RNA storage and delivery

Abstract

The present invention relates to a composition comprising at least one RNA molecule and at least one silica carrier, wherein the silica carrier is a silica particle having a diameter of 0.3 µm - 1000 µm, and wherein the composition further comprises a matrix shell made of a zein or shellac.

Description

Invention Field

[0001] This invention relates to compositions for RNA storage and exogenous delivery to a target, and methods for producing said compositions. Specifically, the compositions comprise RNA and small silicate particles serving as a carrier for said RNA. The compositions can be particularly useful for agricultural use. Background of the Invention RNA has been shown to be very useful in many different industries. One of the industries where RNA is most commonly used is agriculture. Here, RNA / RNAi can be used for pest, insect, and / or virus control. dsRNA can also be used to regulate plant expression to increase growth or alter nutrition. Although RNA has several uses outside of cells, it is known to be relatively unstable and easily degraded, especially outside of cells. For example, unprotected RNA molecules are highly unstable due to RNases, which are ubiquitous in nature. In general, this makes the economical use of RNA, especially in liquid environments, difficult.

[0003] Exogenous application of RNA can generally successfully induce the desired effect. However, the effect for RNA, especially dsRNA, generally declines rapidly because they typically have a half-life of only about 12 to 24 hours in soil, and can have even shorter half-lives depending on the precise environmental conditions under which they are applied.

[0004] Several publications, such as Khandelwal, N., et al., Journal of Environmental Management , 2016, 184(2): 157-169; Worrall, E., et al. Agronomy ,2018, 8(12): 285; Shidore, T., et al. ACS Appl. Nano Mater . 2021, 4(12): 12891–12904 and Niazian, M., et al. Planta , 2021, 254(83) discloses different formulations of RNA. US11286476B2 also provides a method for essentially preserving the bioactivity of dsRNA by maintaining RNA in bacterial cells and then adding a compound and / or acid that functions as a protein- or amine-crosslinker. However, all these methods, as well as those used in the prior art, still have the problem of having to increase the RNA concentration in the final product to ensure that the appropriate amount of RNA reaches the target. Increasing the RNA concentration affects the viscosity of the final product, making its use difficult and cumbersome. Methods known in the art still cannot effectively preserve the active ingredient (i.e., RNA). Furthermore, current methods for RNA preservation and delivery must be stored at low temperatures before use, making storage and delivery expensive and inefficient.

[0005] Therefore, there is still a need in the art for more efficient, simple and inexpensive means of delivering RNA to its target and storing the RNA.

[0006] Invention Description This invention addresses the aforementioned problems by providing a composition comprising at least one RNA molecule and silica particles as a carrier. Specifically, the silica particles have a suitable size that not only binds RNA but also protects it from degradation. Furthermore, the appropriately sized silica particles allow for efficient transport of RNA to its target. The silica particles can be used to increase RNA concentration without gelling. Specifically, the silica particles have an average diameter of 0.3 µm – 1000 µm. Compared to the prior art, this size of silicate particle results in improved RNA handling and storage life. In particular, given the size of the silica particles, increased bioavailability and / or bioavailability of the RNA active ingredient can be achieved over a longer period compared to the prior art.

[0007] According to one aspect of the invention, a composition comprising at least one RNA molecule and at least one silica carrier is provided, wherein the silica carrier is a silica particle with an average diameter of 0.3 µm to 1000 µm, and wherein the composition further comprises a matrix shell made of zein or shellac.

[0008] Specifically, the RNA molecule can be any RNA molecule. More specifically, the RNA molecule is selected from antisense RNA, single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), hairpin RNA (hpRNA), short hairpin RNA (shRNA), small interfering RNA (siRNA), microRNA (miRNA), or combinations thereof. Even more specifically, the RNA molecule is dsRNA. In one instance, the RNA molecule is a repressive RNA molecule. Specifically, the RNA is an isolated RNA molecule. The RNA molecule can be isolated using any method known in the art.

[0009] The term 'carrier' refers to silica or silicate used according to any aspect of the invention, which comprises silica particles. A carrier is capable of transporting RNA according to any aspect of the invention to its final target (e.g., to parts of a plant / crop in agriculture).

[0010] Any type of silica can be used as a silica carrier according to any aspect of the invention. In particular, silica can be used as a hydrophilic carrier with defined water-absorbing properties (maintaining low water activity; aW). In one example, the silica used can be selected from SIPERNAT® 50, SIPERNAT® 50S, SIPERNAT® 22, SIPERNAT® 380, SIPERNAT® 22S, SIPERNAT® 800, SIPERNAT® 880, ZEOLEX® 7, ZEOLEX® 23, and ZEOLEX® 23a. In particular, the silica used according to any aspect of the invention can be precipitated silica. Even more particularly, the silica can be SIPERNAT® 380, a carrier silica for precipitated silica with an ultrafine particle size. Precipitated silica can work more effectively in compositions according to any aspect of the invention due to a variety of reasons, including possibly including pH compared to other silicas.

[0011] The use of a suitable silica carrier allows for good adhesion of RNA droplets to crop leaves. This carrier results in good rain resistance for the RNA on the plant. Even after rain, the RNA remains effective on the crop leaves. The spray droplets of RNA spray become larger and are therefore less prone to drift during the spraying process. The carrier also enables a diffusion effect that evenly covers / distributes the RNA across the entire target surface. Furthermore, the carrier can be sustainably produced from renewable raw materials and is largely biodegradable. Silica carriers therefore exhibit particularly good performance profiles.

[0012] The presence of silica maintains a low aW (average volume) in the final RNA composition. Furthermore, the presence of silica offers the additional benefits of water absorption, good handling properties / flowability, making it suitable and easy to use. Moreover, silica increases the volume of the final product, making its handling easier and more convenient.

[0013] In one example, silica particles may further comprise calcium silicate particles. Calcium silicate carries calcium in its structure. RNA is generally considered to have a partially negative charge. Therefore, stability is achieved through coordination with silica. This coordination also contributes to a certain degree of stability. Thus, for the stability of RNA, the positive charge of calcium silicate can be even more beneficial than that of conventional silicates or silica.

[0014] Different concentrations of silica can be present in the RNA composition. WO2020104612A1 shows a range of silica concentrations that may be applicable according to any aspect of the invention. In particular, the concentration of silica present can be determined by the type of RNA present. In one example, when pure double-stranded RNA is contacted with silica, about 99-70% by weight of silica may be present along with 1-30% by weight of RNA. More particularly, 99-75, 99-80, 99-85, 99-80, 99-90, 99-95% by weight of silica may be present along with 5-30, 10-30, 15-30, 20-30, 25-30, 5-25, 10-25, 15-25, 20-25, 5-20, 10-20, 15-20% by weight of RNA.

[0015] In another instance, when a matrix shell is present, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 5-50, 5-45, 5-40, 5-35, 5-30, 5-25, 5-20, 5-15, 5-10, 10-50, 10-45, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 15-50, 15-45, 15-40, 15-35, 15-30, 15-25, 20-50, 20-45, 20-40, 20-35, or 20-30% by weight of silica are present.

[0016] The silica particles according to any aspect of the invention may have an average diameter size of 0.3µm – 1000µm. Specifically, the average diameter can be 0.3-950, 0.3-900, 0.3-850, 0.3-800, 0.3-750, 0.3-700, 0.3-650, 0.3-600, 0.3-550, 0.3-500, 0.3-450, 0.3-400, 0.3-350, 0.3-300, 0.3-250, 0.3-200, 0.3-150, 0.3-100, 0.3-95, 0.3-90, 0.3-85, 0.3-80, 0.3-75, 0.3-70, 0.3-65, 0.3-60, 0.3-55, 0.3-50, 0.3-45, 0.3- 40, 0.3-35, 0.3-30, 0.3-25, 0.3-20, 0.3-15, 0.3-10, 0.3-950, 0.3-900, 0.3-850, 0.3-800, 0.3-750, 0.3-700, 0.3-650, 0.3-600, 0.3-550, 0.3-500, 0.3-450, 0.3-400, 0.3-350, 0.3-300, 0.3-250, 0.3-200, 0.3-150, 0.3-100, 0.3-95, 0.3-90, 0.3-85, 0.3-80, 0.3-75, 0.3-70, 0.3-65, 0 0.3-60, 0.3-55, 0.3-50, 0.3-45, 0.3-40, 0.3-35, 0.3-30, 0.3-25, 0.3-20, 0.3-15, 0.3-10, 0.5-1000, 0.5-950, 0.5-900, 0.5-850, 0.5-800, 0.5-750, 0.5-700, 0.5-650, 0.5-600, 0.5-550, 0.5-500, 0.5-450, 0.5-400, 0.5-350, 0.5-300, 0.5-250, 0.5-200, 0.5-150, 0.5-100, 0.5-95, 0. 5-90, 0.5-85, 0.5-80, 0.5-75, 0.5-70, 0.5-65, 0.5-60, 0.5-55, 0.5-50, 0.5-45, 0.5-40, 0.5-35, 0.5-30, 0.5-25, 0.5-20, 0.5-15, 0.5-10, 0.5-950, 0.5-900, 0.5-850, 0.5-800, 0.5-750, 0.5-700, 0.5-650, 0.5-600, 0.5-550, 0.5-500, 0.5-450, 0.5-400, 0.5-350, 0.5-300, 0.5-250, 0.5-200、0.5-150、0.5-100、0.5-95、0.5-90、0.5-85、0.5-80、0.5-75、0.5-70、0.5-65、0.5-60、0.5-55、0.5-50、0.5-45、0.5-40、0.5-35、0.5-30、0.5-25、0.5-20、0.5-15、0.5-10、0.75-1000、0.75-950、0.75-900、0.75-850、0.75-800、0.75-750、0.75-700、0.75-650、0.75-600、0.75-550、0.75-500、0.75-450、0.75-400、0.75-350、0.75-300、0.75-250、0.75-200、0.75-150、0.75-100、0.75-95、0.75-90、0.75-85、0.75-80、0.75-75、0.75-70、0.75-65、0.75-60、0.75-55、0.75-50、0.75-45、0.75-40、0.75-35、0.75-30、0.75-25、0.75-20、0.75-15、0.75-10、0.75-950、0.75-900、0.75-850、0.75-800、0.75-750、0.75-700、0.75-650、0.75-600、0.75-550、0.75-500、0.75-450、0.75-400、0.75-350、0.75-300、0.75-250、0.75-200、0.75-150、0.75-100、0.75-95、0.75-90、0.75-85、0.75-80、0.75-75、0.75-70、0.75-65、0.75-60、0.75-55、0.75-50、0.75-45、0.75-40、0.75-35、0.75-30、0.75-25、0.75-20、0.75-15、0.75-10, 2-1000, 2-950, 2-900, 2-850, 2-800, 2-750, 2-700, 2-650, 2-600, 2-550, 2-500, 2-450, 2-400, 2-350, 2-300, 2-250, 2-200, 2-150, 2-100, 2-95, 2-90, 2-85, 2-80, 2-75, 2-70, 2-65, 2-60, 2-55, 2-50, 2-45, 2-40, 2-35, 2-30, 2-25, 2-20, 2-15, 2 -10, 3-1000, 3-950, 3-900, 3-850, 3-800, 3-750, 3-700, 3-650, 3-600, 3-550, 3-500, 3-450, 3-400, 3-350, 3-300, 3-250, 3-200, 3-150, 3-100, 3-95, 3-90, 3-85, 3-80, 3-75, 3-70, 3-65, 3-60, 3-55, 3-50, 3-45, 3-40, 3-35, 3-30, 3-25, 3-20, 3-15, 3-1 0, 4-1000, 4-950, 4-900, 4-850, 4-800, 4-750, 4-700, 4-650, 4-600, 4-550, 4-500, 4-450, 4-400, 4-350, 4-300, 4-250, 4-200, 4-150, 4-100, 4-95, 4-90, 4-85, 4-80, 4-75, 4-70, 4-65, 4-60, 4-55, 4-50, 4-45, 4-40, 4-35, 4-30, 4-25, 4-20, 4-15, 4-10, 5-1000, 5-950, 5-900, 5-850, 5-800, 5-750, 5-700, 5-650, 5-600, 5-550, 5-500, 5-450, 5-400, 5-350, 5-300, 5-250, 5-200, 5-150, 5-100, 5-95, 5-90, 5-85, 5-80, 5-75, 5-70, 5-65, 5-60, 5-55, 5-50, 5-45, 5-40, 5-35, 5-30, 5-25, 5-20, 5-15, or 5-10µm. More specifically, the average diameter of the silica particles can be 0.3-75 µm. Even more specifically, silica particles can have an average diameter of 0.3–10 µm.

[0017] In one example, the average diameter of the silica particles can be approximately 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30. 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 5 3, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 µm.

[0018] As used herein, the term 'about' refers to a variation within 20%. Specifically, as used herein, the term 'about' refers to + / - 20%, more specifically + / - 10%, or even more specifically + / - 5% for a given measurement or value. In some instances, the term 'about' refers to a range of values ​​falling within 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1%, or less of the reference value for that condition. For example, when applied to average diameter dimensions, the term 'about' refers to a range of values ​​similar to the reference value for that condition.

[0019] As used herein, the term 'average diameter' refers to the average diameter of all silica particles in a sample. In one instance, when 10 silica particles are present, the average diameter refers to the average diameter of the silica particles, which is determined by adding the diameters of all 10 silica particles together and then dividing by 10. According to any aspect of the invention, the silica particle size always refers to the average diameter (i.e., the average diameter) of all silica particles present in the RNA composition according to any aspect of the invention. In one instance, each individual silica particle in the RNA composition therefore does not necessarily have a diameter of 0.3–1000 µm. However, the average diameter or mean diameter of all silica particles present in the RNA composition according to any aspect of the invention has a diameter of 0.3–1000 µm.

[0020] The small size of silica particles makes them particularly useful as a carrier and for producing RNA compositions that do not clog the nozzles of agricultural machinery when used in agriculture.

[0021] Any method known in the art can be used to produce silica particles in RNA compositions according to any aspect of the invention. In one example, a steam jet milling process is used to prepare the silica particles. Specifically, the steam jet milling process reduces the size of the original silica particles added into the steam jet mill to smaller particles for use in the RNA composition. Any steam jet mill can be used. Those skilled in the art can vary the speed and / or size of the starting material to obtain silica particles of the required and desired size for the RNA composition according to any aspect of the invention.

[0022] Compositions according to any aspect of the invention may further comprise a matrix shell. The matrix shell, also referred to as a coating, encapsulates all contents. This encapsulation is responsible for the long-term stability of the RNA in the RNA composition according to any aspect of the invention. The matrix shell may be made of zein, shellac, isomaltitol, wax, resin, carbohydrates, proteins, or polymers such as PVOH. In particular, the matrix shell may be made of zein or shellac. Both zein and shellac can increase RNA stability and are particularly useful for protecting and / or maintaining RNA stability during storage.

[0023] The matrix shell can be modulated to trigger release. For example, the shell can be pH-sensitive, such that in the presence of a specific pH, the shell disintegrates and releases RNA from the RNA composition. In another example, the shell can be temperature-sensitive. In particular, coatings including shellac can be suitable for this function.

[0024] RNA compositions according to any aspect of the invention can be completely free of microplastics.

[0025] According to a further aspect of the present invention, a method for producing a composition comprising at least one RNA molecule and a silica carrier is provided, the method comprising the following steps: (a) Combining silica particles with an average diameter of 0.3µm – 1000µm with at least one aqueous RNA molecule and a coating material of zein or shellac.

[0026] Silica particles with an average diameter of 0.3µm–1000µm can be produced using any method known in the art. In one example, silica particles can be produced using a steam jet mill. Any steam jet mill can be used to produce silica particles according to any aspect of the invention. The steam jet milling process reduces the size of the raw silica particles fed into the steam jet mill to smaller particles for use in RNA compositions. Those skilled in the art can vary the speed and / or size of the starting material to obtain silica particles of the required and desired size for RNA compositions according to any aspect of the invention. EP2209739 describes at least one method for producing silica particles with an average diameter of 0.3µm–1000µm, particularly for precipitating silica particles, using a steam jet mill.

[0027] Silica carrier particles from a steam jet mill can then be contacted with liquid RNA molecules. In particular, the desired RNA (to be introduced into the target cell or site) is in liquid form or in an aqueous solution, especially in an extraction buffer or water.

[0028] As used herein, the term "contact" refers to bringing RNA into direct contact with silica particles. This term does not necessarily constitute a step of physically mixing or combining the two components—silica particles and RNA molecules.

[0029] The term "aqueous solution" includes any solution containing water (primarily water) as a solvent, which can be used to at least temporarily maintain RNA in a metabolically active and / or viable state according to any aspect of the invention, and, if necessary, contain any additional substrate. Those skilled in the art are familiar with the preparation of a variety of aqueous media that can be used to extract and / or store RNA, often referred to as culture media. Using a basic culture medium as an aqueous solution is advantageous compared to complex culture media to avoid unnecessary contamination of the product with unwanted byproducts; a basic culture medium is one with a fairly simple composition containing only the minimum set of salts and nutrients essential for maintaining RNA in a metabolically active and / or viable state. Examples of aqueous solutions in which RNA molecules can be present include buffer solutions and water.

[0030] In one example, once the silica carrier and aqueous RNA come into contact with each other and are mixed or combined, the result can be spray-dried to produce a powder. A schematic diagram of a method according to any aspect of the invention is shown in... Figure 1 Provided herein. An optional step of adding a coating is also shown here. The method according to any aspect of the invention is advantageous because it is a one-step method for producing stable and optionally encapsulated RNA.

[0031] The step of spray drying the mixture of RNA and silica particles, and optionally coated particles, reduces the water content of the final mixture or RNA composition according to any aspect of the invention, and this increases the activity of the RNA and therefore its shelf life. Compared with other methods known in the art, spray drying can very rapidly reduce the aW in the RNA composition from 1 to about 0.2-0.3. Using the method according to any aspect of the invention, RNA is successfully formulated into / on the particles (in powder form). Furthermore, using the method according to any aspect of the invention, RNA molecules can be concentrated into / on silica particles, and RNA molecules can optionally be encapsulated on the particles.

[0032] Spray drying according to any aspect of the invention can be performed using any spray dryer. In one example, a Mini Spray Dryer B-290 (Büchi Labortechnik AG, Switzerland) can be used. In particular, RNA compositions can be spray-dried using a spray dryer, thereby obtaining spray-dried RNA compositions according to any aspect of the invention.

[0033] In the spray drying step according to any aspect of the invention, the spray dryer may first be preheated at a very low fan speed. Spray drying can be achieved by allowing the inlet air temperature to be not too high so that RNA can survive above that temperature. In particular, the inlet air temperature may be below or equal to about 80°C, below or equal to about 50°C, or at about 30°C-50°C. Spray drying may be performed using an airflow. The outlet air temperature may be below or equal to about 55°C, below or equal to about 54°C, or 50°C, or at about 30°C-55°C. The drying time can be considered to be directly proportional to the surface area of ​​silica, and the control of water activity is inversely proportional to the surface area of ​​silica. In one example, the spray drying process may be performed using a Büchi B-290 spray dryer at 10% pump power.

[0034] The RNA composition obtained after the spray drying step has a water activity (aW) of approximately 0.01 to 0.5. Water activity (aW) is a thermodynamic parameter. It is a measure of the amount of water that can be used in chemical, biochemical, and microbiological reactions of samples such as aqueous solutions and foods, and can also be used to characterize the composition of a carrier (mixture). Water activity is given as a value and is defined as the ratio of the water vapor pressure (p) above the sample at the same temperature to the water vapor pressure (po) of pure water, i.e., a = p / po. Water activity corresponds to 1 / 100 of the relative equilibrium humidity (RGF). Equilibrium relative humidity is also known as equilibrium relative humidity (ERH). Pure water has a value of 1, and each addition of a water-retaining substance causes the value to drop below 1. Specifically, the spray-dried RNA composition obtained after spray drying has the following water activities (aW): 0.01 to 0.45, 0.01 to 0.4, 0.01 to 0.35, 0.01 to 0.3, 0.01 to 0.25, 0.01 to 0.2, 0.05 to 0.5, 0.05 to 0.45, 0.05 to 0.4, 0.05 to 0.35, 0.05 to 0.3, 0.05 to 0.25. 0.05 to 0.2, 0.05 to 0.15, 0.05 to 0.1, 0.1 to 0.5, 0.1 to 0.45, 0.1 to 0.4, 0.1 to 0.35, 0.1 to 0.3, 0.1 to 0.25, 0.1 to 0.2, 0.1 to 0.15, 0.15 to 0.5, 0.15 to 0.45, 0.15 to 0.4, 0.15 to 0.3, 0.15 to 0.25, or 0.15 to 0.2. More particularly, the spray-dried RNA composition after spray drying has a water activity (aW) of about 0.01, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, or 0.4.

[0035] The method according to any aspect of the invention can be used to produce RNA compositions according to any aspect of the invention.

[0036] According to a further aspect of the present invention, a method for preserving the biological activity of extracellular RNA molecules is provided, the method comprising the following steps: - To bring RNA molecules into contact with a silica carrier and a coating material made of zein or shellac; The silica carrier is silica particles with an average diameter of 0.3µm – 1000µm.

[0037] According to yet another aspect of the invention, the use of compositions according to any aspect of the invention in agriculture is provided.

[0038] RNA compositions, powders, according to any aspect of the invention, can be diluted, for example with water or a suitable diluent or solvent, to form a liquid RNA composition spray before agricultural use (i.e., spraying onto crops and / or seeds). Such diluents may be referred to as adjuvants.

[0039] As used herein, 'adjuvant' refers to a component or substance in a composition according to any aspect of the invention that enhances or modifies the activity of other components (i.e., RNA). In particular, adjuvants according to any aspect of the invention are biocompatible adjuvants (active ingredient mediators) added to the RNA composition to form a liquid composition according to any aspect of the invention. Any adjuvant known in the art can be used in a composition according to any aspect of the invention. Specifically, adjuvants according to any aspect of the invention may be selected from (A), (B), or (C) and mixtures thereof, wherein (A), (B), or (C) is: (A) Polyglycerol esters having the following general formula (I), M j D k T l Formula (I) Where M is [C3H5(OR'')2O 1 / 2 ], D is [C3H5(OR'')1O] 2 / 2 ], T is [C3H5O] 3 / 2 ], j = 1 to 10, preferably 2 to 3, more preferably 2; k = 0 to 10, preferably greater than 0 to 5, more preferably 1 to 3; l = 0 to 3, preferably 0 to 1, more preferably 0; The sum of j+k+l is 1 to 20, preferably 2 to 4, and more preferably 3. Each of the groups R'' is independently selected from acyl groups R'-C (= O)- and H, provided that at least one group R'' is not equal to H; Each of the groups R' is independently selected from monovalent aliphatic, saturated or unsaturated hydrocarbon groups having 3 to 39, preferably 7 to 21, and more preferably 9 to 17 carbon atoms; (B) Polyether-modified siloxanes having formula (II) M 1 o D 1 p D' q Equation (II) Where M 1 For P 1 3SiO 1 / 2 , D1 For P 1 2SiO 2 / 2 D' is P 1 P 2 SiO 2 / 2 , o is 2, p is 0 to 0.1, and q is 1.0 to 1.15. P 1 Independently, it is a hydrocarbon group having 1 to 8 carbon atoms. P 2 Independently having a polyether group of formula (III) —P 3 O[CH2CH2O] m [CH2CH(CH3)O] n P 5 Equation (III) in m ranges from 3.4 to 11.0, and n ranges from 2.5 to 8.0. And the premise is m / n ranges from 1.9 to 2.8. P 3 Independently, it is a divalent hydrocarbon group having 2 to 8 carbon atoms. P 5 For hydrogen; and / or (C) Organically modified polysiloxanes having formula (IV) Formula (IV) Where a+b+c+d+2 = 20 to 210, a is 15 to 205, b is 1 to 12, c is 1 to 12, d is 1 to 12. The R groups are each independently the same or different aliphatic or aromatic hydrocarbon groups having 1 to 10 carbon atoms. Each of the R4 groups can be an independent R, R1, R2, or R3 group, either the same or different. R1, R2, and R3 groups are each independently different polyether groups having the general formula (V). Formula (V) in e is 3 to 11, f is 6 to 30, g is 0 to 15, h is 0 to 5, i is 0 to 5, and R5 can be either the same or different and each can be a methyl, acetyl, or hydrogen group.

[0040] More specifically, the adjuvant may be selected from BREAK-THRU® S 301, BREAK-THRU® SP 133, and BREAK-THRU® S 255. Adjuvants used according to any aspect of the invention result in reduced surface tension at stomata or damaged sites and thus lower repulsion, or improved flow of particles (RNA) through the pores into the plant or parts thereof where RNA is targeted. In particular, the use of the adjuvant allows / accelerates the RNA uptake process. Furthermore, enhanced uptake allows RNA to translocate across the phloem from the leaf to the root, thereby also enhancing nitrogen fixation.

[0041] The use of adjuvants also allows for a uniform distribution of the RNA composition from the upper to the lower surface of the leaf, where most of the (open) stomata are located. This enables faster and more extensive penetration of RNA into the plant tissue. Without the use of biocompatible adjuvants, especially in the case of foliar application, reaching the stomata on the lower surface of the leaf is, if not impossible, particularly difficult. The use of adjuvants with "anti-rinse-off" properties also increases the residence time on the upper surface of the leaf and thus promotes RNA uptake into the plant tissue. The presence of adjuvants reduces premature wash-off of the RNA composition.

[0042] In one instance, the adjuvant is (A): (A) Polyglycerol esters having the following general formula (I), M j D k T l Formula (I) Where M is [C3H5(OR'')2O 1 / 2 ], D is [C3H5(OR'')1O] 2 / 2 ], T is [C3H5O] 3 / 2 ], j = 1 to 10, preferably 2 to 3, more preferably 2; k = 0 to 10, preferably greater than 0 to 5, more preferably 1 to 3; l = 0 to 3, preferably 0 to 1, more preferably 0; The sum of j+k+l is 1 to 20, preferably 2 to 4, and more preferably 3. Each of the groups R'' is independently selected from acyl groups R'-C (= O)- and H, provided that at least one group R'' is not equal to H; Each of the groups R' is independently selected from monovalent aliphatic, saturated or unsaturated hydrocarbon groups having 3 to 39, preferably 7 to 21, and more preferably 9 to 17 carbon atoms.

[0043] In particular, at least one group R'' corresponds to a group having the formula R'—C(O)—.

[0044] More specifically, M, D, and T can be: .

[0045] Even more specifically, the polyglycerol esters of mixtures according to any aspect of the invention have the formula (I(a)): Formula (I(a)) in a = 1 to 10, preferably 2 to 3, and more particularly 2; b = 0 to 10, preferably greater than 0 to 5, and more particularly 1 to 3; The premise is: a + b = 2 to 20, preferably 2 to 4, especially 3; Each of the groups R'' is independently selected from acyl groups R'-C (= O)- and H, provided that at least one group R is not equal to H; wherein each of the groups R' is independently selected from monovalent aliphatic, saturated or unsaturated hydrocarbon groups having 3 to 39, preferably 7 to 21, particularly 9 to 17 carbon atoms.

[0046] The polyglycerol ester of any composition according to any aspect of the invention may have more than one, particularly at least two, and more particularly at least three groups R'' having the formula R'-C (= O)-.

[0047] The group R'' having the formula R'—C(O)— can be independently an acyl group of the same or different saturated or unsaturated fatty acids, wherein the fatty acids comprise 4 to 40 carbon atoms, and in particular, the fatty acids are selected from butyric acid, hexanoic acid, caprylic acid, and capric acid. (decanoic acid), lauric acid (dorsodic acid), myristic acid (tetradecanoic acid), palmitic acid (hexadecanoic acid), stearic acid (octadecanoic acid), arachidic acid (eicosanoic acid), behenic acid (tioctadecanoic acid), creosoteric acid (tetracosanoic acid), palmitoleic acid ((Z)-9-hexadecenoic acid), oleic acid ((Z)-9-hexadecenoic acid), transoleic acid ((E)-9-octadecenoic acid), cis-isooleic acid ((Z)-11-octadecenoic acid), linoleic acid ((9Z,12Z)-9,12-octadecadienoic acid), α-linolenic acid ( (9Z,12Z,15Z)-9,12,15-octadecanotrienoic acid, γ-linolenic acid ((6Z,9Z,12Z)-6,9,12-octadecanotrienoic acid), di-homo-γ-linolenic acid ((8Z,11Z,14Z)-8,11,14-eicosatetraenoic acid), arachidonic acid ((5Z,8Z,11Z,14Z)-5,8,11,14-eicosatetraenoic acid), erucic acid ((Z)-13-docosahexaenoic acid), nervonic acid ((Z)-15-docosahexaenoic acid), ricinoleic acid, hydroxystearic acid, undecanoic acid, and mixtures thereof. In one example, the fatty acid may be a mixture of rapeseed oil fatty acid, soybean fatty acid, sunflower fatty acid, peanut fatty acid, and tall oil fatty acid. In particular, for this background, the fatty acid may be a group of oleic acid. When calculating HLB values, the molar mass of the lipophilic molecular portion is the arithmetic mean of the sum of the molar masses of all groups R' present in the molecule.

[0048] Suitable sources of fatty acids or fatty acid esters, especially glycerides, can be plant or animal fats, oils, or waxes. Examples include lard, beef tallow, goose tallow, duck tallow, chicken tallow, horse tallow, whale oil, fish oil, palm oil, olive oil, avocado oil, kernel oil, coconut oil, palm kernel oil, cocoa butter, cottonseed oil, pumpkin seed oil, corn seed oil, sunflower oil, wheat germ oil, grapeseed oil, soybean oil, peanut oil, lupin oil, rapeseed oil, mustard oil, castor oil, jatropha oil, walnut oil, jojoba oil, lecithin (e.g., lecithin based on soybean, rapeseed, or sunflower), bone oil, claw oil, borage oil, lanolin, emu oil, deer tallow, marmot oil, mink oil, safflower oil, hemp seed oil, pumpkin oil, evening primrose oil, tallow, as well as carnauba wax, beeswax, candelilla wax, crown cocoa wax, sugarcane wax, retamow wax, and caranda wax. Wax, copra wax, Spanish grass wax, alfalfa wax, bamboo wax, Douglas fir wax, cork wax, sisal wax, linseed wax, cotton wax, damar wax, tea wax, coffee wax, rice wax, oleander wax, or wool wax can all be sources of fatty acids or fatty acid esters.

[0049] In particular, the polyglycerol ester compound has formula (I) or (I(a)), has an arithmetic mean of 2.9 to 3.1 groups of formula R'-C (= 0)- and an HLB value of 4 to 6.5.

[0050] More specifically, the polyglycerol ester compound has the formula (I(a)), where the sum of a+b is 3, and has an arithmetic mean of 2.9 to 3.1 groups of the formula R'-C (= 0)- and an HLB value of 4 to 6.5.

[0051] More specifically, the polyglycerol ester compound may have formula (I(a)) having an arithmetic mean of 2.9 to 3.1 groups of formula R'-C (= O)- and an HLB value of 4 to 6.5, wherein the acyl residues of the fatty acid mixture contain oleic acid, stearic acid, palmitic acid and γ-linolenic acid, and said fatty acids particularly constitute at least 85% by weight of the fatty acid mixture.

[0052] In one example, the polyglycerol ester compound may have formula (I(a)) having an arithmetic mean of 2.9 to 3.1 groups of formula R'-C (= 0)- and an HLB value of 4 to 6.5, wherein the acyl residues are derived from a mixture of fatty acids containing oleic acid, stearic acid, palmitic acid and γ-linolenic acid, and said fatty acids particularly constitute at least 85% by weight of the fatty acid mixture.

[0053] In another example, the polyglycerol ester compound used according to any aspect of the invention may have formula (I(a)), having an arithmetic mean of 2.9 to 3.1 groups of formula R'-C (= O)- and an HLB value of 4 to 6.5, wherein the mass fraction of oleoyl residues is at least 75%, particularly 85%, and more particularly 95% of the mass of all acyl residues. Even more particularly, the polyglycerol ester is a triglycerol trioleate.

[0054] A more detailed disclosure of adjuvant (B) is provided at least in US10390530B2.

[0055] In one instance, the adjuvant is (B): (B) Polyether-modified siloxanes having formula (II) M 1 o D 1 p D' q Equation (II) Where M 1 For P 1 3SiO 1 / 2 , D 1 For P 1 2SiO 2 / 2 D' is P 1 P 2 SiO 2 / 2 , o is 2, p is between 0 and 0.1, especially 0 q is 1.0 to 1.15, especially 1.0 to 1.10, and particularly 1.00 to 1.05. P 1 Independently, it is a hydrocarbon group having 1 to 8 carbon atoms, particularly a methyl, ethyl, propyl, or phenyl group, especially a methyl group. P 2 Independently having a polyether group of formula (III) —P 3 O[CH2CH2O] m [CH2CH(CH3)O] n P 5 Equation (III) in m ranges from 3.4 to 11.0, particularly from 3.6 to 9.9, and even more particularly from 4.5 to 8.5. n ranges from 2.5 to 8.0, particularly from 2.7 to 7.5, and even more particularly from 3.0 to 6.0. And the premise is m / n ranges from 1.9 to 2.8. P 3 Independently, it is a divalent hydrocarbon group having 2 to 8 carbon atoms, particularly ethylene, propylene, 1-methylpropene, 1,1-dimethylpropene groups, especially —CH2CH2CH2—. P 5 It is hydrogen.

[0056] The polyether-modified siloxane having formula (II) has a biodegradability of more than 60%, more particularly more than or equal to 63%, and especially more than or equal to 65%, with a maximum of 100%.

[0057] Specifically, it does not contain P. 3 O is calculated and does not contain P. 5 The calculated polyether group has a molar mass M(PE) calculated by 44 g / mol*m + 58 g / mol*n, where the exponents m and n are related to equation (III). More specifically, the values ​​of M(PE) are: a lower limit M(PE) greater than 520 g / mol, particularly greater than 530 g / mol, and even more particularly greater than 535 g / mol; and an upper limit M(PE) less than 660 g / mol, particularly less than 630 g / mol, and even more particularly less than 600 g / mol. Even more specifically, the value of M(PE) is greater than 520 g / mol and less than 660 g / mol, particularly greater than 535 g / mol and less than 600 g / mol. In particular, the sum of m+n is greater than 9 up to 19, more particularly greater than 9.5 up to 15, and even more particularly greater than 10 up to 12.

[0058] In one example, the polyether-modified siloxane used in the composition according to any aspect of the invention is a polyether-modified siloxane having formula (II), wherein the index c is from 1 to 1.05, and wherein the index of the polyether group having formula (III) is 3.4 to 11.0 for m and 2.5 to 8.0 for n. Specifically, the polyether-modified siloxane used in the composition according to any aspect of the invention is a polyether-modified siloxane having formula (II), wherein the index c is from 1 to 1.05, and wherein the m / n ratio is from 1.9 to 2.8. More specifically, the polyether-modified siloxane used in the composition according to any aspect of the invention is a polyether-modified siloxane having formula (II), wherein the index c is from 1 to 1.05, and wherein the molar mass M(PE) of the polyether residues is greater than 520 g / mol and less than 660 g / mol. Even more specifically, the polyether-modified siloxane used in compositions according to any aspect of the invention is a polyether-modified siloxane having formula (II), wherein the index c is from 1 to 1.05, and wherein P 5The group is hydrogen, or the index c is 1 to 1.05, wherein the molar mass M(PE) of the polyether residues is greater than 520 g / mol and less than 660 g / mol, and the P5 group is hydrogen. In particular, the polyether-modified siloxane used in the compositions according to any aspect of the invention is a polyether-modified siloxane having formula (II), excluding any other polyether-modified siloxanes other than those having formula (II).

[0059] A more detailed disclosure of adjuvant (B) is provided at least in US10299471B2.

[0060] In one example, the adjuvant is (C), an organically modified polysiloxane having formula (IV). Formula (IV) Where a+b+c+d+2 = 20 to 210, preferably 30 to 100, especially 40-60. a is 15 to 205, more preferably 35 to 45. b is 1 to 12, preferably 1 to 8, especially 2 to 6. c ranges from 1 to 12, preferably from 1 to 8, and especially from 2 to 6. d is 1 to 12, preferably 1 to 8, especially 2 to 6. The R groups are each independently the same or different aliphatic or aromatic hydrocarbon groups having 1 to 10 carbon atoms, preferably methyl groups. Each of the R4 groups can be an independent R, R1, R2, or R3 group, either the same or different. R1, R2, and R3 groups are each independently different polyether groups having the general formula (V). Formula (V) in e ranges from 3 to 11, with 3 being the preferred value. f is 6 to 30, preferably 10 to 30. g is 0 to 15, preferably 0 to 10. h is between 0 and 5. i is between 0 and 5, and R5 is independently the same or different and each is a methyl, acetyl or hydrogen group, preferably provided that the molecular weight of the polyether group having formula (V) is greater than 200 g / mol, preferably greater than 400 to 2000 g / mol, and the proportion of ethylene oxide in the polyether is greater than 45% by mass, and the percentage of ethylene oxide in the polyether group R2 by mass is at least 9% greater than the percentage of ethylene oxide in the polyether group R1 by mass, in each case based on the polyether group having formula (V), wherein the groups having formula (V) can be formed randomly, in a gradient or block.

[0061] In equation (V), the units specified by the exponent 'g' are those derived from propylene oxide, the units specified by the exponent 'h' are those derived from butane oxide, and the units specified by the exponent 'i' are those derived from phenylene oxide.

[0062] The exponents 'a to d' and 'e to i' can be natural integers or weighted averages. Weighted averages are preferred.

[0063] A more detailed disclosure of adjuvant (C) is provided at least in US8580225B2.

[0064] Compositions according to any aspect of the invention may contain any one of adjuvants (A), (B), or (C). In one example, the composition may contain adjuvants such as (A) and (B), (A) and (C), (B) and (C), or a mixture of (A), (B), and (C). In another example, compositions according to any aspect of the invention may contain more than one adjuvant (A), more than one adjuvant (B), or more than one adjuvant (C).

[0065] Unless otherwise stated, all percentages (%) given are percentages by mass.

[0066] The embodiments described below are intended to illustrate the invention by way of example, and are not intended to limit the invention (the scope of which is obvious from the description and claims as a whole) to the embodiments described in the examples. Brief description of the attached diagram Figure 1 This is a schematic diagram illustrating an example of a method for producing an RNA composition according to any aspect of the present invention.

[0068] Figure 2 These are photographs of the results of scanning electron microscopy (SEM) examination of the composition according to Example 1 in particles without (A) and with (B) shell glue as a matrix.

[0069] Example: Example 1 Method for producing compositions or granules Material GFP dsRNA, 223 bp RNA Greentech LLC 10601 Clarence Dr Ste 250 FriscoTX 75033 Silica with a maximum average diameter of 0.5 µm.

[0070] Analytical methods The samples were separated by HPLC and analyzed by UV detection: Eluent A: 0.1M TEAA-Puffer Eluent B: 75% eluent A and 25% ACN Post: Proswift RP-1S Monolith, 50mm in length, ID 4.6mm Injection volume: 10 μl Flow rate: 1.0 ml / min UV detection: 260nm Column furnace temperature: 50℃ Autosampler temperature: 10℃ Sequence execution time: 30 minutes Balancing time: 7 minutes Syringe flushing medium: Chromatographic water Reservation time: ~14 minutes Gradient curve: .

[0071] spray drying The spray drying process is accomplished using an inlet temperature of 76°C to 80°C for the spray dryer. The outlet temperature is 45°C to 60°C. The use of a two-fluid nozzle with either air or inert gas prevents pressure buildup within the system. The entire process is carried out under a nitrogen atmosphere. The atomization of the mixture within the spray dryer is accomplished with hot drying gas from the top in a concentric flow.

[0072] The actual suspension was then spray-dried in a Buechi B-290 laboratory spray dryer at a gas inlet temperature of 78°C. Spraying was performed using a two-fluid nozzle at an atomization pressure of approximately 1.35 bar. The drying air flow rate was 38 m / s. 3 / hour. The spray rate is approximately 5 mL / min. The set parameters result in an outlet temperature of 53°C.

[0073] Figure 2The results of scanning electron microscopy (SEM) examination of the compositions in the formed particles without (A) and with (B) shells as a matrix are provided.

[0074] Example 2 Stability testing methods The dsRNA reference and dsRNA+silica formulation from Example 1 were tested to determine RNA stability. Particles from Example 1 were sonicated (US) at laboratory temperature for 3 minutes, and then sonicated in water baths at 40°C, 60°C, and 80°C for 1 hour. HPLC results are provided in Table 1 below. As can be seen below, almost all double-stranded RNA was recovered in the presence of silica.

[0075] Table 1. HPLC results of dsRNA stability.

[0076] Example 3 Production and analysis methods for encapsulated compositions and particles Material dsRNA by Genolution Inc. 63, Magokjungang 8-ro 3-gil, Gangseo-gu, Seoul07793 Republic of Korea: It has a short GFP of 240 bp: .

[0077] Silica with a maximum average diameter of 0.5 µm Spray drying of the encapsulation composition The pure dsRNA solution was resuspended in VE water and added to obtain suspensions of Sipernat® 380 containing dsRNA (A), Sipernat® 380 containing dsRNA and shellac solution (C), Sipernat® 380 containing dsRNA and zein solution (G), or Sipernat® 380 containing dsRNA and PVOH solution (K). The suspensions were then spray-dried in a Büchi B-290 laboratory spray dryer at a gas inlet temperature of 80°C. Spraying was performed using a two-fluid nozzle at an atomization pressure of approximately 1.35 bar. The flow rate of the drying air was 38 m / s. 3 / hour. The spray rate was approximately 6 mL / min. The set parameters resulted in an outlet temperature of 50°C. All final product compositions were calculated to have 1 mg dsRNA / g dried final product after treatment.

[0078] Table 2: Products of the spray process.

[0079] Storage method All produced samples and blanks were stored under the following conditions: Pure dsRNA (in buffer) was prepared as a powder (dry powder) (A to K, Table 3) under the following conditions: in BP787 (liquid) (A to K in 1 ml BP787) and in pure water (liquid) (A to K in 1 ml water). - 25℃ and 0.1aw - 54℃ and 0.1aw - 25℃ and 0.62aw - 54℃ and 0.48aw.

[0080] Water activity values ​​are averages. All samples were stored for four weeks and sampled weekly. All final samples were frozen at -50°C immediately after storage and before analysis.

[0081] The samples were processed as follows: All dried samples from the spray process were weighed into Eppis. The amount of dried product in each Eppis ranged from 0.03 g to 1.2 g. For liquid storage, the Eppis were additionally filled with 1 mL of polyether siloxane BP787 (BREAK-THRU® BP787) or 1 mL of pure water and vortexed to homogenize in the liquid for 20 seconds. All Eppis were then stored open in a sealed plastic box on a salt solution to adjust for the specific water activity and atmosphere mentioned above. The boxes were placed and maintained in an oven at 25°C or 54°C. Temperature and water activity (aw) were checked using a digital tracker.

[0082] Analytical methods Equipment used ● CFX96 Touch Real-Time PCR Detection System.

[0083] The analytical method followed the protocol of New England Biolabs (240 County Road Ipswich) Luna® Universal Probe One-Step RT-qPCR Kit Protocol (E3006), with the following modifications: Prior to applying the appropriate protocol, an additional denaturation step is performed to disrupt the double strand of RNA. The sample is denatured at 95°C (PCR module) for 5 minutes and then quickly placed on ice.

[0084] Use the following primers: .

[0085] Example 4 Comparison of coated and uncoated compositions and granules in dry storage The Cq signal is an indicator that is logarithmically correlated with the concentration of detectable dsRNA. A lower Cq signal indicates a higher dsRNA concentration (see Table 3). Table 3: Results from qPCR analysis.

[0086] The results showed that under accelerated, harsh storage conditions for 2 weeks, coating further increased the stability of dsRNA (A17 ↔ C17) compared to particles without any coating.

[0087] Example 5 Comparison of zein-coated compositions and particles in water and BP787 storage The Cq signal is a logarithmic correlation between the Cq signal and the detectable dsRNA concentration. A lower Cq signal indicates a higher dsRNA concentration (see Table 4). Table 4: Results from qPCR analysis.

[0088] The results showed that storage in BP787 was beneficial under accelerated, harsh liquid storage conditions for 4 weeks, and that the zein coating was functional.

Claims

1. A composition comprising at least one RNA molecule and at least one silica carrier, wherein the silica carrier is silica particles with an average diameter of 0.3 µm to 1000 µm, and wherein the composition further comprises a matrix shell made of zein or shellac.

2. The composition according to claim 1, wherein the silica particles have an average diameter of 0.3-75 µm.

3. The composition according to claim 1 or 2, wherein the silica particles have an average diameter of 0.3-10 µm.

4. The composition according to any one of the preceding claims, wherein the silica particles further comprise calcium silicate particles.

5. The composition according to any one of the preceding claims, wherein the RNA is double-stranded RNA.

6. The composition according to any one of the preceding claims, wherein the silica particles are precipitated silica.

7. A method for producing a composition comprising at least one RNA molecule, a silica carrier, and a matrix shell, the method comprising the following steps: (a) Combining silica particles with an average diameter of 0.3µm – 1000µm with at least one liquid RNA molecule and a coating material of zein or shellac.

8. The method of claim 7, wherein the method comprises a further step (b) of spray drying the resulting composition from step (a) to form an RNA composition powder.

9. The method according to claim 7 or 8, wherein the composition is according to any one of claims 1 to 6.

10. A method for preserving the biological activity of extracellular RNA molecules, the method comprising the following steps: - To bring the RNA molecules into contact with the silica carrier and the coating material of zein or shellac; The silica carrier is silica particles with an average diameter of 0.3µm – 1000µm.

11. The method of claim 10, wherein the silica particles further comprise calcium silicate.

12. The method according to claim 10 or 11, wherein the particles have an average diameter of 0.3-75 µm.

13. Use of the composition according to any one of claims 1 to 6 in agriculture.

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