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.

We claim: 
     
         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.