Stabilization of RNA for exogenous RNAi agricultural applications and formulations

Abstract

The composition for providing stability to RNA may include a primary surfactant and a sequestering agent. The primary surfactant may be a non-ionic surfactant. The composition may be in a soluble liquid concentrate form and may be sufficient to provide ambient stability to RNA at room temperature for one year. The composition for delivering RNA to pests via exogenous foliar application of the composition to plants may include RNA, a primary surfactant, and a sequestering agent.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 184,508, filed May 5, 2021, which is incorporated by reference herein in its entirety.

[0002] The present disclosure relates generally to RNA stabilization, and more specifically, to RNA stabilization for exogenous RNAi agricultural applications and formulations. [Background technology]

[0003] RNA interference (RNAi) is a biological process in which selective biological processes controlled by RNA are silenced or altered so that the process is not completed as intended, usually with the effect of death, reduced or stunted growth, reduced virulence, reduced proliferation / reproduction capacity, or elimination of the selected phenotype. In biological systems, the RNAi pathway is initiated by enzymes such as DICER, which cleave dsRNA into shorter 20-25 base pair nucleotides that function as sequence-specific interfering RNAs. This interfering RNA promotes gene silencing through several methods that affect the translation of host genetic material. Because these genetic modifications are highly selective and species-dependent, target organisms can be effectively managed through RNAi technology without the off-target and ecological burdens of traditional chemical pesticides. The active ingredient in RNAi-based technology, or interfering RNA, can be double-stranded RNA (dsRNA) or single-stranded RNA in various forms. Various structures of interfering RNA are known in the prior art, and are discussed, for example, in Ghildiyal and Zamore, Small Silencing RNAs: An Expanding Universe, Nature Vol.10, pp.94-168 (2009).dsRNA is a water-soluble biopolymer of natural origin that has a net negative charge due to the phosphate bonds that form the backbone of dsRNA sequence.Various methods for making RNA are known in the art, and the RNA of the present invention can be produced by any suitable method known in the art. Examples of methods for producing RNA include, but are not limited to, in vitro transcription (IVT), chemical synthesis, microbial fermentation, or cell-free methods such as those described in U.S. Pat. No. 10,858,385 published May 16, 2019 (Publication No. US2019 / 0144489) and U.S. Pat. No. 10,954,541 published October 12, 2017 (Publication No. US2017 / 0292138), each of which is incorporated herein by reference.Examples of RNAi molecules for endogenous delivery for use in the present invention include, but are not limited to, those described in U.S. Pat. No. 11,142,768 published on May 14, 2020 (Publication No. US2020 / 0149044), U.S. Pat. No. 11,185,079 published on March 26, 2020 (Publication No. US2020 / 0093138), and PCT / US / 2021 / 032334 (International Publication No. 2021 / 231791) published on November 18, 2021 (all of which are incorporated by reference herein).

[0004] RNAi technology has been shown to be a selective biological treatment for a wide variety of pests and diseases through interference with internal biological processes. There is a need for delivery of RNAi technology via exogenous delivery in formulations that help maintain and improve RNA stability.

[0005] A discussion of deficiencies and needs that exist in the art prior to the present invention is not an admission that such deficiencies and needs were recognized by those skilled in the art prior to the present disclosure.

[0006] Soluble liquid concentrate (SL) formulations must be stable for use in product applications. Stability in concentrated form must ensure the formulation's response to physical, chemical, microbial, and enzymatic instability. For physical stability, the formulation is evaluated for phase separation by visual or turbidity observation. For chemical stability, active ingredient concentration upon storage at elevated or controlled temperature is evaluated by liquid chromatography or gel electrophoresis. For microbial or enzymatic stability, active ingredient concentration is evaluated after exposure to a known challenge mixture. Summary of the Invention

[0007] Various embodiments relate to a composition for delivering RNA to pests via exogenous foliar application of the formulation to plants. The composition may include RNA, a primary surfactant, and a sequestering agent. According to various embodiments, the primary surfactant may be a non-ionic surfactant.

[0008] Another embodiment relates to a composition for providing stability to RNA. The composition can include a primary surfactant and a sequestering agent. The primary surfactant can be a non-ionic surfactant. The composition can be in a soluble liquid concentrate form and can be sufficient to provide RNA with ambient stability at room temperature for one year.

[0009] These and other features, aspects, and advantages of various embodiments will become better understood with reference to the following description, figures, and claims.

[0010] Many aspects of the disclosure can be better understood with reference to the following drawings. [Brief description of the drawings]

[0011] [Figure 1A] 1 shows examples according to various embodiments, showing photographs of compatible formulations after storage at -10°C for 2 weeks, at 4°C for 2 and 8 weeks, at 40°C for 8 weeks, and at 54°C for 2 weeks. [Figure 1B] 1 shows examples according to various embodiments, showing photographs of compatible formulations after storage at -10°C for 2 weeks, at 4°C for 2 and 8 weeks, at 40°C for 8 weeks, and at 54°C for 2 weeks. [Figure 1C] 1 shows examples according to various embodiments, showing photographs of compatible formulations after storage at -10°C for 2 weeks, at 4°C for 2 and 8 weeks, at 40°C for 8 weeks, and at 54°C for 2 weeks. [Figure 1D] 1 shows examples according to various embodiments, showing photographs of compatible formulations after storage at -10°C for 2 weeks, at 4°C for 2 and 8 weeks, at 40°C for 8 weeks, and at 54°C for 2 weeks. [Figure 2A] 1A-1C are examples according to various embodiments, showing photographs of formulations having ATPLUS® PFA concentrations of 10% and 5% by weight, respectively. [Figure 2B] 1A-1C are examples according to various embodiments, showing photographs of formulations having ATPLUS® PFA concentrations of 10% and 5% by weight, respectively. [Figure 3A]1A-1C are examples according to various embodiments, showing photographs of formulations having ATPLUS® PFA concentrations of 5%, 4.5%, 4.0%, and 3.5% by weight, respectively. [Figure 3B] 1A-1C are examples according to various embodiments, showing photographs of formulations having ATPLUS® PFA concentrations of 5%, 4.5%, 4.0%, and 3.5% by weight, respectively. [Figure 3C] 1A-1C are examples according to various embodiments, showing photographs of formulations having ATPLUS® PFA concentrations of 5%, 4.5%, 4.0%, and 3.5% by weight, respectively. [Figure 3D] 1A-1C are examples according to various embodiments, showing photographs of formulations having ATPLUS® PFA concentrations of 5%, 4.5%, 4.0%, and 3.5% by weight, respectively. [Figure 4] 1 shows an example according to various embodiments, showing a photograph of a formulation without phosphate buffer, showing significant precipitation of dsRNA. [Figure 5A] Examples according to various embodiments show photographs of formulations having phosphate buffer concentrations of 242 mM, 200 mM, 150 mM, 100 mM, and 50 mM, respectively, showing visible precipitation in Figures 5C, 5D, and 5E. [Figure 5B] Examples according to various embodiments show photographs of formulations having phosphate buffer concentrations of 242 mM, 200 mM, 150 mM, 100 mM, and 50 mM, respectively, showing visible precipitation in Figures 5C, 5D, and 5E. [Figure 5C] Examples according to various embodiments show photographs of formulations having phosphate buffer concentrations of 242 mM, 200 mM, 150 mM, 100 mM, and 50 mM, respectively, showing visible precipitation in Figures 5C, 5D, and 5E. [Figure 5D] Examples according to various embodiments show photographs of formulations having phosphate buffer concentrations of 242 mM, 200 mM, 150 mM, 100 mM, and 50 mM, respectively, showing visible precipitation in Figures 5C, 5D, and 5E. [Figure 5E]Examples according to various embodiments show photographs of formulations having phosphate buffer concentrations of 242 mM, 200 mM, 150 mM, 100 mM, and 50 mM, respectively, showing visible precipitation in Figures 5C, 5D, and 5E. [Figure 6A] 1A-1C are examples according to various embodiments, showing photographs of formulations diluted in water at 35 ppm, 342 ppm, 500 ppm, and 1000 ppm, respectively, the photographs being taken immediately after dilution in water. [Figure 6B] 1A-1C are examples according to various embodiments, showing photographs of formulations diluted in water at 35 ppm, 342 ppm, 500 ppm, and 1000 ppm, respectively, the photographs being taken immediately after dilution in water. [Figure 6C] 1A-1C are examples according to various embodiments, showing photographs of formulations diluted in water at 35 ppm, 342 ppm, 500 ppm, and 1000 ppm, respectively, the photographs being taken immediately after dilution in water. [Figure 6D] 1A-1C are examples according to various embodiments, showing photographs of formulations diluted in water at 35 ppm, 342 ppm, 500 ppm, and 1000 ppm, respectively, the photographs being taken immediately after dilution in water. [Figure 6E] 1A-1C are examples according to various embodiments, showing photographs of formulations diluted in water at 35 ppm, 342 ppm, 500 ppm, and 1000 ppm, respectively, the photographs were taken 24 hours after dilution in water. [Figure 6F] 1A-1C are examples according to various embodiments, showing photographs of formulations diluted in water at 35 ppm, 342 ppm, 500 ppm, and 1000 ppm, respectively, the photographs were taken 24 hours after dilution in water. [Figure 6G] 1A-1C are examples according to various embodiments, showing photographs of formulations diluted in water at 35 ppm, 342 ppm, 500 ppm, and 1000 ppm, respectively, the photographs were taken 24 hours after dilution in water. [Figure 6H] 1A-1C are examples according to various embodiments, showing photographs of formulations diluted in water at 35 ppm, 342 ppm, 500 ppm, and 1000 ppm, respectively, the photographs were taken 24 hours after dilution in water. [Figure 7A] Examples according to various embodiments show photographs of formulations containing 200 mM phosphate buffer and 21 mM EDTA diluted in water at 35 ppm, 342 ppm, 500 ppm, and 1000 ppm, respectively, the photographs were taken 24 hours after dilution in water. [Figure 7B] Examples according to various embodiments show photographs of formulations containing 200 mM phosphate buffer and 21 mM EDTA diluted in water at 35 ppm, 342 ppm, 500 ppm, and 1000 ppm, respectively, the photographs were taken 24 hours after dilution in water. [Figure 7C] Examples according to various embodiments show photographs of formulations containing 200 mM phosphate buffer and 21 mM EDTA diluted in water at 35 ppm, 342 ppm, 500 ppm, and 1000 ppm, respectively, the photographs were taken 24 hours after dilution in water. [Figure 7D] Examples according to various embodiments show photographs of formulations containing 200 mM phosphate buffer and 21 mM EDTA diluted in water at 35 ppm, 342 ppm, 500 ppm, and 1000 ppm, respectively, the photographs were taken 24 hours after dilution in water. [Figure 7E] Examples according to various embodiments show photographs of formulations containing 150 mM phosphate buffer and 21 mM EDTA diluted in water at 35 ppm, 342 ppm, 500 ppm, and 1000 ppm, respectively, the photographs were taken 24 hours after dilution in water. [Figure 7F] Examples according to various embodiments show photographs of formulations containing 150 mM phosphate buffer and 21 mM EDTA diluted in water at 35 ppm, 342 ppm, 500 ppm, and 1000 ppm, respectively, the photographs were taken 24 hours after dilution in water. [Figure 7G] Examples according to various embodiments show photographs of formulations containing 150 mM phosphate buffer and 21 mM EDTA diluted in water at 35 ppm, 342 ppm, 500 ppm, and 1000 ppm, respectively, the photographs were taken 24 hours after dilution in water. [Figure 7H] Examples according to various embodiments show photographs of formulations containing 150 mM phosphate buffer and 21 mM EDTA diluted in water at 35 ppm, 342 ppm, 500 ppm, and 1000 ppm, respectively, the photographs were taken 24 hours after dilution in water. [Figure 7I] Examples according to various embodiments show photographs of formulations containing 100 mM phosphate buffer and 21 mM EDTA diluted in water at 35 ppm, 342 ppm, 500 ppm, and 1000 ppm, respectively, the photographs were taken 24 hours after dilution in water. [Figure 7J] Examples according to various embodiments show photographs of formulations containing 100 mM phosphate buffer and 21 mM EDTA diluted in water at 35 ppm, 342 ppm, 500 ppm, and 1000 ppm, respectively, the photographs were taken 24 hours after dilution in water. [Figure 7K] Examples according to various embodiments show photographs of formulations containing 100 mM phosphate buffer and 21 mM EDTA diluted in water at 35 ppm, 342 ppm, 500 ppm, and 1000 ppm, respectively, the photographs were taken 24 hours after dilution in water. [Figure 7L] Examples according to various embodiments show photographs of formulations containing 100 mM phosphate buffer and 21 mM EDTA diluted in water at 35 ppm, 342 ppm, 500 ppm, and 1000 ppm, respectively, the photographs were taken 24 hours after dilution in water. [Figure 7M] Examples according to various embodiments show photographs of formulations containing 20 mM phosphate buffer and 21 mM EDTA diluted in water at 35 ppm, 342 ppm, 500 ppm, and 1000 ppm, respectively, the photographs were taken 24 hours after dilution in water. [Figure 7N] Examples according to various embodiments show photographs of formulations containing 20 mM phosphate buffer and 21 mM EDTA diluted in water at 35 ppm, 342 ppm, 500 ppm, and 1000 ppm, respectively, the photographs were taken 24 hours after dilution in water. [Figure 7O]Examples according to various embodiments show photographs of formulations containing 20 mM phosphate buffer and 21 mM EDTA diluted in water at 35 ppm, 342 ppm, 500 ppm, and 1000 ppm, respectively, the photographs were taken 24 hours after dilution in water. [Figure 7P] Examples according to various embodiments show photographs of formulations containing 20 mM phosphate buffer and 21 mM EDTA diluted in water at 35 ppm, 342 ppm, 500 ppm, and 1000 ppm, respectively, the photographs were taken 24 hours after dilution in water. [Figure 8A] 1 shows examples according to various embodiments, showing photographs of formulations containing 8 g / L dsRNA diluted in water at 35 ppm, 342 ppm, 500 ppm, and 1000 ppm, respectively, the photographs were taken 24 hours after dilution in water. [Figure 8B] 1 shows examples according to various embodiments, showing photographs of formulations containing 8 g / L dsRNA diluted in water at 35 ppm, 342 ppm, 500 ppm, and 1000 ppm, respectively, the photographs were taken 24 hours after dilution in water. [Figure 8C] 1 shows examples according to various embodiments, showing photographs of formulations containing 8 g / L dsRNA diluted in water at 35 ppm, 342 ppm, 500 ppm, and 1000 ppm, respectively, the photographs were taken 24 hours after dilution in water. [Figure 8D] 1 shows examples according to various embodiments, showing photographs of formulations containing 8 g / L dsRNA diluted in water at 35 ppm, 342 ppm, 500 ppm, and 1000 ppm, respectively, the photographs were taken 24 hours after dilution in water. [Figure 8E] 1 shows examples according to various embodiments, showing photographs of formulations containing 4 g / L dsRNA diluted in water at 35 ppm, 342 ppm, 500 ppm, and 1000 ppm, respectively, the photographs were taken 24 hours after dilution in water. [Figure 8F]1 shows examples according to various embodiments, showing photographs of formulations containing 4 g / L dsRNA diluted in water at 35 ppm, 342 ppm, 500 ppm, and 1000 ppm, respectively, the photographs were taken 24 hours after dilution in water. [Figure 8G] 1 shows examples according to various embodiments, showing photographs of formulations containing 4 g / L dsRNA diluted in water at 35 ppm, 342 ppm, 500 ppm, and 1000 ppm, respectively, the photographs were taken 24 hours after dilution in water. [Figure 8H] 1 shows examples according to various embodiments, showing photographs of formulations containing 4 g / L dsRNA diluted in water at 35 ppm, 342 ppm, 500 ppm, and 1000 ppm, respectively, the photographs were taken 24 hours after dilution in water. [Figure 9] 1 is an example according to various embodiments showing high performance liquid chromatography (HPLC) results for chemical stability in various formulations after storage at -10°C, 4°C, 40°C, and 54°C. [Figure 10A] Examples according to various embodiments show photographs of compatible formulations containing SAG 1572™ antifoam after storage at -10°C for 2 weeks, at 4°C for 2 and 8 weeks, at 40°C for 8 weeks, and at 54°C for 2 weeks, demonstrating superior dsRNA stability. [Figure 10B] Examples according to various embodiments show photographs of compatible formulations containing SAG 1572™ antifoam after storage at -10°C for 2 weeks, at 4°C for 2 and 8 weeks, at 40°C for 8 weeks, and at 54°C for 2 weeks, demonstrating superior dsRNA stability. [Figure 11A] Examples according to various embodiments show photographs of compatible formulations containing SAG 1572™ antifoam after storage at -10°C for 2 weeks, at 4°C for 2 and 8 weeks, at 40°C for 8 weeks, and at 54°C for 2 weeks, demonstrating superior dsRNA stability. [Figure 11B] Examples according to various embodiments show photographs of compatible formulations containing SAG 1572™ antifoam after storage at -10°C for 2 weeks, at 4°C for 2 and 8 weeks, at 40°C for 8 weeks, and at 54°C for 2 weeks, demonstrating superior dsRNA stability. [Figure 12A] FIG. 1 shows examples according to various embodiments, showing persistent foam test results for a formulation containing SAG 1572™ antifoam and 8 g / L dsRNA from a TGAI source, diluted 300× and 15× with 342 ppm water, respectively, as specified by CIPAC MT 18, showing the absence of persistent foam and progressive foam reduction upon standing for 1 minute. [Figure 12B] FIG. 1 shows examples according to various embodiments, showing persistent foam test results for a formulation containing SAG 1572™ antifoam and 8 g / L dsRNA from a TGAI source, diluted 300× and 15× with 342 ppm water, respectively, as specified by CIPAC MT 18, showing the absence of persistent foam and progressive foam reduction upon standing for 1 minute. [Figure 12C] FIG. 1 shows examples according to various embodiments, showing persistent foam test results for a formulation containing SAG 1572™ antifoam and 8 g / L dsRNA from a TGAI source, diluted 300× and 15× with 342 ppm water, respectively, as specified by CIPAC MT 18, showing the absence of persistent foam and progressive foam reduction upon standing for 1 minute. [Figure 12D] FIG. 1 shows examples according to various embodiments, showing persistent foam test results for a formulation containing SAG 1572™ antifoam and 8 g / L dsRNA from a TGAI source, diluted 300× and 15× with 342 ppm water, respectively, as specified by CIPAC MT 18, showing the absence of persistent foam and progressive foam reduction upon standing for 1 minute. [Figure 12E] FIG. 1 shows an example according to various embodiments, which shows photographs of persistent foam test results for a formulation containing SAG 1572™ antifoam and 8 g / L dsRNA from a TGAI source, diluted 300× and 15× with 342 ppm water, respectively, as specified by CIPAC MT 18, showing the absence of persistent foam and progressive foam reduction upon standing for 1 minute. [Figure 12F]FIG. 1 shows an example according to various embodiments, which shows photographs of persistent foam test results for a formulation containing SAG 1572™ antifoam and 8 g / L dsRNA from a TGAI source, diluted 300× and 15× with 342 ppm water, respectively, as specified by CIPAC MT 18, showing the absence of persistent foam and progressive foam reduction upon standing for 1 minute. [Figure 13A] 1 shows an example according to various embodiments, showing a photograph of a formulation containing SAG 1572™ antifoam after 8 weeks of storage at 40° C., demonstrating superior dsRNA stability. [Figure 13B] 1 shows an example according to various embodiments, showing a photograph of a formulation containing SAG 1572™ antifoam after 8 weeks of storage at 40° C., demonstrating superior dsRNA stability. [Figure 13C] 1 shows an example according to various embodiments, showing a photograph of a formulation containing SAG 1572™ antifoam after 8 weeks of storage at 40° C., demonstrating superior dsRNA stability. [Figure 13D] 1 shows an example according to various embodiments, showing a photograph of a formulation containing SAG 1572™ antifoam after 8 weeks of storage at 40° C., demonstrating superior dsRNA stability. [Figure 13E] 1 shows an example according to various embodiments, showing a photograph of a formulation containing SAG 1572™ antifoam after 8 weeks of storage at 40° C., demonstrating superior dsRNA stability. [Figure 13F] 1 shows an example according to various embodiments, showing a photograph of a formulation containing SAG 1572™ antifoam after 8 weeks of storage at 40° C., demonstrating superior dsRNA stability. [Figure 13G] 1 shows an example according to various embodiments, showing a photograph of a formulation containing SAG 1572™ antifoam after two weeks of storage at 54° C., demonstrating superior dsRNA stability. [Figure 13H] 1 shows an example according to various embodiments, showing a photograph of a formulation containing SAG 1572™ antifoam after two weeks of storage at 54° C., demonstrating superior dsRNA stability. [Figure 13I]1 shows an example according to various embodiments, showing a photograph of a formulation containing SAG 1572™ antifoam after two weeks of storage at 54° C., demonstrating superior dsRNA stability. [Figure 13J] 1 shows an example according to various embodiments, showing a photograph of a formulation containing SAG 1572™ antifoam after two weeks of storage at 54° C., demonstrating superior dsRNA stability. [Figure 13K] 1 shows an example according to various embodiments, showing a photograph of a formulation containing SAG 1572™ antifoam after two weeks of storage at 54° C., demonstrating superior dsRNA stability. [Figure 13L] 1 shows an example according to various embodiments, showing a photograph of a formulation containing SAG 1572™ antifoam after two weeks of storage at 54° C., demonstrating superior dsRNA stability. [Figure 14A] 13A-13C are examples according to various embodiments, showing photographs of titration assays for formulations containing 0.025% SAG 1572™ antifoam and dsRNA diluted 400× and 100× in 342 ppm water, respectively. [Figure 14B] 13A-13C are examples according to various embodiments, showing photographs of titration assays for formulations containing 0.025% SAG 1572™ antifoam and dsRNA diluted 400× and 100× in 342 ppm water, respectively. [Figure 14C] 13A-13C are examples according to various embodiments, showing photographs of titration assays for formulations containing 0.05% SAG 1572™ antifoam and dsRNA diluted 400× and 100× in 342 ppm water, respectively. [Figure 14D] 13A-13C are examples according to various embodiments, showing photographs of titration assays for formulations containing 0.05% SAG 1572™ antifoam and dsRNA diluted 400× and 100× in 342 ppm water, respectively. [Figure 15] 1 shows examples according to various embodiments, photographs illustrating the physical compatibility and physical stability of SAG1599™ in concentrated formulations. [Figure 16] 1 is an example according to various embodiments, showing a photograph of a formulation containing 0.2% ANTIFOAM GN11P™ after 1 minute of standing. [Figure 17] FIG. 1 is an example according to various embodiments showing the results of unformulated TGAI and formulated TGAI containing dsRNA bioburden levels after contamination with a bacterial cocktail of 107 CFU, where the designations HAL CAN, AL SAC, and AL CAN represent three different yeast sources for the production of TGAI. [Figure 18] FIG. 1 is an example according to various embodiments showing dsRNA degradation profiles for unformulated and formulated dsRNA following contamination with 107 CFU of a bacterial cocktail, where HAL CAN, AL SAC, and AL CAN represent three different yeast sources for the production of TGAI. [Figure 19] FIG. 1 is an example according to various embodiments showing the results of unformulated dsRNA and formulated dsRNA containing dsRNA bioburden levels after contamination with 104 CFU of a fungal cocktail, where HAL CAN, AL SAC, and AL CAN represent three different yeast sources for the production of TGAI. [Figure 20] FIG. 1 is an example according to various embodiments showing dsRNA degradation profiles of unformulated and formulated dsRNA following contamination with 104 CFU of a fungal cocktail, where HAL CAN, AL SAC, and AL CAN represent three different yeast sources for the production of TGAI. [Figure 21A] 1A-1C are examples according to various embodiments, showing photographs of plate-based assays for cetrimonium chloride challenged with B. Licheniformis and E. Coli, respectively. [Figure 21B] 1A-1C are examples according to various embodiments, showing photographs of plate-based assays for cetrimonium chloride challenged with B. Licheniformis and E. Coli, respectively. [Figure 22A] 1A-1C are examples according to various embodiments, showing photographs of plate-based assays for lauryl betaine challenged with B. Licheniformis and E. Coli, respectively. [Figure 22B]1A-1C are examples according to various embodiments, showing photographs of plate-based assays for lauryl betaine challenged with B. Licheniformis and E. Coli, respectively. [Figure 23A] 1A-1C are examples according to various embodiments, showing photographs of plate-based assays for C12 amino oxide challenged with B. Licheniformis and E. Coli, respectively. [Figure 23B] 1A-1C are examples according to various embodiments, showing photographs of plate-based assays for C12 amino oxide challenged with B. Licheniformis and E. Coli, respectively. [Figure 24] FIG. 1 is an example according to various embodiments showing Mg2+ analysis of multiple production lots of TGAI, including TGAI produced with 7 g / L dsRNA containing 9-13 mM Mg2+ with an average content of 10 mM, and TGAI produced with 14 g / L dsRNA containing 15.5-28 mM Mg2+ with an average content of 22.5 mM. [Diagram 25] 1 is an example illustrating dsRNA degradation by nuclease activity. [Figure 26] 1 is an example according to various embodiments showing gel electrophoresis results of dsRNA formulations containing increasing concentrations of EDTA. [Figure 27] 1 is an example according to various embodiments showing gel electrophoresis results of dsRNA formulations containing increasing concentrations of EDTA. [Figure 28] 1 is an example according to various embodiments showing UV-B exposure levels and dsRNA stability after two weeks. [Figure 29] 1 is an example according to various embodiments showing HPLC chromatogram overlays of dsRNA after various UV-B exposures. [Diagram 30] 1 is an example according to various embodiments showing the mass balance of dsRNA, ssRNA, and free nucleotides in a dsRNA sample after two weeks of exposure to UV radiation. [Diagram 31]1 is an example according to various embodiments, where a formulation containing 1.5% lignosulfonate (lanes 2-6) shows retention of the dsRNA band on the gel and some protection from dsRNA degradation from UV irradiation. [Diagram 32] 1 is an example according to various embodiments showing results from HPLC quantification of formulations after exposure to 100 J / cm2 UV-B radiation. [Diagram 33] 1 is an example according to various embodiments showing a comparison of dsRNA stability against UV irradiation on leaf surface versus parafilm. [Diagram 34] 1 shows examples according to various embodiments, showing gel electrophoresis results after UV-B exposure, demonstrating that olefinic and phenyl-based chemistries do not provide any significant protection of dsRNA. [Diagram 35] 1 is an example according to various embodiments showing mortality from a Colorado potato beetle (CPB) bioassay. [Diagram 36] 1 is an example according to various embodiments showing mortality from a Colorado potato beetle (CPB) bioassay of formulations with and without the addition of lignosulfonates. [Figure 37] 1 shows the statistical design of an experimental evaluation of the stability of relative concentrations of cetrimonium hydrochloride and EDTA.

[0012] It should be understood that the various embodiments are not limited to the examples shown in the figures. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] Preamble and Definitions It will be understood that the present disclosure is described to explain the present invention to those skilled in the art, and is not limited to the specific examples or embodiments described. The examples and embodiments are examples of the present invention, and will reveal a much broader scope to those skilled in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. The terminology used herein is only for the purpose of describing the examples and embodiments, and is not intended to be limiting, and it is also understood that the scope of the present disclosure is limited only by the appended claims.

[0014] All features disclosed herein (including the accompanying claims, abstract, and drawings) may be replaced by alternative features serving the same, equivalent, or similar purpose, unless otherwise specified. Thus, unless otherwise specified, each feature disclosed is merely one example of a generic series of equivalent or similar features. The examples and embodiments described herein are for illustrative purposes only, and various modifications or changes in this respect will be suggested to those skilled in the art and should be included within the spirit and scope of the present application. Many variations and modifications can be made to the embodiments of the present disclosure without substantially departing from the spirit and principles of the present disclosure. Such modifications and modifications are intended to be included within the scope of the present disclosure. For example, unless otherwise indicated, the present disclosure is not limited to specific materials, reagents, reactants, manufacturing processes, etc., and as such may vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. The present disclosure also allows for steps to be performed in different order where this is logically possible.

[0015] In this specification, all numerical values, whether expressly stated or not, are assumed to be modified by the term "about". The term "about" generally refers to a range of numerical values ​​that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result). In many cases, the term "about" may include numerical values ​​that are rounded to the nearest significant figure.

[0016] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to a "support" includes a plurality of supports. In this specification and the following claims, reference will be made to a number of terms, which shall be defined to have the following meanings unless a contrary intention is apparent.

[0017] As used herein, the terms "standard temperature and pressure" or "room temperature and / or pressure" generally refer to 25° C. and 1 atmosphere. Standard temperature and pressure may also be referred to as "ambient conditions." Unless otherwise indicated, parts are by weight, temperatures are in degrees C., and pressures are at or near atmospheric pressure. The terms "elevated temperature" or "elevated temperature" generally refer to a temperature of at least 100° C.

[0018] Unless otherwise specified, all percentages indicating the amount of a component in a composition represent the weight percent of the component based on the total weight of the composition. The term "mol percent" or "mole percent" generally refers to the percentage that is the moles of a particular component relative to the total moles in a mixture. The sum of the mole fractions of each component in a solution is equal to 1.

[0019] Where a range of values ​​is provided, it is understood that, unless the context clearly dictates otherwise, each intervening value between the upper and lower limits of that range, to the tenth of the unit of the lower limit, and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any expressly excluded limitations in the stated range. Where a stated range includes one or both of the upper and lower limits, ranges excluding one or both of those included upper and lower limits are also included in the disclosure.

[0020] All publications and patents cited herein are incorporated by reference to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference herein, and are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by prior disclosure. Further, the publication dates provided may be different from the actual publication dates, which may need to be independently confirmed.

[0021] In molecules that have isomers or exhibit one or more chiral centers, for simplicity, only one of the possible variations may be shown. Those skilled in the art will understand that disclosure of all such variations is intended. If a particular variation is preferred, the disclosure will so state.

[0022] As used herein, the term "soluble liquid concentrate" (SL) refers to an aqueous formulation containing a dissolved active ingredient. According to some embodiments, the active ingredient may be a salt.

[0023] As used herein, the term "dsRNA" refers to double-stranded RNA, which is RNA having two complementary strands.

[0024] As used herein, the term "stability" or "instability" when used in reference to RNA refers to the degree to which the chemical makeup or physical state of RNA is subject to change over time from any cause. RNA can be single-stranded RNA or dsRNA. Examples of lack of physical stability can include, but are not limited to, precipitation from the formulation. Examples of lack of chemical stability can include, but are not limited to, degradation of the molecular structure of the interfering RNA by any or all of a variety of factors, such as exposure to ultraviolet (UV) radiation, biological contamination, or chemical contamination. Examples of biological contamination can include, but are not limited to, exposure to bacteria, enzymes, or fungi. Examples of enzymes that can cause biological contamination and thereby affect chemical stability include nucleases that degrade RNA. Examples of chemical contamination can include, but are not limited to, exposure to an inappropriate pH range or chemicals. The physical and / or chemical stability of RNA can also be affected by thermal fluctuations.

[0025] As used herein, the term "half-life of a single-stranded RNA" or "half-life of a dsRNA" refers to the time period required for the stability of a single-stranded RNA or dsRNA to decrease by half.

[0026] As used herein, the term "surfactant" refers to a compound that reduces the surface tension (or interfacial tension) between two liquids, between a gas and a liquid, or between a liquid and a solid. Surfactant molecules can have a hydrophobic portion (hydrophobic) and a hydrophilic portion (hydrophilic).

[0027] As used herein, the terms "primary surfactant" and "secondary surfactant" may be used interchangeably with the terms "first surfactant" and "second surfactant." The terms are used for convenience to refer to and distinguish between various surfactants in a formulation or in a method of preparing a formulation.

[0028] As used herein, the term "biopesticide" refers to a composition that controls or kills pests.

[0029] As used herein, the term "RNAi" refers to RNA interference, a biological process in which RNA molecules inhibit gene expression or translation by neutralizing targeted mRNA molecules.

[0030] As used herein, the term "exogenous application to a plant" refers to the application of an RNAi biopesticide to the outside of a plant. Exogenous application can avoid genetically modifying the plant.

[0031] dsRNA Various embodiments relate to formulations that may include dsRNA, and various embodiments relate to methods of protecting plants from pests or pathogens that may include applying one or more of these formulations that include dsRNA. Various embodiments relate to compositions for providing stability to dsRNA, and various embodiments relate to methods of stabilizing dsRNA that may include combining dsRNA with one or more of these compositions.

[0032] A wide variety of dsRNA can be used according to various embodiments.The dsRNA used in the present disclosure includes, for example, those described in U.S. Patent Nos. 11,185,079 and 11,142,768, and WIPO Publication Nos. 2020 / 123419 and 2021 / 231791, each of which is incorporated herein by reference.The type of dsRNA can vary based on any number of factors, including but not limited to size and structure.

[0033] The size of a dsRNA is typically measured in terms of base pairs. According to various embodiments, the dsRNA that may be used or stabilized according to various embodiments has a number of base pairs in the range of 10 to 1700. Each range described herein is intended to include all numerical values ​​encompassed by that range. Furthermore, additional ranges may be formed from any lower and / or upper limit described herein. For example, the dsRNA that may be used or stabilized according to various embodiments has a number of base pairs within a range having a lower and / or upper limit. A range may include or exclude the lower and / or upper limit. By way of example, and not limitation, the lower and / or upper limits may be selected from 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, and 1700 base pairs. A range formed from a lower limit alone includes at least the lower limit and all numerical values ​​above the lower limit, regardless of whether the values ​​are explicitly recited in this disclosure. A range formed from an upper limit alone includes at least the upper limit and all numerical values ​​below the upper limit, regardless of whether the values ​​are explicitly recited in this disclosure. A range formed from a combination of a lower limit and an upper limit includes at least the lower limit, the upper limit, and all numerical values ​​therebetween, regardless of whether the values ​​are explicitly recited in this disclosure. For example, based on the set of exemplary upper and lower limits explicitly recited above, dsRNAs that may be used or stabilized according to various embodiments have a number of base pairs within a range, such as about 10 to about 1000 base pairs, less than about 10 base pairs, more than about 10 base pairs, less than about 1000 base pairs, or more than about 1000 base pairs. All such ranges are contemplated and are intended to be explicitly disclosed and recited. Each recited value is intended to be modified by the term "about."

[0034] Those skilled in the art will understand that dsRNA exists in a wide variety of structural configurations.These structures can include, but are not limited to, double-stranded structure, paperclip structure, hairpin structure, stem-loop structure, pre-microRNA structure, and artificial microRNA structure.The dsRNA that can be used or stabilized according to various embodiments can have any of these structures.

[0035] single stranded RNA Various embodiments relate to formulations that may include various single-stranded RNAs, such as small interfering RNA, piRNA, or antisense RNA, and various embodiments relate to methods of protecting plants from pests that may include applying one or more of these formulations that include single-stranded RNA. Various embodiments relate to compositions for providing stability to single-stranded RNA, and various embodiments relate to methods of stabilizing single-stranded RNA that may include combining the single-stranded RNA with one or more of these compositions.

[0036] The size of single-stranded RNA is typically measured in terms of nucleotide bases. According to various embodiments, single-stranded RNA that may be used or stabilized according to various embodiments is in the range of 10-1000 bases. Each range described herein is intended to include all numerical values ​​encompassed by that range. Additionally, additional ranges may be formed from any lower and / or upper limits described herein. For example, single-stranded RNA that may be used or stabilized according to various embodiments has a number of bases in a range that has a lower and / or upper limit. A range may include or exclude a lower and / or upper limit. By way of example, and not limitation, the lower and / or upper limit may be 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, and 1000 bases. A range formed from a lower limit alone includes at least the lower limit and all numerical values ​​above the lower limit, regardless of whether the value is explicitly recited in this disclosure. A range formed from an upper limit alone includes at least the upper limit and all numerical values ​​below the upper limit, regardless of whether the value is explicitly recited in this disclosure. A range formed from a combination of a lower limit and an upper limit includes at least the lower limit, the upper limit, and all numerical values ​​therebetween, regardless of whether the value is explicitly recited in this disclosure. For example, based on the set of exemplary upper and lower limits explicitly recited above, single-stranded RNAs that may be used or stabilized according to various embodiments have a number of bases within a range of about 10 to about 1000 bases, less than about 10 bases, more than about 10 bases, less than about 1000 bases, or more than about 1000 bases, etc. All such ranges are contemplated and are intended to be explicitly disclosed and recited. Each recited value is intended to be modified by the term "about".

[0037] Primary Surfactants: Definitions and Examples According to various embodiments, the primary surfactant can be a non-ionic surfactant.As used herein, the term "non-ionic surfactant" refers to a class of surfactants that includes neutrally charged hydrophilic and hydrophobic agents.According to various embodiments, non-ionic surfactants can be selected for use in the formulation based on their compatibility with concentrated formulations, their ability to help stabilize dsRNA or single-stranded RNA when diluted in various water conditions, and their application properties when sprayed on leaf surface.

[0038] Some examples of nonionic surfactants may include, but are not limited to, alkoxylates such as alkoxylated alcohols, alkoxylated phenols, alkoxylated fatty acids, alkoxylated monoalkaolamides, alkoxylated sorbitan esters, and alkoxylated fatty amines. More specific examples of alkoxylates include, but are not limited to, alcohol ethoxylates, alkylphenol ethoxylates, fatty acid ethoxylates, monoalkaolamide ethoxylates, sorbitan ester ethoxylates, and fatty amine ethoxylates. Nonionic surfactants may also include polymeric surfactants, including, but not limited to, ethylene oxide-propylene oxide copolymers. Further examples of nonionic surfactants include, but are not limited to, linear alcohol ethoxylates, branched alcohol ethoxylates, fatty alcohol ethoxylates, alcohol alkoxylates, polyethylene modified fatty acid sorbitan esters, polyalkylglucosides, ethoxylated alkyl polyethylene glycol ethers, alkoxylated alkyl polyethylene glycol ethers, and fatty acid amides.

[0039] Examples of commercially available surfactants that may be classified as non-ionic surfactants for purposes of various embodiments may include, but are not limited to, ATPLUS® PFA, BIO-SOFT® N23-6.5, TWEEN 20™, SYNERGEN® GA, LUTENSOL® TDA8, LUTENSOL® TDA9, T MAZ 20K™, T MAZ 80K™, and AGNIQUE® CSO-36.

[0040] Still further examples of non-ionic surfactants include, but are not limited to, multi-hydroxy products. Examples of multi-hydroxy products may include, but are not limited to, glycol esters, glycerol esters, polyglycerol esters, glucosides, polyglucosides, and sucrose esters. Additional examples of non-ionic surfactants include, but are not limited to, alkyl polyglycosides, CETOMACROGOL 1000™, cetostearyl alcohol, cetyl alcohol, cocamide dea, cocamide mea, decyl glucoside, decyl polyglucose, glycerol monostearate, IGEPAL® CA-630, ISOCETETH-20™, lauryl glucoside, maltoside, monolaurin, mycosubtilin, narrow range ethoxylates, NONIDET These may include, but are not limited to, P-40™ (NP-40), NONOXYNOL-9™ (NP-9), nonoxynol, octaethylene glycol monododecyl ether, n-octyl beta-d-thioglucopyranoside, octyl glucoside, oleyl alcohol, PEG-10 castor flower glyceride, pentaethylene glycol monododecyl ether, polidocanol, poloxamer, poloxamer 407, polyethoxylated tallow amine, polyglycerol polyricinoleate, polysorbate, polysorbate 20, polysorbate 80, sorbitan, sorbitan monolaurate, sorbitan monostearate, sorbitan tristearate, stearyl alcohol, surfactin, TRITON X-100™, and TWEEN 80™.

[0041] Nonoxynol, also known as nonaethylene glycol or polyethylene glycol nonylphenyl ether, is a mixture of nonionic surfactants used as a detergent, emulsifier, wetting agent, or defoamer.

[0042] Poloxamers are nonionic triblock copolymers composed of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)). Poloxamer 407 is a hydrophilic nonionic surfactant of the more general class of copolymers known as poloxamers. Poloxamer 407 is a triblock copolymer consisting of a central hydrophobic block of polypropylene glycol flanked by two hydrophilic blocks of polyethylene glycol (PEG).

[0043] Polysorbates are oily liquids derived from ethoxylated sorbitan (a derivative of sorbitol) esterified with fatty acids. Polysorbate 20 (common commercial brand names include Scattics, Alkest TW20, and Tween 20) is a polysorbate-type nonionic surfactant formed by ethoxylation of sorbitan prior to the addition of lauric acid. Its official IUPAC name is polyoxyethylene (20) sorbitan monolaurate. Polysorbate 80 is a nonionic surfactant and emulsifier derived from polyethoxylated sorbitan and oleic acid. Its official IUPAC name is polyoxyethylene (20) sorbitan monooleate.

[0044] ATPLUS® PFA is a commercially available alkoxylated alcohol, which may be purchased from Croda International Plc. TWEEN 20™ is a commercially available polyoxyethylene sorbitol ester, specifically polysorbate 20. Tween 80™ is a commercially available polysorbate surfactant, specifically polysorbate 80. SYNERGEN® GA is a commercially available sugar-based surfactant containing N-methyl-N-octanoyl / decanoyl glucamine. LUTENSOL® TDA8 and LUTENSOL® TDA9 are commercially available ethoxylated tridecyl alcohols. T MAZ 20K™ and T MAZ 80K™ are commercially available sorbitan monooleates that are ethoxylated with approximately 20 moles of ethylene oxide to obtain a water-soluble oil-water emulsifier. AGNIQUE® CSO-36 is a commercially available ethoxylated castor oil. CETOMACROGOL 1000™ is a commercially available polyethylene glycol hexadecyl ether, a non-ionic surfactant produced by ethoxylation of cetyl alcohol. IGEPAL® CA-630 is a commercially available non-ionic, non-denatured detergent. Its formal IUPAC name is octylphenoxypolyethoxyethanol. ISOCETETH-20™ is a commercially available polyethylene glycol ether produced by ethoxylation of isocetyl alcohol. NONIDET P-40™ (NP-40) is a commercially available non-ionic, non-denatured detergent. Its formal IUPAC name is octylphenoxypolyethoxyethanol. NONOXYNOL-9™ (NP-9) is a commercially available surfactant from the nonoxynol family. Its formal IUPAC name is 2-{2-[2-(2-{2-[2-(2-{2-[2-(4-nonylphenoxy)ethoxy]ethoxy}ethoxy)ethoxy]ethoxy}ethoxy)ethoxy]ethoxy}ethanol. TRITON X-100™ is a commercially available nonionic surfactant with hydrophilic polyethylene oxide chains and aromatic hydrocarbon lipophilic or hydrophobic groups.Its official IUPAC name is 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol.

[0045] Secondary Surfactants: Definitions and Examples According to various embodiments, the secondary surfactant can be a cationic surfactant, a zwitterionic surfactant, and / or an amphoteric surfactant. As used herein, the term "cationic surfactant" refers to a class of surfactants that contain a positively charged hydrophilic agent. As used herein, the term "zwitterionic surfactant" or "amphoteric surfactant" refers to a class of surfactants that contain a hydrophilic agent that has both a positively charged (cationic) center and a negatively charged (anionic) center. The cationic portion is often based on a primary, secondary, or tertiary amine, or a quaternary ammonium cation. The anionic portion can be more variable and includes sulfate, sulfonate, phosphate, carbonate, and other proton-donating moieties.

[0046] According to various embodiments, the secondary surfactant can be an alkyl ammonium halide, such as an alkyl ammonium chloride, and provides antimicrobial protection against dsRNA or single-stranded RNA. Examples of cationic surfactants can include, but are not limited to, behentrimonium chloride, benzalkonium chloride, benzethonium chloride, benzododecinium bromide, bronidox, carbetopendecinium bromide, cetalkonium chloride, cetrimonium bromide, cetrimonium chloride, cetylpyridinium chloride, didecyldimethylammonium chloride, dimethyldioctadecylammonium bromide, dimethyldioctadecylammonium chloride, dioleoyl-3-trimethylammonium propane, domiphen bromide, laurylmethylglucety-10 hydroxypropyldimonium chloride, octenidine dihydrochloride, olaflur, n-oleyl-1,3-propanediamine, pafutoxin, stearalkonium chloride, tetramethylammonium hydroxide, and thonzonium bromide.

[0047] Examples of zwitterionic surfactants may include, but are not limited to, CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) detergent, cocamidopropyl betaine, cocamidopropyl hydroxysultaine, dipalmitoyl phosphatidylcholine, egg lecithin, hydroxysultaine, lecithin, miltefosine, peptitergent, and sodium lauroamphoacetate.

[0048] Lauryl betaine is an amphoteric surfactant derived from N-dodecyl-N,N-dialkanolamine with protein denaturing ability.As used herein, "betaine" refers to any neutral chemical compound that has a positively charged cationic functional group, such as a quaternary ammonium or phosphonium cation (generally an onium ion) that does not have a hydrogen atom, and a negatively charged functional group, such as a carboxylate group, that cannot be adjacent to the cationic site.Betaine is a specific type of zwitterion.

[0049] Examples of amine oxides include pyridine-N-oxide, a water-soluble crystalline solid with a melting point of 62-67° C., and N-methylmorpholine N-oxide, an oxidizing agent. Various embodiments may use C6-C30 amine oxides, for example, C12 amine oxides.

[0050] According to various embodiments, AMMONYX® CETAC-30 and / or MAQUAT® LB may be used as secondary surfactants due to their cationic nature and the role that the ammonium group in each molecule plays in antimicrobial activity. AMMONYX® CETAC-30 is the trade name for cetrimonium chloride, a cationic surfactant with known antimicrobial effects. MAQUAT® LB is lauryl betaine, which due to the physical structure of the betaine, is positively charged at all pH levels and contains an internal ammonium salt with antimicrobial effects.

[0051] Other examples of secondary surfactants can include amine oxides, also known as amine-N-oxides and N-oxides. For example, lauryl dimethylamine oxide (LDAO), also known as dodecyl dimethylamine oxide (DDAO), is an amine oxide-based zwitterionic surfactant.

[0052] Sequestering Agents: Definitions and Examples As used herein, the terms "sequestering agent," "complexing agent," "chelator," "chelating agent," or "sequestering agent" refer to a multidentate ligand capable of forming two or more separate coordinate bonds to a single central atom. According to various embodiments, metal ion chelators can inhibit enzymatic nuclease activity against dsRNA or single-stranded RNA. Examples of sequestering agents include, but are not limited to, citrate, ammonium sulfate, acrylic copolymers (e.g., NOVERITE® K-775), ethylenediaminetetraacetic acid (EDTA), lignosulfonates, sodium lignosulfonate, glutamic acid diacetate (GLDA), diethylenetriaminepentaacetic acid (DTPA), N-carboxymethyliminobis(ethylenenitrilo)tetra(acetic acid), ethylenediamine-N,N'-bis(2-hydroxyphenylacetic acid), N-(2-hydroxyethyl)ethylenediamine-N,N,N'-tiacetic acid, ethylenediamine-N,N'-bis(2-hydroxy-6-methylphenylacetic acid, ethylenediamine-N,N'-bis(4-carboxy-2-hydroxyphenylacetic acid, ethylenediamine-N,N'-bis(2-hydroxy-5-sulfophenylacetic acid).

[0053] According to various embodiments, sequestering agents can be selected for use in the formulation based on their ability to chelate the divalent metal cations required for the enzymatic activity of many common nucleases.For example, EDTA is a sequestering agent that has the ability to chelate the divalent metal cations required for the enzymatic activity of many common nucleases.By chelating these metal ions, any available nucleases cannot perform their functions, and thus dsRNA or single-stranded RNA is protected from enzyme-mediated degradation in concentrated formulations.

[0054] UV Protectants: Definitions and Examples As used herein, the term "UV protectant" or "dispersant surfactant" or "dispersing surfactant" refers to a composition that increases the stability of dsRNA or single-stranded RNA when exposed to ultraviolet radiation. Examples of UV protectants may include, but are not limited to, conjugated aromatic surfactants. For example, UV protectants may include, but are not limited to, polymeric surfactants containing conjugated aromatic functionality as exemplified by lignin, naphthalene-based surfactants such as lignosulfonates, dioctyl sodium sulfosuccinate, and naphthalene sulfonate condensates. Kraft lignin is a type of industrial lignin obtained from kraft pulp, and accounts for about 85% of the total lignin production worldwide. Further examples of UV protection agents may include, but are not limited to, conjugated aromatic surfactants such as lignosulfonates and naphthalenesulfonates, most specifically REAX® 105M, REAX® 910, KRAFTSPERSE® 8828, REAX® 1425E, REAX® 260, MORWET® D-425, MORWET® EFW, and MORWET® IP.

[0055] REAX® 105M is a commercially available highly sufonated low molecular weight kraft lignosulfonate dispersant with low free electrolyte content. REAX® 910 is a commercially available moderately sulfonated kraft lignin dispersant characterized by low free electrolyte content, low conductivity, and near neutral pH. REAX® 1425E is a commercially available ethoxylated kraft lignosulfonate with excellent water solubility. REAX® 260 is a commercially available sodium sulfite lignosulfonate product used as a dispersant in dry formulations such as water dispersible granules or wettable powders. KRAFTSPERSE® 8828 is a commercially available hydrophobic, high molecular weight lignin-based dispersant. MORWET® D-425 is a commercially available sodium salt of a naphthalene sulfonate condensate. MORWET® EFW is a commercially available blend of sodium alkyl naphthalene sulfonates, and MORWET® IP is a commercially available sodium isopropyl naphthalene sulfonate.

[0056] Antifreeze: Definition and Examples As used herein, the term "antifreeze" refers to an additive that reduces the freezing point of water-based liquids. Examples of antifreezes may include, but are not limited to, propylene glycol, methanol, ethanol, propanol, isopropanol, alkylene glycol ethers, alkylene glycol alkyl ethers, and glycerol. According to various embodiments, an antifreeze may be selected for use in a formulation based on its performance at low temperatures to provide stability to the formulation and individual components at temperatures below freezing. For example, propylene glycol is a common alcohol-based solvent used for its excellent performance at low temperatures, thus providing stability to the formulation and individual components at temperatures below freezing.

[0057] Buffers: definition and examples As used herein, the term "buffer" refers to a solution that can resist pH change upon addition of acidic or basic components and functions, and functions to neutralize small amounts of added acid or base, maintaining the pH of the solution for relative stability. According to various embodiments, the buffer can be selected to mimic a neutral pH and help prevent acid / base hydrolysis and subsequent possible degradation of dsRNA or single-stranded RNA in the formulation.

[0058] According to various embodiments, a phosphate-based buffer may be used. For example, according to various embodiments, a potassium phosphate buffer at pH 7 may be used to mimic neutral pH and help prevent possible acid / base hydrolysis and subsequent degradation of dsRNA or single-stranded RNA in the formulation. Another example of a buffer composition is a phosphate-citrate buffer combination at pH 7, according to various embodiments. This composition may be used to mimic neutral pH and help prevent possible acid / base hydrolysis of dsRNA or single-stranded RNA in the formulation, and may provide additional potential chelating effects by including citrate. Another example of a buffer composition is a citrate buffer at pH 6, according to various embodiments. Such a composition may be used to increase chelation through the use of a citrate buffer and improve protection against microbial contamination through the use of a pH of 6.

[0059] Examples of buffer solutions include potassium phosphate, bis-tris(bis-trismethane), ADA (2-[(2-amino-2-oxoethyl)-(carboxymethyl)amino]acetic acid), ACES (2-(carbamoylmethylamino)ethanesulfonic acid), PIPES (1,4-piperazinediethanesulfonic acid), MOPSO (2-hydroxy-3-morpholin-4-ylpropane-1-sulfonic acid), BES (2-[bis(2-hydroxyethyl)amino]ethanesulfonic acid), MOPS (3-morpholinopropane-1-sulfonic acid), TES (2-[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid), HEPES (2-[4-(

[0036] Examples of suitable amines include, but are not limited to, 2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid), DIPSO (3-[bis(2-hydroxyethyl)amino]-2-hydroxy-1-propanesulfonic acid), MOBS (4-(4-morpholinyl)-1-butanesulfonic acid), TAPSO (3-{[1,3-dihydroxy-2-(hydroxymethyl)-2-propanyl]amino}-2-hydroxy-1-propanesulfonic acid), TRIS (3-{1,1,1,5,5,5-hexamethyl-3-[(trimethylsilyl)oxy]-3-trisiloxanyl}propyl methacrylate), and citric acid (2-oxide-1,2,3-propanetricarboxylate).

[0060] Defoamers: Definition and Examples As used herein, the term "defoamer" or "foam suppressant" refers to a compound that reduces and inhibits the formation of foam in industrial process liquids. Examples of defoamers may include, but are not limited to, insoluble oils, polydimethylsiloxanes and other silicones, certain alcohols, stearates, and glycols. Examples of specific commercially available defoamers may include, but are not limited to, SAG 1572™, DREWPLUS™ L-768, ANTIFOAM 8830™, AGNIQUE™ DFM111S, SILFOAM™ SE11, SILFOAM™ SE21, ANTIFOAM 100™, ANTIFOAM HL550™, SAG 1599™, ANTIFOAM 8810™, ANTIFOAM 8820™, and ANTIFOAM GN11P™. According to various embodiments, SAG 1572™ and DREWPLUS® L-768 were selected to reduce persistent foam upon dilution in the spray tank.

[0061] SAG 1572™ and SAG 1599™ are commercially available antifoam emulsions containing polydimethylsiloxane. DREWPLUS® L-768 is a commercially available antifoam emulsion containing polydimethylsiloxane. ANTIFOAM 8830™, ANTIFOAM 100™, ANTIFOAM HL550™, ANTIFOAM 8810™, ANTIFOAM 8820™, and ANTIFOAM GN11P™ are commercially available antifoam emulsions containing polydimethylsiloxane. AGNIQUE® DFM111S is a commercially available antifoam emulsion containing polydimethylsiloxane and propylene glycol. SILFOAM® SE11 and SILFOAM® SE21 are commercially available low viscosity silicone-based antifoam emulsions.

[0062] Biological preservatives: definition and examples As used herein, the term "biological preservative" refers to a substance used to control organisms that are harmful to crops, human or animal health, or cause damage to natural or manufactured products. Examples of biological preservatives may include, but are not limited to, KATHON® CG / ICP, ROCIMA® BT2S, and PROXEL® GXL.

[0063] KATHON® CG / ICP is a broad spectrum fungicide and biocide containing the active substances 5-chloro-2-methyl-4-isothiazolin-3-one (CMIT) and 2-methyl-4-isothiazolin-3-one (MIT). ROCIMA® BT2S and PROXEL® GXL are broad spectrum biocides containing 1,2-benzisothiazolin-3-one (BIT) as the active substance.

[0064] General Considerations Various embodiments provide formulations utilizing dsRNA or single-stranded RNA as active ingredients for use as biopesticides in agricultural markets. According to various embodiments, the formulations may be soluble liquid concentrates. The formulations may provide the necessary physical and chemical stability to the dsRNA or single-stranded RNA such that foliar or other application of the formulations may assist in pest or pathogen control in pest or pathogen management programs via exogenous RNAi. Various embodiments of the formulations perform well in CIPAC / test methods for SL-type formulations for foliar application. Various embodiments of the formulations may improve the chemical and physical stability of the dsRNA or single-stranded RNA to enable stable dsRNA or single-stranded RNA-based agricultural formulations at room temperature and may prevent or limit bacterial and fungal contamination, enzymatic nuclease activity, and RNA degradation due to UV irradiation.

[0065] By utilizing the high specificity and efficacy of exogenously delivered RNA to achieve RNAi in selected genomic targets, selected pest populations can be targeted and eliminated with a significant reduction in harmful biological effects. Leptinotarsa ​​decemlineata has shown increased resistance to current potato pest management programs. The development of SL-type formulations for exogenous delivery of dsRNA to Leptinotarsa ​​decemlineata has demonstrated a high degree of degradation when exposed to common environmental and storage conditions, such as spoilage or non-sterile environments, microbial contamination, and UV irradiation. The present invention aims to provide a formulation that allows RNA to be room temperature stable for exogenous delivery and eliminates or reduces RNA degradation due to contamination or environmental conditions.

[0066] Various embodiments relate to soluble liquid concentrate formulations for the delivery of dsRNA, due to both the inherent negative charge and water solubility of biopolymers. The formulation type is characterized by an aqueous-based concentrate, where the active ingredient is completely solubilized in the formulation medium. The inactive or auxiliary ingredients of the soluble liquid concentrate formulation may include several classes of auxiliary ingredients in addition to the formulation-specific ones that help stabilize or protect the active ingredient from degradation as needed. The formulation design may include auxiliary ingredients designated as antifreeze agents to prevent freezing at low temperatures, one or more broad-spectrum preservative ingredients for microbial and fungal protection, primary surfactants to promote stability and foliar application to plants, antifoam agents to prevent persistent foaming upon dilution in spray tanks, and pH buffers to prevent acid / base hydrolysis of dsRNA, according to various embodiments. However, because dsRNA is a biopolymer and faces specific degradation problems not common to other polymers or small molecules, additional auxiliary components may be added according to various embodiments to include metal ion chelators that serve to inhibit nuclease activity by sequestering divalent metal cations, and anionic dispersant-type surfactants consisting of conjugated aromatic functional groups (preferably polymers) that prevent degradation of dsRNA after foliar application by UV irradiation. Some embodiments further include cationic or zwitterionic agents (also referred to herein as "secondary surfactants") as additional surfactants with antimicrobial effects.

[0067] Various embodiments may provide room temperature stable, soluble liquid concentrate formulations containing dsRNA or single stranded RNA for foliar application. The formulation compositions, according to various embodiments, may promote the stability of RNA in the presence of common contaminants and environmental conditions that would otherwise result in rapid degradation of RNA.

[0068] Various embodiments allow for the production of cost-effective dsRNA or single-stranded RNA formulations that can provide low toxicity and highly selective RNAi treatment for exogenous foliar application.The potential use of RNAi technology as foliar application in agricultural industry is particularly beneficial, as various target species show enhanced resistance to current commercial pest management programs.

[0069] During the formulation development process, each individual component was selected for performance, stability, and compatibility depending on the needs of the formulation design. Table 1 provides a list of the components of an exemplary formulation as well as a brief generalized description of each component's function in the overall formulation. [Table 1]

[0070] Various embodiments relate to a formulation for delivering dsRNA or single-stranded RNA to pests via exogenous foliar application of the formulation to plants. According to various embodiments, the formulation may be a soluble liquid concentrate. According to various embodiments, the formulation may include RNA, a primary surfactant, and a sequestering agent, optionally a secondary surfactant, optionally a UV protection agent, optionally a buffer, optionally a biological preservative, optionally an antifoaming agent, and optionally an antifreeze agent. In some embodiments, the formulation includes RNA, a primary surfactant, and a sequestering agent, optionally a secondary surfactant, optionally a UV protection agent, optionally a buffer, optionally a biological preservative, optionally an antifoaming agent, and optionally an antifreeze agent. In some embodiments, the formulation includes RNA, a primary surfactant, and a sequestering agent, and a UV protection agent, optionally a secondary surfactant, optionally a buffer, optionally a biological preservative, optionally an antifoaming agent, and optionally an antifreeze agent.

[0071] Various embodiments relate to compositions for providing sufficient stability to RNA to facilitate delivery of the RNA to pests via exogenous foliar application of the composition and RNA to plants. The composition may include a primary surfactant, and a sequestering agent, optionally a secondary surfactant, a UV protectant, optionally a buffer, optionally a biological preservative, optionally an antifoaming agent, and optionally an antifreeze agent. The composition may include a primary surfactant, and a sequestering agent, and a secondary surfactant, optionally a UV protectant, optionally a buffer, optionally a biological preservative, optionally an antifoaming agent, and optionally an antifreeze agent. The composition may include a primary surfactant, and a sequestering agent, and a UV protectant, optionally a secondary surfactant, optionally a buffer, optionally a biological preservative, optionally an antifoaming agent, and optionally an antifreeze agent.

[0072] In any of the above formulations or compositions, the RNA can be any suitable dsRNA or RNA as described herein. According to various embodiments of the above formulations, the RNA can be present in an amount of about 0.05 to about 10 percent by weight based on the total weight of the formulation, or 0.25 to about 2 percent by weight based on the total weight of the formulation. According to various embodiments of the above formulations, the dsRNA or single-stranded RNA can be present in an amount within a range having a lower limit and / or an upper limit. The range can include or exclude the lower limit and / or the upper limit.The lower and / or upper limits are about 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.05, 1.1, 1.15, 1.2, 1.25, 1.3, 1.35, 1.4, 1.45, 1.5, 1.55, 1.6, 1.65, 1.7, 1.75, 1.8, 1.85, 1.9, 1.95, 2, 2.05, 2.1, 2.15, 2.2, 2.25, 2.3, 2.35, 2.4, 2.45 ,2.5,2.55,2.6,2.65,2.7,2.75,2.8,2.85,2.9,2.95,3,3.05,3.1,3.15,3.2,3.25,3.3,3.35,3.4,3.45,3.5,3.55,3.6,3.65,3.7,3.75,3.8,3.85,3.9,3.95,4,4.05,4.1,4.15,4.2,4.25,4.3,4.35,4.4,4.45,4.5,4.55,4.6,4.65,4.7,4.75,4.8,4.85,4.9,4.95,5,5.05, 5.1, 5.15, 5.2, 5.25, 5.3, 5.35, 5.4, 5.45, 5.5, 5.55, 5.6, 5.65, 5.7, 5.75, 5.8, 5.85, 5.9, 5.95, 6, 6.05, 6.1, 6.15, 6.2, 6.25, 6.3, 6.35, 6.4, 6.45, 6.5, 6.55, 6.6, 6.65, 6.7, 6.75, 6.8, 6.85, 6.9, 6.95, 7, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35, 7.4, 7.45, 7.5, 7.55, 7.6, 7.6 9.05, 9.1, 9.15, 9.2, 9.25, 9.3, 9.35, 9.4, 9.45, 9.5, 9.55, 9.6, 9.65, 9.7, 9.75, 9.8, 9.85, 9.9, 9.95, and 10 weight percent.For example, according to various embodiments of the above formulations, the dsRNA or single-stranded RNA may be present in an amount of about 0.25 to about 2 weight percent based on the total weight of the formulation, or any combination of the lower and upper limits recited.

[0073] In any of the above formulations or compositions, the primary surfactant may be any suitable primary surfactant or combination of primary surfactants described herein. According to various embodiments of the above formulations or compositions, the primary surfactant may be present in an amount of about 1 to about 10 weight percent based on the total weight of the formulation or composition, or in an amount of about 4 to about 6 weight percent based on the total weight of the formulation or composition. According to various embodiments of the above formulations or compositions, the primary surfactant may be present in an amount within a range having a lower limit and / or an upper limit. The range may include or exclude the lower limit and / or upper limit. The lower limit and / or upper limit may be selected from about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, and 15 weight percent. For example, according to various embodiments of the above formulations or compositions, the primary surfactant may be present in an amount of from about 1 to about 10 weight percent, or from about 4 to about 6 weight percent, or any combination of the recited lower and upper limits, based on the total weight of the formulation or composition.

[0074] In any of the above formulations or compositions, the secondary surfactant may be any suitable secondary surfactant or combination of secondary surfactants described herein. According to various embodiments of the above formulations or compositions, the secondary surfactant may be present in an amount of about 0.01 to about 3 weight percent based on the total weight of the formulation or composition. According to various embodiments of the above or compositions, the secondary surfactant may be present in an amount of about 0.01 to about 1 weight percent based on the total weight of the formulation or composition. According to various embodiments of the above formulations or compositions, the secondary surfactant may be present in an amount within a range having a lower limit and / or an upper limit. The range may include or exclude the lower limit and / or the upper limit. The lower and / or upper limits can be selected from about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, and 5 weight percent. For example, according to various embodiments of the above formulations or compositions, the secondary surfactant may be present in an amount of about 0.01 to about 3 weight percent, or any combination of the lower and upper limits recited, based on the total weight of the formulation or composition. In an additional example, according to various embodiments of the above formulations or compositions, the secondary surfactant may be present in an amount of about 0.01 to about 1 weight percent, or any combination of the lower and upper limits recited, based on the total weight of the formulation or composition.

[0075] In any of the above formulations or compositions, the sequestering agent may be any suitable sequestering agent or combination of sequestering agents described herein. According to various embodiments of the above formulations or compositions, the sequestering agent may be present in an amount of about 0.1 to about 5 weight percent based on the total weight of the formulation or composition. According to various embodiments of the above formulations or compositions, the sequestering agent may be present in an amount of about 0.1 to about 2 weight percent based on the total weight of the formulation or composition. According to various embodiments of the above formulations or compositions, the sequestering agent may be present in an amount within a range having a lower limit and / or an upper limit. The range may include or exclude the lower limit and / or the upper limit. The lower and / or upper limits can be selected from about 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, and 5 weight percent. For example, according to various embodiments of the above formulations or compositions, the sequestering agent may be present in an amount of about 0.1 to about 5 weight percent, or any combination of the lower and upper limits recited, based on the total weight of the formulation or composition. For example, according to various embodiments of the above formulations or compositions, the sequestering agent may be present in an amount of about 0.1 to about 2 weight percent, or any combination of the lower and upper limits recited, based on the total weight of the formulation or composition.

[0076] In any of the above formulations or compositions, the UV protection agent (also known as dispersant surfactant) can be any suitable UV protection agent, dispersant surfactant, or combination thereof described herein, or combinations thereof described herein. According to various embodiments of the above formulations or compositions, the UV protection agent can be present in an amount of about 1 to about 4 weight percent or about 1 to about 2 weight percent, based on the total weight of the formulation or composition. According to various embodiments of the above formulations or compositions, the UV protection agent can be present in an amount within a range having a lower limit and / or an upper limit. The range can include or exclude the lower limit and / or the upper limit. The lower and / or upper limits can be selected from about 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, and 5 weight percent. For example, according to various embodiments of the formulations or compositions described above, the UV protection agent may be present in an amount of about 1 to about 4 weight percent, or about 1 to about 2 weight percent, based on the total weight of the formulation or composition, or any combination of the recited lower and upper limits.

[0077] In any of the above formulations or compositions, the buffer may be any suitable buffer or combination of buffers described herein. According to various embodiments of the above formulations or compositions, the buffer may be present in an amount of about 1 to about 3 weight percent based on the total weight of the formulation or composition. According to various embodiments of the above formulations or compositions, the buffer may be present in an amount within a range having a lower limit and / or an upper limit. The range may include or exclude the lower limit and / or the upper limit. The lower and / or upper limits can be selected from about 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, and 5 weight percent. For example, according to various embodiments of the above formulations or compositions, the buffer may be present in an amount of about 1 to about 3 weight percent, or any combination of the recited lower and upper limits, based on the total weight of the formulation or composition, hi some embodiments, the buffer is present at a concentration of at least about 200 mM or at least about 242 mM.

[0078] In any of the above formulations or compositions, the biological preservative may be any suitable biological preservative or combination of biological preservatives described herein. According to various embodiments of the above formulations or compositions, the biological preservative may be present in an amount of about 0.05 to about 1 weight percent based on the total weight of the formulation or composition. According to various embodiments of the above formulations or compositions, the biological preservative may be present in an amount within a range having a lower limit and / or an upper limit. The range may include or exclude the lower limit and / or the upper limit. The lower and / or upper limits can be selected from about 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, and 5 weight percent. For example, according to various embodiments of the formulations or compositions described above, the biopreservative may be present in an amount of about 0.05 to about 1 weight percent, based on the total weight of the formulation or composition, or any combination of the lower and upper limits recited.

[0079] In any of the above formulations or compositions, the antifoaming agent can be any suitable antifoaming agent or combination of antifoaming agents described herein. According to various embodiments of the above formulations or compositions, the antifoaming agent can be present in an amount of about 0.025 to about 0.2 weight percent based on the total weight of the formulation or composition. According to various embodiments of the above formulations or compositions, the antifoaming agent can be present in an amount of about 0.025 to about 0.1 weight percent based on the total weight of the formulation or composition. According to various embodiments of the above formulations or compositions, the antifoaming agent can be present in an amount within a range having a lower limit and / or an upper limit. The range can include or exclude the lower limit and / or the upper limit. The lower and / or upper limits can be selected from about 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, and 5 percent by weight. For example, according to various embodiments of the above formulations or compositions, the antifoaming agent may be present in an amount of about 0.025 to about 0.1 weight percent, based on the total weight of the formulation or composition, or any combination of the lower and upper limits recited. In additional embodiments, in the above formulations or compositions, the antifoaming agent may be present in an amount of about 0.025 to about 0.2 weight percent, based on the total weight of the formulation or composition, or any combination of the lower and upper limits recited.

[0080] In any of the above formulations or compositions, the antifreeze agent can be any suitable antifreeze agent or combination of antifreeze agents described herein. According to various embodiments of the above formulations or compositions, the antifreeze agent can be present in an amount of about 5 to about 15 percent by weight based on the total weight of the formulation or composition. According to various embodiments of the above formulations or compositions, the antifreeze agent can be present in an amount within a range having a lower limit and / or an upper limit. The range can include or exclude the lower limit and / or the upper limit. The lower limit and / or the upper limit can be selected from about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 percent by weight. For example, according to various embodiments of the above formulations or compositions, the antifreeze agent can be present in an amount of about 5 to about 15 percent by weight based on the total weight of the formulation or composition, or any combination of the lower and upper limits described.

[0081] Various embodiments relate to a method of protecting a plant from a pest, the method comprising exogenously applying to the plant a formulation according to any of the embodiments described herein. The formulation may further comprise water, according to various embodiments. The water may dilute the exogenous RNAi biopesticide by about 15 to about 300 times. The water may dilute the exogenous RNAi biopesticide by a factor within a range having a lower limit and / or an upper limit. The range may include or exclude the lower limit and / or the upper limit. The lower and / or upper limits are about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215 , 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, and 400. For example, according to certain embodiments, the water may be diluted with the exogenous RNAi biopesticide by about 15 to about 300 times, or any combination of the lower and upper limits listed. The formulation may be applied to the foliage of the plant, for example, by spraying.

[0082] Various embodiments relate to methods of stabilizing dsRNA, including combining a dsRNA with a composition to provide the dsRNA with sufficient stability to facilitate delivery of the dsRNA to a pest via exogenous foliar application of the composition and the dsRNA to a plant. In some embodiments, the dsRNA remains in solution and does not precipitate after 2 weeks at 54° C. In some embodiments, the dsRNA shows no evidence of detectable degradation (i.e., within known variations in HPLC) or minimal degradation (less than 5%) when measured by HPLC comparing the initial dsRNA concentration and comparing it to the dsRNA concentration after 2 weeks at 54° C. In some embodiments, the pH of the composition changes by no more than + / - 2 units after 2 weeks at 54° C. In some embodiments, the time periods and temperature conditions for these stability evaluations are 1 year at room temperature, 2 years at room temperature, 4 weeks at 54°C, 8 weeks at 54°C, 2 weeks at -10°C, 2 weeks at 4°C, 2 weeks at 40°C, 4 weeks at 4°C, 4 weeks at 40°C, 8 weeks at 4°C, or 8 weeks at 40°C.

[0083] Table 2 provides details of formulations according to various embodiments. It should be understood that the specific formulations detailed in Table 2 (or any specific formulations detailed herein) do not limit the scope of possible formulations according to various embodiments, and the specific formulations are merely examples. [Table 2]

[0084] In addition to the formulation compositions listed in Table 2, there are additional formulation compositions containing alternative ingredients that further provide the same benefits and perform the intended function of each particular adjunct ingredient. Non-limiting alternative compositions, according to various embodiments, are shown in Tables 3 and 4.

[0085] Table 3 provides exemplary formulation details, according to various embodiments. [Table 3]

[0086] Table 4 provides exemplary formulation details, according to various embodiments. [Table 4]

[0087] Table 5 provides exemplary formulation details, according to various embodiments. [Table 5]

[0088] According to various embodiments, the formulations exhibiting superior chemical and physical stability of dsRNA or single-stranded RNA may include a combination of broad-spectrum biological preservatives, such as a combination of BIT, MIT, and CMIT, to ensure the stability of RNA in solution over time under different environmental conditions and after exposure to common bacterial and fungal contaminants. The formulations exhibiting superior chemical and physical stability of dsRNA or single-stranded RNA may further include a cationic / zwitterionic secondary surfactant, which has been identified in the bacterial challenge test provided in the Examples herein as having a positive impact and antimicrobial activity that cationic surfactants have in the formulation, as well as preventing dsRNA or single-stranded RNA degradation. The formulations exhibiting superior chemical and physical stability of dsRNA or single-stranded RNA may further include a metal ion chelator, such as EDTA, which inhibits nuclease activity. Nuclease activity is a key challenge to RNA stability over time under different environmental conditions and after bacterial or fungal contamination. The inclusion of a metal ion chelator, exemplified by EDTA, helps inhibit nuclease activity by sequestering divalent cations necessary for enzyme nuclease activity. In relation to this TGAI concentration, a minimum concentration of EDTA was determined for dsRNA stability for practical applications, as detailed in the examples provided herein. Formulations exhibiting excellent chemical and physical stability of dsRNA or single-stranded RNA may further include lignosulfonates, or polymer-conjugated aromatic surfactants to prevent RNA degradation by UV irradiation. This prevention of RNA degradation by UV irradiation translates into increased biological efficacy after application and exposure. Formulations exhibiting excellent chemical and physical stability of dsRNA may further include a primary surfactant to stabilize the formulation containing dsRNA and a cationic surfactant. The primary surfactant may help prevent complexation of dsRNA with the cationic surfactant and provide stability to the formulated product. Formulations exhibiting excellent chemical and physical stability of RNA may further include a buffer to prevent acid / base hydrolysis of dsRNA over a variety of storage conditions.Additionally, the examples provided herein show that optimized concentrations of buffers can help maintain the stability of dsRNA in concentrated products and upon dilution, especially under high ionic strength conditions.

[0089] The formulation compositions detailed according to various embodiments, as detailed and tested in the examples provided herein, demonstrate excellent chemical and physical stability attributes that stabilize dsRNA or single stranded RNA as ambient temperature stable formulations. The formulation matrix demonstrates very good compatibility with dsRNA producing a repeatable SL type formulation with complete solubilization of all individual components. The formulation compositions contain several auxiliary ingredients and excipients that work together to impart excellent physical and chemical stability to the dsRNA both in the concentrated product and upon dilution for the intended use. At 100x dilution, these formulations have been shown to be stable across a range of water types and hardness from DI water to 1000 ppm calcium / magnesium water. In most of the various waters, these formulations have been shown to be clear solutions and exhibit what is described as micellar solubilization, while samples at 1000 ppm water exhibit stable nanoparticle precipitates that are in the range of 300-600 nm. These formulations have also been stored at various temperature conditions, including -10°C, 4°C, room temperature, 40°C, and 54°C, and retain their physical and chemical stability over various environmental conditions. After storage at various conditions, they have been analyzed via HPLC for dsRNA stability and, if possible, to determine degradation profiles. This HPLC analysis is known to exhibit a variability of approximately + / -10%, and samples after storage within this known variability compared to the initial concentration demonstrate dsRNA stability in these formulations. The pH of each formulation before and after storage was also measured and no significant changes were demonstrated, indicating chemical and physical stability after various storage conditions. The exemplary formulations contain ATPLUS® PFA as a non-ionic surfactant, which aids in the stability and solubilization of the dsRNA and auxiliary ingredients in the existing formulation matrix, both in concentrated solutions and upon dilution with water. The exemplary formulations also contain a comprehensive preservative package that demonstrates protection of the dsRNA from bacterial and fungal biological contamination. The results of this study show that the selected formulation matrix degrades and destroys several common bacteria and fungi once exposed to this contamination, providing extended stability following bacterial and fungal contamination.Many bacteria and fungi can ingest biopolymers such as dsRNA, promoting bacterial and fungal growth and dsRNA degradation. To prevent this double degradation, the preservative component of the formulation must be able to withstand biological contamination and protect the dsRNA from degradation, and this test demonstrates that this is achieved with selected formulations. The formulations according to various embodiments have a robust design that demonstrates acceptable levels of physical and chemical stability, passes certain regulatory tests specified for soluble liquid concentrate type formulations with agricultural applications, and can protect the active ingredient dsRNA over two years at ambient storage. In the absence of the ability to provide a dsRNA formulation that demonstrates physical and chemical stability as a practical application, there is no formulation to exogenously deliver dsRNA as part of a pest or pathogen management program. The formulation composition described in this disclosure addresses multiple challenges to dsRNA stability in solution as a practical application, enabling the use of dsRNA for commercial applications in agricultural pest programs.

[0090] Various embodiments provide compositions and formulations that can provide chemical and physical stability to RNA to enable practical application of dsRNA or single-stranded RNA in agricultural pest management programs. Individual formulation components have been selected to provide benefits to the stability of the dsRNA formulation, including chemical stability and physical stability, both in concentrated products and upon dilution during application. Many of these components interact with each other and have ratios optimized for stability. Other named components have alternative components that provide similar performance, while others have concentration ranges or alternative components have not been explored.

[0091] Development of soluble liquid concentrate formulations containing dsRNA or single-stranded RNA for practical application as foliar applied agricultural products that are stable in solution and upon dilution for application, and that are resistant to chemical degradation due to time, storage conditions, or contamination. EXAMPLES

[0092] preface The following examples are presented to provide those skilled in the art with a complete disclosure and description of how to carry out the methods, make the compositions and compounds, and use the methods disclosed and claimed herein. Although efforts have been made to ensure accuracy with respect to numerical values ​​(e.g., amounts, temperatures, etc.), some errors and deviations should be accounted for. The purpose of the following examples is not to limit the scope of the various embodiments, but merely to provide examples that illustrate certain embodiments.

[0093] Formulation Method Each probe formulation is prepared via low shear and simple mixing according to the protocols illustrated in the Examples below. Although the Examples provide specific details regarding the amounts and types of ingredients and procedures, one of skill in the art will readily appreciate the wide range of variations suggested by the specific formulation methods set forth.

[0094] Example 1 Formulations according to various embodiments were prepared via low shear and simple mixing according to the following protocol:

[0095] Pre-taring reaction vessels allows proper weight / volume to be added and verified during production.

[0096] To the vessel, add the appropriate weight of dsRNA material to reach a final concentration of 8 g / kg dsRNA, or 0.8% dsRNA.

[0097] Place the lower 3 propeller blades of an overhead mixer unit into the vessel containing the dsRNA and stir at low shear using the minimum speed necessary to create a gentle vortex in the solution, approximately 450 RPM. Maintain gentle stirring throughout the remaining additions to the vessel, increasing the RPM as necessary, but minimally to avoid shearing the dsRNA.

[0098] Add biological preservatives to the container, e.g., 0.1% ROCIMA® BT2S, and 0.05% KATHON® CG / ICP. Stir at 450 RPM for approximately 1 minute until thoroughly mixed.

[0099] Add an antifreeze to the container, for example 10% propylene glycol. Stir the mixture at 500 RPM for approximately 2 minutes until the resulting solution is a clear yellow solution.

[0100] A sequestering agent, for example 1.583% EDTA, is added to the vessel while stirring at 500 RPM.

[0101] Add buffer to the vessel, e.g., 12.10% 2M potassium phosphate buffer pH 7 solution. Upon addition of the buffer and EDTA solution, the formulation will become slightly cloudy but will return to a clear solution after a few minutes of stirring. 600 RPM approximately 5 minutes.

[0102] Add 0.05% antifoam to the container. Various antifoam agents were tested, as detailed in the Examples below. Stir at 600 RPM until thoroughly mixed, resulting in a slightly cloudy yellowish solution in approximately 2 minutes.

[0103] 5% of the primary surfactant is added to the vessel and the RPM is slowly increased to 900 RPM. A variety of primary surfactants were tested, as detailed in the Examples below.

[0104] Add a secondary surfactant to the vessel, for example, up to 0.25% cetrimonium chloride or up to 1% lauryl betaine. With direct addition of cetrimonium chloride or lauryl betaine, a slight local concentration of precipitate will be observed, but will dissolve quickly into the solution. Continue stirring the solution at 900 RPM for approximately 2 minutes until all materials are completely solubilized and the resulting solution is slightly turbid and slightly yellow. If a quaternary ammonium compound (e.g., ATPLUS® PFA) is added as a primary surfactant without the presence of a secondary surfactant, the quaternary ammonium compound may interact with RNA, resulting in immediate instability of dsRNA in the formulation.

[0105] Add up to 1.5% UV protectant (also known as a dispersant surfactant) to the container and allow to mix at 900 RPM for 2 minutes.

[0106] Add the appropriate weight percent of water to the container to reach the target formulation weight. Stir at 500 RPM for approximately 1 minute until the solution is thoroughly mixed.

[0107] The sample is then mixed via low shear until each component is fully incorporated and the resulting liquid should be a translucent, slightly hazy, slightly yellowish liquid with low viscosity. If a naphthalene sulfonate condensate or lignosulfonate is used as the optional UV protectant (also known as a dispersant surfactant), the resulting liquid will be a dark brown liquid with low viscosity.

[0108] The methods described herein or any other methods known in the art can be used to prepare the compositions of the present disclosure using various ingredients in various concentrations.

[0109] Examples 2 to 6: Formulation compatibility These examples demonstrate the importance of the primary surfactant and, according to various embodiments, the buffer as well. Formulation compatibility is characterized by physical observation. As a soluble liquid concentrate type formulation, all components should be easily solubilized in water, and any precipitation, aggregation, phase separation, or miscibility problems are indicators that the formulation components are not compatible. In general, formulations according to various embodiments have been identified to have no compatibility problems and produce finely solubilized formulations with low viscosity. The formulations are mostly translucent, but sometimes slightly turbid, and are clear to yellowish solutions.

[0110] For purposes of these examples, ATPLUS® PFA was used as the primary surfactant and was determined to be stable between a concentration range of 4-6% by weight. Adjusting the concentration of the primary surfactant above or below these specified ranges was demonstrated in these examples to result in a significant amount of precipitation in the concentrated product in compositions having the ingredients at the concentrations listed.

[0111] The formulation composition according to various embodiments may include a buffer such as a phosphate buffer at or about pH 7, or a citrate buffer at or about pH 6 in another embodiment. For the purposes of these examples and elsewhere herein, the term "phosphate buffer" refers to a buffer containing a specific ratio of monobasic potassium phosphate and dibasic potassium phosphate. More specifically, a 0.1-1.1 mass ratio of monobasic potassium phosphate to dibasic potassium phosphate spans a pH range of 6.5-7.5. According to various embodiments, when stored above refrigeration temperatures or when storage temperatures fluctuate significantly, the addition of a buffer is important for the formulation to maintain a pH stable system in preventing acid / base hydrolysis over time as well as during storage under different temperature conditions. In addition to this stability advantage for preventing acid / base hydrolysis, the buffer system provides a particular stability advantage in maintaining the solubility of electrolyte-based systems. The absence or reduction in molar amount of the phosphate buffer may result in precipitation of the concentrated formulation.

[0112] Example 2 1A, 1B, 1C, and 1D are examples according to various embodiments and show photographs of compatible formulations after storage at -10°C for 2 weeks, at 4°C for 2 and 8 weeks, at 40°C for 8 weeks, and at 54°C for 2 weeks.

[0113] The formulations shown in FIGS. 1A, 1B, 1C, and 1D were prepared according to the methods described in Example 1 and had compositions according to Table 6. [Table 6]

[0114] As noted above, the primary surfactant in the formulation samples shown in Figures 1A, 1B, 1C, and 1D was ATPLUS® PFA, an EO:PO modified alcohol ethoxylate surfactant, at a usage rate of 5% by weight in the formulation. The stability and compatibility of formulations containing dsRNA is affected by changes in surfactant chemistry and concentration range within the formulation. Tests were performed using various concentrations of ATPLUS® PFA, and these particular formulations were found to be stable at 5% by weight.

[0115] Example 3 This example demonstrates a formulation with poor compatibility and stability with varying concentrations of a primary surfactant, specifically ATPLUS® PFA.

[0116] 2A and 2B are examples according to various embodiments, showing photographs of formulations having ATPLUS® PFA concentrations of 10% and 5% by weight, respectively, after storage at 54° C. At a concentration of 10% ATPLUS® PFA, a clearly visible precipitate forms in the formulation containing dsRNA. The formulation containing 5% ATPLUS® PFA is found to be stable without any signs of significant precipitation. Similar results were demonstrated at storage conditions ranging from −10° C. to 54° C.

[0117] The formulations shown were prepared according to the method described in Example 1 and had compositions according to Table 7. [Table 7]

[0118] Example 4 This example further illustrates the poor compatibility and stability of the formulation with varying concentrations of the primary surfactant, specifically ATPLUS® PFA.

[0119] 3A, 3B, 3C, and 3D are examples according to various embodiments, showing photographs of formulations having ATPLUS® PFA concentrations of 5%, 4.5%, 4.0%, and 3.5% by weight, respectively. No precipitate was observed at the 5% concentration of ATPLUS® PFA, and small amounts of visible precipitate were observed within acceptable limits in formulations having concentrations of ATPLUS® PFA less than 5% in these tested compositions.

[0120] The formulations shown were prepared according to the method described in Example 1 and had compositions according to Table 8. [Table 8]

[0121] Example 5 This example demonstrates the poor stability caused by the absence of a sequestering agent and the absence of a buffer in the formulation composition of Table 9. Figure 4 shows a photograph of this formulation without a sequestering agent, showing significant precipitation of dsRNA. The formulation shown was prepared according to the method described in Example 1 and had a composition according to Table 9. [Table 9]

[0122] Example 6 This example demonstrates the lower stability due to the concentration of buffer and the concentration of EDTA in the formulation composition.

[0123] FIGS. 5A, 5B, 5C, 5D, and 5E are examples according to various embodiments and show photographs of formulations having phosphate buffer concentrations of 242 mM, 200 mM, 150 mM, 100 mM, and 50 mM, respectively, with FIGS. 5C, 5D, and 5E showing limited amounts of visible precipitation, although within acceptable limits, where the phosphate buffer concentration and EDTA concentration have been significantly reduced.

[0124] The formulations shown were prepared according to the method described in Example 1 and had compositions according to Table 10. [Table 10]

[0125] Examples 7-9: Particle Size and Water Hardness Stability Particle size measurement is an important physical parameter and is used to determine whether a formulation containing dsRNA is stable upon dilution. To determine whether a formulation is stable, samples are diluted in CIPAC standard water specified by CIPAC MT 18 as 35 ppm water, 342 ppm water, 500 ppm water, and 1000 ppm water at spray application rates of 4:1 Mg 2+ :Ca 2+ It is roughly composed of an ionic solution. Upon dilution, the sample is left for 24 hours to observe any physical instability, and then the particle size is collected via DLS measurement using a Malvern Zetasizer NanoDS, or other suitable DLS device. The physical stability test is determining the optimal ratio between dsRNA supplied as technical grade active ingredient (TGAI) and the buffer system, especially when diluted with water having high ionic content.

[0126] Example 7 Selected formulation probes were diluted in water at concentrations equivalent to maximum field use rates using an application volume of 20 gallons per acre. Each water condition tested was prepared according to the CIPAC MT 18 standard method. Particle size measurements and physical observations were made on samples immediately after dilution and after 24 hours. Results of successful formulations are presented in this example, showing visual appearance and median particle size via DLS. [Table 11]

[0127] Table 11 shows the particle size, polydispersity, and count rate measured by dynamic light scattering for selected formulation dilutions under various aqueous conditions. The low count rate and Z-average particle size of 310-330 nm indicate that the diluted formulation has a very low concentration of particles and is stable in solution at room temperature.

[0128] Figures 6A, 6B, 6C, 6D are examples according to various embodiments, showing photographs of formulations diluted in water at 35 ppm, 342 ppm, 500 ppm, and 1000 ppm, respectively, the photographs were taken immediately after dilution in water, Figures 6E, 6F, 6G, and 6H are examples according to various embodiments, showing photographs of formulations diluted in water at 35 ppm, 342 ppm, 500 ppm, and 1000 ppm, respectively, the photographs were taken 24 hours after dilution in water.

[0129] The formulations shown were prepared according to the method described in Example 1 and had compositions according to Table 12. [Table 12]

[0130] Example 8 An additional aspect to formulation stability upon dilution is the ratio of phosphate buffer to TGAI concentration. Evidence shows that maximizing the ratio between buffer concentration and TGAI input source provides greater stability to the formulation upon dilution with high ionic strength (hard) water. This effect on stability upon dilution with high ionic strength water is in contrast to the stability of concentrated formulations, which show greater stability when the ratio of buffer to TGAI is minimized. An optimized ratio between buffer and TGAI (a mass ratio of TGAI to buffer composition greater than 0.2) has been selected for the exemplified composition providing stability in concentrated formulations and dilution with various ionic strength waters.

[0131] Figures 7A, 7B, 7C, and 7D are examples according to various embodiments, showing photographs of formulations including 200 mM phosphate buffer and 21 mM EDTA diluted in water at 35 ppm, 342 ppm, 500 ppm, and 1000 ppm, respectively, taken 24 hours after dilution in water. Figures 7E, 7F, 7G, and 7H are examples according to various embodiments, showing photographs of formulations including 150 mM phosphate buffer and 21 mM EDTA diluted in water at 35 ppm, 342 ppm, 500 ppm, and 1000 ppm, respectively, taken 24 hours after dilution in water. Figures 7I, 7J, 7K, and 7L are examples according to various embodiments, and show photographs of formulations including 100 mM phosphate buffer and 21 mM EDTA diluted in water at 35 ppm, 342 ppm, 500 ppm, and 1000 ppm, respectively, taken 24 hours after dilution in water. Figures 7M, 7N, 7O, and 7P are examples according to various embodiments, and show photographs of formulations including 20 mM phosphate buffer and 21 mM EDTA diluted in water at 35 ppm, 342 ppm, 500 ppm, and 1000 ppm, respectively, taken 24 hours after dilution in water.

[0132] As shown, decreasing the concentration of phosphate buffer and increasing the ratio between TGAI and buffer results in better solution stability in high ionic strength water, as shown by the disappearance of precipitate in 1000 ppm water after 24 hours, viewing the sample from left to right.

[0133] The formulations shown were prepared according to the method described in Example 1 and had compositions according to Table 13. [Table 13]

[0134] Example 9 This example provides observations on the effect on formulation stability upon dilution with high ionic strength water with respect to the ratio of TGAI to buffer at a dsRNA concentration of 4 g / L. When the dsRNA concentration is doubled to 8 g / L in the current composition, the TGAI input concentration is also roughly doubled. As a result of this increase in input TGAI, the ratio between buffer and TGAI is also increased as a result, with an improvement between otherwise comparable formulation compositions. An example of this between the corresponding TGAI inputs at 4 g / L dsRNA and 8 g / L dsRNA with the same concentration of phosphate buffer is shown in the figure below.

[0135] Figures 8A, 8B, 8C, and 8D show examples of formulations containing 8 g / L dsRNA diluted in water at 35 ppm, 342 ppm, 500 ppm, and 1000 ppm, respectively, according to various embodiments, and the photographs were taken 24 hours after dilution in water. Figures 8E, 8F, 8G, and 8H show examples of formulations containing 4 g / L dsRNA diluted in water at 35 ppm, 342 ppm, 500 ppm, and 1000 ppm, respectively, according to various embodiments, and the photographs were taken 24 hours after dilution in water. These figures show the difference in solution stability in high ionic strength water as a result of the ratio of TGAI to phosphate buffer. Samples with roughly a two-fold increase in the ratio of TGAI to phosphate buffer do not show the same precipitation after 24 hours after dilution in 1000 ppm water.

[0136] The indicated formulations were prepared according to the method described in Example 1 and had the compositions shown in Table 14. [Table 14]

[0137] The importance of buffer concentration on solution stability upon dilution can be summarized as follows: A general trend was observed indicating that increasing the ratio of TGAI to phosphate concentration results in an improvement in product stability upon dilution when using high ionic strength water. A decrease in phosphate buffer concentration results in a condition where there is a maximized ratio between TGAI and buffer, which aids in solution stability upon dilution with high ionic strength water. It is important to note that this effect of phosphate buffer concentration on stability in solution upon dilution is opposite to the effect of phosphate buffer concentration on stability in concentrated products. With these effects in mind, a concentration of 242 mM "maximizes" stability in concentrated solutions but "minimizes" stability in solution upon dilution with high ionic strength water. A decrease in phosphate buffer concentration positively impacts stability in solution upon dilution with high ionic strength water but negatively impacts stability in concentrated solutions. An increase in phosphate buffer concentration negatively impacts stability in solution upon dilution with high ionic strength water but positively impacts stability in concentrated solutions, therefore an optimized balance of high ionic strength water with 242 mM phosphate buffer was selected for stability in both conditions. This effect, and observations of this general trend, are shown in Table 15. [Table 15]

[0138] Table 15 demonstrates the clear effect of phosphate buffer concentration on formulation stability in the concentrated product and upon dilution in high ionic strength water.

[0139] Examples 10 to 12: Storage stability: physical and chemical stability after storage Shelf life stability and accelerated storage testing is performed by placing aliquots (minimum 50 mL) of each potential formulation at the following environmental conditions: -10°C, 4°C, room temperature, 40°C, 54°C, and freeze / thaw temperature cycling between -10°C and 40°C every 24 hours. Samples are stored at these conditions for a minimum of 2 weeks and 8 weeks at 40°C before analysis, which is determined by physical and chemical stability compared to the original sample or an aliquot from the sample stored at 4°C. Chemical stability is determined by quantification by HPLC utilizing a solid phase extraction-based HPLC method. Chemical stability is also verified using Ribogreen reagent, a nucleic acid binding reagent, and fluorescence spectroscopy according to the manufacturer's protocols. Physical stability is determined by noting any color change, precipitation, aggregation, phase separation, bacterial or fungal growth, change in pH, change in turbidity, syneresis or clearing on top, and change in particle size upon dilution.

[0140] Formulated dsRNA has been shown to be highly stable after accelerated storage conditions and over a temperature range of -10°C to 54°C. Unformulated dsRNA does not show this same stability to high temperatures, degrading rapidly within 2 weeks of storage at 54°C. Such accelerated storage conditions are frequently used to test stability at less extreme temperatures over extended periods of time. For example, 2 weeks at 54°C is commonly used to represent 1 year of storage at room temperature. (See United States Environmental Protection Agency November 16,2012 Memorandum regarding Accelerated Storage Stability and Corrosion Characteristics Study Protocol).

[0141] Example 10 (Comparative) This comparative example demonstrates that unformulated dsRNA does not show this same stability to high temperatures, and degrades rapidly within 2 weeks at 54°C. Table 16 shows the chemical degradation of unformulated dsRNA when stored at high temperatures. Of note is the significant degradation profile shown for unformulated dsRNA stored at 54°C for 2 weeks. [Table 16]

[0142] Example 11 The chemical stability of the probe formulations is demonstrated by HPLC analysis before and after storage under the listed environmental conditions, the results of which are shown in Figure 9 and Table 17.

[0143] FIG. 9 is an example according to various embodiments and shows high performance liquid chromatography (HPLC) results for chemical stability in various formulations after storage at −10° C., 4° C., 40° C., and 54° C. Samples were collected for formulations containing 8 g / L and 4 g / L of dsRNA. The formulations show no degradation of dsRNA over the tested time courses at accelerated storage conditions: 8 weeks at 40° C., 2 weeks at 54° C., 2 weeks at −10° C., and 2 and 8 weeks at 4° C. Thus, the results show that all formulations tested remain stable at room temperature for at least one year.

[0144] The formulations shown were prepared according to the method described in Example 1 and had compositions according to Table 22.

[0145] Table 17 shows data that demonstrates the chemical stability of dsRNA via HPLC after the above storage conditions.As shown in this table, HPLC measurement of dsRNA shows less than 10% dsRNA degradation in formulation samples, compared to unformulated dsRNA in Table 16, which showed a significantly higher degradation rate.These measurements in the formulation composition are within the expected 10% variation in HPLC measurement, indicating that dsRNA degradation is minimal. [Table 17]

[0146] Example 12 The formulated samples were also evaluated for any changes in pH after storage, and the results are shown in Table 18. Changes in pH are indicative of physical or chemical instability and are not exhibited by the exemplary formulation compositions. [Table 18]

[0147] Table 18 shows data on pH stability of formulated dsRNA after accelerated storage conditions. No significant changes in pH after various storage conditions are observed from the initial sampling conditions. Indications of physical and chemical stability, as well as prevention of potential acid / base hydrolysis of dsRNA during storage in the formulation.

[0148] The above formulations were prepared according to the method described in Example 1 and had the compositions according to Table 19. [Table 19]

[0149] Examples 13-18: Sustained foaming test The foam persistence of each formulation at the maximum indicated magnification as a diluted system is measured according to CIPAC MT 47. The graduated cylinder used in this experiment is a 250 mL cylinder conforming to the requirements specified in the test method. This is reported as the maximum volume after 1 minute of standing.

[0150] Example 13 Formulations containing dsRNA for foliar application have been developed to stabilize the dsRNA for storage stability and use as diluted spray solutions. The use of surfactants and other stabilizers in the formulation to prevent dsRNA degradation and to allow room temperature stable formulations containing dsRNA results in a fairly persistent foam when diluted into a spray solution for foliar application. The addition of antifoam type compounds in the formulation composition is required to reduce or eliminate this persistent foam upon dilution for practical application. Typical antifoam agents have been found to exhibit significant stability challenges when formulated with dsRNA and other auxiliary ingredients, and can be classified as aqueous-based formulations with high electrolyte content. An exemplary formulation composition contains antifoam type components that show excellent stability in dsRNA formulations over a variety of storage conditions, while also providing excellent foam reduction upon dilution in spray solutions. This compatibility in the formulation and the results of persistent foam testing are shown in this example.

[0151] 10A, 10B, 11A, and 11B are examples according to various embodiments and show photographs of compatible formulations containing SAG 1572™ antifoam after storage at -10°C for 2 weeks, at 4°C for 2 and 8 weeks, at 40°C for 8 weeks, and at 54°C for 2 weeks, demonstrating superior dsRNA stability.

[0152] The above formulations were prepared according to the method described in Example 1 and had the compositions according to Table 20. [Table 20]

[0153] Example 14 This example demonstrates further results of compatibility and sustained foam testing in various formulations. Figures 12A and 12B show examples of sustained foam testing results for a formulation containing SAG 1572™ antifoam and 8 g / L dsRNA from a TGAI source, as specified by CIPAC MT 18, diluted 300x and 15x with 342 ppm water, respectively, showing the absence of sustained foam and the foam decreasing stepwise after 1 minute of rest. Figures 12C and 12D show examples of sustained foam testing results for a formulation containing SAG 1572™ antifoam and 8 g / L dsRNA from a Halifax-CAN TGAI source, as specified by CIPAC MT 18, diluted 300x and 15x with 342 ppm water, respectively, showing the absence of sustained foam and the foam decreasing stepwise after 1 minute of rest. 12E and 12F are examples according to various embodiments and show photographs of persistent foam test results for a formulation containing SAG 1572™ antifoam and 8 g / L dsRNA from a TGAI source diluted 300× and 15× with 342 ppm water, respectively, as specified by CIPAC MT 18, showing the absence of persistent foam and progressive foam reduction upon standing for 1 minute.

[0154] The above formulations were prepared according to the method described in Example 1 and had the compositions according to Table 21. [Table 21]

[0155] Example 15 This example demonstrates that the results obtained for formulations containing SAG 1572™ antifoaming agent are also stored at 54°C for 2 weeks and 40°C for 8 weeks, completing accelerated storage conditions. These formulations show excellent stability of dsRNA and remain physically stable over the time course tested. Any slight precipitation events, mainly from emulsified silicone-based oils used as antifoaming agents, are dramatically minimized in the formulations and, if present, can pass through a 150um sieve.

[0156] Figures 13A, 13B, 13C, 13D, 13E, 13F, according to various embodiments, show photographs of formulations with SAG 1572™ antifoam after 8 weeks of storage at 40° C., demonstrating superior dsRNA stability. Figures 13G, 13H, 13I, 13J, 13K, 13L, according to various embodiments, show photographs of formulations with SAG 1572™ antifoam after 2 weeks of storage at 54° C., demonstrating superior dsRNA stability.

[0157] The above formulations were prepared according to the method described in Example 1 and had the compositions according to Table 22. [Table 22]

[0158] Example 16 This example shows results for a formulated composition containing SAG 1572™ at a specified use rate of 0.05% in the formulation. This concentration was identified after titration studies aimed at reducing the total concentration of antifoam in the formulation. This minimum use rate was identified by evaluating the effectiveness of foam reduction in solution upon dilution, the results of which can be seen in the figure below.

[0159] 14A and 14B are examples according to various embodiments, showing photographs of titration assays for formulations containing 0.025% SAG 1572™ antifoam and dsRNA diluted 400× and 100× with 342 ppm water, respectively. Figures 14C and 14D are examples according to various embodiments, showing photographs of titration assays for formulations containing 0.05% SAG 1572™ antifoam and dsRNA diluted 400× and 100× with 342 ppm water, respectively.

[0160] The above formulations were prepared according to the method described in Example 1 and had the compositions according to Table 23. [Table 23]

[0161] Example 17 As illustrated in Examples 14-16, SAG 1572™ showed the best stability and effectiveness results as an antifoam agent in formulations according to various embodiments, although several other antifoam ingredients were tested for compatibility and foam reduction. The examples demonstrate that SAG 1599™ provides greater stability to the compositions tested than SAG 1572™.

[0162] Figure 15 is an example according to various embodiments and shows photographs illustrating the incompatibility and physical stability of SAG 1599™ in concentrated formulations. The aggregation of silicone and oil particles in the formulations during storage highlights the stability challenges of formulating oil-based antifoam ingredients into high salt aqueous formulations.

[0163] The above formulations were prepared according to the method described in Example 1 and had the compositions according to Table 24. [Table 24]

[0164] Example 18 This example demonstrates that ANTIFOAM GN11P™ provides reduced foam reduction effectiveness compared to SAG 1572™.

[0165] Figure 16 is an example according to various embodiments, showing a photograph of a formulation containing 0.2% ANTIFOAM GN11P™ after 1 minute of standing. No significant foam reducing properties were observed for this formulation as prepared, and it was determined that the antifoam component was not as effective in solution as other options.

[0166] The above formulations were prepared according to the method described in Example 1 and had the compositions according to Table 25. [Table 25]

[0167] Examples 19-22: Bacterial and fungal biological contamination load testing Bacterial and fungal biological contamination tests were performed with these formulations to evaluate their inherent ability to withstand bacterial and fungal contamination that may occur if the container is not sealed and contaminated prior to use. These assays were performed using a modified version of the United States Pharmacopeia Chapter 51: Antimicrobial Effectiveness Testing. In this test method, each formulation is subjected to a bacterial or fungal cocktail challenge with and without the addition of selected biocidal and fungicidal preservatives KATHON® CG / ICP and ROCIMA® BT2S, and selected organisms are introduced into the material to evaluate growth or reduction in organism population. For the fungal cocktail, the following fungal cultures were selected, including Aspergilus niger (filamentous morph), Candida albicans (yeast morph), Penicilium commune (common contaminant, filamentous), and Aurebasidium pullulans (common contaminant, dimorphic). The following bacterial cultures were selected for the bacterial cocktail, including Escherichia coli (a common contaminant), Pseudomonas fluorescens, Bacillus licheniformis (a spore-forming, heat-resistant bacterium that excretes proteases and RNAses), and Serratia marcescens. To perform this test, 40 mL of each preparation is aliquoted into sterile conicals, with and without preservatives, and samples are spiked with either the bacterial or fungal cocktail. Both the bacterial and fungal cocktails are spiked at 1 x 10 6 CFU / mL and 1×10 5The formulation is spiked at 1% (v / v) with a target concentration of greater than CFU / mL. Each sample is stored at 30°C for 4 weeks to promote bacterial and fungal growth. Aliquots are taken for testing consecutively on days 0, 1, 7, 14, 21, and 28 to assess the bacterial and fungal concentrations in the sample. Bacterial counts are performed by spreading the aliquots to be tested on tryptic soy plates, incubating at 30°C for 1-3 days, checking daily for bacterial growth and then obtaining a total cell count. Fungal counts are performed by spreading the aliquots to be tested on Sabouraud dextrose agar plates, incubating at 25°C for 3-5 days, and checking daily for bacterial growth and then obtaining a total cell count. Biological contamination is recorded in CFU / mL, determined by the total number of colonies counted multiplied by the dilution factor and, if necessary, divided by the total volume of the sample in milliliters.

[0168] Formulations containing dsRNA are particularly susceptible to degradation of the dsRNA from bacterial and fungal contamination over time during storage. The formulations developed contain a preservative and surfactant package that inhibits bacterial and fungal degradation of the dsRNA during ambient storage. Unformulated dsRNA can be used for up to 10 4 When exposed to fungal contamination at CFU levels, dsRNA shows significant degradation within 21 days of storage. 7 When exposed to CFU levels of bacterial contamination, dsRNA shows complete degradation within 21 days of storage. dsRNA treated with the additional cationic surfactant package not being developed for this formulation shows complete degradation within 10 days of storage. 4 When exposed to CFU levels of fungal contamination, the unformulated dsRNA or TGAI treated with a preservative package, but not the additional cationic surfactant package developed for this formulation, shows no significant degradation of the dsRNA after 42 days of storage. 7 When exposed to CFU levels of bacterial contamination, dsRNA shows significant degradation after 14 days of storage. All formulated dsRNA treated with the preservative and surfactant packages specific to the exemplified formulation compositions exhibited significant degradation after 10 days of storage. 4Fungal contamination and 10 CFU 7 This demonstrates superior stability of dsRNA when contaminated with CFU bacterial contamination. The degradation profiles of unformulated dsRNA treated with the preservative package, or TGAI, but not the additional cationic surfactant package, demonstrate the necessity and utility of the cationic surfactant package as a necessary component to ensure complete protection of dsRNA from degradation due to bacterial contamination. This data set highlights the benefit of the additional preservative component, especially when using a traditional broad-spectrum preservative package consisting of BIT, CMIT, and MIT. HPLC quantification and bioburden spore count measurements demonstrate that formulations containing dsRNA can withstand significant bacterial and fungal contamination, both in eliminating bioburden levels within 1-7 days and preventing any dsRNA degradation over 42 days of storage at 37°C. This differs from unformulated dsRNA treated with preservative alone, which shows bacterial and fungal spore degradation between 1-7 days but dsRNA degradation due to bacterial contamination after 14 days. This demonstrates the particular stability advantage to dsRNA that the formulation provides in both knocking down the spore count for biological contamination and protecting the dsRNA from degradation by biological contamination over the same period. The following examples show the bioburden levels after bacterial and fungal challenge, as well as the dsRNA degradation profile after bacterial and fungal challenge for unformulated and formulated dsRNA.

[0169] Example 19 FIG. 17 is an example according to various embodiments and shows results for unformulated dsRNA (first column), dsRNA formulated with KATHON® CG / ICP and ROCIMA® BT2S (columns 2-4), and dsRNA formulated as shown in Table 26 (columns 5-7). The figure shows that the use of various packages reduces the 10% yield of the bacterial cocktail. 7 Demonstrate usefulness in reducing bioburden levels following contamination with CFU. [Table 26]

[0170] Example 20 FIG. 18 is an example according to various embodiments. 7 Table 26 shows the dsRNA degradation profiles of unformulated dsRNA (labeled "TGAI HAL CAN no bio+BC"), dsRNA formulated with ROCIMA® BT2S and KATHON® CG / ICP (labeled "TGAI...BC"), and formulated dsRNA (labeled "FORM...") shown in Table 26 after contamination with CFU of bacterial cocktail. HAL CAN, AL SAC, AL CAN represent three different yeast sources for the production of TGAI. These results show that ROCIMA® BT2S and KATHON® CG / ICP alone had some effectiveness in clearing bacterial infection over time, but were not effective by themselves in providing stability to dsRNA due to residual bacterial nuclease activity that persisted even after the bacteria were cleared. The table further demonstrates that samples formulated with a sequestering agent inhibited nuclease activity and, in combination with a secondary surfactant, provided robust stability after bacterial challenge after 42 days of storage at 37°C after contamination.

[0171] Example 21 FIG. 19 is an example according to various embodiments and shows results for unformulated dsRNA (first column), dsRNA formulated with KATHON® CG / ICP and ROCIMA® BT2S (columns 2-4), and dsRNA formulated as shown in Table 26 (columns 5-7). The figure shows that the use of various packages reduces the overall dsRNA concentration by 10%. 4 We demonstrated that HAL CAN, AL SAC, and AL CAN represent three different yeast sources for the production of TGAI, which can help reduce bioburden levels after contamination with a fungal cocktail of CFUs.

[0172] Example 22 FIG. 20 is an example according to various embodiments, showing dsRNA degradation profiles for unformulated dsRNA (labeled "TGAI HAL CAN no bio+ BC"), dsRNA formulated with ROCIMA® BT2S and KATHON® CG / ICP (labeled "TGAI...BC"), and formulated dsRNA shown in Table 26 (labeled "FORM...") after contamination with 10^4 CFU of fungal cocktail. HAL CAN, AL SAC, AL CAN represent three different yeast sources for the production of TGAI. These results demonstrate that ROCIMA® BT2S and KATHON® CG / ICP alone were effective in removing fungal contamination, and although they were not as effective as the more formulations in Table 26 that contained sequestering agents and secondary surfactants, they provided acceptable protection from nuclease activity.

[0173] Examples 23-25: Bacterial Loading Plate Assay and Water Activity Determination The addition of cationic surfactants is utilized as a key component of the overall preservative package due to the desired antimicrobial effect of alkylammonium chlorides in aqueous solutions. Bacterial and fungal degradation of dsRNA is a significant barrier to the delivery of dsRNA-containing solutions in practical applications, and the inclusion of these types of auxiliary components is important for dsRNA stability. To effectively screen formulations for the inclusion of cationic surfactant components and to determine the minimum inhibitory concentrations of those components in the formulation, a plate-based assay was developed. The plate-based assay was performed using 1×10 Escherichia Coli as the bacterial organism. 8 CFU / mL, and Bacillus Licheniformis at 1×10 6CFU / mL counts were used and performed in microtiter plates with tryptic soy broth. Ammonium-containing formulation components were screened at different concentrations using stepwise reduction to determine the minimum inhibitory concentration of each component for both organisms. Each formulation was serially diluted into a microtiter plate and a 10uL spike of each culture was added before spotting samples onto 150mm tryptic soy agar plates using a plate replicator tool. After completing concentration spotting for each formulation component, the tryptic soy plates were incubated overnight at 30C and imaged after 18 hours of incubation. Biocontrol properties and minimum inhibitory concentrations were determined for each component when the spots yielded individual colonies as opposed to a complete microlaw of bacterial growth.

[0174] To further characterize the formulations for inherent biocontrol properties, water activity levels were measured. Water activity levels were measured using a Novasine Lab Master Neo. Water activity is an important measure and is used to determine the amount of free water in the formulation and compare it to the known minimum water activity level for growth of fouling organisms.

[0175] The results of the bacterial contamination challenge test demonstrate the benefit of the additional preservative ingredient, included as an addition to the cationic surfactant. A plate-based assay was developed to screen ingredients and concentrations to demonstrate biocontrol efficacy and identify minimum use rates in the formulation. Three different ingredients were selected for testing, covering three different chemical classes. Cetrimonium chloride was selected to represent alkylammonium chloride, a traditional cationic surfactant approved for food use. Lauryl betaine was selected to represent betaines, a type of zwitterionic compound that exhibits ammonium functionality across all pH due to the position of the internal salt within the structure. MACAT® AO-12 was selected as an amine oxide, a type of surfactant that contains amine functionality but is not present as a charged species like the cationic or zwitterionic ingredients. The results from the plate-based assay show that the cationic surfactant provides the greatest benefit to the organisms tested and has a minimum use rate of 0.05% in the formulation. Lauryl betaine was shown to provide protection against organisms to a lesser extent than cetrimonium chloride and had a minimum recommended use rate of 0.75% in the formulation. Amine oxide surfactants were not shown to demonstrate any biocontrol against E. coli, demonstrating the specific need for zwitterionic, or more preferably cationic, surfactants for improved biocontrol properties against contamination. The results of these assays are presented in the Examples below.

[0176] Example 23 Figures 21A and B are examples according to various embodiments, showing photographs of plate-based assays for cetrimonium chloride challenged with B. Licheniformis and E. Coli, respectively. The minimum inhibitory concentration of cetrimonium chloride was determined to be 0.05% in the formulation. The absence of micro-lawns of growth within the spotted plates indicates inhibitory control of the organisms.

[0177] The above formulations were prepared according to the method described in Example 1 and had the compositions according to Table 27. [Table 27]

[0178] Example 24 22A and 22B are examples according to various embodiments, showing photographs of plate-based assays for lauryl betaine challenged with B. Licheniformis and E. Coli, respectively. The minimum inhibitory concentration of lauryl betaine was determined to be 0.75% in the formulation. The absence of micro-lawns of growth within the spotted plates indicates inhibitory control of the organisms.

[0179] The above formulations were prepared according to the method described in Example 1 and had the compositions according to Table 28. [Table 28]

[0180] Example 25 23A and 23B are examples according to various embodiments, showing photographs of plate-based assays for C12 amine oxide challenged with B. Licheniformis and E. Coli, respectively. The minimum inhibitory concentration of C12 amine oxide was determined to be 0.5% in the formulation. The absence of micro-lawns of growth within the spotted plates indicates inhibitory control of the organisms.

[0181] The above formulations were prepared according to the method described in Example 1 and had the compositions according to Table 29. [Table 29]

[0182] Example 26 In addition to the biocontrol properties due to the specific nature of the cationic surfactant and the broad spectrum preservative package, the formulations were analyzed for water activity levels. Each organism requires a specific concentration of free water to promote and encourage bacterial or fungal growth. Due to the high levels of salt and other surfactants in the formulation, the moisture levels of the formulations were measured to directly determine the inherent biocontrol properties of the formulation and to demonstrate the advantage of the preservative package to prevent dsRNA degradation due to microbial contamination, which can occur if the container is unsealed and contaminated prior to use. A listing of the water activity levels required to grow various organisms, and the water activity of several formulations, is shown in Table 30. [Table 30]

[0183] Table 30 shows the water activity levels of several tested formulations. Surfactant concentrations were tested at 5% and 10%, and dsRNA concentrations were tested at 4 g / L and 8 g / L. The results of this assay demonstrate that the formulations have very low water activity levels of 0.944-0.951 and contain some inherent biocontrol properties against several organisms. However, several additional common organisms have lower water activity levels, highlighting the need for additional preservative components. In addition, no significant differences are shown between surfactants with increasing dsRNA concentrations.

[0184] The above formulations were prepared according to the method described in Example 1 and had the compositions according to Table 31. [Table 31]

[0185] For reference, Table 32 provides the water activity levels for several common organisms. The minimum water activity levels required for growth are listed. [Table 32]

[0186] Examples 27 to 29: EDTA concentration and enzyme nuclease decomposition test To determine the stability of dsRNA in formulations containing various concentrations of EDTA, two separate nuclease challenge test assays using benzonase were developed. EDTA is used in formulations containing dsRNA to stabilize dsRNA against nuclease activity and prevent degradation over various storage conditions as well as contamination. The standard nuclease activity test utilized a 3 hour incubation period at 37C with 0.3U / uL benzoase and formulations containing dsRNA. EDTA was used to quench nuclease activity after 3 hours, and samples were then run as untreated and treated samples on a 1.2% agarose gel using electrophoresis. Quantitative determination of dsRNA in samples treated with 0.3U / uL benzonase was also performed by HPLC. Degradation of dsRNA was assessed qualitatively by the disappearance of the corresponding molecular weight band in the gel and quantitatively by HPLC analysis. An ultranuclease challenge assay was developed to rapidly test dsRNA formulations against a significant excess of benzonase in 1 hour. This test protocol was used to screen EDTA concentrations and determine the minimum concentration of EDTA required to inhibit nuclease activity in formulated samples. The ultranuclease activity test utilized a 1 hour incubation period at 37C with formulations containing 30U / uL benzoase and dsRNA. EDTA was used to quench nuclease activity after 1 hour, and samples were then run as untreated and treated samples on a 1.2% agarose gel using electrophoresis. Quantitative determination of dsRNA in samples treated with 30U / uL benzonase was also performed by HPLC. Degradation of dsRNA was assessed qualitatively by the disappearance of the corresponding molecular weight band in the gel, and quantitatively by HPLC analysis.

[0187] Enzymatic nuclease activity has been determined to be a significant cause of dsRNA degradation and instability of solutions containing dsRNA over a variety of storage conditions. Many nucleases require divalent metal ions to catalyze their enzymatic processes, and therefore room temperature stable formulations containing dsRNA require a metal ion chelator to be present to sequester the divalent metal ions from the system and inhibit nuclease activity. Because dsRNA is a polyanion and exists as a double stranded nucleic acid helix with a phosphate backbone, dsRNA itself requires Mg as the counter ion species associated with the phosphate backbone in aqueous solution. 2+ In addition, the cell-free reactions utilized to produce dsRNA require the use of free divalent metal ions such as Mg to construct the dsRNA molecule. 2+ The presence of divalent metal ions during the production of dsRNA creates an inherent susceptibility to nuclease activity in TGAI. The practical production of dsRNA utilizing this cell-free reaction is prohibitive to substantial dialysis, and counterion replacement methods are commonly used to remove divalent ions at the lab scale. Therefore, containing an appropriate concentration of a chelator such as EDTA is critical to the stability of dsRNA within the formulation.

[0188] Example 27 Multiple production lots of TGAI were tested using MgCl to understand the concentration range of these divalent ions in TGAI at a particular target dsRNA concentration. 2+ The content was analyzed and can be seen in FIG. 24. More specifically, FIG. 24 shows an example according to various embodiments, which shows a sample containing 9-13 mM Mg with an average content of 10 mM. 2+ TGAI produced with 7 g / L dsRNA and 15.5 to 28 mM Mg with an average content of 22.5 mM. 2+ The Mg content of several production lots of TGAI, including TGAI produced with 14 g / L dsRNA, 2+ The analysis is presented.

[0189] Example 28 The initial concentration of EDTA at 13.8 mM demonstrated sufficient protection of dsRNA in the end-use product of 4 g / L, and when formulated, it was determined that it was insufficient to protect dsRNA at higher concentrations. Further reduction of EDTA concentration in the formulated dsRNA product only resulted in more complete degradation of dsRNA through nuclease activity. This trend and the results of dsRNA degradation by uninhibited nuclease activity are shown in FIG. 25. More specifically, FIG. 25 is an example according to various embodiments, showing dsRNA degradation by nuclease activity. It was shown that formulations with reduced concentrations of metal ion chelators provide insufficient inhibition of benzonase, a non-specific nuclease. Further reduction of EDTA below 8 mM for the tested composition resulted in complete degradation, while higher concentrations of EDTA were not sufficient to provide complete protection for the compositions tested in this example, but altered the reaction rate and resulted in only partial degradation over time.

[0190] The above formulations were prepared according to the method described in Example 1 and had the compositions according to Table 33. [Table 33]

[0191] From this data, it was determined that, according to various embodiments, it may be beneficial to increase the EDTA concentration in the formulation to above 13.8 mM (>0.4% by weight), and also demonstrated the general advantage of including a metal ion chelator in the compositions of the present disclosure.

[0192] Example 29 A separate nuclease loading assay was developed using a large excess of benzonase and 22.6 mM Mg 2+ For a representative lot of TGAI containing Mg, the minimum level of EDTA required to provide dsRNA stability from nuclease-mediated degradation was determined. Results from this assay were consistent with the Mg 2+It was shown that an approximately 2 mM increase from a 1:1 molar equivalent of EDTA to ion was required to conclusively inhibit nuclease-mediated degradation of dsRNA. The results of this ultranuclease loading assay are shown in Figures 26 and 27.

[0193] FIG. 26 is an example according to various embodiments, showing gel electrophoresis results of dsRNA formulations containing increasing concentrations of EDTA. The TGAI lot used to manufacture these formulations contains 22.6 mM Mg 2+ A significant improvement in stability against nuclease susceptibility is observed when the EDTA concentration is increased above 20 mM. A molar excess of EDTA at 35 mM clearly demonstrates complete qualitative stability, preserving the integrity of the dsRNA against nuclease activity.

[0194] 27 is an example according to various embodiments, showing gel electrophoresis results of dsRNA formulations containing increasing concentrations of EDTA. Gel electrophoresis of dsRNA formulations containing increasing concentrations of EDTA. The TGAI lots used to manufacture these formulations contain 22.6 mM Mg 2+ and stability against nuclease activity is observed when the EDTA concentration increases by approximately 2 mM molar equivalent, or 22.6 mM.

[0195] The above formulations were prepared according to the method described in Example 1 and had the compositions according to Table 34. [Table 34]

[0196] Initial nuclease loading assays, ultranuclease loading assays, and Mg loading assays collected for several production lots of TGAI. 2+ Combining the data from the concentration data, it has been determined that, according to various embodiments, about 35 mM EDTA is an appropriate concentration to include in formulations containing dsRNA to provide stability from nuclease activity and help prevent degradation of the dsRNA under practical storage conditions.

[0197] Examples 30 to 36: UV irradiation stability test UV-B irradiation levels were monitored externally over the course of two weeks in Boston, MA. Collected energy levels were used to determine UV-B exposure levels over the course of 1, 7, and 14 days. Naked dsRNA was subjected to UV irradiation over the course of two weeks in Boston, MA. The integrity and degradation profile of the dsRNA over this time course was assessed by HPLC quantification. Naked dsRNA exposed under environmental conditions for one week was determined to be completely degraded, with the total UV-B exposure to the dsRNA over this period being 79 J / cm. 2 It was determined that.

[0198] The sample containing dsRNA was placed in a UV irradiation chamber and exposed to 100 J / cm 2 The samples were conditioned with 100 ug of UV-B radiation. This level of radiation was determined to be at least equivalent to or exceeding the average level of average radiation exposure measured over the course of a week in the normal environment of Boston, MA. The samples were prepared both on parafilm-lined plates to determine the inherent photostability of the samples, as well as in plants to determine any effects of photostability at the leaf surface. Each sample was conditioned as nine replicates of 5 ug of dsRNA. After conditioning with UV radiation, the samples were reconstituted in 200 uL of water and pooled for analysis.

[0199] Pooled samples of UV-conditioned material were analyzed by gel electrophoresis to qualitatively determine both the dsRNA recovery and integrity of the samples. Results were generated using an Invitrogen E-Gel Flash electrophoresis system.

[0200] Pooled samples of the UV-conditioned material were analyzed by HPLC to quantitatively determine recovery of dsRNA and post-exposure quantitation of dsRNA.

[0201] Bioassays of the samples were performed in the laboratory using spread leaf application. Twenty Colorado potato beetle larvae were used per treatment. Samples were treated with 0 J / cm as a positive control. 2 of UV radiation, determined to be 100 J / cm2 equivalent to a one-week exposure period under normal environmental conditions. 2 of UV irradiation, and 200 J / cm as a negative control with complete dsRNA degradation under these conditions. 2 The difference in mortality between the formulations at different levels of exposure and the composition containing the additional UV protectant dispersant type surfactant was determined after 9 days of treatment.

[0202] A summary of the chemicals tested and the level of UV protection provided to the dsRNA following exposure is provided in Table 35. [Table 35]

[0203] Example 30 UV irradiation levels were measured over the course of two weeks on a rooftop in Boston, MA. Quantitation of dsRNA in samples exposed to these environmental conditions was monitored by HPLC to determine exposure limits and degradation effects. dsRNA was found to be completely degraded after one week via HPLC determination, which was 76 J / cm. 2 It was determined that this corresponds to a UV-B irradiation of 131 J / cm 2 After 2 weeks of exposure, equivalent to 1000 nm of UV-B exposure, dsRNA quantification decreased to 0 ng / μL.

[0204] FIG. 28 is an example according to various embodiments showing UV-B exposure levels and dsRNA stability after two weeks.

[0205] The above formulations were prepared according to the method described in Example 1 and had the compositions according to Table 36. [Table 36]

[0206] Example 31 Inspection of individual chromatograms collected for samples tested after UV exposure demonstrated that although quantification of dsRNA via HPLC had decreased to 0 ng / uL, the dsRNA, ssRNA, and free nucleotide peaks showed significant distortion and shift. This shift and distortion of the RNA type peak demonstrates that some levels of dsRNA remain after 7-14 days, but the chemical shift indicates random loss of size and base pairs in the sample. Mixing the dsRNA and ssRNA peaks indicates that significant degradation and conformational changes to dsRNA occur after 7-14 days of UV exposure, although it is unclear whether this degradation results in loss of efficacy.

[0207] FIG. 29 is an example according to various embodiments, showing HPLC chromatogram overlays of dsRNA after various UV-B exposures. The peak intensity of dsRNA is significantly reduced after 1 day of exposure. The dsRNA and ssRNA peaks begin to spread and mix after 7-14 days of exposure to UV radiation. The formulations described were prepared according to the method described in Example 1 and had the compositions specified in Example 30.

[0208] Example 32 Figure 30 is an example according to various embodiments, showing the mass balance of dsRNA, ssRNA, and free nucleotides in a dsRNA sample after two weeks of exposure to UV light. The significant decrease in dsRNA, the slight increase in ssRNA, and the significant increase in free nucleotides indicate degradation of dsRNA. The formulations described were prepared according to the method described in Example 1 and had the composition specified in Example 30.

[0209] Example 33 An alternative formulation containing a lignosulfonate dispersant type surfactant was developed. Lignosulfonates consist of a polymer network containing sulfonated conjugated aromatic functionality. It was hypothesized that this polymer network of conjugated aromatic functionality would have a significant beneficial impact on UV protection by light absorption of UV radiation. Five formulations were made with various lignosulfonates, all containing different molecular weights, degrees of sulfonation, and sites of sulfonation, and tested for protection from UV radiation against an exemplary formulation that does not contain this type of surfactant. These formulations were dried on a parafilm plate and then irradiated in a UV chamber at 100 J / cm. 2 The samples were exposed to 1000 ng of UV-B radiation. The samples were rehydrated with water and then analyzed by gel electrophoresis to determine qualitative dsRNA degradation and via HPLC to determine quantitative dsRNA degradation. The gel and HPLC results can be seen in Figures 31 and 32.

[0210] 31 is an example according to various embodiments, where a formulation containing 1.5% lignosulfonate (lanes 2-6) shows retention of the dsRNA band on the gel and some protection from dsRNA degradation from UV irradiation, whereas a formulation not containing lignosulfonate shows complete loss of the dsRNA band on the gel and dsRNA degradation.

[0211] FIG. 32 shows an example according to various embodiments, where 100 J / cm 2 1 shows results from HPLC quantification of formulations following exposure to UV-B radiation. Formulations containing lignosulfonate surfactants demonstrate greater than 60% improvement in dsRNA stability.

[0212] The above formulations were prepared according to the method described in Example 1 and had the compositions according to Table 37. [Table 37]

[0213] Example 34 Formulation samples containing lignosulfonate were also dried on leaves to understand if any interaction with the leaf surface further improved the stability of dsRNA when exposed to UV radiation. A comparison between formulations exposed on Parafilm plates and on leaf surfaces is shown in Figure 33. More specifically, Figure 33 is an example according to various embodiments, showing a comparison of leaf surface vs. Parafilm for dsRNA stability against UV radiation. The leaf surface appears to provide additional protection for dsRNA from UV radiation, improving the stability of formulations containing dsRNA.

[0214] The above formulation was prepared according to the method described in Example 1 and had the composition according to Example 34.

[0215] Example 35 Several other chemical classes similar to lignosulfonates have been explored as potential UV-absorbing surfactants, including aromatic-based chemistries, conjugated surfactants, and olefin-based surfactants. Examples of these types of chemistries are found in naphthalene sulfonates, alkyl phenyl ethoxylates, and methyl oleyl taurate, and gel electrophoresis results for these types of additives are shown in FIG. 34. More specifically, FIG. 34 shows examples according to various embodiments of gel electrophoresis results after UV-B exposure, showing that olefin-based and phenyl-based chemistries do not provide any significant protection of dsRNA. Other conjugated chemistries, exemplified by the MORWET® series, show slight improvements in dsRNA stability after UV-B exposure, but the benefits are not as significant as those of the lignosulfonate chemical class.

[0216] The above formulation was prepared according to the method described in Example 1 and had the composition of the previous embodiment, but with the 1.5% dispersing surfactant replaced by these additional ingredients.

[0217] Example 36 Qualitative and quantitative analysis of dsRNA formulations exposed to UV-B irradiation showed some level of dsRNA degradation, with the amount of degradation dependent on the class of surfactant used in the formulation. Formulations were UV-B irradiated and then used in bioassays to determine if the level of degradation observed across samples resulted in a loss of efficacy. Formulations were provided with and without the addition of lignosulfonate, with no exposure as a positive control and 200 J / cm for complete degradation as a negative control. 2 and without the drying and rehydration steps as a positive control to determine improvement in efficacy as a result of the UV protectant. The results of the Colorado potato beetle bioassay are shown in Figures 35 and 36.

[0218] Figure 35 is an example according to various embodiments, showing mortality from a Colorado potato beetle (CPB) bioassay. Formulations not exposed to UV-B radiation demonstrated control with over 80% mortality, while exposure to 100 J / cm2 reduced mortality to 47% and exposure to 200 J / cm2 reduced mortality to 25%, equivalent to untreated controls. Evaluation of formulations exposed to UV-B radiation demonstrates that the degradation of dsRNA observed in gels and HPLC translates into a significant loss of efficacy in the bioassay.

[0219] The above formulation was prepared according to the method described in Example 1 and had the composition specified in Example 30.

[0220] FIG. 36 is an example according to various embodiments showing mortality from a Colorado potato beetle (CPB) bioassay of formulations with or without lignosulfonate. Formulations with and without lignosulfonate were exposed to UV-B radiation and fed to CPB larvae. Several formulations containing lignosulfonate and exposed to 100 J / cm2 of UV-B radiation provided similar or better performance compared to standard dsRNA formulations without UV-B exposure. All formulations containing lignosulfonate and exposed to 100 J / cm2 showed improved efficacy compared to the same formulations without lignosulfonate. The bioassay data confirms that the protection against dsRNA degradation observed in the same samples in qualitative and quantitative gel and HPLC assays provides improved efficacy compared to samples that did not show protection against dsRNA degradation from UV-B radiation.

[0221] The above formulations were prepared according to the method described in Example 1 and had the compositions according to Table 38. [Table 38]

[0222] Example 37 Compositions according to Table 39 were prepared. Samples incorporating a variety of different dsRNA sequences were tested for stability over various time periods and temperatures, as shown in Tables 40A, 40B, and 40C. One sample contained a 497 bp dsRNA targeting a gene in the fungus Erysiphe necator (Table 40A), one sample contained a 478 bp dsRNA targeting a gene in the fungus Botrytis cinerea, and one sample contained a 450 bp dsRNA targeting a gene in the fungus Botrytis cinerea.

[0223] To determine physical stability, samples were observed with the naked eye for any visual separation and the samples were gently inverted to check for sediment. All samples were observed to be homogeneous demonstrating the physical stability of all samples under all conditions tested.

[0224] To assess chemical stability, the initial pH was set to approximately 6 with citrate buffer, and the pH of each sample was measured at various time points using an electronically calibrated pH meter as described in CIPAC MT 75.3. All samples demonstrated strong chemical stability at all time points, with pH varying by up to 0.16, 0.31, and 0.29 for the three samples shown in Figures 40A, 40B, and 40C, respectively, well below the threshold for a chemically stable composition.

[0225] To evaluate physical stability after dilution in water, the concentrated products were diluted to the target percentages at the water hardness levels listed in Figures 40A, 40B, and 40C. The water was prepared according to an ASTM method similar to that of ASTM E1945-02. The samples were mixed briefly and then observed for any turbidity or precipitation, similar to that of method CIPAC MT 41. No turbidity or precipitation was observed for any sample for any period or temperature, demonstrating the stability of the diluted products.

[0226] To assess the chemical stability of the compositions, the total dsRNA content was measured by HPLC at a target concentration of 0.8% w / w or 8.24 g / L. The variability of the analytical method can be estimated to be plus or minus 15%. The HPLC results were confirmed by gel electrophoresis. No dsRNA degradation below 7.0 g / L was observed at any time point, demonstrating the stability of all samples tested. [Table 39] [Table 40] [Table 41] [Table 42]

[0227] Example 38 This example deals with a multi-parameter investigation of factors leading to the physical stability of dsRNA concentration, primary detergent, secondary detergent, and sequestrant. JMP (version 15.2.0, SAS Institute, Cary NC, 18) was used to construct a statistical design of experiment within the following parameters: [Table 43]

[0228] Within this range of parameters tested, dsRNA concentration did not have a significant effect on the physical stability of the composition after storage. Cetrimonium chloride had a significant effect on stability, with the combination of low EDTA and high cetrimonium chloride resulting in instability, as shown in the dark gray area of ​​Figure 37, which shows the relationship between EDTA and cetrimonium chloride. Figure 39 illustrates the significant relationship between EDTA and cetrimonium chloride on the stability of the formulation. At the highest level tested of ATPLUS® PFA (10% by weight), minimal precipitation was observed under some conditions, but it was within acceptable limits and was not a significant factor in the DOE. The DOE confirms that within the range of compositions tested, physically stable mixtures with dsRNA can be made with all compositions except those with low EDTA and high cetrimonium chloride concentrations.

[0229] Example 39 Compositions according to Table 19 were prepared and tested at various time periods and temperature conditions to evaluate various matrices for stability. Samples were first evaluated for pH, density of dsRNA, dilution stability, and foaming. The remaining samples were sealed in containers with aluminum and stored at various conditions and time periods, including 1 year and 2 years at room temperature, and 4 weeks at 54°C (simulating at least 2 years of storage at room temperature). Samples were then evaluated to confirm stability. As shown in Table 42, these samples remained within or minimally outside the 10% detection range of the HPLC method, demonstrating minimal or no detectable degradation of dsRNA due to nucleases, fungal contamination, or bacterial contamination. pH levels were well maintained even within the 1 unit threshold for stability, with the pH varying up to 0.19 units from the initial pH of 7. Physical stability was evaluated by examining the color of the concentrated samples and evaluating precipitation in both the stored concentrated samples and 18 hours after diluting the concentrated samples. Samples were clear or almost clear in appearance. No precipitation was observed in the samples before or after dilution. Foaming was tested according to CPAC MT47 and all samples were below the standard 15% foam / solution v / v after 1 minute. [Table 44]

[0230] The dimensions and values ​​disclosed herein should not be understood as being strictly limited to the exact numerical values ​​recited. Instead, unless otherwise specified, each such dimension is intended to mean both the recited value and a functionally equivalent range surrounding that value. For example, a dimension disclosed as "40 mm" is intended to mean "about 40 mm."

[0231] All documents cited herein, including any cross-referenced or related patents or applications, as well as any patent applications or patents to which this application claims priority or benefit, are incorporated herein by reference in their entirety, unless expressly excluded or otherwise limited. The citation of any document is not an admission that it is prior art with respect to any invention disclosed or claimed herein, or that it alone, or in any combination with any other reference or references, teaches, suggests, or discloses any such invention. Furthermore, to the extent that any meaning or definition of a term in this document conflicts with a meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to that term in this document shall apply.

[0232] While particular embodiments of the present disclosure have been illustrated and described, it would be obvious to those skilled in the art that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended in the appended claims to cover all such changes and modifications that are within the scope of this invention.

Claims

[Claim 1] 1. A composition for delivering RNA to a pest via exogenous foliar application of the composition to a plant, comprising: RNA, a primary surfactant, said primary surfactant being a non-ionic surfactant; and a sequestering agent.

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