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WO2025098983A1 - Stockage et distribution d'arn - Google Patents

Stockage et distribution d'arn Download PDF

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Publication number
WO2025098983A1
WO2025098983A1 PCT/EP2024/081167 EP2024081167W WO2025098983A1 WO 2025098983 A1 WO2025098983 A1 WO 2025098983A1 EP 2024081167 W EP2024081167 W EP 2024081167W WO 2025098983 A1 WO2025098983 A1 WO 2025098983A1
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WIPO (PCT)
Prior art keywords
rna
silica
composition
acid
silica particles
Prior art date
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PCT/EP2024/081167
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English (en)
Inventor
Max Braun
Stefan GILCH
Thomas Rieger
Mathias DERNEDDE
Johannes ÖHRLEIN
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Evonik Operations GmbH
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Evonik Operations GmbH
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Publication of WO2025098983A1 publication Critical patent/WO2025098983A1/fr
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/60—Isolated nucleic acids

Definitions

  • the invention relates to a composition for storage and exogeneous delivery of RNA to a target and the method for producing the composition.
  • the composition comprises RNA and small silicate particles used as a carrier for the RNA.
  • the composition may especially be useful for use in agriculture.
  • BACKGROUND OF THE INVENTION RNA has been shown to be very useful in many different industries. In one industry where RNA is very commonly used is in agriculture.
  • RNA/RNAi can be used against pest, insect, and/or virus control.
  • dsRNA can also be used in modulation of plant expression for increased growth, or nutritional change.
  • RNA has several uses outside of the cell, it is known to be relatively unstable and easily degradable, especially outside of the cell.
  • unprotected RNA molecules are highly unstable due to the ubiquitous RNases in nature. This in general makes an economical application of RNA, especially in the liquid environment difficult.
  • Exogeneous application of RNA is generally capable of successfully bringing about the desired effect.
  • the effect generally rapidly declines with the RNA, particularly dsRNA since they typically having a half-life of only about 12 to 24 hours in soil for example, and further depending on the precise environmental conditions in which it is administered may have even shorter half-life than that.
  • US11286476B2 also provides methods of substantially retaining the biological activity of dsRNA by maintaining the RNA in a bacterial cell and then adding a compound having the function of a protein—or amine—cross linking agent and/or an acid.
  • the present invention solves the problems above by providing a composition comprising at least one molecule of RNA and silica particles as carriers.
  • the silica particles are of a suitable size that not only binds the RNA but also protects the RNA from degradation.
  • the suitably sized silica 202300100 Foreign Filing 2 particles enable the RNA to be transported efficiently to its target.
  • Silica particles can be utilized to increase RNA concentration without gelling.
  • the silica particles have a mean diameter of 0.3 ⁇ m – 1000 ⁇ m. The silicate particles of this size lead to improved handling and shelf life of the RNA compared to the prior art.
  • RNA active ingredient and/or bioavailability over a longer period may be achieved in comparison with the prior art in view of the size of the silica particles.
  • a composition comprising at least one molecule of RNA and at least one silica carrier, wherein the silica carrier is silica particles with a mean diameter of 0.3 ⁇ m – 1000 ⁇ m and wherein the composition further comprises a matrix shell made of Zein, or Shellac.
  • the molecule of RNA may be any RNA molecule.
  • the RNA molecule is selected from the group consisting of an antisense RNA, a single-stranded RNA (ssRNA), a double- stranded RNA (dsRNA), a hairpin RNA (hpRNA), a short-hairpin RNA (shRNA), a small-interfering RNA (siRNA), a microRNA (miRNA), or combinations thereof. Even more in particular, the RNA molecule is a dsRNA. In one example, the RNA molecule is an inhibitory RNA molecule. In particular, the RNA is an isolated RNA molecule. The RNA molecule may be isolated using any method known in the art.
  • carrier refers to silica or silicate used according to any aspect of the present invention comprising silica particles.
  • the carrier is able to transport the RNA according to any aspect of the present invention to its final target (for example in agriculture to a part of the plant/ crop).
  • Any type of silica may be used as a silica carrier according to any aspect of the present invention.
  • silica may be used as a defined hydrophilic carrier with water-absorbing properties (maintenance of low water activity; aW).
  • the silica used may be selected from the group consisting of SIPERNAT® 50, SIPERNAT® 50S, SIPERNAT® 22, SIPERNAT® 380, SIPERNAT® 22S, SIPERNAT® 800, SIPERNAT® 880, ZEOLEX® 7, ZEOLEX ® 23, and ZEOLEX® 23a.
  • the silica used according to any aspect of the present invention may be a precipitated silica.
  • the silica may be SIPERNAT® 380, a carrier silica with an ultra-fine particle size precipitated silica.
  • the precipitated silica may work more effectively in the composition according to any aspect of the present invention due to many reasons, including possibly the pH of the precipitated silica compared to others.
  • the use of the right silica carrier allows for good adhesion of the drops of the RNA onto the leaves of the crops.
  • This carrier leads to good rain resistance of the RNA on the plants.
  • the RNA are still present and effective on the leaves of the crops even after rain.
  • the spray droplets of the RNA spray become larger and are therefore less prone to drifting off during the spraying process.
  • the carrier also enables the spreading effect which equally covers/distributes the RNA all over the surface of the target.
  • the carrier can be produced sustainably from renewable raw materials and is also largely biodegradable.
  • the silica carrier therefore shows a particularly good profile of properties. 202300100 Foreign Filing 3
  • the presence of silica maintains the low aW of the final product, the composition of RNA.
  • the presence of silica has the added benefit of being able to absorb water, has good handling properties/ flowability making it suitable and easy to use. Further, silica increases the volume of the final product making the handling of the final product also easier and more convenient.
  • the silica particles may further comprise calcium silicate particles. Calcium Silicates carry a Calcium in their structure. RNA normally is thought of having a negative partial charge. Therefore, the stability is realized due to a coordinated binding to the silicas. This coordination also gives certain stability. Therefore, for stabilization of RNA, the positive charge of Calcium silicate may be even more beneficial than classic silicate or silica. A varied concentration of silica may be present in the composition of RNA.
  • WO2020104612A1 shows a range of concentrations of silica that may be applicable according to any aspect of the present invention.
  • the concentration of silica present may be determined by the type of RNA present.
  • about 99-70 wt% of silica may be present with 1-30 wt% of RNA. More in particular, 99-75, 99-80, 99-85, 99-80, 99-90, 99-95 wt% silica 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 wt% RNA.
  • silica particles according to any aspect of the present invention may have a mean diameter size of 0.3 ⁇ m – 1000 ⁇ m.
  • the mean diameter size may 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, 0.3-300
  • the mean diameter size of the silica particles may be 0.3-75 ⁇ m. Even more in particular, the silica particles may have a mean diameter of 0.3-10 ⁇ m. In one example, the mean diameter size of the silica particle may be about 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, 2829, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 ⁇ m.
  • the term ‘about’ as used herein refers to a variation within 20 percent.
  • the term “about” as used herein refers to +/- 20%, more in particular, +/-10%, even more in particular, +/- 5% of a given measurement or value.
  • the term “about” refers to a range of values that fall within 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 percent or less of the stated reference value for that condition.
  • the term “about”, as applied to the mean diameter size refer to a range of values that are similar to the stated reference value for that condition.
  • the term ‘mean diameter size’ refers to the average of the diameters of all the silica particles in a sample.
  • the mean diameter size refers to the average diameter of the silica particle, determined by adding all the diameters of the 10 silica particles and dividing by 10.
  • the silica particle size always refers to the mean diameter size (i.e. the average diameter size) of all the silica particles present in the RNA composition according to any aspect of the present invention.
  • each individual silica particle in the RNA composition therefore does not always have the diameter of 0.3-1000 ⁇ m.
  • the average or mean diameter of all the silica particles present in the RNA composition according to any aspect of the present invention has a diameter of 0.3-1000 ⁇ m.
  • the small size of the silica particles is especially useful for being a carrier and for producing RNA compositions that do not block the nozzle of agricultural machines when used in agriculture. Any method known in the art may be used to produce the silica particles in the RNA composition according to any aspect of the present invention.
  • the silica particles are made using a 202300100 Foreign Filing 5 steam jet milling process.
  • the steam jet milling process reduces the size of the original silica particles added into the steam jet mill to smaller particles to be used in the RNA composition.
  • Any steam jet mill may be used.
  • a skilled person is capable of varying the speed and/or size of the starting material to obtain silica particles of the required and desired size needed for the RNA composition according to any aspect of the present invention.
  • the composition according to any aspect of the present invention may further comprise a matrix shell.
  • the matrix shell may also be known as a coating, encloses everything. This encapsulation is responsible for the long term stability of the RNA in the RNA composition according to any aspect of the present invention.
  • the matrix shell may be made of Zein, Shellac, Isomalt, Waxes, Resins, carbohydrates, proteins, or Polymers like PVOH.
  • the matrix shell may be made of Zein, or Shellac. Both Zein and Shellac are able to increase the stability of the RNA and is especially useful in protecting and/or maintaining the stability of the RNA in storage.
  • the matrix shell may be regulated for a triggered release.
  • the shell may be pH sensitive such that in the presence of a particular pH, the shell disintegrates and the RNA from the RNA composition may be released.
  • the shell may be temperature-sensitive.
  • a coating including shellac may be suitable for this function.
  • the RNA composition according to any aspect of the present invention may be completely microplastic- free. According to a further aspect of the present invention, there is provided a method of producing a composition comprising at least one molecule of RNA and a silica carrier, the method comprising the steps of: (a) combining silica particles with a mean diameter of 0.3 ⁇ m – 1000 ⁇ m with at least one aqueous RNA molecule and a coating material of Zein or Shellac.
  • the silica particles with a mean diameter of 0.3 ⁇ m – 1000 ⁇ m may be produced using any method known in the art.
  • the silica particles may be produced using a steam jet mill. Any steam jet mill may be used to produce the silica particles used according to any aspect of the present invention.
  • the steam jet milling process reduces the size of the original silica particles added into the steam jet mill to smaller particles to be used in the RNA composition.
  • a skilled person is capable of varying the speed and/or size of the starting material to obtain silica particles of the required and desired size needed for the RNA composition according to any aspect of the present invention.
  • EP2209739 describes at least one method for producing silica particles with a mean diameter of 0.3 ⁇ m – 1000 ⁇ m, particularly precipitated silica particles using a steam jet mill.
  • the silica carrier particles from the steam jet mill may then be brought into contact with the liquid RNA molecule.
  • the desired RNA (to be introduced to the target cell or location) is in a liquid form or in an aqueous solution, particularly in the extractant buffer or water.
  • contacting means bringing about direct contact the RNA and the silica particles.
  • the term does not necessarily constitute the step of physically mixing or combining the two components- silica particles and RNA molecules.
  • an aqueous solution comprises any solution comprising water, mainly water as solvent that may be used to keep the RNA according to any aspect of the present invention, at least temporarily, in a metabolically active and/or viable state and comprises, if such is necessary, any additional substrates.
  • the person skilled in the art is familiar with the preparation of numerous aqueous mediums, usually referred to as media that may be used to extract and/or store RNA. It is advantageous to use as an aqueous solution a minimal medium, i.e. a medium of reasonably simple composition that comprises only the minimal set of salts and nutrients indispensable for keeping the RNA in a metabolically active and/or viable state, by contrast to complex mediums, to avoid dispensable contamination of the products with unwanted side products.
  • aqueous solutions in which the RNA molecule may be found include buffer and water.
  • the silica carrier and the aqueous RNA are brought into contact with one another and mixed or combined, the result may be spray dried to produce a powder.
  • An schematic drawing of the method according to any aspect of the present invention is provided in Figure 1. Here, the optional step of adding a coating is also shown.
  • the method according to any aspect of the present invention is advantageous as it is a one-step process for producing stabilised and optionally encapsulated RNA.
  • RNA is successfully formulated in/on particles (powder form) using the method according to any aspect of the present invention.
  • RNA molecules can be concentrated in/on silica particles and RNA molecules can be optionally encapsulated on particles using the method according to any aspect of the present invention.
  • the spray-drying according to any aspect of the present invention may be carried out using any spray- dryer.
  • a spray-dryer Mini Spray Dryer B-290 (Büchi Labortechnik AG, Switzerland) may be used.
  • the RNA composition may be spray-dried by a spray-dryer, whereby the spray- dried RNA composition according to any aspect of the present invention may be obtained.
  • the spray dryer may first be preheated with a very low fan speed. Spray drying may be achieved by allowing the inlet air temperature to be not so high so that RNA will survive the temperature. In particular, the inlet air temperature may be less than or equal to about 80°C, less than or equal to about 50°C, at about 30°-50°C.
  • the spray drying may be with gas flow.
  • the outlet air temperature may be less than or equal to about 55°C, less than or equal to about 54, or 50°C, at about 30°-55°C.
  • the drying time may be considered to be proportional to the surface area of the silica and the control of water 202300100 Foreign Filing 7 activity is inversely proportional to surface area of the silica.
  • the spray-drying process may be carried out using Büchi B-290 spray dryer with 10% pump power.
  • the composition of RNA composition obtained after step of spray drying has a water activity (aW) of about 0.01 to 0.5.
  • the activity of water (aW) is a thermodynamic parameter.
  • the water activity corresponds to 1/100 of the relative equilibrium humidity (RGF).
  • Equilibrium relative humidity is also referred to as equilibrium relative humidity (ERH).
  • Pure water has a value of 1 and every addition of water-binding substances causes the value to drop below 1.
  • the spray-dried RNA composition obtained after spray drying has a water activity (aW) of 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.
  • aW water activity
  • 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.
  • aW water activity
  • the method according to any aspect of the present invention may be used to produce the RNA composition according to any aspect of the present invention.
  • a method of preserving the biological activity of an extracellular RNA molecule comprising the step of: - contacting the RNA molecule with a silica carrier and a coating material made of Zein or Shellac; wherein the silica carrier is silica particles with a mean diameter of 0.3 ⁇ m – 1000 ⁇ m.
  • RNA composition according to any aspect of the present invention a powder
  • a suitable diluent or solvent to form a liquid RNA composition spray before being used in agriculture (i.e. Sprayed on crops and/or seeds).
  • This diluent may be called an adjuvant.
  • An ‘adjuvant’ as used herein, refers to an ingredient or a substance in the composition according to any aspect of the present invention that increases or modifies the activity of the other ingredients namely, the RNA.
  • the adjuvants according to any aspect of the present invention are biocompatible adjuvant (active ingredient mediator) that are added to the RNA composition to form a liquid composition according to any aspect of the present invention.
  • biocompatible adjuvant active ingredient mediator
  • Any adjuvant known in the art may be used in the composition according to any aspect of the present invention.
  • the adjuvants may be selected from the group consisting of BREAK-THRU® S 301, BREAK-THRU® SP 133, BREAK-THRU® S 255.
  • the adjuvants used according to any aspect of the present invention leads to a reduction of the surface tension at the stomata or where there are injuries and thus to a lower rejection or an improved flow of the particles (RNA) through the orifices into the plant or part thereof where the RNA is targeted.
  • the use of adjuvants enables/ accelerates the process of RNA uptake.
  • enhanced uptake also allows translocation of the RNA across the phloem from the leaf to the root, from where nitrogen fixation can also be enhanced
  • adjuvants also allows a uniform distribution of the RNA composition from the upper leaf surface to the lower leaf surface where most (opened) stomata are present. Thus, faster and more widespread penetration of RNA into plant tissue is enabled. Without the use of biocompatible adjuvants, especially in the case of foliar application, reaching the stomata on the underside of the leaf would be particularly hard if not impossible.
  • 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.
  • At least one radical R’’ corresponds to a radical of the formula R′—C(O)—.
  • M, D and T may be:
  • the radicals R’’ of the formula R′—C(O)— may be independent of each another identical or different acyl radicals of saturated or unsaturated fatty acids, where the fatty acids include 4 up to 40 carbon atoms, particularly, the fatty acids are selected from the group consisting of butyric acid (butanoic acid), caproic acid (hexanoic acid), caprylic acid (octanoic acid), capric acid (decanoic acid), lauric acid (dodecanoic acid), myristic acid (tetradecanoic acid), palmitic acid (hexadecanoic acid), stearic acid (octadecanoic acid), arachidic acid (eicosanoic acid), behenic acid (docosanoic acid), lig
  • the fatty acid may be a mixture of rapeseed oil acids, soya fatty acids, sunflower fatty acids, peanut fatty acids and tall oil fatty acids.
  • the fatty acids may be radicals of oleic acid.
  • the molar mass of the lipophilic molecule moiety is the arithmetic mean of the total of the molar masses of all of the radicals R′ which are present in the molecule.
  • Sources of suitable fatty acids or fatty acid esters, especially glycerides can be vegetable or animal fats, oils or waxes.
  • the polyglycerol ester is triglycerol trioleate.
  • a more thorough disclosure of the adjuvant (B) is provided at least in US10390530B2.
  • the adjuvant is (B): (B) polyether-modified siloxanes of formula (II) M 1 o D 1 p D′q Formula (II) wherein, M 1 is P 1 3SiO1/2, D 1 is P 1 2SiO2/2, D′ is P 1 P 2 SiO2/2, o is 2, p is between 0 and 0.1, particularly 0 q is between 1.0 and 1.15, particularly between 1.0 and 1.10, especially particularly between 1.00 and 1.05, P 1 are independently hydrocarbyl having 1 to 8 carbon atoms, particularly methyl, ethyl, propyl or phenyl radicals, especially particularly methyl radicals, P 2 are independently a polyether radical of the formula (III) —P 3 O[CH2CH2O]m[CH2CH(CH3)O]nP 5 Formula (III) where m is from 3.4 to 11.0, particularly 3.6 to 9.9, more particularly 4.5 to 8.5, 202300100 Foreign Filing 13 n is from 2.5
  • the polyether-modified siloxanes of formula (II) have a biodegradability of greater than 60%, more particularly of greater than or equal to 63% and especially particularly of greater than or equal to 65%, the maximum value being 100%.
  • the polyether radical calculated without P 3 O and calculated without P 5 , has a molar mass M(PE) calculated by 44 g/mol*m+58 g/mol*n where the indices m and n relate to formula (III).
  • the values of M(PE) are: lower limits M(PE) greater than 520 g/mol, particularly greater than 530 g/mol, more particularly greater than 535 g/mol; upper limit M(PE) less than 660 g/mol, particularly less than 630 g/mol, more particularly less than 600 g/mol. Even more in particular, the value of M(PE) is greater than 520 g/mol and less than 660 g/mol, especially greater than 535 g/mol and less than 600 g/mol. In particular, the sum total of m+n is greater than 9 up to 19, more in particular, than 9.5 up to 15 and even more in particular greater than 10 up to 12.
  • the polyether-modified siloxane used in the composition according to any aspect of the present invention is a polyether-modified siloxane of the formula (II) with an index c between 1 and 1.05, where the indices of the polyether radical of formula (III) are m from 3.4 to 11.0 and n from 2.5 to 8.0.
  • the polyether-modified siloxane used in the composition according to any aspect of the present invention is a polyether-modified siloxane of the formula (II) with an index c between 1 and 1.05, where the ratio m/n is 1.9 to 2.8.
  • the polyether-modified siloxane used in the composition according to any aspect of the present invention is a polyether-modified siloxane of the formula (II) with an index c between 1 and 1.05, where the molar mass of the polyether residue M(PE) is greater than 520 g/mol and less than 660 g/mol.
  • the polyether-modified siloxane used in the composition according to any aspect of the present invention is a polyether-modified siloxane of the formula (II) with an index c between 1 and 1.05, where the P 5 radical is hydrogen or with an index c between 1 and 1.05, where the molar mass of the polyether residue M(PE) is greater than 520 g/mol and less than 660 g/mol and the P5 radical is hydrogen.
  • the polyether-modified siloxane used in the composition according to any aspect of the present invention is a polyether-modified siloxane of the formula (II) does not include any further polyether-modified siloxanes apart from those of formula (II).
  • the units designated by the index ‘g’ are those which have originated from propylene oxide
  • the units designated by the index ‘h’ are those which have originated from butylene oxide
  • the units designated by the index ‘i’ are those which have originated from styrene oxide.
  • the indices ‘a to d’ and ‘e to i’ may be natural whole numbers, or weight averages.
  • the indices are preferably weight averages.
  • a more thorough disclosure of the adjuvant (C) is provided at least in US8580225B2.
  • the composition according to any aspect of the present invention may comprise any one of the adjuvants (A), (B) or (C).
  • the composition may comprise a mixture of adjuvants such as (A) and (B), (A) and (C), (B) and (C) or (A), (B) and (C).
  • the composition according to any aspect of the present invention may comprise more than one adjuvant (A) or more than one adjuvant (B) or more than one adjuvant (C). Unless stated otherwise, all percentages (%) given are percentages by mass.
  • FIGURES Figure 1 is a schematic drawing of an example of the method according to any aspect of the present invention for producing RNA composition.
  • Figure 2 are pictures of the Scanning electron microscopy (SEM) results of the composition according to example 1 in particles without (A) and with (B) shellac as the matrix shell.
  • the outlet temperature was between 45°C and 60°C.
  • the two-fluid nozzle using air or inert gas did not build up pressure within the system.
  • the whole process was conducted under nitrogen atmosphere.
  • the atomization of the mixture was done in co-current flow with the hot drying gas from the top in the spray- dryer.
  • 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 done using a two-fluid-nozzle at an atomizing pressure of approximately 1,35 bar.
  • the flow rate of the drying air was 38 m 3 /h.
  • the spraying rate was approximately 5 mL/min.
  • the set parameters result in an outlet temperature of 53°C.
  • the dsRNA reference and the dsRNA+silica formulation from example 1 were tested to determine the stability of the RNA.
  • the particle from example 1 was ultrasonicated (US) for 3min at laboratory temperature, 40°C, 60°C and 80°C for 1h in a water bath.
  • the HPLC results are provided in Table 1 below. As can be seen below, almost all the double stranded RNA is recovered in the presence of silica. Table 1.
  • the suspension was then spray dried in a Büchi B-290 laboratory spray dryer at a gas inlet temperature of 80°C. Spraying was done using a two-fluid-nozzle at an atomizing pressure of approximately 1,35 bar. The flow rate of the drying air was 38 m3/h. The spraying 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 1mg dsRNA per g dry final product after processing.
  • Analytical method Used equipment ⁇ CFX96 Touch Real-Time PCR Detection System The analytical method was followed using the protocol of the New England Biolabs (240 County Road Ipswich) Luna® Universal Probe One-Step RT-qPCR Kit Protocol (E3006) with following changes: Before the respective protocols were applied, an additional denaturation step was carried out beforehand to break up the double strand of the RNA. The samples were melted for 5 minutes at 95°C (PCR block) and then quickly placed on ice.
  • Example 5 Comparison of composition and particles with zein coating in water and BP787 storage
  • the Cq Signal is an indicator correlating logarithmical with the detectable dsRNA concentration.
  • Table 4 Results from qPCR analytics The results show that over the time of 4 weeks of accelerated harsh liquid storage conditions a storage in BP787 is beneficial and that the zein coating is functional.

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  • Agricultural Chemicals And Associated Chemicals (AREA)

Abstract

L'invention concerne une composition comprenant au moins une molécule d'ARN et au moins un support de silice, le support de silice étant des particules de silice ayant un diamètre de 0,3 µm à 1 000 µm et la composition comprenant en outre une enveloppe de matrice constituée de zéine ou de gomme-laque.
PCT/EP2024/081167 2023-11-07 2024-11-05 Stockage et distribution d'arn Pending WO2025098983A1 (fr)

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EP23208115.8 2023-11-07
EP23208115 2023-11-07

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WO2025098983A1 true WO2025098983A1 (fr) 2025-05-15

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