WO2021204594A1 - Nucleic acid chaperone mixtures for insect control - Google Patents

Abstract

The present invention provides a composition for the stabilisation of an RNA interference (RNAi) agent against degradation by insect saliva or gut enzymes, the composition comprising a) one or more RNA interference (RNAi) agents, and b) a chaperone moiety comprising a polyfunctional acidic compound having at least 3 net negative charges, or its corresponding salt.

Description

NUCLEIC ACID CHAPERONE MIXTURES FOR INSECT CONTROL

Field of the Invention

The present invention relates to compositions comprising nucleic acids and to methods for enhancing delivery into insects.

Background of the Invention

Economic agricultural losses due to insects are today considerable for many staple crops such as corn, soybeans, peas, and cotton. Pests that threaten natural and cultivated ecosystems even represent a mounting concern due to climate changes and to the increasing world trade and travel, which are now known to induce modification of the geographic distribution areas of insect species. Indeed, pest species are expected to become new threats for agriculture and hence economy in regions where such insects were not endemic and where natural parasites or pathogens may be absent. Infestations with such pests may become particularly pronounced in monocultures, where the insect pests may not only directly damage the crops, but also act as vectors introducing a variety of fungal or bacterial pathogens. While the traditional synthetic pesticides to combat these pests are under pressure, other methods focused on delivering insecticidal activity by microorganisms or genes derived from microorganisms, for instance by expressing them in transgenic plants.

Bacterial toxins have been used due to their pesticidal activity against a broad range of insect pests including Lepidoptera, Diptera, Coleoptera, and Hemiptera. Hence an approach for insect control was to genetically engineer crops such as corn and cotton plants producing insecticidal proteins derived from Bacillus, which have provided farmers with an alternative to traditional insect- control methods. However, such insecticidal proteins expressed were found to only protect plants from a relatively narrow range of pests. Moreover, these modes of insecticidal activity provide varying levels of specificity and, in some cases, may cause undesired environmental consequences. Yet further, with the adoption of transgenic controls for major lepidopteran pests in crops, chemical insecticides are no longer used and hence hemipterans (Heteroptera), in particular stinkbugs have become major secondary pests. In particular, no successful practical use of a transgenic control of stinkbugs has thus far been described or adopted. This may be due in part to the extra oral digestion employed by stinkbugs where digestive enzymes are injected into the host plant prior to feeding, thereby digesting insecticidal proteins.

RNAi agents as control agents have been seen as a potential environmentally friendly approach to reduce the pest populations. Herein, insecticidal double-stranded ribonucleic acid (dsRNA) compounds are used, which may be used to silence vital genes in the insect organisms through the mechanism of RNA interference (RNAi), for example as disclosed in US20090238805A1. However, it was found that a major obstacle for the technology resides in the fact that insect saliva not only is able to digest proteins, but also often contains highly active ribonucleases, thereby severely reducing the effect of RNAi by degrading the dsRNA compounds.

Accordingly, there is an immediate need for alternative methods to control such pests. Summary of the Invention

Applicants now surprisingly found that a composition formed by adding a chaperone compound comprising polyanionic compounds to the RNAi agents, the latter were protected from enzymatic degradation in insect saliva, while retaining sufficient bioavailability for manifesting a control of the insect population. Accordingly, the present invention provides methods and compositions employing an RNAi silencing agent in combination with a chaperone component, which mixture is capable of decreasing the expression of a target sequence in the pest after ingestion.

Accordingly, in a first aspect, the present invention relates to a composition for the stabilization of an RNA interference (RNAi) agent against degradation by insect saliva or gut enzymes, the composition comprising a combination comprising a) one or more RNA interference (RNAi) agents, and b) a chaperone moiety comprising a polyfunctional acidic compound having at least three net negative charges, or its corresponding salt.

In a further aspect, the present invention provides compositions and methods for delivering nucleic acid molecules to an insect cell, in particular of Pentatomidae plant pest cells including for example, cells of a Nezara viridula (southern green stinkbug), Oebalus pugnax (rice stinkbug), Oebalus insularis, Chinavia hilaris or Acrosternum hilare (green stinkbug), Bagrada hilaris (bagrada bug), Proxys punctulatus (black stinkbug), Piezodorus guildinii (redbanded stinkbug), Euschistus servus (brown stinkbug), Euchistus tristigmus (dusky stinkbug), Euschistus herns (neotropical brown stink bug), melacanthus and Dichelops furcatus (green-belly stink bugs) and/or Halymorpha halys (brown marmorated stinkbug) plant pests.

In yet a further aspect, the present invention provides compositions and methods for enhancing delivery of nucleic acids into insect cells and for modifying expression of target genes in the cells.

In yet a further aspect, the present invention also provides a fast screening method for the efficacy of chaperone RNAi agent stabilization.

In yet a further aspect, the present invention also provides a method for controlling insects, comprising applying a composition comprising a combination comprising a) one or more RNA interference (RNAi) agents, and b) a chaperone moiety comprising a polyfunctional acidic compound, or its corresponding salt, to an insect or its environment, an insect food, a plant or plant propagation material or the locus where the plant or plant propagation material is planted.

In yet a further aspect, the present invention also provides a method for reducing degradation of double stranded RNA in or on an insect, comprising the step of adding to a composition comprising the double stranded RNA a polyfunctional acidic compound or its corresponding salt.

In yet a further aspect, the present invention also provides a method for reducing degradation of double stranded RNA in or on an insect, which comprises applying a composition comprising a combination comprising a) one or more RNA interference (RNAi) agents, and b) a chaperone moiety comprising a polyfunctional acidic compound, or its corresponding salt, to an insect or their environment, an insect food, a plant or plant propagation material or the site where the plant or plant propagation material is planted.

In yet a further aspect, the present invention also provides a method for determining the stability of a RNAi agent against insecticidal enzymes, comprising a) providing a mixture containing a chaperone and the RNAi agent and insect enzymes, b) incubating the mixture for a suitable period of time, and c) performing an electrophoresis to determine degradation of the starting RNAi agent. Short Description of the Figures

Figure 1 shows a saliva degradation gel electrophoresis assay, employing a sodium polyacrylate chaperone composition, with increasing concentrations of dsRNA agents and stink bug saliva, and after incubation for 24h at 25°C.

Figure 2 shows a saliva degradation gel electrophoresis assay, employing a sodium pyrophosphate chaperone composition, with increasing concentrations of dsRNA agents and stink bug saliva, and after incubation for 24h at 25°C.

Figure 3 shows a saliva degradation gel electrophoresis assay, employing a poly(4- styrenesulfonic acid-co-maleic acid), sodium salt, composition, with increasing concentrations of dsRNA agents and stink bug saliva, and after incubation for 24h at 25°C.

Figure 4 shows a saliva degradation gel electrophoresis assay, employing a monosodium glutamate chaperone composition, with increasing concentrations of dsRNA agents and stink bug saliva, and after incubation for 24h at 25°C.

Figure 5 shows a saliva degradation gel electrophoresis assay, employing a potassium glyphosate chaperone composition, with increasing concentrations of dsRNA agents and stink bug saliva, and after incubation for 24h at 25°C.

Detailed Description of the Invention The present invention relates to a composition for the stabilisation of an RNA interference (RNAi) agent against degradation by insect saliva or gut enzymes, wherein the composition comprises a) one or more RNA interference (RNAi) agents, and b) a chaperone moiety comprising a polyfunctional acidic compound having at least 3 net negative charges, or its corresponding salt.

In another embodiment, a composition and method for controlling a pest expressing nucleases in their saliva or gut, and where the nuclease activity limits the ability of orally acquired dsRNA to cause gene silencing.

In another embodiment, a composition and method for controlling a pest expressing nucleases in their saliva or gut, and where the nuclease activity limits the ability of orally acquired dsRNA to cause gene silencing, such as insect plant pests, such as for example, Anthonomus grandis (boll weevil), are provided.

In another embodiment, a composition and method for controlling a pest expressing nucleases in their saliva or gut, and where the nuclease activity limits the ability of orally acquired dsRNA to cause gene silencing, such as a plant pest of the order Hemiptera, such as, for example, Lygus lineolaris (tarnished plant bug), Nasonovia ribisnigri, Aphis gossypii (melon and cotton aphid), Myzus persicae (peach-potato aphid), Bemisia tabaci (silverleaf whitefly), Empoasca fabae (potato leafhopper) and/or Nilaparvata lugens (brown planthopper), are provided.

In an embodiment, compositions and methods for controlling a pest, such as a harmful insect. Such insects can occur in any taxonomic group of insects, but are especially commonly found in the beetles (Coleoptera), two-winged insects (Diptera) and butterflies (Lepidoptera). Preferably, compositions and methods for controlling pentatomidae plant pest, such as, for example, a Nezara viridula, Oebalus pugnax, Oebalus insularis, Chinavia hilaris, Bagrada hilaris, Proxys punctulatus, Acrosternum hilare, Piezodorus guildini, Euschistus servus, Euchistus tristigmus, Euschistus herns, melacanthus and Dichelops furcatus (green-belly stink bugs) and/or Halymorpha halys plant pest, are provided.

Other targets include any pests that express nucleases in their saliva, and where the nuclease activity limits the ability of orally acquired dsRNA to cause gene silencing.

The method comprises feeding to a pest a composition comprising an RNAi Agent stabilized by a chaperone component, wherein the composition, when ingested by the pest, reduces the level of a target sequence in the pest and thereby controls the pest.

Further provided are methods to protect a plant from a pest, comprising applying a composition according to the invention onto a plant, a plant part or the locus of a plant, as part of a topical formulation. When the pest ingests the silencing element, the level of the target sequence is decreased in the pest and the pest is controlled. Double-stranded ribonucleic acid (dsRNA) is used to silence vital genes in organisms through the mechanism of RNA interference (RNAi). This mechanism can be exploited to control insect pests in the field. A major obstacle of this technology for stink bugs is that dsRNA is readily degraded by nucleases found in the stink bug saliva. By adding a variety of polyanionic polymers to a dsRNA solution, we have shown that dsRNA is protected from enzymatic degradation in stink bug saliva while still remaining bioavailable for control of the insect.

In a particularly preferred embodiment, the present invention involves adding polyacidic chaperone compounds to in vitro transcribed (IVT) dsRNA or dsRNA from a dilute bacterial lysate solution, to protect the dsRNA from salivary nucleases in insects, preferably in stink bugs.

The addition of polyanions was found to protect dsRNA in an agarose gel-based saliva degradation assay.

Examples for suitable chaperone compounds include compounds having at least three, preferably more than three acid functionalities. The term "Acid functionality" herein refers to a Bronsted acid, such as carboxylates, sulfonates, sulphates, phosphates, phosphonates and so on, thereby forming polycarboxylates, polysulfonates, polysulfates, polyphosphates, polyphosphonates, wherein each acid group present in the chaperone compound contributes to the net negative charge.

Net negative charge is defined as a group having a net negative charge in a basic solution. These compounds may be supplied in acidic form or neutralized with non-polymeric bases.

Suitable polyanionic compounds include, but are not limited to: (concentrations shown to inhibit degradation of 200 ng/ul IVT dsRNA after 24 hours in parentheses): Sodium polyacrylate (> 0.016% w/w); Sodium chondroitin sulfate (> 0.25% w/w); Sodium dextran sulfate (> 0.004% w/w) ); poly (vinyl sulfonic acid) (> 0.016% w/w); Sodium polyphosphate (> 0.25% w/w); Sodium pyrophosphate (> 1.0% w/w); Sodium hexametaphosphate (> 0.25% w/w); Disodium EDTA (> 0.125% w/w); Tetrasodium EGTA (> 0.125% w/w); Poly (aspartic acid) (> 2.0% w/w); Sodium citrate dihydrate (> 0.5% w/w); Potassium poly (sulfopropyl acrylate) (> 0.125% w/w); Sodium poly (methacrylic acid) (> 0.5% w/w); Sodium poly (4-styrenesulfonic acid-co-maleic acid) (> 0.125% w/w); Powerblox D-518 (sodium polyacrylate from Dow) (> 0.005% w/w); Ultrazine NA (lignosulfonate from Borregaard) (> 0.016% w/w); Polyfon H (lignosulfonate from Ingevity) (> 0.016% w/w); MorWet D390 (naphthalene sulfonate condensate from Nouryon) (> 0.016% w/w).

The polyacidic chaperone compounds above were found effective, from which we deduce that all polyanions (polycarboxylates, polysulfonates, polysulfates, polyphosphates, polyphosphonates, etc.) will have a similar effect. Polyfunctional acidic chaperone compounds may be effective when present in an amount of for example, at least 0.0001% w/w, or at least 0.001% w/w, or at least 0.002% w/w, or at least 0.003% w/w, or at least 0.004% w/w, or at least 0.005% w/w, or at least 0.01% w/w, or at least 0.016% w/w, or at least 0.02% w/w, or at least 0.10% w/w, or at least 0.20% w/w, or at least 0.25% w/w, or at least 0.5% w/w, or at least 1% w/w, or at least 1.5% w/w, or at least 2.0% w/w, or at least 2.5% w/w, or at least 5.0% w/w.

Polyfunctional acidic chaperone compounds may be preferably effective when present in a defined range of, for example, from 0.005% to 5.0% w/w, or from 0.005% to 2.0% w/w, or from 0.005% to 1.0% w/w, or from 0.016% to 5.0% w/w, or from 0.016% to 2.0% w/w, or from 0.016% to 1.0% w/w, or from 0.125% to 5.0% w/w, or from 0.125% to 2.0% w/w, or from 0.125% to 1.0% w/w.

Contrarily, compounds tested with less than three acid moieties did not exhibit an inhibitory effect in the agarose gel-based saliva degradation assay. These comparative examples included monosodium glutamate and potassium glyphosate, even when applied to the high concentration of up to 2% w/w. (At physiological or neutral pH, glyphosate has net two negative charges.) The gel electrophoresis results were then confirmed in a plant bioassay.

In a preferred embodiment the present invention provides for a composition for delivering an insecticidal double-stranded RNA function inhibitor, hereafter referred to as "RNAi inhibitor" to an insect cell.

It further advantageously relates to the delivery of the inhibitor resulting in inhibition of target gene expression by causing degradation of RNA. Inhibitors may be selected from siRNA, dsRNA, antisense nucleic acid, ribozymes, RNA polymerase III transcribed DNAs, and the like. A preferred inhibitor is dsRNA.

In a preferred embodiment, a process for delivery of an inhibitor to a cell of an insect for the purposes of inhibition of gene expression comprising providing an inhibitor compound, contacting the inhibitor compound with a chaperone composition, and delivering the inhibitor to a crop or the area or locus of a crop likely to be, or already infected with an insect pest.

A variety of chaperone compounds can be used in conjunction with the RNAi agents to mediate the stabilization. In a preferred embodiment, the present invention provides a process for delivering an inhibitor to an animal cell comprising; mixing an inhibitor compound and an effective amount of a polyacidic compound in a solution, and associating the combination with an insect animal cell, and delivering the inhibitor to the interior of an insect cell. The inhibitor then inhibits expression of a gene in the cell. The term "deliver" herein means that the RNAi agent becomes associated with the cell thereby altering the endogenous properties of the cell by inhibiting expression of a gene. ln a preferred embodiment, a combination of two or more inhibitors are delivered together or sequentially to enhance inhibition of target gene expression.

In a preferred embodiment, an inhibitor may be delivered to a cell in an insect for the purposes of inhibiting a target gene to provide a lethal, growth and/or development limiting effect.

In a further embodiment, an inhibitor is delivered to an insect cell for the purpose of facilitating take-up of a different insecticidally or otherwise agrochemically active ingredient, for instance by reduction of the expression of an enzyme able to digest or otherwise inactivate the active ingredient.

Further objects, features, and advantages of the invention will be apparent from the following detailed description when taken in conjunction with the accompanying figures.

The composition comprising one or more RNAi agent(s) and a chaperone compound described herein are preferably formulated into sprayable or otherwise applicable compositions. It is hence also an object of the invention to provide improved compounds, compositions, and formulations for RNAi agent delivery. It is further an object of the invention to provide methods of making improved compounds, compositions, and formulations for temporospatial RNAi agent delivery.

An RNA function inhibitor ("RNAi agent") herein comprises any nucleic acid or nucleic acid analog containing a sequence ("inhibiting sequence") whose presence or expression in a cell causes the degradation of or inhibits the function or translation of a specific cellular RNA, usually a mRNA, in a sequence- specific manner. Inhibition of RNA can thus effectively inhibit expression of a gene from which the RNA is transcribed.

Inhibitor compounds may advantageously be selected from the group comprising: siRNA, interfering RNA or RNAi, dsRNA, RNA Polymerase III transcribed DNAs, ribozymes, and antisense nucleic acid, or artificial nucleic acid. SiRNA comprises a double stranded structure typically containing 15-50 base pairs and preferably 21-25 base pairs and having a nucleotide sequence identical or nearly identical to an expressed target gene or RNA within the cell. Antisense polynucleotides include, but are not limited to: morpholinos, 2'-0-methyl polynucleotides, DNA, RNA and the like. RNA polymerase III transcribed DNAs contain promoters, such as the U6 promoter. These DNAs can be transcribed to produce small hairpin RNAs in the cell that can function as siRNA or linear RNAs that can function as antisense RNA. The inhibitor may be polymerized in vitro, recombinant RNA, contain chimeric sequences, or derivatives of these groups. The inhibitor may contain ribonucleotides, deoxyribonucleotides, synthetic nucleotides, or any suitable combination such that the target RNA and/or gene is inhibited. In addition, these forms of nucleic acid may be single, double, triple, or quadruple stranded. A delivered inhibitor can stay within the cytoplasm or nucleus. The inhibitor can be delivered to a cell to inhibit expression of an endogenous or exogenous nucleotide sequence or to affect a specific physiological characteristic not naturally associated with the cell. An inhibitor may advantageously be delivered to a cell in order to produce a cellular change that is insecticidal, or is stopping the proliferation or development of the insect into a different developmental stage.

The term nucleic acid, or polynucleotide, is a term of art that refers to a string of at least two nucleotides. Nucleotides are the monomeric units of nucleic acid polymers. Polynucleotides with less than 120 monomeric units are often called oligonucleotides. Natural nucleic acids have a deoxyribose- or ribose-phosphate backbone while artificial polynucleotides may be polymerized in vitro and contain the same or similar bases but may contain other types of backbones. These backbones include: PNAs (peptide nucleic acids), phosphorothioates, phosphorodiamidates, morpholinos, and other variants of the phosphate backbone of native nucleic acids. Bases include purines and pyrimidines, which further include the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs. Synthetic derivatives of purines and pyrimidines include, but are not limited to, modifications which place new reactive groups on the base such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkylhalides. The term base encompasses any of the known base analogs of DNA and RNA including, but not limited to: 4- acetylcytosine, 8-hydroxy-N-6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5- (carboxyhydroxyl-methyl) uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2- thiouracil, 5-carboxymethyl-aminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1- methyladenine, 1-methylpseudo-uracil, 1-methylguanine, 1-methylinosine, 2,2-dimethyl-guanine, 2- methyladenine, 2-methylguanine, 3-methyl-cytosine, 5-methylcytosine, N6-methyladenine, 7- methylguanine, 5-methylaminomethyluracil, 5-methoxy-amino-methyl-2-thiouracil, beta-D- mannosylqueosine, 5'-methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6- isopentenyladenine, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5- methyluracil, N-uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine.The term includes deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). DNA may be in form of cDNA, in vitro polymerized DNA, plasmid DNA, parts of a plasmid DNA, genetic material derived from a virus, linear DNA, chromosomal DNA, an oligonucleotide, antisense DNA, or derivatives of these groups. RNA may be in the form of tRNA (transfer RNA), snRNA (small nuclear RNA), rRNA (ribosomal RNA), mRNA (messenger RNA), antisense RNA, siRNA (small interfering RNA), dsRNA (double stranded RNA), RNAi, ribozymes, in vitro polymerized RNA, or derivatives of these groups. The polyacidic compound may be selected from polymers. A polymer is a molecule built up by repetitive bonding together of smaller units called monomers. Small polymers having 2 to about 80 monomers can be called oligomers. The polymer can be linear, branched network, star, comb, or ladder type. The polymer can be a homopolymer in which a single monomer is used or can be copolymer in which two or more monomers are used. Types of copolymers include alternating, random, block and graft.

The main backbone chain of a polymer is composed of the atoms whose bonds are required for propagation of polymer length. The side or pendant chains of a polymer are usually composed of the atoms whose bonds are not required for propagation of polymer length. As used herein, the term "polymer backbone" or "backbone" refers to that portion of the polymer which is a continuous chain comprising the bonds which are formed between monomers upon polymerization. The composition of the polymer backbone can be described in terms of the identity of the monomers from which it is formed, without regard to the composition of branches, or side chains, off of the polymer backbone. Thus, poly(acrylic acid) is said to have a poly(ethylene) backbone which is substituted with carboxylic acid (— C(O)OH) groups as side chains.

A "pendant" group is a moiety which forms a side chain or a portion of a side chain attached to the polymer backbone. A pendant group can, for example, be bonded directly to one or more atoms within the polymer backbone or can be connected to the polymer backbone by way of a spacer group. To those skilled in the art, there are several categories of polymerization processes that can be utilized in the described process. The polymerization can be chain or step. This classification description is more often used than the previous terminology of addition and condensation polymerization. "Most step-reaction polymerizations are condensation processes and most chain- reaction polymerizations are addition processes" (M. P. Stevens Polymer Chemistry: An Introduction New York Oxford University Press 1990). Template polymerization can be used to form polymers from daughter polymers.

Step polymerization. In step polymerization, the polymerization occurs in a stepwise fashion. Polymer growth occurs by reaction between monomers, oligomers and polymers. No initiator is needed since the same reaction occurs throughout and there is no termination step so that the end groups are still reactive. The polymerization rate decreases as the functional groups are consumed.

Chain polymerization. In chain- reaction polymerization growth of the polymer occurs by successive addition of monomer units to limited number of growing chains. The initiation and propagation mechanisms are different and there is usually a chain-terminating step. The polymerization rate remains constant until the monomer is depleted. Monomers containing vinyl, acrylate, methacrylate, acrylamide, methacrylamide groups can undergo chain reaction, which can be radical, anionic, or cationic. Chain polymerization can also be accomplished by cycle or ring opening polymerization. Several different types of free radical initiators can be used that include peroxides, hydroxy peroxides, and azo compounds such as 2,2'- Azobis(-amidinopropane) dihydrochloride (AAP). A wide variety of monomers can be used in the polymerization processes.

The polymers may also contain cleavable groups either in the main chain or in side chains. Cleavable groups include but are not restricted to disulfide bonds, diols, diazo bonds, ester bonds, sulfone bonds, acetals, ketals, enol ethers, enol esters, enamines and imines. Preferred cleavable groups include groups that are pH labile.

The polymers may have other functional groups or modifications that increase their utility. These groups can be incorporated into monomers prior to polymer formation or attached to the polymer after its formation.

As discussed above, the polymers utilized in the subject composition comprise pendant acid or anionic groups or moieties, which act as Bronsted acids. Suitable acid functional groups or moieties include carboxylic acid, sulfonic acid, phosphonic acid, phosphoric acid, hydrosulfates, hydrophosphates, sulfamic acid and boronic acid groups, sulfuric acid groups, sulfonic acid groups, phenolic groups, sulfamic acid groups, boronic acid groups, or mixtures thereof.

The acid groups can also be present in the conjugate base form in combination with a suitable cation, i.e. as a salt.

In a preferred embodiment, the polymer comprises a copolymer characterized by a first monomer or repeat unit having a pendant acid functional group and a second monomer or repeat unit having a pendant hydrophobic group. In another embodiment, the polymer is characterized by a monomer or repeat unit having both a pendant acid functional group and a pendant hydrophobic group. The polymer may, optionally, be further characterized by a monomer or repeat unit comprising a neutral hydrophilic group, such as a hydroxyl group or an amide group.

The acid-functionalized monomer comprises a pendant acid functional group, such as a carboxylic acid group, a sulfonic acid group, a hydrosulfate group, a phosphonic acid group, a sulfamic acid group, a hydrophosphate group or a boronic acid group. Acid functional groups are referred to herein as the acid protonated form or partially protonated form. However, it is to be understood that any acid functional group can also exist in the conjugate base or deprotonated form in combination with a pharmaceutically acceptable cation. The polymer to be administered can include acid functional groups in either the protonated form, the deprotonated form or a combination thereof. Suitable cations include alkali metal ions, such as sodium, potassium and cesium ions, alkaline earth ions, such as calcium and magnesium ions, transition metal ions and unsubstituted and substituted (primary, secondary, tertiary and quaternary) ammonium ions.

The acid functional group can be directly bonded to the polymer backbone or can be attached to the polymer backbone via a spacer group. The spacer group is a component of the polymer side chain and connects the acid functional group to the polymer backbone.

The spacer group can be linear, branched or cyclic, aliphatic, aromatic or partially aromatic and partially aliphatic. Suitable aliphatic spacer groups include normal or branched, saturated or partially unsaturated hydrocarbyl groups, including alkylene groups, for example, polymethylene groups such as — (Chhj_n— , wherein n is an integer from 1 to about 20, and cycloalkylene groups, such as the 1,4-cyclohexylene group. Suitable aromatic spacer groups include ortho-, meta- and para-phenylene groups, naphthylene groups and biphenylene groups. The polymer may be homopolymer or a copolymer, a naturally occuring polymer that has been modified, such as for instance lignocellulose of polysaccharides, or a polymer prepared for this purpose, e.g. such as a poly(meth)acrylate or polyvinyl compounds such as an acid-functionalized polyvinyl acetate or alcohol.

A polyelectrolyte, or polyion, is a polymer possessing more than one charge, i.e. the polymer contains groups that have either gained or lost one or more electrons. A polyanion is a polyelectrolyte containing a net negative charge. The polyanion can contain monomer units that are charge negative, charge neutral, or charge positive, however, the net charge on the polymer must be negative. A polyanion can also mean a non-polymeric molecule that contains two or more negative charges.

Applicants have now shown that a combination of polyanionic, in particular polyacidic polymers, whether obtained naturally or synthetic, may be effective RNAi agent chaperons, allowing the RNAi agents to access the insect cells without inactivation by ribonucleases in the insect saliva or gut. Oligomers of nucleic acids are polyanions. It is plausible that the active sites of enzymes that degrade oligomers of nucleic acids are themselves cationic, such that the enzymes have electrostatically-driven affinity to their target substrate.

It is also possible that this invention could be used to protect dsRNA against other enzymes such as those found in the soil or other environments.

Figure 1 shows example results of saliva degradation assay (sodium polyacrylate: PAA-Na). Herein, increasing concentrations of compounds were spiked with dsRNA and stink bug saliva, at a 1:5 dilution, and were incubated for 24 hours at 25°C. Appropriate controls were included. Gel electrophoresis was then run at 100V, 40 min in TAE, and the resultant bands visualized with SYBR Gold. Positive degradation control (PC) was shown with enzyme + dsRNA. Figure 2 shows example results of saliva degradation assay (sodium pyrophosphate). Herein, increasing concentrations of compounds were spiked with dsRNA and stink bug saliva, at a 1:5 dilution, and were incubated for 24 hours at 25°C. Appropriate controls were included. Gel electrophoresis was then run at 100V, 40 min in TAE, and the resultant bands visualized with SYBR Gold. Positive degradation control (PC) was shown with enzyme + dsRNA.

Figure 3 shows example results of saliva degradation assay (poly(4-styrenesulfonic acid-co- maleic acid), sodium salt: "additive"). Herein, increasing concentrations of compounds were spiked with dsRNA and stink bug saliva, at a 1:5 dilution, and were incubated for 24h at 25°C. Appropriate controls were included. Gel electrophoresis was then run at 100V, 40 min in TAE, and the resultant bands visualized with SYBR Gold. Positive degradation control (PC) was shown with enzyme + dsRNA.

Figure 4 shows comparative example results of saliva degradation assay (monosodium glutamate: "MSG"). Herein, increasing concentrations of compounds were spiked with dsRNA and stink bug saliva, at a 1:5 dilution, and were incubated for 24 hours at 25°C. Appropriate controls were included. Gel electrophoresis was then run at 100V, 40 min in TAE, and the resultant bands visualized with SYBR Gold. Positive degradation control (PC) was shown with enzyme + dsRNA. MSG does not protect dsRNA from salivary enzymes at any of the tested concentrations.

Figure 5 shows comparative example results of saliva degradation assay (potassium glyphosate: "PMGK"). Herein, increasing concentrations of compounds were spiked with dsRNA and stink bug saliva, at a 1:5 dilution, and were incubated for 24 hours at 25°C. Appropriate controls were included. Gel electrophoresis was then run at 100V, 40 min in TAE, and the resultant bands visualized with SYBR Gold. Positive degradation control (PC) was shown with enzyme + dsRNA. PMGK does not protect dsRNA from salivary enzymes at any of the tested concentrations.

These results indicate effective delivery of RNAi agents to a number of different insects in vivo.

The RNAi agents and chaperone compounds may further be used in combination with any other type(s) of insect and pest control agent, whether conventional organic chemistry or biologically derived, as would be obvious to one skilled in the art.

The foregoing is considered as illustrative only of the principles of the invention. Furthermore, since numerous modifications and changes will readily occur to those skilled in the art, it is not desired to limit the invention to the exact construction and operation shown and described. Therefore, all suitable modifications and equivalents fall within the scope of the invention.

Claims

Claims

1. A composition for the stabilisation of an RNA interference (RNAi) agent against degradation by insect saliva or gut enzymes, the composition comprising a) one or more RNA interference (RNAi) agents, and b) a chaperone moiety comprising a polyfunctional acidic compound having at least 3 net negative charges, or its corresponding salt.

2. The composition according to claim 1, comprising a. of from 0.5 wt % to 40 wt % of the RNAi agent; and b. of from 0.005 wt % to 2.0 wt % of the polyfunctional acidic composition stabilizer or a corresponding salt.

3. The composition according to claim 1 or claim 2, wherein the polyfunctional acidic compound comprises a multitude of Bronsted acid moieties.

4. The composition according to anyone of claims 1 to 3, wherein the Bronsted acid moieties include carboxylic acid moieties, phosphoric acid moieties, phosphonic acid moieties, sulfuric acid moieties, sulfonic acid moieties, phenolic moieties, sulfamic acid moieties, boronic acid moieties, or salts thereof including carboxylate moieties, phosphate moieties, phosphonate moieties, phenolate moieties, sulfate, sulfonate, sulfamate and/or boronate moieties.

5. The composition according to any one of the previous claims, wherein the polyfunctional acidic compound is present in an amount suitable to suppress RNAi agent degradation in the insect saliva or insect gut.

6. The composition according to any one of the previous claims, wherein the polyfunctional acidic stabilizer comprises a backbone that is covalently linked to pendant acid groups and/or pendant acid salt groups,

7. The composition according to claim 6, wherein the backbone comprises an optionally substituted (poly)ether, (poly)ester, (poly)alkylene, (poly)(meth)acrylate, and/or (poly)vinyl structure.

8. The composition according to any one of the previous claims, wherein the polyfunctional acidic stabilizer is a water-soluble polymer containing pendant acid groups and/or pendant acid salt groups with a molecular weight (M_w ) of at least 190 g/mol.

9. The composition according to any one of claims 1 to 7, wherein the polyfunctional acidic stabilizer is a water-soluble polymer containing pendant acid groups and/or pendant acid salt groups with an average molecular weight (Mw ) in the range of from 1,000 to 10,000 g/mol.

10. The composition according to claim 9, wherein the water-soluble polymer has a polydispersity index, as defined by weight average molecular weight over number average molecular weight (Mw/Mn) of less than 2, preferably of less than 1.5.

11. The composition according to any one of the previous claims, wherein the polyfunctional acidic compound comprises at least three acidic moieties.

12. The composition according to any one of the previous claims, wherein the polyfunctional acidic compound is selected from citric acid, etidronic acid, ethylene diamine tetra methylene phosphonic acid, ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), hexametaphosphiric acid, polyphosphoric acid, pyrophosphoric acid, poly aspartic acid, poly (sulfopropyl) acrylic acid, poly (methacrylic acid), polyacrylic acid, poly (4-styrenesulfonic acid-co-maleic acid, poly (acrylic acid-co-maleic acid), chondroitin sulfate, dextran sulfate, lignosulfonate, poly(styrenesulfonic) acid, and/or poly(vinylsulfonic acid), and or salts thereof, or combinations thereof.

13. A composition according to any one of the previous claims, wherein the RNAi agent comprises double stranded RNA.

14. A composition according to claim 13, wherein the double stranded RNA comprises small interfering RNA or insecticidal double stranded RNA.

15. A composition according to claim 13 or claim 14, wherein the insecticidal double stranded RNA is insecticidal to insects of the order Hemiptera.

16. A composition according to any one of the previous claims, wherein the composition is a plant-protecting composition.

17. A composition according to any one of the previous claims, comprising further at least one agriculturally acceptable carrier and, optionally, a surfactant and/or one or more formulation adjuvants.

18. A sprayable preparation comprising the composition according to any one of the preceding claims, an aqueous phase and a solid, or dissolved solid, phase.

19. A method of delivering an RNAi agent to an insect, the method comprising administering the composition according to any one of claims 1 to 17 or the preparation of claim 18 to the plant locus, plant leaves or stems, or plant propagation material, to obtain a coated or treated material suitable as insect food.

20. The method according to claim 19, wherein the insect food is a plant, a plant part, a synthetic insect food source, an insect bait or an insect trap.

21. A method for controlling insects, comprising applying a composition according to any one of claims 1 to 20 to an insect or its environment, an insect food, a plant or plant propagation material or the locus where the plant or plant propagation material is planted.

22. A method for reducing degradation of an RNAi agent in or on an insect, comprising the step of adding to a composition comprising the RNAi agent a polyfunctional acidic compound or its corresponding salt.

23. A method of claim 22, wherein the final concentration of the composition contains the polyfunctional acidic compound or its corresponding salt in the range of from 0.005% to 2.0% by weight.

24. A method according to claim 22 or 23, wherein the polyfunctional acidic compound or its corresponding salt comprises respectively a polycarboxylate compound, or a salt thereof.

25. A method according to claim 22 or 23, wherein the polyfunctional acidic compound is selected from citric acid, etidronic acid, ethylene diamine tetra methylene phosphonic acid), ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), hexametaphosphate, polyphosphate, pyrophosphate, poly aspartic acid, poly (sulfopropyl) acrylate), poly (methacrylic acid), polyacrylic acid, poly (4-styrenesulfonic acid-co-maleic acid, poly (acrylic acid-co-maleic acid), chondroitin sulfate, dextran sulfate, lignosulfonate, poly(styrenesulfonic) acid, and/or poly(vinylsulfonic acid), and or a salt thereof.

26. The method according to any one of claims 22 to 25, wherein the RNAi agent comprises double stranded RNA.

27. The method according to claim 26, wherein the double stranded RNA comprises small interfering RNA or insecticidal double stranded RNA.

28. The method according to claim 27, wherein the insecticidal double stranded RNA is insecticidal to insects of the order Hemiptera.

29. The method according to any one of claims 22 to 28 wherein reducing degradation comprises reducing the degradation of the RNAi agent of from 1 % to 100 %.

30. The method according to any one of claims 22 to 29 wherein reducing degradation of the RNAi agent in or on an insect comprises extracellular fluid, preferably comprising saliva.

31. The method for reducing degradation of the RNAi agent in or on an insect, which comprises applying a composition according to claims 1 to 17 to an insect or their environment, an insect food, a plant or plant propagation material or the site where the plant or plant propagation material is planted.

32. The method for determining the stability of a RNAi agent against insecticidal enzymes, comprising a) providing a mixture containing a chaperone moiety and the RNAi agent and insect enzymes, b) incubating the mixture for a suitable period of time, and c) performing an electrophoresis to determine degradation of the starting RNAi agent.