Gene silencing

Abstract

The present invention provides for methods of delivering polynucleotides to subterranean plant pests. In particular, the present invention relates to methods of reducing the binding of a composition, comprising polynucleotide and a cationic polymer, to soil by substantially quenching positively charged residues with a quenching agent. The invention also comprises compositions comprising a cationic polymer, a polynucleotide and a quenching agent.

Claims

1. A method of inhibiting or reducing the binding of a composition, comprising a complex formed between a cationic polymer and a polynucleotide, to negatively charged molecules present in soil comprising including in the composition a quenching agent capable of neutralising exposed-surface positive charges on the complex.

2. The method according to claim 1, wherein the polynucleotide is a dsRNA, the polymer is a polyamine, and the quenching agent is a mono-aldehyde.

3. The method according to claim 1, wherein the polymer is linear or branched polyethyleneimine, polypeptide or cationic polysaccharide.

4. The method according to claim 1, wherein the negatively charged molecules comprise humic acid.

5. A method of controlling a subterranean plant pest infestation comprising ; a) providing a composition comprising a complex formed between a cationic polymer and a dsRNA, b) neutralising exposed-surface positive charges of the complex of a) with a quenching agent and c) applying the composition of b) to soil, wherein said dsRNA effects post-transcriptional silencing of a target gene in said subterranean plant pest.

6. The method according to claim 5, wherein the subterranean plant pest is selected from the group consisting of Diabrotica virgifera virgifera (Western corn rootworm), Diabrotica barberi (Northern corn rootworm), Diabrotica undecimpunctata howardi (Southern corn rootworm), Diabrotica virgifera zeae (Mexican corn rootworm), Diabrotica speciosa (cucurbit beetle), nematodes, wireworms and grubs and appropriate soil pathogens such as bacteria and fungi.

7. The method according to claim 6, wherein said subterranean plant pest is a Diabrotica insect.

Description

EXAMPLE 1

(1) Control sample A. 10 μL of IVT dsRNA at nominal 1 mg/mL was combined with 13 μL of 10% solution of bPEI (branched polyethyleneimine) 60 kDa in PBS buffer. This was diluted to 500 μL total volume in PBS, then combined with 10 mg colloidal silica [Aerosil 200, Evonik]. The sample was spun briefly in a centrifuge at 2000 rpm and the supernatant collected. 1 μL of 1% heparin. Na salt was added to de-complex any bPEI-dsRNA. When run on an electrophoresis gel no band was visible corresponding to the dsRNA, indicating that all of the complex had bound to the colloidal silica.

(2) Test sample B. This sample was prepared in the same way as control sample A, except that before combining with colloidal silica, 25 μL of a quench pentanal 10% solution in PBS buffer was added and allowed to react under ambient conditions for 1 hour. In this case a band was visible corresponding to free dsRNA and the band intensity indicated that about 25% of the total dsRNA had not bound to the colloidal silica in the form of bPEI-dsRNA complex.

EXAMPLE 2

(3) PEI (=Polyethyleneimine) coated cells may improve stability of dsRNA in soil. However, PEI coating of cells may cause the cells to adsorb strongly to soil due to interaction of amine groups to acid groups in soil. This may impair soil mobility and delivery of the dsRNA to CRW. By reacting the PEI coat with glutaraldehyde or monoaldehyde (pentanal), the free amine groups on the PEI may be quenched so that their interaction with soil is reduced. However, quenching with glutaraldehyde leads to some degree of flocculation, which may impair soil mobility. As shown below, monoaldehyde treatment does not lead to flocculation, and may therefore be a preferred way to quench the free amines. 1. Intact cells (previously treated with 0.002% w/w per ODU glutaraldehyde and then washed) were resuspended at 12 ODU/mL in PBS buffer and divided into 1 mL aliquots. 2. BPEI 25 kD solutions were prepared at 2% w/w BPEI (pH ˜11) and at 1.25% w/w BPEI (pH˜7). 3. Each intact cell aliquot was treated as follows with BPEI solutions, and observations recorded: Aliquot 1: Added BPEI (pH 11) to 0.1% w/w final BPEI concentration, then treated with 0.2% glutaraldehyde. Immediate severe flocculation was observed. Aliquot 2: Added BPEI (pH 11) to 0.1% w/w final BPEI concentration, then treated with 0.2% pentanal. No flocculation was observed. Aliquot 3: Added BPEI (pH 7) to 0.1% w/w final BPEI concentration, then treated with 0.2% glutaraldehyde. Immediate flocculation was observed, not as severe as aliquot 1, particle size of flocs was smaller as assessed visually. Aliquot 4: Added BPEI (pH 7) to 0.1% w/w final BPEI concentration, then treated with 0.2% pentanal. No flocculation was observed.

EXAMPLE 3

(4) Modification of Polyethyleneimine (PEI)

(5) PEGylation of PEI by epoxy-functionalized PEG

(6) PEG-diglycidyl ether (500D) was first quenched with Tris-HCl (pH7) to target quenching of half of the epoxy groups in the di-functionalized PEG, to yield a mono-functionalized PEG. This was done by mixing PEG-diglycidyl ether of 20% Tris-HCl solution (pH 7) in a ratio targeting reaction with half of the epoxy groups on the PEG, followed by overnight incubation at elevated temperature. The mon-functionalized PEG thus obtained was mixed with branched PEI solution (adjusted to pH 7) in ratios targeting various levels of PEGylation of the primary amines on the PEI (nominally targeting 30%, 60% and 90% quenching of the primary amines). The actual degree of quenching of primary amines on the PEI was determined by performing a colorimetric TNBSA (2,4,6-trinitrobenzene sulfonic) assay. The actual PEGylation levels obtained were as follows (Table 1) and as expected are somewhat different from the nominal target for several possible reasons, including that the conversion of di-functional PEG to mono-functional was not a specific reaction, and because of steric hindrance blocking availability of the primary amines. The difference between nominal and actual degree of quenching does not affect the outcome of the experiment because it was designed to span a range of actual degree of quenching.

(7) TABLE-US-00001 TABLE 1 Targeted quenching of PEI Measured quenching of PEI primary amines primary amines 30% 11% 60% 28% 90% 22%
Modification of PEIs by Diethylpyrocarbonate

(8) Primary amines on PEI were quenched by reaction with diethylpyrocarbonate (DEPC) by direct addition of DEPC to PET solutions adjusted to pH 7 to target varying levels of quenching of primary amines by DEPC (notably targeting 25%, 50% and 75% quenching). The actual degree of quenching of primary amines on the PEI was determined by performing a colorimetric TNBSA (2,4,6-trinitrobenzene sulfonic) assay. The actual levels of DEPC modification measured were as follows (Table 2), and as discussed above the discrepancy between targeted and actual degree of quenching does not affect the outcome of the experiment.

(9) TABLE-US-00002 TABLE 2 Targeted quenching of PEI Measured quenching of PEI primary amines primary amines 25% 30% 50% 40% 75% 50%

EXAMPLE 4

(10) Evaluating Binding Affinities of Modified Vs. Unmodified PEI to Soil

(11) Unmodified PEI solutions and solutions of PEI modified by PEGylation or DEPC were incubated with soil in a ratio of 2:1 (solution: soil) wt/wt for 1 hour. The concentrations of solutions tested varied from 0-0.08% of PEI. After soil incubation, the soil was separated from the PEI solutions by centrifugation. The unbound PEI remaining in the solution was assessed by the TNBSA assay. The binding profile of the PEI to soil was represented by binding isotherms (FIG. 1.). The level of primary amine quenching on the PEI is indicated the numbers in parentheses in the legend.

EXAMPLE 5

(12) Binding Affinity of dsRNA-PEI Complexes

(13) E. coli cell lysate expressing dsRNA was mixed with PEI solutions as follows: lysates were diluted to 25 OD/mL and PEI solutions were added to a final concentration of 0.75% to the lysates. The lysate dsRNA-PEI complex solutions were further diluted 2.5-fold and incubated with soil in a 2:1 solution: soil wt/wt ratio for 1 hour. Unformulated lysate solution was used as a control. The soil was subsequently pelleted by centrifugation and the unbound total RNA in the supernatant was extracted from the complexes by addition of a poly-anion to decomplex the dsRNA from the PEI, followed by RNAZol RT extraction. The total RNA concentration present in the supernatant of the samples was quantified by UV-Vis spectroscopy (FIG. 2.).

EXAMPLE 6

(14) Evaluating Stability of Lysate-dsRNA in Complexes Made with Modified PEIs

(15) E. coli cell lysate expressing dsRNA was mixed with PEI solutions as follows: lysates were diluted to 50 OD/mL and PEI solutions were added to a final concentration of 0.5% (1×PEI) or 1.5% (3×PEI) to the lysates. The lysate dsRNA-PEI complex solutions were incubated with RNAse III overnight at 37° C. Total RNA was extracted from the complexes by addition of a poly-anion to decomplex the dsRNA from the PEI, followed by RNAZol RT extraction. The integrity of the dsRNA was evaluated using agarose gel electrophoresis (FIGS. 3 and 4.)

EXAMPLE 7

(16) Evaluating Bioavailability of dsRNA in Complexes Made with Modified PEIs

(17) Artificial western corn rootworm diet-incorporation bioassay was conducted on the complexes made with the modified PEIs and the E. coli lysate expressing dsRNA. The lysate formulations were diluted using RNAse free water and mixed very well with equal amount of the diet (0.5 ml diet and 0.5 ml diluted formulation solutions) by vortex-mixing until homogeneous. Three replicates were conducted for each formulation type and for each replicate plate, approximately 12 neonatal larvae were added. Plates were incubated at room temperature. Mortality was recorded every other day for the duration of the assay. The final mortality rates are represented in FIGS. 5 and 6 and show improved activity of the quenched complexes This finding was also confirmed by bioassay in soil.

FIGURE LEGENDS

(18) FIG. 1. Both PEGylated and DEPC-treated PEIs have lowered binding affinities compared to unmodified PEI.

(19) FIG. 2. Complexes made with PEGylated PEIs bind less strongly to soil, hence remain in the supernatant after soil incubation. The higher the degree of PEGylation, the lower the binding affinity of the complex to soil.

(20) FIG. 3. Stability of lysate dsRNA in complexes made with modified PEIs (PEGylated PEIs). dsRNA is stable in complexes made with PEGylated PEIs.

(21) FIG. 4. Stability of lysate dsRNA in complexes made with modified PEIs (DEPC-treated PEIs). dsRNA is stable in complexes made with DEPC-treated PEIs.

(22) FIG. 5. Mortality of CRW is improved when dsRNA is complexed with PEGylated PEI, as compared to complexes made with unmodified PEI, showing improved bioavailability of the dsRNA in modified complexes.

(23) FIG. 6. Mortality of CRW is improved when dsRNA is complexed with DEPC-PEI, as compared to complexes made with unmodified PEI, suggesting improved bioavailability of the dsRNA in modified complexes. Higher modification levels on the PEI leads to improved bioavailability.