism may also be, or be produced by, an expression vector that
expresses a double-stranded RNA. Such RNA molecules may be provided
as cells that harbor the expression vector. Thus, the molecules or
composition can be one or more prokaryotic host cells (such as
bacterial cells), eukaryotic host cells (such as yeast cells) or
host organisms that express the double-stranded RNA. In such
embodiments, the double-stranded RNA typically is expressed from a
recombinant construct, which construct includes at least one
regulatory sequence operably linked to the nucleotide sequence
which is complementary to at least part of the nucleotide sequence
of a selected gene or genes to be knocked down. Such expression
vectors, cells and organisms are well known in the art, for
example, as described in WO 2001/088121 and WO 2000/001846, both of
Devgen NV.
[0054] In addition, any suitable double-stranded RNA fragment
capable of directing RNAi or RNA-mediated gene silencing or
inhibition of a pest and/or pathogen gene may be used in the
methods of the invention.
[0055] In more detailed terms, the invention provides for host
cells and/or RNA molecules comprising a nucleotide sequence that is
the RNA complement of or that represents the RNA equivalent of at
least part of the nucleotide sequence of a target gene from a
target pest and/or pathogen organism, as produced by transcription
of a nucleic acid molecule.
[0056] The term "complementary" relates to any of DNA-DNA
complementarity, DNA-RNA complementarity, and RNA-RNA
complementarity. In analogy herewith, the term "RNA complement" or
"RNA equivalent" substantially means that in the DNA sequence(s),
the base "T" may be replaced by the corresponding base "U" normally
present in ribonucleic acids.
[0057] In preferred embodiments as shown in the Examples below, the
RNA molecules used as the second active component or expressed by
any of the cell(s) therein, is any of SEQ ID NOs 1 to 6. For
instance, SEQ ID NO 1 is the partial sense strand of a gene that is
a GTPase activator of Leptinotarsa decemlineata (see also
WO2007/083193 from DEVGEN, NV). For application in the present
invention are included: all dsRNA molecules comprising (or being
complementary to) at least part of any of SEQ ID NOs 1 to 6, as
well as all partial sequences/fragments that show at least 80%
identity, more preferably at least 85% identity, still more
preferably at least 90% identity, still more preferably at least
95% identity, still more preferably at least 98% identity, and most
preferably at least 99% identity to any of SEQ ID NOs 1 to 6 (over
the length of the fragment). Percent identity can be calculated
using methods well known in the art, for example, as described in
Dufresne et al 2002 Nature Biotechnol. 20: 1269-1271. Also included
for use as the second active component are orthologous sequences of
other insects, nematodes or fungi (see also WO2007/083193 from
DEVGEN, NV). In addition, any of the sequences with SEQ ID NOs 1 to
2481, any complements or fragments thereof as described in the
WO2007/083193 publication from applicant can be used in the methods
and compositions described in the present application and included
herein by reference.
[0058] The "second active component" of the compositions and
methods of the invention may comprise isolated double-stranded RNA
comprising annealed complementary strands, one of which has a
nucleotide sequence which is complementary to at least part of a
target nucleotide sequence of a gene of a pest or pathogen. The
gene may be any of the (target) genes known in the art, or a part
thereof that exerts the same function. Preferred target sequences
are exemplified in WO2007/083193.
[0059] The double-stranded RNA comprises annealed complementary
strands, one of which has a nucleotide sequence which corresponds
to a target nucleotide sequence of the gene to be knocked down. The
other strand of the double-stranded RNA is able to base-pair, at
least in part, with the first strand.
[0060] The present invention relates to any gene of interest (which
may be referred to herein as the "target gene") that can be knocked
down.
[0061] The (target) gene(s) against which the "second active
component," or the dsRNA, is directed is/are selected as being
essential for the viability, growth, development or reproduction of
the cell or organism. The cell or organism that is targeted by the
RNA molecules of the invention preferably is a pest or pathogen
species. Pests or pathogens may be, for example, selected from
fungi, insects and nematodes.
[0062] The terms "knockdown of gene expression", "inhibition of
gene expression" and the like are used interchangeably and refer to
a measurable or observable reduction in gene expression or a
complete abolition of detectable gene expression, at the level of
protein product and/or mRNA product from the (target) gene.
Knockdown or inhibition of gene expression is "specific" when
knockdown or inhibition of the (target) gene occurs without
manifested effects on other genes of the targeted cell or
organism.
[0063] The term "knockdown of gene expression" implies reduced
expression of one or more genes of an organism due to the action of
a dsRNA such as a short DNA or RNA oligonucleotide with a sequence
complementary to a gene or its mRNA transcripts. During a gene
knockdown event, the binding of this dsRNA to the gene or its
transcripts causes decreased expression through blocking of
transcription.
[0064] Depending on the nature of the (target) gene, knockdown or
inhibition of gene expression in cells of an organism such as a
pest or pathogen can be confirmed by phenotypic analysis of a cell
or the whole pest or pathogen or by measurement of mRNA or protein
expression using molecular techniques such as RNA solution
hybridization, nuclease protection, Northern hybridization, reverse
transcription polymerase chain reaction, gene expression monitoring
with a microarray, antibody binding, enzyme-linked immunosorbent
assay (ELISA), Western blotting, radioimmunoassay (RIA), other
immunoassays, or fluorescence-activated cell analysis (FACS).
[0065] For targeting pests or pathogens, the "gene" or "target
gene" may be essentially any gene that it is desirable to be
inhibited because it interferes with growth or pathogenicity or
infectivity of the pest or pathogen. For instance, if the method of
the invention is to be used to prevent insect growth and/or
infestation then it is preferred to select a (target) gene which is
essential for viability, growth, development or reproduction of the
insect, or any gene that is involved with the insect's ability to
infest, such that specific inhibition of the (target) gene leads to
a lethal phenotype or decreases or stops insect infestation.
[0066] According to one non-limiting embodiment, the (target) gene
is such that when its expression is knocked down or inhibited using
the method of the invention, the pest or pathogen is killed, or the
reproduction or growth of the pest or pathogen is stopped or
retarded. This type of (target) gene is considered to be essential
for the viability of the pest or pathogen and is referred to as
essential genes. Therefore, the present invention encompasses a
method as described herein, wherein the (target) gene is an
essential gene.
[0067] According to a further non-limiting embodiment, the (target)
gene is such that when it is knocked down using the method of the
invention, the infestation or infection by the pest or pathogen,
the damage caused by the pest or pathogen, and/or the ability of
the pest or pathogen to infest or infect host organisms and/or
cause such damage, is reduced. The terms "infest" and "infect" or
"infestation" and "infection" are generally used interchangeably
throughout. This type of (target) gene is considered to be involved
in the pest's ability to infest or in the pathogenicity or
infectivity of the pathogen. Therefore, the present invention
extends to methods as described herein, wherein the (target) gene
is involved in the pest's ability to infest or in the pathogenicity
or infectivity of the pathogen. The advantage of choosing the
latter type of (target) gene is that the pest or pathogen is
blocked to infest or infect further organisms and to form further
generations.
[0068] According to one embodiment, (target) genes are conserved
genes, pest-specific genes or pathogen-specific genes.
[0069] The expression "target region" or "target nucleotide
sequence" of the pest/pathogen (target) gene may be any suitable
region or nucleotide sequence of the gene. The target region should
comprise at least 17, at least 18 or at least 19 consecutive
nucleotides of the (target) gene, more preferably at least 20 or at
least 21 nucleotide and still more preferably at least 22, 23 or 24
nucleotides of the (target) gene.
[0070] It is preferred that at least part of the double-stranded
RNA will share 100% sequence identity with the target region of the
pest or pathogen (target) gene. However, it will be appreciated
that 100% sequence identity over the whole length of the double
stranded RNA is not essential for functional RNA inhibition. RNA
sequences with insertions, deletions, and single point mutations
relative to the target sequence have also been found to be
effective for RNA inhibition. The terms "corresponding to" or
"complementary to" are used interchangeably herein, and when these
terms are used to refer to sequence correspondence between the
double-stranded RNA and the target region of the (target) gene,
they are to be interpreted accordingly, i.e. as not absolutely
requiring 100% sequence identity. However, the percent sequence
identity between the double-stranded RNA and the target region will
generally be at least 50% to 85% identical, preferably at least
90%, 95%, 96%, or more preferably at least 97%, 98% and still more
preferably at least 99%. One preferred way of calculating sequence
identity is as described in Dufresne et al 2002 Nature Biotechnol.
20: 1269-1271. Preferred sequences that are useful in accordance
with the invention to produce a dsRNA for use in the compositions
and methods described herein are any of SEQ ID NO 1 to 6, or any of
SEQ ID NOs 1 to 2481, any complements or fragments thereof as
described in the WO2007/083193.
[0071] Although the dsRNA contains a sequence which corresponds to
the target region of the (target) gene it is not absolutely
essential for the whole of the dsRNA to correspond to the sequence
of the target region. For example, the dsRNA may contain short
non-target regions flanking the target-specific sequence, provided
that such sequences do not affect performance of the dsRNA in RNA
inhibition to a material extent.
[0072] The dsRNA may contain one or more substitute bases in order
to optimize performance in RNAi. It will be apparent to the skilled
reader how to vary each of the bases of the dsRNA in turn and test
the activity of the resulting siRNAs (e.g. in a suitable in vitro
test system) in order to optimize the performance of a given
dsRNA.
[0073] The dsRNA may further contain DNA bases, non-natural bases
or non-natural backbone linkages or modifications of the
sugar-phosphate backbone, for example, to enhance stability during
storage or enhance resistance to degradation by nucleases.
[0074] It has also recently been suggested that synthetic RNA
duplexes consisting of either 27-mer blunt or short hairpin (sh)
RNAs with 29 bp stems and 2-nt 3' overhangs are more potent
inducers of RNA interference than conventional 21-mer siRNAs. Thus,
molecules based upon the targets identified above and being either
27-mer blunt or short hairpin (sh) RNAs with 29-bp stems and 2-nt
3' overhangs are also included within the scope of the
invention.
[0075] Therefore, in one embodiment, the double-stranded RNA
fragment (or region) will itself preferably be at least 17 base
pairs (bp) in length, preferably 18 or 19 bp in length, more
preferably at least 20 bp, more preferably at least 21 bp, or at
least 22 bp, or at least 23 bp, or at least 24 bp, 25 bp, 26 bp or
at least 27 bp in length. The expressions "double-stranded RNA
fragment" or "double-stranded RNA region" refer to a small entity
of the double-stranded RNA corresponding with (part of) the
(target) gene.
[0076] Generally, the double stranded RNA is preferably between
about 17-15000 bp, even more preferably between about 80-12000 bp
and most preferably between about 17-27 bp or between about 80-1000
bp; such as double stranded RNA regions of about 17 bp, 18 bp, 19
bp, 20 bp, 21 bp, 22 bp, 23 bp, 24 bp, 25 bp, 27 bp, 50 bp, 80 bp,
100 bp, 150 bp, 200 bp, 250 bp, 300 bp, 350 bp, 400 bp, 450 bp, 500
bp, 550 bp, 600 bp, 650 bp, 700 bp, 900 bp, 100 bp, 1100 bp, 1200
bp, 1300 bp, 1400 bp, 1500 bp, 2000 bp, 2500 bp, 3000 bp, 4500 bp,
5000 bp, 5500 bp, 6500 bp, 7000 bp, 8000 bp, 9000 bp, 10000 bp,
11000 bp, 12000 bp, 13000 bp, 14000 bp or 15000 bp. Exemplary
sequences that can be used to produce double-stranded RNA are SEQ
ID NOs 1 to 6, or any of SEQ ID NOs 1 to 2481, any complements or
fragments thereof as described in the WO2007/083193.
[0077] The double-stranded RNA may be fully or partially
double-stranded. Partially double-stranded RNAs may include short
single-stranded overhangs at one or both ends of the
double-stranded portion, provided that the RNA is still capable of
being taken up by the pest or pathogen and directing RNAi. The
double-stranded RNA may also contain internal non-complementary
regions.
[0078] The "second active component" of compositions and methods of
the invention encompasses the simultaneous or sequential provision
of two or more different double-stranded RNAs or RNA constructs to
the same pest or pathogen, so as to achieve knockdown or inhibition
of multiple (target) genes or to achieve a more potent inhibition
of a single (target) gene.
[0079] Alternatively, multiple targets are hit by the provision of
one double-stranded RNA that hits multiple target sequences.
Alternatively, a single target is more efficiently inhibited by the
presence of more than one copy of the double-stranded RNA fragment
corresponding to the (target) gene. Thus, in one embodiment of the
invention, the double-stranded RNA construct comprises multiple
dsRNA regions, at least one strand of each dsRNA region comprising
a nucleotide sequence that is complementary to at least part of a
target nucleotide sequence of a pest or pathogen (target) gene.
According to the invention, the dsRNA regions in the RNA construct
may be complementary to the same or to different (target) genes
and/or the dsRNA regions may be complementary to (target) genes
from the same or from different pest or pathogen species. The use
of such dsRNA constructs in an application on a cell or organism
(such as a plant) or a substrate susceptible to pest or pathogen
infestation and/or infection, can establish a more potent
resistance to a single pest or pathogen or to multiple pest or
pathogen species.
[0080] In one embodiment, the double-stranded RNA region comprises
multiple copies of the nucleotide sequence that is complementary to
the (target) gene. The "second active component" of compositions of
the invention thus encompasses isolated double-stranded RNA
constructs comprising at least two copies of said nucleotide
sequence complementary to at least part of a nucleotide sequence of
a pest or pathogen target.
[0081] The term "multiple" in the context of the present invention
means at least two, at least three, at least four, at least five,
at least six, etc.
[0082] DsRNA that hits more than one of the above-mentioned
targets, or a combination of different dsRNA against different of
the above mentioned targets are developed and used in the methods
of the present invention. Alternatively, the dsRNA hits more than
one target sequence of the same (target) gene.
[0083] Accordingly, the "second active component" of the present
invention extends to isolated dsRNAs or RNA constructs wherein the
dsRNA comprises annealed complementary strands, one of which has a
nucleotide sequence which is complementary to at least part of a
target nucleotide sequence of a pest or pathogen (target) gene, and
which comprises the RNA equivalents of at least two nucleotide
sequences independently chosen from each other, or preferably at
least three, four or five, independently chosen nucleotide
sequences or fragments thereof of at least 17 (contiguous)
basepairs in length, preferably at least 18, 19, 20 or 21, more
preferably at least 22, 23 or 24 basepairs in length thereof, or
more preferably at least 50, 100 or 150 bp thereof.
[0084] At least two nucleotide sequences may be derived from any of
the (target) genes herein described. According to one preferred
embodiment, the dsRNA hits at least one (target) gene that is
essential for viability, growth, development or reproduction of the
pest or pathogen and hits at least one gene involved in the pest's
ability to infest or pathogenicity or infectivity of the pathogen
as described hereinabove. Alternatively, the dsRNA hits multiple
genes of the same category, for example, the dsRNA hits at least
two essential genes or at least two genes involved in. According to
a further embodiment, the dsRNA hits at least two (target) genes,
which (target) genes are involved in a different cellular function.
The dsRNA regions (or fragments) in the double-stranded RNA may be
combined as described in detail in WO2006/046148 the contents of
which are incorporated herein by reference, which RNA constructs
can be used in the methods and compositions of the invention.
[0085] Preferably, all double-stranded RNA regions of the "second
active component" of the invention comprise at least one strand
that is complementary to at least part or a portion of the
nucleotide sequence of any of the (target) genes herein described.
However, provided one of the double-stranded RNA regions comprises
at least one strand that is complementary to a portion of the
nucleotide sequence of any one of the (target) genes herein
described, the other double-stranded RNA regions may comprise at
least one strand that is complementary to a portion of any other
pest or pathogen (target) gene (including known (target)
genes).
[0086] Also provided in the "second active component" of described
compositions and methods are isolated double-stranded RNA
constructs, further comprising at least one additional sequence and
optionally a linker. In one embodiment, the additional sequence is
chosen from the group comprising (i) a sequence facilitating
large-scale production of the dsRNA construct; (ii) a sequence
effecting an increase or decrease in the stability of the dsRNA;
(iii) a sequence allowing the binding of proteins or other
molecules to facilitate uptake of the RNA construct by pests or
pathogens; (iv) a sequence which is an aptamer that binds to a
receptor or to a molecule on the surface or in the cytoplasm of a
pest or pathogen to facilitate uptake, endocytosis and/or
transcytosis by the pest or pathogen; or (v) additional sequences
to catalyze processing of dsRNA regions. In one embodiment, the
linker is a conditionally self-cleaving RNA sequence, preferably a
pH-sensitive linker or a hydrophobic sensitive linker. In another
embodiment, the linker is an intron.
[0087] In one embodiment of the "second active component," multiple
dsRNA regions of the double-stranded RNA construct are connected by
one or more linkers. In another embodiment, the linker is present
at a site in the RNA construct, separating the dsRNA regions from
another region of interest. Different linker types for use in the
dsRNA molecules of the invention are described in detail in
WO2006/046148 the contents of which are incorporated herein by
reference.
[0088] In one particular embodiment of the "second active
component" of the invention, dsRNA constructs are provided with an
aptamer to facilitate uptake of the dsRNA by the cell or organism,
such as a pest or pathogen. The aptamer is designed to bind a
substance which is taken up by the pest or pathogen. Such
substances may be from a pest or pathogen or plant origin. One
specific example of an aptamer, is an aptamer that binds to a
transmembrane protein, for example a transmembrane protein of a
pest or pathogen. Alternatively, the aptamer may bind a (plant)
metabolite or nutrient which is taken up by the pest or pathogen.
Aptamers for use in the present invention are in detail described
in WO2006/045590 the contents of which are incorporated herein by
reference.
[0089] Without wishing to be bound by any particular theory or
mechanism, it is thought that "second active component," the
double-stranded RNAs, are taken up by a pest or pathogen from their
immediate environment in the presence of the "first active
component" of the invention. Double-stranded RNAs taken up into the
gut and transferred to the gut epithelial cells are then processed
within the cell into short double-stranded RNAs, called small
interfering RNAs (siRNAs), by the action of an endogenous
endonuclease. The resulting siRNAs then mediate RNAi via formation
of a multi-component RNase complex termed the RISC or RNA
interfering silencing complex.
[0090] The dsRNA may be formed from two separate (sense and
antisense) RNA strands that are annealed together by (non-covalent)
basepairing. Alternatively, the dsRNA may have a foldback stem-loop
or hairpin structure, wherein the two annealed strands of the dsRNA
are covalently linked. In this embodiment the sense and antisense
stands of the dsRNA are formed from different regions of single
polynucleotide molecule that is partially self-complementary. RNAs
having this structure are convenient if the dsRNA is to be
synthesized by expression in vivo, for example in a host cell or
organism, or by in vitro transcription. The precise nature and
sequence of the "loop" linking the two RNA strands is generally not
material to the invention, except that it should not impair the
ability of the double-stranded part of the molecule to mediate
RNAi. The features of "hairpin" or "stem-loop" RNAs for use in RNAi
are generally known in the art (see for example WO 99/53050, the
contents of which are incorporated herein by reference). In other
embodiments of the invention, the loop structure may comprise
linker sequences or additional sequences as described above.
[0091] The double-stranded RNA or construct may be prepared in a
manner known per se. For example, double-stranded RNAs may be
synthesized in vitro using chemical or enzymatic RNA synthesis
techniques well known in the art. In one approach the two separate
RNA strands may be synthesized separately and then annealed to form
double-strands. In another embodiment, double-stranded RNAs or
constructs may be synthesized by intracellular expression in a host
cell or organism from one or more suitable expression vectors. This
approach is discussed in further detail below.
[0092] The amount of the "second active component," e.g. the
double-stranded RNA, with which the pest or pathogen is in contact
is such that specific knockdown of the one or more (target) genes
is achieved. The RNA may be introduced in an amount which allows
delivery of at least one copy. However, in certain embodiments
higher doses (e.g., at least 5, 10, 100, 500 or 1000 copies) of
dsRNA may yield more effective inhibition. For any given pest or
pathogen gene target the optimum amount of dsRNA for effective
inhibition may be determined by routine experimentation.
[0093] The pest or pathogen can be contacted with the sulfated
polysaccharides and/or glycosaminoglycans together with the dsRNA
in any suitable manner, permitting direct uptake of the dsRNA by
the pest or pathogen. For example, the pest or pathogen can be
contacted with the dsRNA in pure or substantially pure form, for
example, an aqueous solution containing the dsRNA. In this
embodiment, the pest or pathogen may be simply "soaked" with an
aqueous solution comprising the dsRNA. In a further embodiment the
pest or pathogen can be contacted with the dsRNA by spraying the
pest or pathogen with a liquid composition comprising the
double-stranded RNA.
[0094] Alternatively, the sulfated polysaccharides and/or
glycosaminoglycans and/or the double-stranded RNA may be linked to
a food component of the pests or pathogens, such as a food
component for a mammalian pathogenic pest, in order to increase
uptake of the dsRNA by the pest or pathogen.
[0095] The compositions of the invention can include various
amounts and ratios of the "two active components." For example, the
"first active component," one or more sulfated polysaccharides
and/or glycosaminoglycans, can be present in an amount of between
about 0.000001%-99% by weight of the composition (W/W), preferably
0.00001%-99% by weight (W/W), more preferably, 0.0001%-99% by
weight (W/W), still more preferably 0.0002%-99% by weight (W/W).
The "second active component," the RNA molecule, can be present in
an amount of between about 0.0000000001%-99% by weight (W/W) of the
composition, preferably 0.000000001%-99% by weight (W/W), more
preferably 0.00000001%-99% by weight (W/W). Higher relative weight
percentages may be used when the second active component is
considered to be cells or organisms that express dsRNA, such as a
bacterium harboring an expression vector that is constructed to
produce dsRNA complementary to a (target) gene. Lower relative
weight percentages may be used when the second active component is
synthetic molecules, such as a synthetic short oligonucleotide
(e.g., double-stranded or hairpin RNA) that is complementary to a
(target) gene. The referenced amounts can be applied or
administered in one or more applications or doses given over
time.
[0096] The invention also provides methods for making compositions
of at least two active components, including (1) an amount of one
or more sulfated polysaccharides and/or glycosaminoglycans as
described herein and (2) a molecule(s) or compositions that reduce
expression of a selected gene or genes by RNA interference. The
amount of the one or more sulfated polysaccharides and/or
glycosaminoglycans is sufficient to be effective to increase the
RNAi effect of the molecule(s) or composition on the selected gene
or genes.
[0097] Methods for increasing an RNA interference effect of a
molecule or composition that reduces expression of a selected gene
or genes in cell(s) or an organism also are provided. The methods
generally include contacting cell(s) or organism with molecules or
a composition that reduces expression of a selected gene or genes
by RNA interference and an amount of one or more sulfated
polysaccharides and/or glycosaminoglycans that is effective to
enhance the RNAi effect of the molecules or composition. The
properties of the RNAi molecules or compositions and the sulfated
polysaccharides and/or glycosaminoglycans are described elsewhere
herein.
[0098] The methods may be practiced by contacting the cells or
organisms with the two active components in combination or in any
order, as long as at least some of the sulfated polysaccharides
and/or glycosaminoglycans are present at the same time as at least
some of the RNAi molecules. Thus, the cell(s) or organism can be
contacted with the one or more sulfated polysaccharides and/or
glycosaminoglycans before or after contacting the cell(s) or
organism with the RNAi molecules. The cell or organism also can be
contacted with the one or more sulfated polysaccharides and/or
glycosaminoglycans simultaneously with the RNAi molecules.
Combinations of the contacting schemes can be used also. For
example, the cell or organism can be contacted with the one or more
sulfated polysaccharides and/or glycosaminoglycans before,
simultaneously with and after contacting the cell(s) or organism
with the RNAi molecules.
[0099] Contacting the cell(s) or organism with the RNAi molecules
and the sulfated polysaccharides and/or glycosaminoglycans can be
carried out by any convenient method(s) known to the skilled
person. For example, the molecules or composition and the one or
more sulfated polysaccharides and/or glycosaminoglycans can be
applied by soaking or spraying onto the cell(s) or organism. When
the organism is a human or animal, i.e., for therapeutic use, the
first and second active components can be administered to the human
or animal by standard routes of administration as are well known in
the art. Additional methods will be known to the skilled
person.
[0100] In other embodiments, the pest or pathogen may be contacted
with a composition containing the two active components of the
invention. The composition may, in addition to the two active
components, contain further excipients, diluents or carriers.
Preferred features of such compositions are discussed in more
detail below.
[0101] The sulfated polysaccharides and/or glycosaminoglycans
and/or the double-stranded RNA may also be incorporated in the
medium in which the pest or pathogen grows or in or on a material
or substrate that is infested or infected by the pest or pathogen,
respectively, or impregnated in a substrate or material susceptible
to infestation or infection by pest or pathogen.
[0102] The composition may be provided in a form wherein it
actively expresses the "second active component," the
double-stranded RNA, e.g. the composition may be a cell harboring
an expression vector expressing dsRNA. The cell may be a eukaryotic
or prokaryotic cell. Alternatively, the cell may be "capable of
expressing", meaning that it is transformed with a transgene which
encodes the desired dsRNA but that the transgene is not active in
the cell when (and in the form in which) the cell is supplied. In
preferred embodiments, when the "second active component" comprises
eukaryotic or prokaryotic cells harboring or expressing the dsRNA
is killed or heat-inactivated prior to application on the plant,
the animal or human or the substrate to be treated or to be applied
to.
[0103] Therefore, according to another embodiment, a recombinant
DNA construct is provided comprising the nucleotide sequence
encoding the dsRNA or dsRNA construct according to the present
invention operably linked to at least one regulatory sequence.
Preferably, the regulatory sequence is selected from the group
comprising constitutive promoters or inducible promoters as are
well known in the art and as are described herein.
[0104] The (target) gene may be any (target) gene known to persons
skilled in the art. The term "regulatory sequence" is to be taken
in a broad context and refers to a regulatory nucleic acid capable
of effecting expression of the sequences to which it is operably
linked.
[0105] Encompassed by the aforementioned term are promoters and
nucleic acids or synthetic fusion molecules or derivatives thereof
which activate or enhance expression of a nucleic acid, so called
activators or enhancers. The term "operably linked" as used herein
refers to a functional linkage between the promoter sequence and
the gene of interest, such that the promoter sequence is able to
initiate transcription of the gene of interest.
[0106] By way of example, the transgene nucleotide sequence
encoding the double-stranded RNA could be placed under the control
of an inducible or growth or developmental stage-specific promoter
which permits transcription of the dsRNA to be turned on, by the
addition of the inducer for an inducible promoter or when the
particular stage of growth or development is reached.
[0107] In yet other embodiments of the present invention, other
promoters useful for the expression of "the second active
component," the dsRNA, are used and include, but are not limited
to, promoters from an RNA Pol I, an RNA Pol II, an RNA Pol III, T7
RNA polymerase or SP6 RNA polymerase. Vectors comprising these
promoters and cells comprising these vectors are described in
WO2000/001846 and WO 2001/088151. These promoters are typically
used for in vitro-production of dsRNA, which dsRNA is then included
in a pesticidal, insecticidal, nematicidal or fungicidal agent, for
example, in a pesticidal, insecticidal, nematicidal or fungicidal
liquid, spray or powder.
[0108] Optionally, one or more transcription termination sequences
may also be incorporated in the recombinant construct of the
invention. The term "transcription termination sequence"
encompasses a control sequence at the end of a transcriptional
unit, which signals 3' processing and polyadenylation of a primary
transcript and termination of transcription. Vectors comprising
these terminators and cells comprising these vectors are described
in WO 2001/088151.
[0109] Additional regulatory elements, such as transcriptional or
translational enhancers, may be incorporated in the expression
construct.
[0110] Accordingly, the present invention also encompasses a cell
comprising any of the nucleotide sequences or recombinant DNA
constructs described herein. The invention further encompasses
prokaryotic cells (such as, but not limited to, gram-positive and
gram-negative bacterial cells) and eukaryotic cells (such as, but
not limited to, yeast cells, plant cells, mammalian cells or human
cells). Preferably, said cell is a bacterial cell or a plant
cell.
[0111] Accordingly, the second active component as used herein
encompasses a cell (e.g., a bacterial or eukaryotic cell)
comprising any of the nucleotide sequences encoding the dsRNA or
dsRNA construct as described herein. The present invention also
encompasses a cell (e.g., Gram-negative and Gram-positive bacteria,
such as, but not limited to, Escherichia spp. (e.g. E. coli),
Bacillus spp. (e.g. B. thuringiensis), Rhizobium spp.,
Lactobacilllus spp., Lactococcus spp., etc); or eukaryotic cell
such as a yeast cell (e.g. Saccharomyces spp.) comprising any of
the nucleotide sequences encoding the dsRNA or dsRNA constructs
described herein. Preferably, these cells comprise a recombinant
construct wherein the nucleotide sequence encoding the dsRNA or
dsRNA construct according to the present invention is operably
linked to at least one regulatory element as described above.
[0112] The bacterial cells as used in the compositions described
herein are preferably inactivated when used in an environment where
contact with humans or other mammals is likely. Inactivation may be
achieved by any means, such as by heat treatment, phenol or
formaldehyde treatment for example, or by mechanical treatment.
[0113] The compositions of the present invention optionally also
can include at least one suitable carrier, excipient or diluent.
The composition may contain further components which serve to
stabilize the dsRNA and/or prevent degradation of the dsRNA during
prolonged storage of the composition. The composition may still
further contain components, in addition to the first active
component, which enhance or promote uptake of the second active
component (e.g., dsRNA) by the pest or pathogen. These may include,
for example, chemical agents which generally promote the uptake of
RNA into cells, e.g. lipofectamine etc.
[0114] The composition containing the two active components may be
in any suitable physical form for application to pests or
pathogens, to substrates, to cells, or administration to organisms
susceptible to infestation or infected by pests or pathogens.
[0115] The invention also provides kits that include containers of
the two active components described herein. For example, a kit of
the invention can include a first container containing an RNA
molecule(s) or composition that reduces expression of a selected
gene or genes by RNA interference in a cell or organism and a
second container containing an amount of one or more sulfated
polysaccharides and/or glycosaminoglycans. The amount of the one or
more sulfated polysaccharides and/or glycosaminoglycans is
sufficient to increase the RNAi effect of the molecule(s) or
composition.
[0116] Thus it is contemplated that the composition of the
invention may be supplied as a "kit-of-parts" comprising the dsRNA
in one container and an amount of one or more sulfated
polysaccharides and/or glycosaminoglycans in a second container
and, optionally, one or more suitable diluents or carriers for the
foregoing components in one or more separate containers. In these
embodiments the dsRNA may be supplied in a concentrated form, such
as a concentrated aqueous solution. It may even be supplied in
frozen form or in freeze-dried or lyophilized form. The latter may
be more stable for long term storage and may be defrosted and/or
reconstituted with a suitable diluent immediately prior to use.
[0117] The kit may be supplied with suitable instructions for use.
The instructions may be printed on suitable packaging in which the
other components are supplied or may be provided as a separate
entity, which may be in the form of a sheet or leaflet for example.
The instructions may be rolled or folded for example when in a
stored state and may then be unrolled and unfolded to direct use of
the remaining components of the kit.
[0118] The term "pest" as used herein includes a variety of types
of pests such as insects. The term "pathogen" as used herein
includes a variety of types of pathogens such as nematodes and
fungi. The terms "pests" and "pathogens" may imply numerous members
from a single species or numerous members from a combination of
species comprising insects, nematodes, or fungi.
[0119] In preferred, but non-limiting, embodiments of the invention
the pest or pathogen is chosen from the group consisting of:
[0120] (1) an insect, nematode, or fungus which is a plant pest or
pathogen,
[0121] (2) an insect, nematode, or fungus capable of infesting,
infecting or injuring humans and/or animals;
[0122] and
[0123] (3) an insect, nematode, or fungus that causes unwanted
damage to substrates or materials, such as insects that attack
foodstuffs, seeds, wood, paint, plastic, clothing etc.
[0124] An insect can be any insect, meaning any organism belonging
to the Kingdom Animals, more specific to the Phylum Arthropoda, and
to the Class Insecta or the Class Arachnida. The methods of the
invention are applicable to all insects and that are susceptible to
gene silencing by RNA interference and that are capable of
internalizing double-stranded RNA from their immediate
environment.
[0125] In one embodiment of the invention, the insect may belong to
the following orders: Acari, Araneae, Anoplura, Coleoptera,
Collembola, Dermaptera, Dictyoptera, Diplura, Diptera, Embioptera,
Ephemeroptera, Grylloblatodea, Hemiptera, Homoptera, Hymenoptera,
Isoptera, Lepidoptera, Mallophaga, Mecoptera, Neuroptera, Odonata,
Orthoptera, Phasmida, Plecoptera, Protura, Psocoptera,
Siphonaptera, Siphunculata, Thysanura, Strepsiptera, Thysanoptera,
Trichoptera, and Zoraptera.
[0126] In preferred, but non-limiting, embodiments of the invention
the insect may be one or more of the following non-limiting
list:
[0127] (1) an insect which is a plant pest, such as, but not
limited, to Nilaparvata spp. (e.g. N. lugens (brown planthopper));
Laodelphax spp. (e.g. L. striatellus (small brown planthopper));
Nephotettix spp. (e.g. N. virescens or N. cincticeps (green
leafhopper), or N. nigropictus (rice leafhopper)); Sogatella spp.
(e.g. S. furcifera (white-backed planthopper)); Blissus spp. (e.g.
B. leucopterus leucopterus (chinch bug)); Scotinophora spp. (e.g.
S. vermidulate (rice blackbug)); Acrosternum spp. (e.g. A. hilare
(green stink bug)); Parnara spp. (e.g. P. guttata (rice skipper));
Chilo spp. (e.g. C. suppressalis (rice striped stem borer), C.
auricilius (gold-fringed stem borer), or C. polychrysus
(dark-headed stem borer)); Chilotraea spp. (e.g. C. polychrysa
(rice stalk borer)); Sesamia spp. (e.g. S. inferens (pink rice
borer)); Tryporyza spp. (e.g. T. innotata (white rice borer), or T.
incertulas (yellow rice borer)); Cnaphalocrocis spp. (e.g. C.
medinalis (rice leafroller)); Agromyza spp. (e.g. A. oryzae
(leafminer), or A. parvicornis (corn blot leafminer)); Diatraea
spp. (e.g. D. saccharalis (sugarcane borer), or D. grandiosella
(southwestern corn borer)); Narnaga spp. (e.g. N. aenescens (green
rice caterpillar)); Xanthodes spp. (e.g. X. transversa (green
caterpillar)); Spodoptera spp. (e.g. S. frugiperda (fall armyworm),
S. exigua (beet armyworm), S. litura (Oriental leafworm), S.
littoralis (climbing cutworm) or S. praefica (western yellowstriped
armyworm)); Mythimna spp. (e.g. Mythmna (Pseudaletia) seperata
(armyworm)); Helicoverpa spp. (e.g. H. zea (corn earworm), H.
armigera); Colaspis spp. (e.g. C. brunnea (grape colaspis));
Lissorhoptrus spp. (e.g. L. oryzophilus (rice water weevil));
Echinocnemus spp. (e.g. E. squamos (rice plant weevil)); Diclodispa
spp. (e.g. D. armigera (rice hispa)); Oulema spp. (e.g. O. oryzae
(leaf beetle); Sitophilus spp. (e.g. S. oryzae (rice weevil));
Pachydiplosis spp. (e.g. P. oryzae (rice gall midge)); Hydrellia
spp. (e.g. H. griseola (small rice leafminer), or H. sasakii (rice
stem maggot)); Chlorops spp. (e.g. C. oryzae (stem maggot));
Diabrotica spp. (e.g. D. virgifera (western corn rootworm), D.
barberi (northern corn rootworm), D. undecimpunctata howardi
(southern corn rootworm), D. virgifera zeae (Mexican corn
rootworm); D. balteata (banded cucumber beetle)); Ostrinia spp.
(e.g. O. nubilalis (European corn borer)); Agrotis spp. (e.g. A.
ipsilon (black cutworm)); Elasmopalpus spp. (e.g. E. lignosellus
(lesser cornstalk borer)); Melanotus spp. (wireworms); Cyclocephala
spp. (e.g. C. borealis (northern masked chafer), or C. immaculata
(southern masked chafer)); Phaedon spp. (e.g. P. cochleariae
(mustard leaf beetle)); Epilachna spp. (e.g. E. varivestis (Mexican
bean beetle)); Popillia spp. (e.g. P. japonica (Japanese beetle));
Chaetocnema spp. (e.g. C. pulicaria (corn flea beetle));
Sphenophorus spp. (e.g. S. maidis (maize billbug)); Rhopalosiphum
spp. (e.g. R. maidis (corn leaf aphid)); Anuraphis spp. (e.g. A.
maidiradicis (corn root aphid)); Melanoplus spp. (e.g. M.
femurrubrum (redlegged grasshopper) M. differentialis (differential
grasshopper) or M. sanguinipes (migratory grasshopper)); Hylemya
spp. (e.g. H. platura (seedcorn maggot)); Anaphothrips spp. (e.g.
A. obscrurus (grass thrips)); Solenopsis spp. (e.g. S. milesta
(thief ant)); or spp. (e.g. T. urticae (twospotted spider mite), T.
cinnabarinus (carmine spider mite); Helicoverpa spp. (e.g. H. zea
(corn earworm), or H. armigera (cotton bollworm)); Pectinophora
spp. (e.g. P. gossypiella (pink bollworm)); Earias spp. (e.g. E.
vittella (spotted bollworm)); Heliothis spp. (e.g. H. virescens
(tobacco budworm)); Anthonomus spp. (e.g. A. grandis (boll
weevil)); Pseudatomoscelis spp. (e.g. P. seriatus (cotton
fleahopper)); Trialeurodes spp. (e.g. T. abutiloneus (banded-winged
whitefly) T. vaporariorum (greenhouse whitefly)); Bemisia spp.
(e.g. B. argentifolii (silverleaf whitefly)); Aphis spp. (e.g. A.
gossypii (cotton aphid)); Lygus spp. (e.g. L. lineolaris (tarnished
plant bug) or L. hesperus (western tarnished plant bug));
Euschistus spp. (e.g. E. conspersus (consperse stink bug));
Chlorochroa spp. (e.g. C. sayi (Say stinkbug)); Nezara spp. (e.g.
N. viridula (southern green stinkbug)); Thrips spp. (e.g. T. tabaci
(onion thrips)); Frankliniella spp. (e.g. F. fusca (tobacco
thrips), or F. occidentalis (western flower thrips)); Leptinotarsa
spp. (e.g. L. decemlineata (Colorado potato beetle), L. juncta
(false potato beetle), or L. texana (Texan false potato beetle));
Lema spp. (e.g. L. trilineata (three-lined potato beetle)); Epitrix
spp. (e.g. E. cucumeris (potato flea beetle), E. hirtipennis (flea
beetle), or E. tuberis (tuber flea beetle)); Epicauta spp. (e.g. E.
vittata (striped blister beetle)); Empoasca spp. (e.g. E. fabae
(potato leafhopper)); Myzus spp. (e.g. M. persicae (green peach
aphid)); Paratrioza spp. (e.g. P. cockerelli (psyllid)); Conoderus
spp. (e.g. C. falli (southern potato wireworm), or C. vespertinus
(tobacco wireworm)); Phthorimaea spp. (e.g. P. operculella (potato
tuberworm)); Macrosiphum spp. (e.g. M. euphorbiae (potato aphid));
Thyanta spp. (e.g. T. pallidovirens (redshouldered stinkbug));
Phthorimaea spp. (e.g. P. operculella (potato tuberworm)); Keiferia
spp. (e.g. K. lycopersicella (tomato pinworm)); Limonius spp.
(wireworms); Manduca spp. (e.g. M. sexta (tobacco hornworm), or M.
quinquemaculata (tomato hornworm)); Liriomyza spp. (e.g. L.
sativae, L. trifolli or L. huidobrensis (leafminer)); Drosophila
spp. (e.g. D. simulans, D. yakuba, D. pseudoobscura, D. virilis or
D. melanogaster (fruitflies)); Atherigona spp. (e.g. A. soccata
(shoot fly); Carabus spp. (e.g. C. granulatus); Chironomus spp.
(e.g. C. tentanus); Ctenocephalides spp. (e.g. C. felis (cat
flea)); Diaprepes spp. (e.g. D. abbreviatus (root weevil)); Ips
spp. (e.g. I. pini (pine engraver)); Tribolium spp. (e.g. T.
castaneum (red floor beetle)); Glossina spp. (e.g. G. morsitans
(tsetse fly)); Anopheles spp. (e.g. A. gambiae str. PEST (malaria
mosquito) or A. albimanus (malaria mosquito); Acyrthosiphon spp.
(e.g. A. pisum (pea aphid)); Apis spp. (e.g. A. melifera (honey
bee)); Homalodisca spp. (e.g. H. coagulata (glassy-winged
sharpshooter)); Aedes spp. (e.g. Ae. aegypti (yellow fever
mosquito)); Bombyx spp. (e.g. B. mori (silkworm)); Locusta spp.
(e.g. L. migratoria (migratory locust)); Boophilus spp. (e.g. B.
microplus (cattle tick))s Acanthoscurria spp. (e.g. A. gomesiana
(red-haired chololate bird eater)); Diploptera spp. (e.g. D.
punctata (pacific beetle cockroach)); Heliconius spp. (e.g. H.
erato (red passion flower butterfly), H. melpomene (postman
butterfly) or H. himera); Plutella spp. (e.g. P. xylostella
(diamontback moth)); Armigeres spp. (e.g. A. subalbatus);
Culicoides spp. (e.g. C. sonorensis (biting midge)); Biphyllus spp.
(e.g. B. lunatus (skin beetle)); Mycetophagus spp (e.g. M.
quadripustulatus); Hydropsyche spp (caddisflies); Oncometopia spp.
(e.g. O. nigricans (sharpshooter)); Papilio spp. (e.g. P. dardanus
(swallowtail butterfly)); Antheraea spp. (e.g. A. yamamai (Japanese
oak silkmoth); Trichoplusia spp. (e.g. T. ni (cabbage looper));
Callosobruchus spp. (e.g. C. maculatus (cowpea weevil));
Rhynchosciara spp. (e.g. R. Americana (fungus gnat)); Sphaerius
spp. (minute bog beatle); Ixodes spp. (e.g. I. scapularis
(black-legged tick)); Diaphorina spp. (e.g. D. citri (asian citrus
psyllid)); Meladema spp. (e.g. M. coriacea (Black Predacious Diving
Beetle); Rhipicephalus spp. (e.g. R. appendiculatus (brown ear
tick)); Amblyomma spp. (e.g. A. americanum (lone star tick);
Toxoptera spp. (e.g. T. citricida (brown citrus aphid); Hister
spp.; Dysdera spp. (e.g. D. erythrina (cell spider)), Lonomia spp.
(e.g. L. obliqua (caterpillar)); and Culex spp. (e.g. C. pipiens
(house mosquito)):
[0128] (2) an insect capable of infesting or injuring humans and/or
animals such as, but not limited to, those with piercing-sucking or
chewing mouthparts or stings, as found in Hemiptera and some
Hymenoptera and Diptera such as mosquitos, bees, wasps, lice, fleas
and ants as well as members of the Arachnidae, such as ticks and
mites;
[0129] and
[0130] (3) an insect that causes unwanted damage to substrates or
materials, such as insects that attack foodstuffs, seeds, wood,
paint, plastic, clothing etc. Insects or arachnid examples of such
pests include household insects, ecto-parasites and insects and/or
arachnids relevant for public health and hygiene such as, by way of
example and not limitation, flies, spider mites, thrips, ticks, red
poultry mite, ants, cockroaches, termites, crickets including
house-crickets, silverfish, booklice, beetles, earwigs, mosquitos
and fleas.
[0131] The term "insect" encompasses insects of all types and at
all stages of development, including egg, larval or nymphal, pupal
and adult stages.
[0132] Additionally, the pathogen may be a fungus or fungi. The
fungus or fungi may be one or more of the following not-limiting
list:
[0133] (1) a fungal cell of, or a cell derived from a plant
pathogenic fungus, such as but not limited to Acremoniella spp.,
Alternaria spp. (e.g. Alternaria brassicola or Alternaria solani),
Ascochyta spp. (e.g. Ascochyta pisi), Botrytis spp. (e.g. Botrytis
cinerea or Botryotinia fuckeliana), Cladosporium spp., Cercospora
spp. (e.g. Cercospora kikuchii or Cercospora zaea-maydis),
Cladosporium spp. (e.g. Cladosporium fulvum), Colletotrichum spp.
(e.g. Colletotrichum lindemuthianum), Curvularia spp., Diplodia
spp. (e.g. Diplodia maydis), Erysiphe spp. (e.g. Erysiphe graminis
f. sp. graminis, Erysiphe graminis f. sp. hordei or Erysiphe pisi),
Erwinia armylovora, Fusarium spp. (e.g. Fusarium nivale, Fusarium
sporotrichioides, Fusarium oxysporum, Fusarium graminearum,
Fusarium germinearum, Fusarium culmorum, Fusarium solani, Fusarium
moniliforme or Fusarium roseum), Gaeumanomyces spp. (e.g.
Gaeumanomyces graminis f. sp. tritici), Gibberella spp. (e.g.
Gibberella zeae), Helminthosporium spp. (e.g. Helminthosporium
turcicum, Helminthosporium carbonum, Helminthosporium mavdis or
Helminthosporium sigmoideum), Leptosphaeria salvinii, Macrophomina
spp. (e.g. Macrophomina phaseolina), Magnaportha spp. (e.g.
Magnaporthe oryzae), Mycosphaerella spp., Nectria spp. (e.g.
Nectria heamatococca), Peronospora spp. (e.g. Peronospora
manshurica or Peronospora tabacina), Phoma spp. (e.g. Phoma betae),
Phakopsora spp. (e.g. Phakopsora pachyrhizi), Phymatotrichum spp.
(e.g. Phymatotrichum omnivorum), Phytophthora spp. (e.g.
Phytophthora cinnamomi, Phytophthora cactorum, Phytophthora
phaseoli, Phytophthora parasitica, Phytophthora citrophthora,
Phytophthora megasperma fsp. soiae or Phytophthora infestans),
Plasmopara spp. (e.g. Plasmopara viticola), Podosphaera spp. (e.g.
Podosphaera leucotricha), Puccinia spp. (e.g. Puccinia sorghi,
Puccinia striiformis, Puccinia graminis f. sp. tritici, Puccinia
asparagi, Puccinia recondita or Puccinia arachidis), Pythium spp.
(e.g. Pythium aphanidermatum), Pyrenophora spp. (e.g. Pyrenophora
tritici-repentens or Pyrenophora teres), Pyricularia spp. (e.g.
Pyricularia oryzae), Pythium spp. (e.g. Pythium ultimum),
Rhincosporium secalis, Rhizoctonia spp. (e.g. Rhizoctonia solani,
Rhizoctonia oryzae or Rhizoctonia cerealis), Rhizopus spp. (e.g.
Rhizopus chinensid), Scerotium spp. (e.g. Scerotium rolfsii),
Sclerotinia spp. (e.g. Sclerotinia sclerotiorum), Septoria spp.
(e.g. Septoria lycopersici, Septoria glycines, Septoria nodorum or
Septoria tritici), Thielaviopsis spp. (e.g. Thielaviopsis
basicola), Tilletia spp., Trichoderma spp. (e.g. Trichoderma
virde), Uncinula spp. (e.g. Uncinula necator), Ustilago maydis
(e.g. corn smut), Venturia spp. (e.g. Venturia inaequalis or
Venturia pirina) or Verticillium spp. (e.g. Verticillium dahliae or
Verticillium albo-atrum);
[0134] (2) a fungal cell of, or a cell derived from a fungus
capable of infesting humans and/or animals such as, but not limited
to, Candida spp., particularly Candida albicans; Dermatophytes
including Epidermophyton spp., Trichophyton spp, and Microsporum
spp. (particularly Microsporum canis or Microsporum gypseum);
Aspergillus spp. (particularly Aspergillus flavus, Aspergillus
fumigatus, Aspergillus nidulans, Aspergillus niger or Aspergillus
terreus); Blastomyces dermatitidis; Paracoccidioides brasiliensis;
Coccidioides immitis; Cryptococcus neoformans; Histoplasma
capsulatum Var. capsulatum or Var. duboisii; Sporothrix schenckii;
Fusarium spp.; Scopulariopsis brevicaulis; Trichophyton
mentagrophytes, Fonsecaea spp.; Penicillium spp.; or Zygomycetes
group of fungi (particularly Absidia corymbifera, Rhizomucor
pusillus or Rhizopus arrhizus);
[0135] (3) a fungal cell of, or a cell derived from a fungus that
causes unwanted damage to substrates or materials, such as fungi
that attack foodstuffs, seeds, wood, paint, plastic, clothing etc.
Examples of such fungi are the moulds, including but not limited to
Stachybotrys spp., Aspergillus spp., Alternaria spp., Cladosporium
spp., Penicillium spp. or Phanerochaete chrysosporium.
[0136] Furthermore, the pathogen may be a nematode or nematodes.
The nematode or nematodes may be one or more of the following
not-limiting list:
[0137] (1) a nematode which is a plant pathogenic nematode, such as
but not limited to Root Knot Nematodes (Meloidogyne spp.) in rice
(e.g. M. incognita, M. javanica or M. graminicola), in soybean
(e.g. M. incognita or M. arenaria), in cotton (e.g. M. incognita),
in potato (e.g. M. chitwoodi or M. hapla), in tomato (e.g. M.
chitwoodi), in tobacco (e.g. M. incognita, M. javanica or M.
arenaria), and in corn (e.g. M. incognita); Cyst Nematodes
(Heterodera spp.) in rice (e.g. H. oryzae), in soybean (e.g. H.
glycines) and in corn (e.g. H. zeae); Cyst nematodes (Globodera
spp.) in potato (e.g. G. pallida or G. rostochiensis); Reniform
Nematodes (Rotylenchulus spp.) in cotton (e.g. R. reniformis); Root
lesion nematodes (Pratylenchus spp.) in banana (e.g. P. coffeae or
P. goodeyi); Burrowing Nematodes (Radopholus spp.) in banana (e.g.
R. similis); Other rice damaging nematodes such as rice root
nematode (Hirschmaniella spp., e.g. H. oryzae);
[0138] (2) a nematode capable of infesting humans such as, but not
limited to: Enterobius vermicularis, the pinworm that causes
enterobiasis; Ascaris lumbridoides, the large intestinal roundworm
that causes ascariasis; Necator and Ancylostoma, two types of
hookworms that cause ancylostomiasis; Trichuris trichiura, the
whipworm that causes trichuriasis; Strongyloides stercoralis that
causes strongyloidiasis; and Trichonella spirae that causes
trichinosis; Brugia malayi and Wuchereria bancrofti, the filarial
nematodes associated with the worm infections known as lymphatic
filariasis and its gross manifestation, elephantiasis, and
Onchocerca volvulus that causes river blindness. Transfer of
nematodes to humans may also occur through blood-feeding mosquitoes
which have fed upon infected animals or humans; or a nematode
capable of infesting animals such as, but not limited to: dogs
(Hookworms e.g. Ancylostoma caninum or Uncinaria stenocephala,
Ascarids e.g. Toxocara canis or Toxascaris leonina, or Whipworms
e.g. Trichuris vulpis), cats (Hookworms e.g. Ancylostoma
tubaeforme, Ascarids e.g. Toxocara cati), fish (herring worms or
cod worms e.g. Anisakid, or tapeworm e.g. Diphyllobothrium), sheep
(Wire worms e.g. Haemonchus contortus) and cattle
(Gastro-intestinal worms e.g. Ostertagia ostertagi, Cooperia
oncophora);
[0139] (3) a nematode that causes unwanted damage to substrates or
materials, such as nematodes that attack foodstuffs, seeds, wood,
paint, plastic, clothing etc. Examples of such nematodes include
but are not limited to Meloidogyne spp. (e.g. M. incognita, M.
javanica, M. arenaria, M. graminicola, M. chitwoodi or M. hapla);
Heterodera spp. (e.g. H. oryzae, H. glycines, H. zeae or H.
schachtii); Globodera spp. (e.g. G. pallida or G. rostochiensis);
Ditylenchus spp. (e.g. D. dipsaci, D. destructor or D. angustus);
Belonolaimus spp.; Rotylenchulus spp. (e.g. R. reniformis);
Pratylenchus spp. (e.g. P. coffeae, P. goodeyi or P. zeae);
Radopholus spp. (e.g. R. Similis); Hirschmaniella spp. (e.g. H.
oryzae); Aphelenchoides spp. (e.g. A. besseyi); Criconemoides spp.;
Longidorus spp.; Helicotylenchus spp.; Hoplolaimus spp.; Xiphinema
spp.; Paratrichodorus spp. (e.g. P. minor); Tylenchorhynchus
spp;
[0140] and virus transmitting nematodes (e.g. Longidorus macrosoma:
transmits prunus necrotic ring spot virus, Xiphinema americanum:
transmits tobacco ring spot virus, Paratrichadorus teres: transmits
pea early browning virus, or Trichodorus similis: transmits tobacco
rattle virus).
[0141] In one specific embodiment, the composition including the
two active components is a pharmaceutical or veterinary composition
for treating or preventing insect disease or infections of humans
or animals, respectively. Such compositions will comprise at least
one double-stranded RNA or RNA construct, or nucleotide sequence or
recombinant DNA construct encoding the double-stranded RNA or RNA
construct, wherein the double-stranded RNA comprises annealed
complementary strands, one of which has a nucleotide sequence which
corresponds to a target nucleotide sequence of an insect (target)
gene that causes the disease or infection and an amount of one or
more sulfated polysaccharides and/or glycosaminoglycans that
effectively increases the RNAi effect of the dsRNA, and,
optionally, at least one carrier, excipient or diluent suitable for
pharmaceutical use.
[0142] The composition may be a composition suitable for topical
use, such as application on the skin of an animal or human, for
example as liquid composition to be applied to the skin as drops,
gel, aerosol, or by brushing, or a spray, cream, ointment, etc. for
topical application or as transdermal patches.
[0143] Other conventional pharmaceutical dosage forms may also be
produced, including tablets, capsules, pessaries, transdermal
patches, suppositories, etc. The chosen form will depend upon the
nature of the target pest or pathogen and hence the nature of the
disease it is desired to treat.
[0144] In another specific embodiment, the composition including
the two active components may be, or be used in, a coating that can
be applied to a substrate in order to protect the substrate from
infestation by a pest and/or to prevent, arrest or reduce pest
growth on the substrate and thereby prevent damage caused by the
pest or pathogen. In this embodiment, the composition can be used
to protect any substrate or material that is susceptible to
infestation by or damage caused by a pest or pathogen, for example
foodstuffs and other perishable materials, and substrates such as
wood. Houses and other wood products can be destroyed by termites,
powder post beetles, and carpenter ants. The subterranean termite
and Formosan termite are the most serious pests of houses in the
southern United States and tropical regions. Any harvested plant or
animal product can be attacked by insects. Flour beetles, grain
weevils, meal moths and other stored product pests will feed on
stored grain, cereals, pet food, powdered chocolate, and almost
everything else in the kitchen pantry that is not protected. Larvae
of clothes moths eat clothes made from animal products, such as
fur, silk and wool. Larvae of carpet beetles eat both animal and
plant products, including leather, fur, cotton, stored grain, and
even museum specimens. Book lice and silverfish are pests of
libraries. These insects eat the starchy glue in the bindings of
books. Other insects that have invaded houses include cockroaches
which eat almost anything. Cockroaches are not known to be a
specific transmitter of disease, but they contaminate food and have
an unpleasant odor. They are very annoying, and many pest control
companies are kept busy in attempts to control them. The most
common cockroaches in houses, grocery stores, and restaurants
include the German cockroach, American cockroach, Oriental
cockroach, and brown banded cockroach.
[0145] The nature of the excipients and the physical form of the
composition may vary depending upon the nature of the substrate
that is desired to treat. For example, the composition may be a
liquid that is brushed or sprayed onto or imprinted into the
material or substrate to be treated, or a coating that is applied
to the material or substrate to be treated.
[0146] The present invention further encompasses a method for
treating and/or preventing pest infestation on a substrate
comprising applying an effective amount of any of the compositions
described herein to said substrate.
[0147] The invention further encompasses a method for treating
and/or preventing a disease or condition, comprising administering
to a subject in need of such treatment and/or prevention, any of
the compositions as herein described, said composition comprising
an amount of one or more sulfated polysaccharides and/or
glycosaminoglycans that is effective to increase the RNAi effect of
dsRNA, and at least one double-stranded RNA or double stranded RNA
construct comprising annealed complementary strands, one of which
has a nucleotide sequence which is complementary to at least part
of a nucleotide sequence of a pest or pathogen (target) gene that
causes the insect disease or condition.
[0148] Therapeutic use of the compositions described herein
includes administering to an individual in a effective dose
sufficient to treat a disorder by knocking down expression of a
(target) gene. The effective amount may vary according to a variety
of factors such as the gene or genes targeted, the particular
composition administered, the individual's condition, weight, sex
and age. Other factors include the route of administration.
Pharmaceutical compositions may be administered to the individual
by a variety of routes such as by topical, parenteral, enteral,
transdermal, cutaneous, subcutaneous, intravenous, intraperitoneal,
intramuscular or oral routes. In addition, co-administration or
sequential administration of the two active components or other
agents may be desirable.
[0149] In another embodiment of the invention the compositions are
used as a pesticide, insecticide, nematicide or fungicide for a
plant or for propagation or reproductive material of a plant, such
as on seeds. As an example, the composition can be used as a
pesticide, insecticide, nematicide or fungicide by spraying or
applying it on plant tissue or spraying or mixing it on the soil
before or after emergence of the plantlets.
[0150] In yet another embodiment, the present invention provides a
method for treating and/or preventing insect growth and/or insect
infestation of a plant or propagation or reproductive material of a
plant, comprising applying an effective amount of any of the
compositions herein described to a plant or to propagation or
reproductive material of a plant.
EXAMPLES
[0151] The following examples illustrate the present invention in
more detail. These examples are meant to be exemplary and not as
limitations of the present invention. A range of compounds were
tested for enhanced RNAi-induced lethality with each compound
coadministered with the target double-stranded RNA in feeding
bioassays against larvae of the Colorado potato beetle
(Leptinotarsa decemlineata), the mustard leaf beatle (Phaedon
cochleariae) and the nematode worm Caenorhabditis elegans. The
sulfated polysaccharide, dextran sulfate, substantially increased
the RNAi-induced lethality of target dsRNA when the two components
were ingested. Ingestion of target dsRNA in the presence of the
glycosaminoglycan, heparin, also increased insect mortality when
compared to target dsRNA alone.
Example 1
The Effects of Dextran Sulfate (.+-.8 kDa) at Different
Concentrations
[0152] In a Colorado potato beetle (CPB) larvae bioassay, target
Ld105 dsRNA (SEQ ID NO:1) was tested at one amount (50 ng) in the
presence of dextran sulfate (average Mw.+-.8 kDa; Sigma cat. no.
D4911) at different amounts ranging from 50 ng to 50 .mu.g per
larvae. The bioassay was typically performed in an 48-well-plate
containing 500 .mu.l of artificial diet per well. Thirty .mu.l of
test solution consisting of 5 .mu.l Ld105 (SEQ ID NO 1) dsRNA
(starting concentration 10 ng/.mu.l) and 25 .mu.l of dextran
sulfate (of varying concentrations in Milli-Q water) was applied
topically to the diet and the diet left to dry for about two hours
in the laminar flow at room temperature. One 2.sup.nd stage CPB
larva was placed in each well. Per treatment, 24 insects were
tested. The plates were incubated in the insect rearing chamber at
25.degree. C. and .+-.50% RH, with a photoperiod of 16 hours
light/8 hours dark. Mortality was scored over the course of the
experiment. The Kaplan-Meier estimator was used to plot the
survival curves and the survival curves were compared using the
logrank .chi..sup.2 test.
[0153] The time-to-kill, i.e. the time it takes to reach a certain
mortality number in each treatment, was reduced when target dsRNA
was accompanied with dextran sulfate, as shown in FIG. 1. In this
bioassay, at day 7 (i.e. 7 days post infestation on artificial
diet), approximately 60% of larvae had been killed in the
treatments of dsRNA with dextran sulfate at 5 or 50 .mu.g, whereas
approx. 30% (i.e. half) of larvae died on diet containing target
dsRNA alone. In this bioassay, it took at least an extra 5 days for
the number of dead CPB larvae to reach 60% in the treatment of
dsRNA alone; at this time, day 12 onwards, more than 90% of CPB
larvae were killed in the treatments of dsRNA with an amount of 5
or 50 .mu.g dextran sulfate. Very little or no toxicity was
associated with dextran sulfate alone.
[0154] In this bioassay, overall potency was increased. Over the
course of the whole bioassay, the percentage of CPB larval
survivors was substantially lower where these insects have been
feeding on a diet containing target dsRNA with dextran sulfate at
the two highest concentrations than with those on a diet with
target dsRNA alone (.chi..sup.2=8.558 and P-value=0.0034 for 5
.mu.g dextran sulfate supplement, and .chi..sup.2=8.764 and
P-value=0.0031 for 50 .mu.g dextran sulfate supplement).
[0155] Thus, it was shown that the sulfated polysaccharide, dextran
sulfate, substantially increased the RNAi-induced lethality of
target dsRNA when the two components were ingested.
Example 2
The Effects of Fucoidan at Different Concentrations
[0156] In a CPB bioassay (set-up described in Example 1), target
Ld105 dsRNA (SEQ ID NO:1) was tested at one quantity (50 ng) in the
presence of fucoidan (average Mw.+-.20 kDa; Sigma cat. no. F5631)
at different amounts ranging from 50 ng to 50 .mu.g per larva.
[0157] The time-to-kill was observed to be reduced with target
Ld105 dsRNA in the presence of fucoidan when compared to target
dsRNA alone, as is shown in FIG. 2. In this bioassay, at day 7,
approx. 70% of larvae were killed in the treatment of dsRNA with 50
.mu.g fucoidan whereas approx. 45% of larvae died on diet
containing target dsRNA alone. Very little or no toxicity was
associated with fucoidan alone.
Example 3
The Effects of Polyinosine at Different Concentrations
[0158] In a CPB bioassay (set-up described in Example 1 but with
polyinosine dissolved in 0.9 M NaCl), target Ld105 dsRNA (SEQ ID
NO:1) was tested at one quantity (50 ng) in the presence of the
nucleotide polyinosine (poly I; Sigma cat. no. P4154) at different
amounts ranging from 50 ng to 50 .mu.g per larva. Very little or no
toxicity was associated with poly I alone.
[0159] No significant effects (P-value>0.05) were obtained, as
is shown in FIG. 3.
Example 4
The Effects of Different Concentrations of Target dsRNA and One
Concentration of Dextran Sulfate (.+-.8 kDa)
[0160] In a CPB bioassay (set-up described in Example 1), target
Ld105 dsRNA (SEQ ID NO:1) was tested at different amounts ranging
from 1 pg to 1 .mu.g in the presence of 50 .mu.g dextran sulfate
per larva.
[0161] The time-to-kill was reduced for treatments with dsRNA at 1
.mu.g or 100 ng and 50 .mu.g dextran sulfate when compared to
target dsRNA alone treatment, as is shown in FIG. 4. At day 7, CPB
mortality with dsRNA alone was 29 and 38% for 1 .mu.g and 100 ng of
Ld105 dsRNA, respectively, whereas in the presence of dextran
sulfate the number of dead larvae had approx. doubled to 79 and
63%, respectively. Very little or no toxicity was associated with
dextran sulfate alone.
[0162] Over the course of the bioassay, the CPB survival numbers of
the 1 .mu.g and 100 ng target dsRNA with dextran sulfate treatments
were significantly reduced when compared to target dsRNA alone at
the same quantities (.chi..sup.2=12.56 and P-value=0.0004, and
.chi..sup.2=5.671 and P-value=0.0173, respectively).
Example 5
The Effects of Dextran Sulfate (.+-.1400 kDa) at Different
Concentrations
[0163] In a CPB bioassay (set-up described in Example 1), target
Ld105 dsRNA (SEQ ID NO:1) was tested at one quantity (50 ng) in the
presence of dextran sulfate (average Mw.+-.1400 kDa; MP Biomedicals
cat. no. 193992) at different amounts ranging from 50 ng to 50
.mu.g per larva.
[0164] The time-to-kill was reduced for treatment dsRNA with 50
.mu.g dextran sulfate, as is shown in FIG. 5. In this bioassay, at
day seven, 75% of larvae had been killed in the treatments of dsRNA
with dextran sulfate at 50 .mu.g, whereas 33% of larvae died on
diet containing target dsRNA alone. Very little or no toxicity was
associated with dextran sulfate alone.
[0165] In this bioassay, overall potency was increased. Over the
course of the whole bioassay, the percentage of CPB larval
survivors was substantially lower where these insects have been
feeding on a diet containing target dsRNA with dextran sulfate at
the highest concentration (i.e. 50 .mu.g) than with those on a diet
with target dsRNA alone (.chi..sup.2=6.680 and P-value=0.0097).
Lower amounts (i.e. 5 .mu.g, 500 ng or 50 ng) of dextran sulfate
with target dsRNA did not significantly increase CPB larval
mortality when compared to target dsRNA alone
(P-value>0.05).
Example 6
The Effects of Heparin at Different Concentrations
[0166] In a CPB bioassay (set-up described in Example 1), target
Ld105 dsRNA (SEQ ID NO:1) was tested at one quantity (50 ng) in the
presence of heparin (MP Biomedicals cat. no. 194114) at different
amounts ranging from 50 ng to 50 .mu.g per larva.
[0167] The time-to-kill was reduced for treatment dsRNA with 50
.mu.g heparin, as is shown in FIG. 6. In this bioassay, at day
seven, approx. 21% of larvae had been killed in the treatments of
dsRNA with heparin at 50 .mu.g, whereas approx. 8% of larvae died
on diet containing target dsRNA alone. Dextran sulfate at 50 .mu.g
per larva with target dsRNA was included in this bioassay and
resulted in 50% mortality at day 7. Very little or no toxicity was
associated with heparin and dextran sulfate (see Example 1)
alone.
[0168] Over the course of the whole bioassay, the percentage of CPB
larval survivors was substantially lower where these insects have
been feeding on a diet containing target dsRNA with heparin at the
highest concentration (i.e. 50 .mu.g) than with those on a diet
with target dsRNA alone (.chi..sup.2=4.995 and P-value=0.026).
Lower concentrations (i.e. 5 .mu.g, 500 ng or 50 ng) of heparin
with target dsRNA did not significantly increase CPB larval
mortality when compared to target dsRNA alone (P-value>0.05).
Target dsRNA with 50 .mu.g dextran sulfate was more toxic than
Ld105 dsRNA alone (.chi..sup.2=22.24 and P-value<0.0001) or with
the highest concentration of heparin tested (.chi..sup.2=8.566 and
P-value=0.0034).
[0169] Thus, it was shown that the sulfated glycosaminoglycan,
heparin, substantially increased the RNAi-induced lethality of
target dsRNA when the two components were ingested.
Example 7
The Effects of Chondroitin Sulfate and Hyaluronic Acid at One
Concentration
[0170] In a CPB bioassay (set-up described in Example 1), target
Ld105 dsRNA (SEQ ID NO:1) was tested at one quantity (50 ng per
larva) in the presence of 50 .mu.g chondroitin sulfate (Sigma cat.
no. C3788) or hyaluronic acid (Sigma cat. no. 53747).
[0171] A strong tendency for a reduction in time-to-kill was
observed (FIG. 7) with target Ld105 dsRNA in the presence of either
chondroitin sulfate or hyaluronic acid when compared to Ld105 dsRNA
alone.
Example 8
The Effects of Dextran Sulfate (.+-.8 kDa) on Bacterial-Expressed
dsRNA Activity
[0172] Heat-killed recombinant Escherichia coli with expressed
double-stranded RNA of target Ld105 (designated as pGBNJ003) or
control (designated as pGN29) was tested at different amounts in
the presence of 50 .mu.g dextran sulfate (Mw.+-.8 kDa) in a CPB
bioassay (set-up described in Example 1). Preparation of the
recombinant bacteria is described in the international publication
WO 2007/080127 (see pages 55-56).
[0173] The time-to-kill was substantially reduced for each
treatment with pGBNJ003 plus dextran sulfate when compared to
pGBNJ003 only treatments (FIG. 8). For example, at day 10, dextran
sulfate increased CPB larval mortality from 31% to 88% for pGBNJ003
at 0.25 U, from 62% to 88% for pGBNJ003 at 008 U, and from 13% to
56% for pGBNJ003 at 0.027 U (where 1 unit (U) corresponds to the
amount of bacteria present in 1 mL culture with an OD600 nm value
of 1 prior to heat-inactivation).
[0174] Over the course of the bioassay, the CPB survival numbers in
all treatments of pGBNJ003 with dextran sulfate were significantly
reduced when compared to pGBNJ003 alone at the respective
concentrations (.chi..sup.2=10.93 and P-value=0.0009 for 0.25 U,
.chi..sup.2=7.524 and P-value=0.0061 for 0.08 U, .chi..sup.2=11.33
and P-value=0.0008 for 0.027 U). No toxicity was observed with
pGN29 controls in the absence or presence of dextran sulfate.
[0175] Thus, it was demonstrated that dextran sulfate substantially
increased RNAi-induced lethality of bacterial-expressed target
dsRNA when both components were ingested by the CPB larvae.
Example 9
The Effects of Dextran Sulfate (.+-.8 kDa) with dsRNAs from
Different Targets
[0176] In one CPB bioassay (set-up described in Example 1),
different target dsRNAs (SEQ ID NO:1, 2, 3, 4; Ld105 dsRNA=SEQ ID
NO:1, Ld013=SEQ ID NO: 2, Ld009=SEQ ID NO: 3 and Ld248=SEQ ID NO:
4) were tested at one quantity (100 ng) in the presence of dextran
sulfate (50 .mu.g).
[0177] Over the course of the bioassay, the percentages of CPB
larval survivors were substantially lower where these insects have
been feeding on a diet containing any of the target dsRNAs in
combination with dextran sulfate than those on a diet with the
respective target dRNAs alone (Ld009: .chi..sup.2=15.70 and
P-value<0.0001; Ld013: .chi..sup.2=14.50 and P-value=0.0001;
Ld105: .chi..sup.2=20.57 and P-value<0.0001; Ld248:
.chi..sup.2=13.45 and P-value=0.0002), as is shown in FIG. 9. The
effects observed with Ld105 dsRNA plus dextran sulfate in this
assay were comparable to those observed in another assay shown in
FIG. 4 (of Example 4); the target dsRNA alone treatment responses
were different between the two assays as a consequence of
variabilities between assays.
[0178] Thus, it was demonstrated that the sulfated polysaccharide,
dextran sulfate, substantially increased the RNAi-induced lethality
of different target dsRNAs when the two components were
ingested.
Example 10
The Effects of Dextran Sulfate (.+-.8 kDa) Using an Alternative
Bioassay System
[0179] An alternative bioassay was used using potato leaf discs
rather than artificial diet as food source for CPB. Discs of
approximately 0.9 cm in diameter (or 0.64 cm.sup.2) were cut out
off leaves of 4- to 6-week-old potato plants using a suitably sized
cork borer. Treated leaf discs were prepared by applying 2 .mu.l of
a solution comprising 100 ng target Ld105 dsRNA (SEQ ID NO:1) and
50 .mu.g dextran sulfate on the adaxial leaf surface. The leaf
discs were allowed to dry and placed individually in 24 wells of a
48-well multiwell plate. A single second-larval stage CPB was
placed into each well and the plate covered with a plastic lid.
Twenty-four larvae were tested per treatment. The plates containing
the larvae on leaf discs were kept in the insect rearing chamber as
described in Example 1. After 24 hours, the larvae were transferred
to fresh, untreated leaf discs and placed in the chamber for a
further 24 hours. This was repeated once after which the CPB larvae
were transferred on to artificial diet where they were kept until
the end of the bioassay.
[0180] The time-to-kill was reduced for treatments with dsRNA at
100 ng or 10 ng and 50 .mu.g dextran sulfate when compared to
target dsRNA alone treatment, as is shown in FIG. 10. At day 8, CPB
mortality with dsRNA alone was 33 and 21% for 100 and 10 ng of
Ld105 dsRNA, respectively, whereas in the presence of dextran
sulfate the number of dead larvae had approx. doubled to 71 and
58%, respectively. Very little toxicity was associated with dextran
sulfate alone.
[0181] Over the course of the bioassay, the CPB survival numbers of
the 100 and 10 ng target dsRNA with dextran sulfate treatments were
significantly reduced when compared to target dsRNA alone at the
same concentrations (.chi..sup.2=5.071 and P-value=0.0243, and
.chi..sup.2=15.42 and P-value<0.0001, respectively).
Example 11
Priming Insects with Dextran Sulfate for Increased RNAi-Induced
Killing
[0182] In one CPB bioassay, target Ld105 dsRNA (SEQ ID NO:1) was
tested at one quantity (1 .mu.g) following a short exposure to
dextran sulfate only. The bioassay set-up was similar to that
described in Example 1, except that the larvae were first fed diet
with topically applied dextran sulfate (DS; 50 .mu.g), or water,
for two full days before transferring them to fresh diet with
topically applied Ld105 dsRNA, dextran sulfate or water only, for
the remainder of the assay.
[0183] The time-to-kill was reduced for treatment with dsRNA after
first fed with dextran sulfate (i.e. DS/dsRNA), as is shown in FIG.
11. In this bioassay, at day eight, approx. 58% of larvae had been
killed in the DS/dsRNA treatment, whereas approx. 38% of larvae
died in the water/dsRNA treatment. Very little or no toxicity was
observed in the water/water and DS/DS treatments.
[0184] Over the course of the whole bioassay, there was a tendency
towards a lower percentage of CPB larval survivors in the DS/dsRNA
treatment when compared to the water/dsRNA treatment.
Example 12
The Effects of Dextran Sulfate (.+-.8 kDa) on RNAi-Induced Killing
of the Mustard Leaf Beetle
[0185] To test the effects of dextran sulfate on the RNAi-induced
killing of another insect species, namely, the mustard leaf beetle
(Phaedon cochleariae; MLB), a leaf-disc assay was used. Discs of
approximately 1.1 cm in diameter (or 0.95 cm.sup.2) were cut out
off leaves of 4- to 6-week-old oilseed rape plants using a suitably
sized cork borer. Treated leaf discs were prepared by applying 25
.mu.l of a 0.05% Triton X-100 solution comprising 50 ng target
Pc105 dsRNA (SEQ ID NO: 5) and 50 ng dextran sulfate on the adaxial
leaf surface. The droplet was spread over the leaf disc surface and
the leaf discs were allowed to dry. The leaf discs were placed
individually in wells of a 128-well multiwell plate
(CD-International) containing 1 ml of gellified 2% agar. A single
second-larval stage MLB was placed into each well and the plate
covered with a plastic lid. Thirty larvae were tested per
treatment. The plates containing the larvae on leaf discs were kept
in the insect rearing chamber as described for CPB in Example 1.
The larvae were allowed to consume the whole treated leaf disc
before transferring them onto fresh untreated leaf discs.
Refreshing of the leaf material was repeated until the end of the
bioassay.
[0186] The time-to-kill was reduced for treatment dsRNA with
dextran sulfate, as is shown in FIG. 12. In this bioassay, at day
four, approx. 90% of larvae had been killed in the treatment dsRNA
with dextran sulfate, whereas approx. 60% of larvae died on leaf
discs treated with target dsRNA alone.
[0187] Over the course of the whole bioassay, the percentage of MLB
larval survivors was substantially lower where these insects have
been feeding on leaf discs treated with target dsRNA plus dextran
sulfate than those on leaf discs with target dsRAN alone
(.chi..sup.2=7.995 and P-value=0.0047).
[0188] Thus, it was shown that dextran sulfate substantially
increased the RNAi-induced lethality of target dsRNA when the two
components were ingested by an insect species other than the
Colorado potato beetle, namely, the mustard leaf beetle.
Example 13
The Effects of Dextran Sulfate (.+-.8 kDa) on RNAi-Induced
Phenotype of a Non-Insect Species, the Nematode Caenorhabditis
elegans
[0189] The effects of dextran sulfate on the RNAi-induced
phenotypes of a non-insect species, namely, the nematode
Caenorhabditis elegans, were tested in a soaking assay. Wild-type
worms were chunked from a stock plate and adults were bleached to
obtain a well-staged population. The assay was set up in a V-shaped
96-well plate, with each well containing 20 .mu.l of solution
comprising 50 ng/.mu.l target rps-14 dsRNA (SEQ ID NO:6), various
concentrations of dextran sulfate (0 mg/ml, 0.25 mg/ml, 0.50 mg/ml
and 1 mg/ml) and 50 first-larval stage nematodes. Each treatment
was performed in triplicate. The plate containing the worms was
incubated at 25.degree. C. for overnight soaking After 17 hours,
the worms were transferred to 3.5 cm agar-plates seeded with OP50
E. coli and incubated at 20.degree. C. for a further 2 days. After
this growing period, the assay was assessed by counting the
different life stages for all treatments.
[0190] Treatments with 50 ng/.mu.l dsRNA and dextran sulfate
resulted in a reduction in numbers of adults and L4 stage larvae,
while the number of L3 stage and dead larvae increased when
compared to target dsRNA alone treatment, as shown in FIG. 13. The
number of adults and L4 stage larvae decreased from 63% and 19%,
respectively, to 48% and 11%, respectively, in the dsRNA plus 1
mg/ml dextran sulfate treatment. Number of L3 stage and dead larvae
increased from 11% and 7%, respectively, to 28% and 13%,
respectively, in the dsRNA plus 1 mg/ml dextran sulfate treatment.
No toxicity was associated with dextran sulfate alone (data not
shown).
[0191] In this bioassay, an increase in developmental delay of the
C. elegans population was observed when worms were soaked in target
dsRNA in the presence of dextran sulfate compared to the target
dsRNA alone treatment.
[0192] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
[0193] All references disclosed herein are incorporated by
reference in their entirety for the purposes indicated herein.
Sequence CWU 1
1
611504DNALeptinotarsa decemlineata 1gcttgttgcc cccgaatgcc
ttgatagggt tgattacctt tgggaagatg gtccaagtgc 60acgaactagg taccgagggc
tgcagcaaat cttacgtttt ccgagggacg aaagacctca 120cagctaagca
agttcaagag atgttggaag tgggcagagc cgcagtaagt gctcaacctg
180ctcctcaaca accaggacaa cccatgaggc ctggagcact ccagcaagct
cctacgccac 240caggaagcag gttccttcaa cccatctcga aatgcgacat
gaacctcact gatcttattg 300gagagttgca aagagaccca tggcctgtcc
accaaggcaa atgcgccctt agatcgaccg 360ggacagcttt atcgatagcc
attgggttgt tggagtgcac atacgccaat actggtgcca 420gggtcatgct
attcgttgga ggaccttgct ctcaaggccc tggtcaagtc ttgaatgatg
480atctgaagca acctatcaga tctcaccacg acatccaaaa agacaatgcc
aaatacatga 540agaaagcaat caagcactat gataatttag cgatgagagc
agcaacgaat ggccactgcg 600ttgacatata ttcatgcgct ttggatcaga
caggattgat ggagatgaaa cagtgttgta 660attcaacagg gggacatatg
gtcatgggcg actcgttcaa ttcttccctg ttcaagcaaa 720cgttccagcg
catattttcg aaagatcaga aaaacgagct gaagatggca tttaatggta
780ctctggaggt caagtgttcc agggagttga aaattcaagg cggtattgga
tcttgtgttt 840cgttgaatgt gaagaatcct ttggtttccg acaccgaaat
aggaatgggt aacacggtcc 900agtggaaaat gtgtacggta actccaagta
ctaccatggc cttgttcttc gaggtcgtca 960accaacattc cgctcccata
cctcaagggg gaaggggctg catacagttc atcacgcaat 1020atcagcatgc
tagtggccag aagaggatcc gagtaacgac agttgctaga aactgggccg
1080atgcttccgc taatatacat catgtcagtg ctggattcga tcaggaggca
gccgcagtga 1140taatggcgag gatggcagtt tacagagcgg aatcagacga
tagccctgat gttttgagat 1200gggtcgatag gatgttgata cgtctgtgcc
agaaattcgg cgaatataac aaggacgacc 1260cgaattcgtt ccgcttgggc
gaaaacttca gcctctaccc gcagttcatg taccatttga 1320gaaggtcaca
gttcctgcag gtgtttaaca attctcccga cgaaacgtcc ttctacaggc
1380acatgcttat gcgcgaagac ctcacgcagt cgctgatcat gatccagccg
atactctaca 1440gctacagttt caatggacca ccagaacctg tgcttttgga
tacgagttcc atccaacccg 1500atag 15042395DNALeptinotarsa decemlineata
2ggccccaaga agcatttgaa gcgtttgaat gccccaaaag catggatgtt ggataaattg
60ggaggtgttt tcgcacctcg cccatctaca ggacctcaca aattgcgaga gtctttgccc
120ttggtgatct tcctacgtaa ccgattgaag tatgctttga ctaacagcga
agttactaag 180attgttatgc aaaggttaat caaagtagat ggaaaagtga
ggaccgactc caattaccct 240gctgggttta tggatgttat taccattgaa
aaaactggtg aatttttccg actcatctat 300gatgttaaag gacgatttgc
agtgcatcgt attactgctg aggaagcaaa gtacaaacta 360tgcaaagtca
ggaggatgca aactggcccc aaagg 3953203DNALeptinotarsa decemlineata
3cgccggagag tttttgtcag cttcttcaaa agctttgcgc aagttactct cagactcgcc
60agcgagtttg ctcatgatct ccggcccgtt tatcaagaag aagaacgccc cagtctcatt
120agccacggcg cgagcaatca gggtcttacc cgtaccaggg ggaccataca
gcagtatacc 180cctagggggc ttcacgccga tag 2034967DNALeptinotarsa
decemlineata 4gggagcagac gatcggttgg ttaaaatctg ggactatcaa
aacaaaacgt gtgtccaaac 60cttggaagga cacgcccaaa acgtaaccgc ggtttgtttc
caccctgaac tacctgtggc 120tctcacaggc agcgaagatg gtaccgttag
agtttggcat acgaatacac acagattaga 180gaattgtttg aattatgggt
tcgagagagt gtggaccatt tgttgcttga agggttcgaa 240taatgtttct
ctggggtatg acgagggcag tatattagtg aaagttggaa gagaagaacc
300ggcagttagt atggatgcca gtggcggtaa aataatttgg gcaaggcact
cggaattaca 360acaagctaat ttgaaggcgc tgccagaagg tggagaaata
agagatgggg agcgtttacc 420tgtctctgta aaagatatgg gagcatgtga
aatataccct caaacaatcc aacataatcc 480gaatggaaga ttcgttgtag
tatgcggaga cggcgaatat atcatttaca cagcgatggc 540tctacggaac
aaggcttttg gaagcgctca agagtttgtc tgggctcagg actccagcga
600gtatgccatt cgcgagtctg gttccacaat tcggatattc aaaaacttca
aagaaaggaa 660gaacttcaag tcggatttca gcgcggaagg aatctacggg
ggttttctct tggggattaa 720atcggtgtcc ggtttaacgt tttacgattg
ggaaactttg gacttggtga gacggattga 780aatacaaccg agggcggttt
attggtctga cagtggaaaa ttagtctgtc tcgcaacgga 840ggacagctac
ttcatccttt cttatgattc ggagcaagtt cagaaggcca gggagaacaa
900tcaagtcgca gaggatggcg tagaggccgc tttcgatgtg ttgggggaaa
tgaacgagtc 960tgtccga 96751546DNAPhaedon cochleariae 5gctcagccta
ttaccgccca acgcgttgat tggattgatc acgttcggaa aaatggtgca 60agtccacgaa
ctgggtaccg aaggctgcag caagtcgtac gtgttctgtg gaacgaaaga
120tctcaccgcc aagcaagtcc aggagatgtt gggcattgga aaagggtcac
caaatcccca 180acaacagcca gggcaacctg ggcggccagg gcagaatccc
caagctgccc ctgtaccacc 240ggggagcaga ttcttgcagc ccgtgtcaaa
atgcgacatg aacttgacag atctgatcgg 300ggagttgcag aaagaccctt
ggcccgtaca tcagggcaaa agacctctta gatccacagg 360cgcagcattg
tccatcgctg tcggcctctt agaatgcacc tatccgaata cgggtggcag
420aatcatgata ttcttaggag gaccatgctc tcagggtccc ggccaggtgt
tgaacgacga 480tttgaagcag cccatcaggt cccatcatga catacacaaa
gacaatgcca agtacatgaa 540gaaggctatc aaacattacg atcacttggc
aatgcgagct gccaccaaca gccattgcat 600cgacatttac tcctgcgccc
tggatcagac gggactgatg gagatgaagc agtgctgcaa 660ttccaccgga
gggcacatgg tcatgggcga ttccttcaat tcctctctat tcaaacaaac
720cttccagcga gtgttctcaa aagacccgaa gaacgacctc aagatggcgt
tcaacgccac 780cttggaggtg aagtgttcca gggagttaaa agtccaaggg
ggcatcggct cgtgcgtgtc 840cttgaacgtt aaaagccctc tggtttccga
tacggaacta ggcatgggga atactgtgca 900gtggaaactt tgcacgttgg
cgccgagctc tactgtggcg ctgttcttcg aggtggttaa 960ccagcattcg
gcgcccatac cacagggagg caggggctgc atccagctca tcacccagta
1020tcagcacgcg agcgggcaaa ggaggatcag agtgaccacg attgctagaa
attgggcgga 1080cgctactgcc aacatccacc acattagcgc tggcttcgac
caagaagcgg cggcagttgt 1140gatggcccga atggccggtt acaaggcgga
atcggacgag actcccgacg tgctcagatg 1200ggtggacagg atgttgatca
ggctgtgcca gaagttcgga gagtacaata aagacgatcc 1260gaattcgttc
aggttggggg agaacttcag tctgtatccg cagttcatgt accatttgag
1320acggtcgcag tttctgcagg tgttcaataa ttctcctgat gaaacgtcgt
tttataggca 1380catgctgatg cgtgaggatt tgactcagtc tttgatcatg
atccagccga ttttgtacag 1440ttacagcttc aacgggccgc ccgagcctgt
gttgttggac acaagctcta ttcagccgga 1500tagaatcctg ctcatggaca
ctttcttcca gatactcatt ttccat 15466351DNACaenorhabditis elegans
6ccgctcgtaa gggaaaggct aaggaggaac aggctgtcgt gtcccttgga ccacaggcca
60aagaaggaga gctcatcttc ggagtcgctc acatctttgc ttcgttcaac gacactttcg
120tccacatcac cgatatctca ggacgtgaaa ccatcgttcg agttaccgga
ggaatgaagg 180tcaaggccga tcgtgacgag tcatcgccat acgctgctat
gctcgccgct caagacgtcg 240ctgatcgttg caaacaactc ggaatcaacg
ctcttcacat caagcttcgt gctactggag 300gaaccagaac caagacccca
ggaccaggag ctcagtctgc tcttcgtgcc c 351
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