RNA delivery composition, method for producing RNA delivery composition, method for rearing aquatic animal, and aquatic animal

Abstract

To provide an RNA delivery composition that enables uptake of RNA effective for prevention and / or treatment of viral infections in aquatic animals including crustaceans into the body through oral administration.SOLUTION: The present invention for solving the above problem is an RNA delivery composition comprising a basic polymer and an RNA having 20 or more bases, wherein the basic polymer is a polymer having a repeating unit containing a basic amino acid and having a pKa of 7.0 or higher. It is preferable that the basic amino acid is a lysine derivative.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a composition for RNA delivery, a method for producing a composition for RNA delivery, a method for raising aquatic animals, and aquatic animals. [Background technology]

[0002] Aquaculture is one of the fastest-growing food animal production sectors in the world. This growth is attributed in large part to the decline of wild fisheries resources and the increasing demand for seafood in the United States, Europe, Japan, and other regions. To increase their competitiveness, aquaculture producers are adopting higher stocking densities and higher feeding rates, which increase the risk of viral infections and subsequent secondary bacterial infections. Approximately 10% of farmed shrimp are known to be culled due to viral infections, resulting in significant economic losses. For example, white spot syndrome virus (WSSV) is one of the most devastating infectious pathogens in shrimp aquaculture. WSSV infections have become pandemics, resulting in billions of dollars of losses worldwide. This virus infects a wide range of crustaceans, including freshwater shrimp, lobsters, freshwater crabs, and several species of marine crabs.

[0003] To combat these infectious diseases, aquaculture farmers have taken measures such as draining the water from farms and drying them in the sun, thoroughly cleaning with chlorine, reducing the rearing density of shrimp and other fish, and conducting PCR tests on crustaceans living in the surrounding area to detect viral infections early. However, these measures do not currently prevent viral infections. Currently, there are no commercially available vaccines or treatments for these viruses in the aquaculture of shrimp and other crustaceans. In particular, it is generally recognized that vaccines in the traditional sense are ineffective against crustaceans, as they lack the specific immune systems found in vertebrates.

[0004] It is known that the intracellular machinery necessary to mount a gene-specific RNA interference (RNAi) response is present in the shrimp. This shrimp RNAi response mechanism can be used as a means to interfere with the replication of viruses and other pathogens. Injection of dsRNA or hairpin dsRNA targeting viral RNA into shrimp has been shown to interfere with viral replication and to have at least short-term effects against diseases caused by white spot syndrome virus (WSSV) (Non-Patent Document 1), taura syndrome virus (TSV) (Non-Patent Document 2), yellow head virus (YHV) (Non-Patent Document 3), and gill-associated virus (Non-Patent Document 4).

[0005] Although there is ample experimental evidence that dsRNA is effective against viral diseases in shrimp as described above, solutions for effective dsRNA delivery are not yet fully developed. U.S. Patent Publication No. 2014 / 0371295 (Patent Document 1) discloses methods for delivering dsRNA targeting pathogenic organisms to shrimp and other aquatic invertebrates via injection or ingestion using various forms of encapsulation. U.S. Patent Publication No. 2005 / 0080032 (Patent Document 2) discloses a biological delivery method using microorganisms genetically engineered to express dsRNA targeting pathogenic / parasitic microorganisms in marine invertebrates. Furthermore, U.S. Patent Publication No. 2019 / 0175518 (Patent Document 3) describes the reduction of pathological virus levels by feeding farmed crustaceans chitosan-RNA nanoparticles containing partially acetylated chitosan and RNA targeting the gene product of a pathological virus. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] U.S. Patent Publication No. 2014 / 0371295 [Patent Document 2] U.S. Patent Publication No. 2005 / 0080032 [Patent Document 3] U.S. Patent Publication No. 2019 / 0175518 [Non-patent literature]

[0007] [Non-Patent Document 1] Journal of Virology,79(21):13561-13571, 2005 [Non-patent document 2] Journal of Virology,78(19):10442-10448, 2004 [Non-patent document 3] Biochemical and Biophysical Research Communications,341(2):351-356,2006 [Non-patent document 4] Diseases of Aquatic Organisms,95(1):19-30,2011 Summary of the Invention [Problem to be solved by the invention]

[0008] Because injection is inefficient, oral administration of drugs and other substances to crustaceans is desirable, with the drug being incorporated into feed or other foods. However, oral administration poses various problems, such as elution of the active ingredient into the breeding water, degradation by gastric juice, and insufficient intestinal absorption, which have not yet been fully resolved by the above-mentioned prior art. Therefore, the present invention aims to provide an RNA delivery composition that can orally administer RNA effective for preventing and / or treating viral infections in aquatic animals such as crustaceans. More specifically, the present invention aims to provide an RNA delivery composition that effectively prevents elution of the active ingredient, RNA, into the breeding water, inhibits degradation by gastric juice, and promotes uptake in the small intestine, ultimately allowing sufficient RNA to be internalized. The present invention also aims to provide a method for producing the RNA delivery composition, a method for raising aquatic animals using the RNA delivery composition, and aquatic animals raised using this method. [Means for solving the problem]

[0009] Under these circumstances, the inventors conducted extensive research and discovered that the above problems could be solved by creating a composition for RNA delivery containing RNA as an active ingredient and a basic polymer, thereby completing the present invention.

[0010] The gist of the present invention is as follows. [1] A polymer comprising a basic polymer and an RNA having 20 or more bases; The composition for RNA delivery, wherein the basic polymer is a polymer having a repeating unit containing a basic amino acid and has a pKa of 7.0 or higher. [2] The composition for RNA delivery according to [1], wherein the basic amino acid is a lysine derivative. [3] The composition for RNA delivery according to [1] or [2], wherein the basic polymer contains a compound represented by the following formula (I) or a salt thereof: [ka] (In the above formula, n is an integer of 6 to 75; X is hydrogen, —C(═NH)—NH, or C(═O)—CHR—NH; and R is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted alkyl ether, substituted or unsubstituted alkylthioether, substituted or unsubstituted hydroxyalkyl, substituted or unsubstituted hydroxyaryl, substituted or unsubstituted sulfanylalkyl, substituted or unsubstituted sulfanylaryl, substituted or unsubstituted alkylthioalkyl, substituted or unsubstituted alkylthioaryl, substituted or unsubstituted aminoalkyl, substituted or unsubstituted aminoaryl, substituted or unsubstituted aminocarbonylalkyl, substituted or unsubstituted aminocarbonylaryl, substituted or unsubstituted guanidylalkyl, or substituted or unsubstituted guanidylaryl, provided that multiple Xs may be the same or different.) [4] The composition for RNA delivery according to [3], wherein R is hydrogen, methyl, isopropyl, isobutyl, sec-butyl, benzyl, hydroxymethyl, 1-hydroxyethyl, p-hydroxyphenylmethyl, aminocarbonylmethyl, aminocarbonylethyl, sulfanylmethyl, methanethioethyl, 1H-imidazol-4-ylmethyl, 4-aminobutyl, 3-guanidinopropyl, or 3-indolylmethyl. [5] The composition for RNA delivery according to [3], wherein R is isobutyl or benzyl. [6] The composition for RNA delivery according to any one of [1] to [5], wherein the Mn of the basic polymer is 1,000 to 50,000. [7] The composition for RNA delivery according to any one of [1] to [6], wherein the RNA is double-stranded RNA (dsRNA). [8] The RNA delivery composition according to any one of [1] to [7], wherein the ratio of the basic polymer (wt%) to the RNA having 20 or more bases (wt%) in the RNA delivery composition (RNA / basic polymer) is 0.5 to 50. [9] The composition for RNA delivery according to any one of [1] to [8], which is in the form of a pharmaceutical or feed.

[10] The composition for RNA delivery according to [9], which is in the form of a medicine or feed for aquatic animals.

[11] The composition for RNA delivery according to

[10] , wherein the aquatic animal is a crustacean.

[12] A process comprising a step of mixing an aqueous solution of a basic polymer and an aqueous solution of RNA having 20 or more bases; A method for producing a composition for RNA delivery, wherein the basic polymer is a polymer having a repeating unit containing a basic amino acid and has a pKa of 7.0 or higher.

[13] A method for raising an aquatic animal, comprising a step of feeding the aquatic animal the composition for RNA delivery according to

[10] or

[11] .

[14]

[13] Aquatic animals reared according to the method described in. [Effects of the Invention]

[0011] The RNA delivery composition of the present invention contains RNA effective against viral infections in aquatic animals such as crustaceans, and a basic polymer, thereby preventing RNA elution in breeding water, suppressing its degradation in gastric juice, and promoting uptake in the small intestine, allowing it to be sufficiently taken up into the body even by oral administration. As a result, the RNA delivery composition of the present invention can be suitably used for the prevention and / or treatment of viral infections. Furthermore, raising aquatic animals using the above-described RNA delivery composition of the present invention can effectively prevent and / or treat viral infections in aquatic animals, thereby greatly contributing to solving the problem of viral infections in aquaculture. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 shows the results of confirming the stability of double-stranded RNA (SEQ ID NO: 1) at 50°C. [Figure 2] FIG. 2 shows the degree of deacetylation of chitosan_H52. [Figure 3] FIG. 3 shows the results of investigating the effect of injecting compositions for RNA delivery (comparative examples and examples) on suppressing mRNA expression. [Figure 4]Figure 4 shows the results of examining the effect of differences in the modification of basic amino acids in a basic polymer on the mRNA expression inhibitory effect by oral administration of the composition for RNA delivery.

Mode for Carrying Out the Invention

[0013] Hereinafter, the present invention will be described in detail. In this specification, molecular biological techniques can be carried out by methods described in general experimental books known to those skilled in the art or methods analogous thereto, unless otherwise specified. Also, the terms used in this specification are construed in the meaning commonly used in the technical field, unless otherwise mentioned.

[0014] <Composition for RNA Delivery> The composition for RNA delivery of the present invention contains a basic polymer and an RNA having 20 or more bases, and the basic polymer is a polymer having a repeating unit containing a basic amino acid and has a pKa of 7.0 or more. In the composition for RNA delivery of the present invention, since the basic polymer is a polymer having a repeating unit containing a basic amino acid and has a pKa of 7.0 or more, the electrostatic interaction between the basic polymer and the RNA becomes stronger and the binding force is enhanced. Therefore, the above-mentioned RNA as an active ingredient is hardly eluted in water or the like. Further, since the basic polymer is a polymer containing a basic amino acid, it is hardly decomposed by enzymes in gastric juice, while in the small intestine, it becomes easily decomposed by digestive enzymes that decompose basic amino acid residues, and the above-mentioned RNA is efficiently taken up in the intestinal tract. Therefore, even by oral administration, RNA can be sufficiently taken into the body.

[0015] (Basic Polymer) The basic polymer in the present invention is a polymer having a repeating unit containing a basic amino acid and has a pKa of 7.0 or more.

[0016] Examples of the basic amino acid include lysine derivatives, histidine derivatives, and arginine derivatives. Among these, lysine derivatives are preferred from the viewpoint of excellent electrostatic interaction with RNA.

[0017] The polymer having a repeating unit containing a basic amino acid preferably contains a compound represented by the following formula (I) or a salt thereof. [ka]

[0018] In the above formula, n is an integer of 6 to 75, preferably 15 to 50, and more preferably 22 to 33.

[0019] In the above formula, X is hydrogen, -C(=NH)-NH2, or C(=O)-CHR-NH2; R is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted alkyl ether, substituted or unsubstituted alkylthioether, substituted or unsubstituted hydroxyalkyl, substituted or unsubstituted hydroxyaryl, substituted or unsubstituted sulfanylalkyl, substituted or unsubstituted sulfanylaryl, substituted or unsubstituted alkylthioalkyl, substituted or unsubstituted alkylthioaryl, substituted or unsubstituted aminoalkyl, substituted or unsubstituted aminoaryl, substituted or unsubstituted aminocarbonylalkyl, substituted or unsubstituted aminocarbonylaryl, substituted or unsubstituted guanidylalkyl, or substituted or unsubstituted guanidylaryl, provided that multiple Xs may be the same or different.

[0020] The above-mentioned R is preferably hydrogen, methyl, isopropyl, isobutyl, sec-butyl, benzyl, hydroxymethyl, 1-hydroxyethyl, p-hydroxyphenylmethyl, aminocarbonylmethyl, aminocarbonylethyl, sulfanylmethyl, methanethioethyl, 1H-imidazol-4-ylmethyl, 4-aminobutyl, 3-guanidinopropyl, or 3-indolylmethyl, and more preferably isobutyl or benzyl.

[0021] In the polymer, the amino groups in the repeating units may be partially or entirely modified with other organic groups as described above. Examples of the modification include guanidine modification (when X is -C(=NH)-NH2 in the above formula), phenylalanine modification (when X is -C(=O)-CHR-NH2 and R is benzyl in the above formula), leucine modification (when X is -C(=O)-CHR-NH2 and R is isobutyl in the above formula), and histidine modification (when X is -C(=O)-CHR-NH2 and R is 1H-imidazol-4-ylmethyl in the above formula). Among these, guanidine modification, phenylalanine modification, and leucine modification are preferred, and guanidine modification is more preferred, from the viewpoint of oral uptake efficiency in the body when used in the RNA delivery composition of the present invention. The proportion of repeating units having the modified amino group in the entire polymer may be 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more. The proportion of repeating units having the modified amino group in the entire polymer is 0% to 100%, preferably 10% to 100%, more preferably 30% to 100%, even more preferably 50% to 100%, even more preferably 70% to 100%, and particularly preferably 80% to 100%.

[0022] An example of the above modification method is the guanidine modification method shown in the following scheme.

[0023] [ka]

[0024] The number average molecular weight (Mn) of the basic polymer in the present invention is 1,000 to 50,000, preferably 2,000 to 20,000, and more preferably 3,000 to 10,000.

[0025] The number average molecular weight (Mn) is the average molecular weight given by Mn = ΣMiNi / ΣNi when there are Ni molecules with molecular weight Mi. Measurement methods include osmotic pressure, boiling point elevation, coagulation drop method, reverse phase chromatography, and GFC (Gel Filtration Chromatography). When measuring the number average molecular weight using the GFC method, the molecular weight distribution is measured using high performance liquid chromatography, and the number average molecular weight can be calculated from a calibration curve by analyzing the data using a GFC analysis system.

[0026] The pKa of the basic polymer of the present invention is 7.0 or higher, and from the viewpoint of oral uptake efficiency when used in the RNA delivery composition of the present invention, it is preferably 7.4 or higher, and more preferably 7.5 or higher.

[0027] (RNA) The RNA contained in the RNA delivery composition of the present invention has a base number of 20 or more. The RNA may have a base number of 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 150 or more, 200 or more, 250 or more, 300 or more, 350 or more, 400 or more, 450 or more, 500 or more, 550 or more, 600 or more, 650 or more, 700 or more, 750 or more, 800 or more, 850 or more, 900 or more, or 1000 or more. The number of bases in the RNA may be 2000 or less, 1500 or less, 1200 or less, 1000 or less, 800 or less, 700 or less, 650 or less, 600 or less, 550 or less, 500 or less, 450 or less, 400 or less, 350 or less, 300 or less, 250 or less, 200 or less, 150 or less, 120 or less, 100 or less, 80 or less, 60 or less, or 50 or less. The number of bases in the RNA contained in the RNA delivery composition of the present invention ranges from 20 to 2000, preferably from 20 to 1500, more preferably from 20 to 1000, even more preferably from 20 to 800, and particularly preferably from 20 to 700.

[0028] The RNA contained in the RNA delivery composition of the present invention is not particularly limited as long as it contains four bases, adenine, guanine, cytosine, and uracil, and has a total of 20 or more bases. It may be single-stranded RNA or double-stranded RNA. Among these, preferred examples of the RNA contained in the RNA delivery composition of the present invention include inhibitory RNA (RNAi) and exogenous mRNA.

[0029] Inhibitory RNA (RNAi) is RNA that can suppress the expression of a target gene. As used herein, "inhibitory RNA (RNAi)" refers to an RNA molecule that can induce RNAi (RNA interference) in vivo and suppress (silence) the expression of the target gene through degradation of the transcription product of the gene (Fire A. et al., Nature 391, 806-811 (1998), etc.). Specific examples of RNAi molecules include siRNA and shRNA. "siRNA" is a double-stranded RNA formed by hybridization of an antisense strand containing a sequence complementary to a portion of the mRNA sequence of a target gene with a sense strand containing a sequence complementary to the antisense strand (homologous to a portion of the sequence of the target gene). "shRNA" refers to a single-stranded RNA in which the sense and antisense strands of the siRNA are linked by a short spacer sequence having an appropriate sequence. In other words, within a single molecule of shRNA, the sense and antisense regions base pair with each other to form a stem structure, and at the same time, the spacer sequence forms a loop structure, resulting in the entire molecule forming a hairpin-type stem-loop structure.

[0030] Examples of the target gene include exogenous pathogenic viruses that can infect or have infected a subject, and disease-related endogenous genes that a subject is suffering from or may suffer from. Various viruses exist for each subject as the exogenous pathogenic virus. For example, when the subject is a crustacean such as shrimp, examples of the exogenous pathogenic virus include white spot syndrome virus (WSSV), taura syndrome virus (TSV), yellow head virus (YHV), and gill-associated virus. Examples of the disease-related endogenous gene include various oncogenes. Non-limiting examples are shown in the table below.

[0031] [Table 1]

[0032] [Table 2]

[0033] As used herein, "inhibition of target gene expression" refers to not only 100% inhibition of target gene expression, as determined based on the expression level of the gene's mRNA or protein, but also 75% or more, 50% or more, or 20% or more inhibition compared to when no RNAi molecule is introduced or when an unrelated control RNAi molecule is introduced. mRNA expression levels can be measured, for example, by Northern hybridization or real-time PCR, and protein expression levels can be appropriately measured by those skilled in the art, for example, by Western blotting, ELISA, or protein activity assays. Specific methods for measuring gene expression levels are also described in Green, M.R. and Sambrook, J. (2012) Molecular Cloning: A Laboratory Manual, Fourth Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

[0034] Those skilled in the art can appropriately design the sequence of an RNAi molecule based on the nucleotide sequence of a target gene. For example, the antisense strand may be designed to contain a sequence complementary to a part of the coding region or 5' or 3' untranslated region (UTR) of the target gene mRNA, and the sense strand may be designed to contain a sequence complementary to the antisense strand.

[0035] As used herein, "complementary" refers to a relationship in which two bases can undergo base pairing (e.g., Watson-Crick type), such as the relationship between adenine and thymine or uracil, and the relationship between cytosine and guanine. As used herein, "complementary" preferably refers to perfect complementarity, but does not necessarily mean perfect complementarity; as long as the RNAi molecule retains its ability to suppress target gene expression, it may contain one or more (e.g., 1 to 5 or 1 to 3) mismatches. A mismatch refers to a relationship other than the relationship between adenine and thymine or uracil, or the relationship between cytosine and guanine.

[0036] It is generally known that RNAi molecules such as siRNAs have high RNAi activity when they have a single-stranded portion (overhang) of several nucleotides (e.g., 2 to 5 nucleotides) at their ends. Therefore, the RNAi molecules used in the present invention preferably have an overhang of several deoxyribonucleotides or ribonucleotides at their ends. For example, the RNAi molecule may have a 3' overhang of two nucleotides. Specifically, the RNAi molecule may have a 3' overhang consisting of two ribonucleotides (e.g., AU or AG). The number of bases in the sense strand and antisense strand constituting the RNAi molecule is as described above, and they may be the same length or different lengths.

[0037] All nucleotides in an RNAi molecule are preferably ribonucleotides, but several (e.g., 1 to 5, 1 to 3, or 1 to 2) may be deoxyribonucleotides. In addition to natural nucleotides, the nucleotides in an RNAi molecule may also be modified nucleotides containing groups such as halogen (fluorine, chlorine, bromine, or iodine), methyl, carboxymethyl, or thio groups, for example, to improve the stability of the RNAi molecule.

[0038] The sense and antisense strands constituting the RNAi molecule can be appropriately produced using a commercially available nucleic acid synthesizer. The produced sense and antisense strands may be mixed, preferably in an equimolar ratio, and hybridized to each other to produce the RNAi molecule. Alternatively, the RNAi molecule may be produced using the contract manufacturing services of manufacturers (e.g., BioSpring, Takara Bio, Sigma-Aldrich, GENOLUTION (Korea), etc.).

[0039] When the RNA delivery composition of the present invention contains exogenous mRNA, the mRNA may be one that encodes a disease therapeutic protein, a defective protein, an immune protein (vaccine), etc. When the exogenous mRNA is delivered into the body by the RNA delivery composition of the present invention, the protein encoded by the mRNA can be expressed in the body, resulting in disease therapeutic effects, vaccine effects, etc.

[0040] Herein, the expression of a protein encoded by an mRNA in vivo can be determined using the expression level of the protein as an indicator, which can be appropriately measured by those skilled in the art, for example, by Western blotting, ELISA, or protein activity measurement.

[0041] The ratio of the content of the basic polymer (wt%) to the content of the RNA having 20 or more bases (wt%) in the RNA delivery composition of the present invention (RNA / basic polymer) is 0.5 to 50, preferably 1 to 40, and more preferably 1.5 to 30. By keeping the content ratio of RNA / basic polymer within the above range, the efficiency of RNA uptake into the body can be improved when the RNA delivery composition of the present invention is orally ingested.

[0042] The RNA delivery composition of the present invention may contain, in addition to the essential components of a basic polymer and an RNA having 20 or more bases, other components depending on the need and purpose.

[0043] The composition for RNA delivery of the present invention may be used in various forms depending on the purpose, for example, in the form of pharmaceuticals such as therapeutic drugs and vaccines, foods, feeds, etc. Among these, it is preferably used in the form of pharmaceuticals and feeds.

[0044] Since the RNA delivery composition of the present invention can suppress the elution of RNA into water, it is preferably used as a medicine or feed for aquatic animals that require water for rearing. Examples of the aquatic animals include, but are not limited to, animals that live in freshwater, i.e., animals reared in freshwater, and animals that live in seawater, i.e., animals reared in seawater. "Water" in this specification includes freshwater, seawater, brackish water, etc.

[0045] The above-mentioned freshwater animals are not particularly limited, whether they are for ornamental or edible purposes, and examples include shrimp such as giant river prawn, Yamato swamp shrimp, southern swamp shrimp, red fire shrimp, red lure shrimp, and white glove shrimp; shellfish such as pond snails, stone snails, and kanoko shellfish; crabs; crayfish; edible fish such as carp, char, trout, yamame trout, and loach; ornamental fish such as tropical fish, goldfish, medaka, and arowana; reptiles such as turtles; and amphibians such as frogs and salamanders.

[0046] The above-mentioned seawater-dwelling animals are not particularly limited, but examples include ornamental or edible shrimp such as kuruma prawn, vannamei shrimp, Penaeus monodon, red-spotted sea shrimp, white-spotted sea shrimp, Suzaku shrimp, frisoid shrimp, otohime shrimp, coral shrimp, spiny lobster, and lobster, as well as fish, shellfish, crabs, starfish, sea urchins, sea cucumbers, sea squirts, and hermit crabs.

[0047] Crustaceans are particularly preferred as aquatic animals for which the RNA delivery composition of the present invention can be used as a pharmaceutical or feed, and shrimp are particularly preferred.

[0048] <Pharmaceutical Composition> The present invention also provides a pharmaceutical composition for treating and / or preventing a disease, comprising a basic polymer and RNA having 20 or more bases, wherein the basic polymer is a polymer having a repeating unit containing a basic amino acid and has a pKa of 7.0 or higher. The pharmaceutical composition described here can also be considered an embodiment of the above-mentioned RNA delivery composition. Therefore, the description of the above-mentioned RNA delivery composition can be applied to the basic polymer and RNA contained in the pharmaceutical composition of the present invention. In this specification, "treatment" means curing, alleviating, or ameliorating a disease or symptom, and "prevention" means preventing, suppressing, or delaying the onset of a disease or symptom.

[0049] The target disease is not particularly limited as long as the pharmaceutical composition of the present invention is effective, and examples include pathogenic viral infections, gene-related diseases, etc. The pharmaceutical composition of the present invention can be used for the prevention and / or treatment of these target diseases.

[0050] The pharmaceutical composition may contain any formulation auxiliary commonly used in the field of formulation. Examples of the formulation auxiliary include various pharmaceutically acceptable drug carriers or additives, such as carriers (solid or liquid carriers), excipients, stabilizers, emulsifiers, surfactants, binders, disintegrants, lubricants, odorants, solubilizers, suspending agents, coating agents, colorants, flavoring agents, preservatives, and buffers. Specific examples of the formulation auxiliary include water, saline, other aqueous solvents, pharmaceutically acceptable organic solvents, mannitol, microcrystalline cellulose, starch, glucose, calcium, polyvinyl alcohol, collagen, polyvinylpyrrolidone, carboxyvinyl polymers, sodium alginate, water-soluble dextran, water-soluble dextrin, sodium carboxymethyl starch, gum arabic, pectin, xanthan gum, casein, gelatin, agar, propylene glycol, glycerin, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, sorbitol, and lactose. The formulation adjuvants may be selected appropriately or in combination depending on the dosage form of the formulation.

[0051] The pharmaceutical composition according to the present invention can be administered orally or parenterally (for example, by injection), but oral administration is particularly preferred from the viewpoint of being suitable for use in aquatic animals.

[0052] Dosage forms suitable for oral administration include, for example, solid preparations (including tablets, pills, sublingual preparations, capsules, troches, drops, and forms mixed into feed, etc.), granules, powders, powders, liquid preparations, etc. Solid preparations may be in the form of dosage forms coated with coatings known in the art, such as sugar-coated tablets, gelatin-coated tablets, enteric-coated tablets, film-coated tablets, double tablets, or multi-layer tablets. Such coatings may be intended, for example, to release the active ingredient at a desired location in the body or to enhance the absorption of the active ingredient.

[0053] For parenteral administration, a dosage form suitable for each administration method can be used as appropriate. Examples of dosage forms suitable for parenteral administration include injections, inhalants, suspensions, emulsions, creams, pastes, gels, ointments, drops, topical preparations, eye drops, nasal drops, and plasters.

[0054] The pharmaceutical compositions of the present invention can be administered to a living body in a pharmaceutically effective amount for the treatment and / or prevention of a target disease. As used herein, the term "pharmaceutically effective amount" refers to a dose of the RNA molecule contained in the pharmaceutical composition of the present invention that is necessary to treat or prevent the target disease and that has little or no harmful side effects on the living body to which it is administered. The specific dosage is determined for each individual subject based on the type or severity of the disease, general health, age, sex, body weight, tolerance to treatment, and other factors. For example, when the pharmaceutical compositions of the present invention are administered orally, they may be administered in an amount that provides, for example, 0.001 to 1,000 mg / kg body weight / day, 0.01 to 100 mg / kg body weight / day, or 0.1 to 10 mg / kg body weight / day of the RNA molecule. The pharmaceutical composition of the present invention can be administered in a single dose, or can be administered to a subject in several or several dozen doses at regular time intervals, for example, at intervals of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 6 months, or 1 year.

[0055] The pharmaceutical composition of the present invention may be administered in combination with other drugs. When used in combination with other drugs, they may be used as a combined preparation for simultaneous administration or as separate preparations combined for independent administration. Combination includes simultaneous administration and sequential administration.

[0056] The subject to which the pharmaceutical composition of the present invention is administered may be an animal, such as an aquatic animal (for detailed examples, see the above section "Composition for RNA delivery").

[0057] The present invention also provides a method for treating and / or preventing a disease, which comprises administering the pharmaceutical composition of the present invention to a subject in need thereof. It also provides a method for raising an aquatic animal, which comprises a step of having the subject ingest the pharmaceutical composition (RNA delivery composition) of the present invention, and an aquatic animal raised by this method.

[0058] The present invention also provides a method for delivering RNA using a composition for RNA delivery, which comprises a basic polymer and RNA having 20 or more bases, wherein the basic polymer is a polymer having a repeating unit containing a basic amino acid and having a pKa of 7.0 or higher. Furthermore, the present invention also provides a method for inhibiting RNA elution using a polymer having a repeating unit containing a basic amino acid and having a pKa of 7.0 or higher, and an invention for an RNA elution inhibitor comprising a polymer having a repeating unit containing a basic amino acid and having a pKa of 7.0 or higher.

[0059] <Manufacturing method> The present invention also provides a method for producing the above-mentioned RNA delivery composition of the present invention. The production method of the present invention comprises a step of mixing an aqueous solution of a basic polymer and RNA having 20 or more bases, wherein the basic polymer is a polymer having a repeating unit containing a basic amino acid and has a pKa of 7.0 or higher. Note that, since the present invention is an invention of a method for producing the above-mentioned RNA delivery composition of the present invention, the descriptions of the RNA delivery composition can be applied as is to each component contained in the RNA delivery composition (basic polymer, RNA having 20 or more bases, etc.).

[0060] In the step of mixing the basic polymer and the aqueous solution of RNA having 20 or more bases, the components are mixed by a conventional method to form a complex of the basic polymer and RNA having 20 or more bases. In this step, the basic polymer and the aqueous solution of RNA having 20 or more bases may be directly mixed, and then other components may be mixed as needed, or the basic polymer and other components may be mixed, and then the aqueous solution of RNA having 20 or more bases may be added and mixed.

[0061] The production method of the present invention will be described below with reference to specific examples, but the production method of the present invention is not limited to the following methods.

[0062] In the production method of the present invention, a commercially available 25% polylysine solution (manufactured by JNC) or the like may be used as the basic polymer, or a solution modified with guanidine according to the above-mentioned scheme may be used. The basic polymer is appropriately diluted with a buffer solution, and the dsRNA solution is further diluted with a buffer solution. The dsRNA solution is added to the basic polymer solution in several batches and mixed. After allowing to stand at 4°C to 37°C, preferably at room temperature, for 12 to 24 hours, the disappearance of free dsRNA is confirmed by electrophoresis. This process yields a complex of the basic polymer and dsRNA (one embodiment of the RNA delivery composition of the present invention). The concentration of the basic polymer after dilution when preparing the complex is preferably 0.01% to 1.0%, more preferably 0.1% to 0.5%, and even more preferably about 0.2%. The concentration of the dsRNA solution after dilution is preferably 10 μg / mL to 1 mg / mL, more preferably 50 μg / mL to 300 μg / mL, and even more preferably about 100 μg / mL. By setting each concentration within the above range, the basic polymer does not aggregate, and a complex that exhibits the effects of the present invention can be efficiently prepared.

[0063] The complex obtained as described above may be dried by any drying method, such as lyophilization or vacuum drying. Lyophilization is preferred. The dried complex may be further processed, such as pulverization or granulation. Sterile water is added to the obtained powder, and the mixture is then added to other compositions, such as feed, depending on the intended purpose to produce the desired composition. For example, the powder may be added to sterilized water and mixed, and then the mixture may be added to pulverized aquatic animal feed (Higashimaru Co., Ltd., etc.) and further mixed to form a paste. The paste may be pelletized and then dried at 40°C to 80°C, preferably 50°C to 60°C, for 1 to 5 hours, preferably 3 hours, to produce a compound feed (one embodiment of the RNA delivery composition of the present invention). [Example]

[0064] 1. Preparation of double-stranded RNA (dsRNA) Total RNA was extracted from the gills of white shrimp (Litopenaeus vannamei) using the RNeasy Mini Kit (Qiagen). The resulting total RNA was reverse transcribed into cDNA using SuperScript III First-Strand Synthesis SuperMix (Thermo Fisher Scientific). The original template DNA was prepared using a primer pair complementary to the resulting cDNA [forward primer Rab7-Fw 5'-TGGGTAACAAGATTGATCTGGAG-3' (SEQ ID NO: 2) and reverse primer Rab7-Rv 5'-CATCCTGTTTAGCCTTGTTGTCA-3' (SEQ ID NO: 3)] and KOD FX (Toyobo). To 1 μL of the original template DNA solution diluted 100-fold, 4.8 μL of sterile water, 10 μL of 2x buffer for KOD Fx, 2 μL each of a primer pair [forward primer Rab7-Fw-T 5′-GGATCCTAATACGACTCACTATAGGTGGGTAACAAGATTGATCTGGAG-3′ (SEQ ID NO: 4) and reverse primer Rab7-Rv 5′-CATCCTGTTTAGCCTTGTTGTCA-3′ (SEQ ID NO: 3)], and 0.2 μL of KOD Fx were added, and the mixture was heat-denatured at 96°C for 2 minutes. After 20 cycles of 98°C for 10 seconds, 60°C for 30 seconds, and 68°C for 30 seconds, this cycle was repeated to prepare a template sense strand solution. A primer pair [forward primer Rab7-Fw 5'-TGGGTAACAAGATTGATCTGGAG-3' (SEQ ID NO: 2) and reverse primer Rab7-Rv-T 5'-GGATCCTAATACGACTCACTATAGGCATCCTGTTTAGCCTTGTTGTCA-3' (SEQ ID NO: 5)] was used to prepare an anti-sense template solution in the same manner as the sense template solution. Each template solution was purified using Proreinase K and a NucleoSpin Gel & PCR clean up column (Takara Bio).The concentration of the resulting solution was measured using a NanoDrop™ and adjusted to 1 μg / 10 μL with nuclease-free water. Then, 10 μL of Express T7 2x Buffer (Promega) and 2 μL of Enzyme Mix T7 Express (Promega) were added and incubated at 37°C for 30 minutes to obtain a sense strand RNA solution and an anti-sense strand RNA solution. 19 μL of the sense strand RNA solution and 19 μL of the anti-sense strand RNA solution were mixed and incubated at 70°C for 10 minutes and at 25°C for 20 minutes. Then, 2 μL of RQ1 RNase-Free DNase solution and 2 μL of 200-fold diluted RNase A solution were added and the mixture was left to stand at 37°C for 30 minutes. After incubation, 4 μL of 3M sodium acetate solution and 100 μL of ethanol were added, mixed by inversion, and then left to stand at 4°C for 15 minutes. The mixture was then centrifuged at 15,000 g for 15 minutes. After removing the supernatant using a pipette, 500 μL of 70% ethanol was added, and the mixture was left to stand at 25°C for 15 minutes and centrifuged at 15,000 g for 15 minutes. After removing the supernatant using a pipette, the mixture was air-dried at 25°C for 15 minutes, and 100 μL of sterile water was added to obtain a double-stranded RNA solution. The size of the obtained double-stranded RNA solution (approximately 237 bp, SEQ ID NO: 1) was confirmed by electrophoresis using a 1.5% agarose gel. In the sequence listing, "u (uracil)" in the RNA is represented as "t (thymine)," but the sequence of SEQ ID NO: 1 as RNA is as follows: (In the sequence listing, "u (uracil)" is represented as "t (thymine).")

[0065] [Table 3]

[0066] 2. Confirmation of the stability of the double-stranded RNA solution The prepared dsRNA (SEQ ID NO: 1) was diluted with sterile water to a concentration of 400 μg / mL. The prepared dsRNA solution was aliquoted into microtubes and heated in a dry bath incubator (50°C) for 0, 2, and 4 hours. It was also stored at 4°C for 0, 18, 46, and 84 days. The dsRNA contained in the dsRNA solution heated at 50°C for the specified time and stored at 4°C for the specified period was analyzed by HPLC. The results of the heating test are shown in Figure 1. As shown in Figure 1, the peak shape and detection area of ​​the dsRNA did not change with the heating treatment time, confirming the stability of the dsRNA at 50°C. The results of the test for storage at 4°C are also shown in Table 4 below. As shown in Table 4 below, there was no change in peak area even after storage at 4°C for 84 days, confirming stability. The HPLC conditions were as follows:

[0067] HPLC conditions: Column: DNAPac RS LC (2.1 mm x 25 cm, 4 μm, Thermo Fisher Scientific) Mobile phase A: 25 mmol / L phosphoric acid - 77 mmol / L sodium hydroxide aqueous solution Mobile phase B: 25 mmol / L phosphoric acid - 77 mmol / L sodium hydroxide - 0.5 M sodium perchlorate aqueous solution Gradient: 1-45%B (0-10 min), 45%B (10-11 min) Flow rate: 0.3mL / min Column temperature: 25℃ Detector: UV (260 nm) Injection volume: 1μL (0.4mg / mL) Retention time of double-stranded RNA: 8-9 minutes (sense and antisense strands)

[0068] [Table 4]

[0069] 3. Confirmation of the mRNA expression suppression effect by injection of double-stranded RNA The prepared double-stranded RNA (SEQ ID NO: 1) was diluted with 2% sodium chloride solution to concentrations of 500 μg / mL, 50 μg / mL, 5 μg / mL, and 0.5 μg / mL to prepare each solution. Each solution was administered to five vannamei shrimp at a weight ratio of 1000 ppm, 100 ppm, 10 ppm, or 1 ppm. The group administered with 2% sodium chloride was used as a control. Forty-eight hours after administration, the gills were removed and immersed in 1 mL of RNAlater (Thermo Fisher Scientific) overnight. The gills were removed from the RNAlater, and total RNA was extracted from them using an RNeasy Mini Kit (Qiagen). The concentration of the resulting total RNA solution was measured using a NanoDrop (Thermo Fisher Scientific). The effect of inhibiting mRNA expression was confirmed according to the procedures in Comparative Examples 1-3 described below. The results are shown in Table 5 below. Vannamei shrimp recognize the injected dsRNA as a foreign substance and produce an enzyme to degrade it. The dsRNA used in this test has the same base sequence as the Rab7 protein mRNA, so the degradative enzyme recognizes the Rab7 protein mRNA as dsRNA and degrades it, resulting in a decrease in the amount of Rab7 protein mRNA.

[0070] [Table 5]

[0071] As shown in Table 5, when the dose of dsRNA was 10 ppm or higher, the amount of Rab7 protein mRNA was significantly reduced compared to the control, and the effect was dose-dependent. As described above, the effect of injecting double-stranded RNA (dsRNA) to suppress mRNA expression was confirmed.

[0072] 4. Confirmation of the mRNA expression suppression effect by oral administration of double-stranded RNA 4-1. Target experiment (Subject Experiment 1-1) Raising Vannamei shrimp using compound feed and extraction of total RNA Five vannamei shrimp weighing 0.5-2.0 g were acclimated to 0.5% artificial seawater prepared from 105 g of Lacy Marine II (Iwaki) and 20 L of purified water. After feeding on a vannamei shrimp formula feed (Higashimaru), the gills were removed and immersed overnight in 1 mL of RNAlater (Thermo Fisher Scientific). The gills were removed from the RNAlater, and total RNA was extracted from them using an RNeasy Mini Kit (Qiagen). The concentration of the resulting total RNA solution was measured using a NanoDrop (Thermo Fisher Scientific).

[0073] (Subject Experiment 1-2) Analysis of mRNA expression levels The resulting total RNA was diluted with RNase-free water (Takara Bio) to a concentration of 125 ng / μL and reverse-transcribed to cDNA using ReverTra Ace qPCR RT Master Mix (Toyobo). The resulting cDNA was mixed with a complementary primer pair [forward primer Rab7-Fw 5'-GGGATACAGCTGGTCAAGAAA-3' (SEQ ID NO: 6) and reverse primer Rab7-Rv 5'-CGAGAGACTTGAAGGTATTGGG-3' (SEQ ID NO: 7)] and KOD SYBR qPCR Mix (Toyobo). The Ct_Rab7 value for Rab7 mRNA was measured using a StepOnePlus real-time PCR system (Thermo Fisher Scientific). A complementary primer pair [forward primer EF1a-Fw 5'-GTGGAGACCTTCCAACAGTATG-3' (SEQ ID NO: 8) and reverse primer EF1a-Rv 5'-CCTTCTTGTTGACCTCCTTGAT-3' (SEQ ID NO: 9)] and KOD SYBR qPCR Mix (Toyobo Co., Ltd.) were added to the cDNA, and the Ct_EF1a value for EF1a mRNA was measured using a StepOnePlus real-time PCR system (Thermo Fisher Scientific Co., Ltd.). The average of the Ct_EF1a value minus the Ct_Rab7 value was used as the ΔCt_Control value.

[0074] 4-2. Comparative Example Test Comparative Example 1: (Comparative Example 1-1) Preparation of compound feed 7.5 mL of a double-stranded RNA (SEQ ID NO: 1) solution adjusted to 40 μg / mL with 0.2 M sodium acetate solution adjusted to pH 4.6 was dispensed into a centrifuge tube, frozen, and then lyophilized to remove the solvent. 1 mL of sterile water was added to the resulting powder, which was then mixed. 1.5 g of crushed vannamei shrimp feed (Higashimaru Co., Ltd.) was then added and further mixed to create a paste. The paste was formed into pellets and dried at 50°C for 3 hours to obtain a compound feed.

[0075] (Comparative Example 1-2) Rearing of Vannamei shrimp using compound feed and extraction of total RNA Five whiteleg shrimp weighing 0.5 to 2.0 g were acclimatized to 0.5% artificial seawater prepared from 105 g of Lacy Marine II (Iwaki) and 20 L of purified water. The shrimp were fed the compound feed prepared in Comparative Example 1-1 at 5% by mass of the shrimp weight per day for five days. After that, the shrimp were raised without food for one day, and the gills were removed and immersed in 1 mL of RNAlater (Thermo Fisher Scientific) overnight. The gills were removed from the RNAlater, and total RNA was extracted from them using an RNeasy Mini Kit (Qiagen). The concentration of the resulting total RNA solution was measured using a NanoDrop (Thermo Fisher Scientific).

[0076] (Comparative Example 1-3) Measurement of mRNA expression level and evaluation of efficacy The resulting total RNA was diluted with RNase-free water (Takara Bio) to a concentration of 125 ng / μL and reverse-transcribed to cDNA using ReverTra Ace qPCR RT Master Mix (Toyobo). A complementary primer pair [forward primer Rab7-Fw 5'-GGGATACAGCTGGTCAAGAAA-3' (SEQ ID NO: 6) and reverse primer Rab7-Rv 5'-CGAGAGACTTGAAGGTATTGGG-3' (SEQ ID NO: 7)] and KOD SYBR qPCR Mix (Toyobo) were added to the resulting cDNA, and the Ct_Rab7 value for Rab7 mRNA was measured using a StepOnePlus real-time PCR system (Thermo Fisher Scientific). Furthermore, a complementary primer pair [forward primer EF1a-Fw 5'-GTGGAGACCTTCCAACAGTATG-3' (SEQ ID NO: 8) and reverse primer EF1a-Rv 5'-CCTTCTTGTTGACCTCCTTGAT-3' (SEQ ID NO: 9)] and KOD SYBR qPCR Mix (Toyobo Co., Ltd.) were added to the cDNA, and the Ct_EF1a value for EF1a mRNA was measured using a StepOnePlus real-time PCR system (Thermo Fisher Scientific). The ΔCt value was calculated by subtracting the Ct_EF1a value from the Ct_Rab7 value. The mRNA expression level was calculated using the following formula, and the mean, standard deviation, and significance with the control experiment were evaluated using a Welch t-test with levels of 0.5% and 5%. The ΔCt_Control value is the value from the control experiment described above.

[0077]

number

[0078] Comparative Example 2: (Comparative Example 2-1) Preparation of compound feed 0.75 mL of a 400 μg / mL double-stranded RNA (SEQ ID NO: 1) solution was sprinkled onto 1.5 g of formulated feed for vannamei shrimp (Higashimaru Co., Ltd.) to allow it to soak in. The soaked feed was dried under reduced pressure at 50°C for 3 hours.

[0079] (Comparative Example 2-2) Rearing of Vannamei shrimp using compound feed and extraction of total RNA Five whiteleg shrimp weighing 0.5-2.0 g were acclimated to 0.5% artificial seawater prepared from 105 g of Lacy Marine II (Iwaki) and 20 L of purified water. The shrimp were fed the formulated feed prepared in Comparative Example 2-1 at 12.5% ​​by weight of the shrimp for one day. After that, the shrimp were reared without food for one day, fed with 6% of the shrimp's weight per day of formulated whiteleg shrimp feed (Higashimaru) for four days, and then reared without food for another day. The gills were then removed and immersed in 1 mL of RNAlater (Thermo Fisher Scientific) overnight. The gills were removed from the RNAlater, and total RNA was extracted from them using an Rneasy Mini Kit (Qiagen). The concentration of the resulting total RNA solution was measured using a NanoDrop (Thermo Fisher Scientific).

[0080] (Comparative Example 2-3) Measurement of mRNA expression level and evaluation of efficacy The same procedure as in Comparative Example 1-3 was followed.

[0081] Comparative Example 3: (Comparative Example 3-1) Preparation of Chitosan_H52 A 500 mL round-bottom flask equipped with a stirring blade was charged with 309.9 g of purified water, 4.0 g of chitosan_H76 (Sigma-Aldrich), and 4.0 g of acetic acid (Fujifilm Wako Pure Chemical Industries, Ltd.), and stirred until a homogeneous solution was obtained. 0.7 g of acetic anhydride (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to the resulting solution and stirred at room temperature for 15 minutes, followed by the dropwise addition of 44.0 g of 10% potassium hydroxide (Fujifilm Wako Pure Chemical Industries, Ltd.). 0.3 g of acetic anhydride (Fujifilm Wako Pure Chemical Industries, Ltd.) was added and stirred at room temperature for 10 minutes, followed by the addition of 0.7 g of 10% potassium hydroxide (Fujifilm Wako Pure Chemical Industries, Ltd.) and stirring for 90 minutes. The reaction solution was transferred to a cellulose dialysis tube (Kenis) and dialyzed eight times against purified water. The dialyzed solution was lyophilized to obtain the target product (4.0 g, 86% yield). The pKa value was 6.47. 1H-NMR(400MHz,D2O+DCl,81℃)δ:5.04(brs,1H of CHNH2),4.76(brs,1H of CHNHCOCH3),4.06-3.75(m,6H of Acetylglucosamine unit & 5H of Glucosamine unit),3.36(brs,1H of CHCHNH2),2.20(s,3H of NHCOCH3), FT-IR(ATR method):3367,2880,1655,1561,1376,1314,1153,1074,898,617cm -1

[0082] (Comparative Example 3-2) Measurement of Deacetylation Degree 30 mg of chitosan_H52 obtained in Comparative Example 3-1 was weighed out and dissolved in 1.96 mL of heavy water and 0.04 mL of 20 wt% deuterium chloride. 1H-NMR was measured at 81°C (Figure 2), and the degree of deacetylation was calculated from the ratio of the CH3 peak area of ​​the acetylglucosamine unit to the peak areas of the acetylglucosamine unit and the glucosamine unit as follows: Degree of deacetylation = 1 - {(peak area at δ = 2.20 / 3) / (peak area at δ = 4.06 to 3.36 / 6)} =1-{(1.1859 / 3) / ((4.4855+0.4593) / 6)} =1-(0.3953 / 0.8241) =0.52

[0083] (Comparative Example 3-3) Preparation of the complex The chitosan_H52 prepared in Comparative Example 3-1 was dissolved in 0.2 M sodium acetate buffer adjusted to pH 4.6 to prepare a 0.2 wt% H52 solution. The dsRNA solution (1 μg / μL) was diluted with 0.2 M sodium acetate buffer adjusted to pH 4.6 to prepare a 100 μg / mL dsRNA solution. 14 mL of the 100 μg / mL dsRNA solution was added to 21 mL of the 0.2 wt% chitosan_H52 solution in four separate additions and mixed. After allowing to stand overnight at room temperature, the disappearance of the dsRNA band was confirmed by electrophoresis.

[0084] (Comparative Example 3-4) Measurement of the amount of double-stranded RNA eluted from the complex 1 mL of the complex solution was placed in a disposable homogenizer (Nippi) and freeze-dried. 300 μL of artificial seawater of the specified concentration (0 wt%, 0.5 wt%, 3.0 wt%) was added to the dried complex, heated at 30°C for 3 hours, and then homogenized for 1 minute. The filtrate was collected using a centrifugal filter (0.45 μm, PVDF, Merck) and a small benchtop centrifuge (As One). The detection area of ​​dsRNA in the filtrate was analyzed by HPLC.

[0085] (Comparative Example 3-5) Preparation of compound feed Chitosan_H52 was dissolved in 0.2 M sodium acetate adjusted to pH 4.6 to prepare a 0.2 mass percent solution. 3 mL of 100 μg / mL double-stranded RNA (SEQ ID NO: 1) was added in four portions to 4.5 mL of the prepared solution, mixed, and then allowed to stand at room temperature for 1 hour. The resulting suspension was frozen and then lyophilized to remove the solvent. 1 mL of sterilized water was added to the resulting powder and mixed, followed by the addition of 1.5 g of crushed vannamei shrimp formula feed (Higashimaru Co., Ltd.) and further mixing to prepare a paste. The paste was molded into pellets and then air-dried at 60°C for 3 hours to obtain a formula feed.

[0086] (Comparative Example 3-6) Rearing of Vannamei shrimp using compound feed and extraction of total RNA The compound feed obtained in Comparative Example 3-5 was used, and the same procedure as in Comparative Example 1-2 was followed.

[0087] (Comparative Examples 3-7) Measurement of mRNA Expression Levels and Evaluation of Efficacy The total RNA solution obtained in Comparative Example 3-6 was used, and the same procedure as in Comparative Example 1-3 was followed.

[0088] Comparative Example 4: (Comparative Example 4-1) Preparation of compound feed 0.75 mL of a 12 mg / mL chitosan_H52 solution was sprinkled onto 1.5 g of formulated feed for vannamei shrimp (Higashimaru Co., Ltd.) to allow the feed to be impregnated. The impregnated feed was dried under reduced pressure at 50°C for 3 hours. 0.75 mL of a 400 μg / mL double-stranded RNA (SEQ ID NO: 1) solution was sprinkled onto the resulting feed to allow the feed to be impregnated. The impregnated feed was dried under reduced pressure at 50°C for 3 hours.

[0089] (Comparative Example 4-2) Rearing of Vannamei shrimp using compound feed and extraction of total RNA The compound feed obtained in Comparative Example 4-1 was used, and the same procedure as in Comparative Example 2-2 was followed.

[0090] (Comparative Example 4-3) Measurement of mRNA expression level and evaluation of efficacy The total RNA solution obtained in Comparative Example 4-2 was used, and the same procedure as in Comparative Example 1-3 was followed.

[0091] Comparative Example 5: (Comparative Example 5-1) Preparation of the complex Chitosan_H76 (Sigma-Aldrich) was used, and the same procedure as in Comparative Example 3-3 was carried out. The pKa value of chitosan_H76 was 6.46, the number average molecular weight (Mn) was 59,000, and the weight average molecular weight (Mw) was 470,000.

[0092] (Comparative Example 5-2) Measurement of the amount of double-stranded RNA eluted from the complex The composite obtained in Comparative Example 5-1 was used in the same procedure as in Comparative Example 3-4.

[0093] (Comparative Example 5-3) Preparation of compound feed Chitosan_H76 (Sigma-Aldrich) was dissolved in 0.2 M sodium acetate adjusted to pH 4.6 to prepare a 0.2% by mass solution. 3 mL of 100 μg / mL double-stranded RNA (SEQ ID NO: 1) was added in four portions to 4.5 mL of the prepared solution, mixed, and then allowed to stand at room temperature for 1 hour. 11 mg of phosphoric acid (Fujifilm Wako Pure Chemical Industries, Ltd.) was then added and allowed to stand for 12 hours. The resulting suspension was frozen, lyophilized, and the solvent was distilled off. 1.0 mL of sterilized water was added to the resulting powder, followed by mixing. 1.5 g of crushed vannamei shrimp formula feed (Higashimaru Co., Ltd.) was added and further mixed to prepare a paste. The paste was molded into pellets and air-dried at 60°C for 3 hours to obtain a formula feed.

[0094] (Comparative Example 5-4) Rearing of Vannamei shrimp using compound feed and extraction of total RNA The compound feed prepared in Comparative Example 5-3 was used, and the same procedure as in Comparative Example 1-2 was followed.

[0095] (Comparative Example 5-5) Measurement of mRNA expression level and evaluation of efficacy The total RNA obtained in Comparative Example 5-4 was used in the same procedure as in Comparative Example 1-3.

[0096] Comparative Example 6: (Comparative Example 6-1) Preparation of compound feed Chitosan_H76 (Sigma-Aldrich) was dissolved in 0.2 M sodium acetate adjusted to pH 4.6 to prepare a 0.2 mass percent solution. 1.5 mL of 100 μg / mL double-stranded RNA (SEQ ID NO: 1) was added in four portions to 2.3 mL of the prepared solution, mixed, and then allowed to stand at room temperature for 1 hour. The resulting suspension was frozen and then lyophilized to remove the solvent. 1.0 mL of sterilized water was added to the resulting powder and mixed, followed by 1.5 g of crushed vannamei shrimp formula feed (Higashimaru Co., Ltd.) and further mixing to prepare a paste. The paste was molded into pellets and then air-dried at 60°C for 3 hours to obtain a formula feed.

[0097] (Comparative Example 6-2) Rearing of Vannamei shrimp using compound feed and extraction of total RNA The compound feed prepared in Comparative Example 6-1 was used, and the same procedure as in Comparative Example 1-2 was followed.

[0098] (Comparative Example 6-3) Measurement of mRNA expression level and evaluation of efficacy The total RNA obtained in Comparative Example 6-2 was used, and the same procedure as in Comparative Example 1-3 was followed.

[0099] 4-3. Testing of Examples Example 1: (Example 1-1) Preparation of the complex A 25% polylysine solution (manufactured by JNC Corporation) was diluted with pure water to 0.2%, and then prepared in the same manner as in Comparative Example 3-3. The polylysine used here had a pKa value of 7.51, a number average molecular weight (Mn) of 5,400, and a weight average molecular weight (Mw) of 6,900.

[0100] (Example 1-2) Measurement of the amount of double-stranded RNA eluted from the complex The composite obtained in Example 1-1 was measured in the same manner as in Comparative Example 3-4.

[0101] (Example 1-3) Preparation of compound feed The preparation was carried out in the same manner as in Comparative Example 3-5, except that chitosan_H52 was replaced with a 25% polylysine solution (manufactured by JNC Corporation).

[0102] (Example 1-4) Rearing of Vannamei shrimp using formulated feed and extraction of total RNA The compound feed prepared in Example 1-3 was used, and the same procedure as in Comparative Example 1-2 was followed.

[0103] (Example 1-5) Measurement of mRNA expression level and evaluation of efficacy The total RNA obtained in Example 1-4 was used, and the same procedure as in Comparative Example 1-3 was followed.

[0104] Example 2: (Example 2-1) Preparation of compound feed The same procedure as in Comparative Example 4-1 was repeated except that chitosan_H52 was replaced with a 25% polylysine solution (manufactured by JNC Corporation).

[0105] (Example 2-2) Rearing of Vannamei shrimp using formulated feed and extraction of total RNA The compound feed prepared in Example 2-1 was used, and the same procedure as in Comparative Example 4-2 was followed.

[0106] (Example 2-3) Measurement of mRNA expression level and evaluation of efficacy The total RNA obtained in Example 2-2 was used, and the same procedure as in Comparative Example 1-3 was followed.

[0107] Example 3: (Example 3-1) Preparation of compound feed 0.75 mL of a 600 μg / mL polylysine solution was sprinkled onto 1.5 g of formulated feed for white shrimp (Higashimaru Co., Ltd.) to allow the feed to be impregnated. The impregnated feed was dried under reduced pressure at 50°C for 3 hours. 0.75 mL of a 200 μg / mL double-stranded RNA (SEQ ID NO: 1) solution was sprinkled onto the resulting feed to allow the feed to be impregnated. The impregnated feed was dried under reduced pressure at 50°C for 3 hours.

[0108] (Example 3-2) Rearing of Vannamei shrimp using formulated feed and extraction of total RNA The compound feed prepared in Example 3-1 was used, and the same procedure as in Comparative Example 4-2 was followed.

[0109] (Example 3-3) Measurement of mRNA expression level and evaluation of efficacy The total RNA obtained in Example 3-2 was used, and the same procedure as in Comparative Example 1-3 was followed.

[0110] Example 4: (Example 4-1) Synthesis of diBoc guanidyl polylysine N,N'-Di-Boc-1H-pyrazole-1-carboxamidine (0.99 g, Tokyo Chemical Industry Co., Ltd.) was dissolved in N,N'-dimethylformamide (4 mL, Fujifilm Wako Pure Chemical Industries, Ltd.) and acetonitrile (5 mL, Fujifilm Wako Pure Chemical Industries, Ltd.), and then 25% polylysine solution (1.62 g, JNC Corporation) was added and stirred overnight at room temperature. The precipitated solid was collected by filtration and dried under reduced pressure to obtain the target product (0.89 g, 80% yield). 1H-NMR(400MHz,DMSO-d6,22℃)δ:11.45(s,1H),8.67-8.65(m,1H),8.20(brs,1H),4.53-4.52(m,1H),3.07-2.99(m,2H),1.68-1.16(m,6H),1 .47(s,9H),1.37(s,9H), 13C-NMR(100MHz,DMSO-d6,22℃)δ:169.6,162.9,154.5,151.9,82.9,78.1,53.0,38.4,32.5,28.6,27.8,27.5,21.7

[0111] (Example 4-2) Synthesis of guanidyl polylysine hydrochloride DiBoc guanidyl polylysine (0.35 g) was added to 2 M hydrogen chloride in isopropanol (3.5 mL, Fujifilm Wako Pure Chemical Industries, Ltd.) and stirred under reflux for 8 hours. After cooling to room temperature, the precipitate was collected by filtration and dried under reduced pressure to obtain the target product (0.15 g, 74% yield). The pKa value of the resulting guanidyl polylysine was >10. H-NMR (400 MHz, DMSO-d6, 22°C) δ: 8.43 (brs, 1H), 7.89-7.88 (m, 1H), 7.67 (brs, 2H), 7.11 (brs, 2H), 4.17 (brs, 1H), 3.08 (brs, 2H), 1.75-1.56 (m, 2H), 1.48-1.44 (m, 2H), 1. 36-1.18(m,2H), 13C-NMR(100MHz,DMSO-d6,21℃)δ:169.6,156.6,54.0,38.2,31.5,2 8.2,22.1, FT-IR (ATR method): 3262, 3157, 2939, 2867, 1776, 1655, 1552, 1376, 1248, 1102cm -1

[0112] (Example 4-3) Preparation of the complex The same procedure as in Comparative Example 3-3 was repeated except that chitosan_H52 was replaced with the guanidyl polylysine hydrochloride prepared in Example 4-2.

[0113] (Example 4-4) Measurement of the amount of double-stranded RNA eluted from the complex The composite obtained in Example 4-3 was measured in the same manner as in Comparative Example 3-4.

[0114] (Example 4-5) Preparation of compound feed The same procedure as in Comparative Example 3-5 was repeated except that chitosan_H52 was replaced with the guanidyl polylysine hydrochloride prepared in Example 4-2.

[0115] (Examples 4-6) Rearing of Vannamei shrimp using formulated feed and extraction of total RNA The compound feed prepared in Example 4-5 was used, and the same procedure as in Comparative Example 1-2 was followed.

[0116] (Example 4-7) Measurement of mRNA expression level and evaluation of efficacy The total RNA obtained in Example 4-6 was used, and the same procedure as in Comparative Example 1-3 was followed.

[0117] Example 5: (Example 5-1) Preparation of compound feed The same procedure as in Comparative Example 4-1 was carried out using the guanidyl polylysine hydrochloride prepared in Example 4-2.

[0118] (Example 5-2) Rearing of Vannamei shrimp using formulated feed and extraction of total RNA The compound feed prepared in Example 5-1 was used, and the same procedure as in Comparative Example 4-2 was followed.

[0119] (Example 5-3) Measurement of mRNA expression level and evaluation of efficacy The total RNA obtained in Example 5-2 was used, and the same procedure as in Comparative Example 1-3 was followed.

[0120] Example 6: (Example 6-1) Preparation of compound feed The same procedure as in Example 3-1 was carried out using the guanidyl polylysine hydrochloride prepared in Example 4-2.

[0121] (Example 6-2) Rearing of Vannamei shrimp using formulated feed and extraction of total RNA The compound feed prepared in Example 6-1 was used, and the same procedure as in Comparative Example 4-2 was followed.

[0122] (Example 6-3) Measurement of mRNA expression level and evaluation of efficacy The total RNA obtained in Example 6-2 was used, and the same procedure as in Comparative Example 1-3 was followed.

[0123] Example 7: (Example 7-1) Synthesis of N-(N,-tert-butoxycarbonyl-phenylalanyl)polylysine A 25% polylysine solution (1.59 g, manufactured by JNC Corporation) was dissolved in N,N'-dimethylformamide (4 mL, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) and acetonitrile (5 mL, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and then Nt-butoxycarbonyl-L-phenylalanine N-hydroxysuccinimide ester (1.13 g, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added and stirred at room temperature for 17 hours. The precipitated solid was collected by filtration and dried under reduced pressure to obtain the target product (1.18 g, 100% yield). 1H-NMR(400MHz,HFIP-d2,22℃)δ:7.39-7.30(m,3H),7.23-7.21(m,2H),4 .44-4.35(m,1H),4.26(brs,1H),3.46-3.03(m,4H),1.84-1.27(m,6H),1. 43(s,9H), 13C-NMR(100MHz,HFIP-d2,22℃)δ:176.0,174.7,159.4,137.6, 131.3,131.1,129.7,85.4,58.6,56.5,42.1,39.9,33.5,30.5,28.9,25.1

[0124] (Example 7-2) Synthesis of phenylalanyl polylysine hydrochloride N-(N,-tert-butoxycarbonyl-phenylalanyl)polylysine (0.50 g) was added with 2 M hydrogen chloride in isopropanol (5.0 g, Fujifilm Wako Pure Chemical Industries, Ltd.) and stirred under reflux for 4 hours. After cooling to room temperature, the precipitate was collected by filtration and dried under reduced pressure to obtain the target product (0.39 g, yield 93%). 1H-NMR(400MHz,DMSO-d6,22℃)δ:8.90-8.88(m,3H),8.40(brs,3H),8.07(brs,1H),7.37-7.23(m,5H) ,4.22-4.18(m,2H),3.20-3.16(m,1H),3.04-3.01(m,3H),1.65-1.56(m,2H),1.37-1.26(m,4H),13C-N MR(100MHz,DMSO-d6,22℃)δ:170.8,167.6,135.0,129.7,128.4,127.1,53.3,53.1,38.7,38.4,31.9, 28.7,22.7, FT-IR (ATR method):3257,3057,3033,2937,2865,1652,1548,1498,1456,1377,1254,747,701cm -1

[0125] (Example 7-3) Preparation of the complex The same procedure as in Comparative Example 3-3 was repeated except that chitosan_H52 was replaced with the phenylalanyl polylysine hydrochloride prepared in Example 7-2.

[0126] (Example 7-4) Measurement of the amount of double-stranded RNA eluted from the complex The composite obtained in Example 7-3 was measured in the same manner as in Comparative Example 3-4.

[0127] (Example 7-5) Preparation of compound feed The same procedure as in Comparative Example 3-5 was repeated except that chitosan_H52 was replaced with the phenylalanyl polylysine hydrochloride prepared in Example 7-2.

[0128] (Example 7-6) Rearing of Vannamei shrimp using formulated feed and extraction of total RNA The compound feed prepared in Example 7-5 was used, and the same procedure as in Comparative Example 1-2 was followed.

[0129] (Example 7-7) Measurement of mRNA expression level and evaluation of efficacy The total RNA obtained in Example 7-6 was used, and the same procedure as in Comparative Example 1-3 was followed.

[0130] Example 8: (Example 8-1) Preparation of compound feed The same procedure as in Comparative Example 6-1 was repeated, except that chitosan_H76 was replaced with the phenylalanyl polylysine hydrochloride prepared in Example 7-2.

[0131] (Example 8-2) Rearing of Vannamei shrimp using formulated feed and extraction of total RNA The compound feed prepared in Example 8-1 was used, and the same procedure as in Comparative Example 4-2 was followed.

[0132] (Example 8-3) Measurement of mRNA expression level and evaluation of efficacy The total RNA obtained in Example 8-2 was used, and the same procedure as in Comparative Example 1-3 was followed.

[0133] Example 9: (Example 9-1) Preparation of compound feed The same procedure as in Example 3-1 was repeated, except that chitosan_H52 was replaced with the phenylalanyl polylysine hydrochloride prepared in Example 7-2.

[0134] (Example 9-2) Rearing of Vannamei shrimp using formulated feed and extraction of total RNA The compound feed prepared in Example 9-1 was used, and the same procedure as in Comparative Example 4-2 was followed.

[0135] (Example 9-3) Measurement of mRNA expression level and evaluation of efficacy The total RNA obtained in Example 9-2 was used, and the same procedure as in Comparative Example 1-3 was followed.

[0136] Example 10: (Example 10-1) Synthesis of N-(N,-tert-butoxycarbonyl-leucinyl)polylysine A 25% polylysine solution (1.64 g, manufactured by JNC Corporation) was dissolved in N,N'-dimethylformamide (4 mL, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) and acetonitrile (5 mL, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and then Nt-butoxycarbonyl-L-leucine N-hydroxysuccinimide ester (1.06 g, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added and stirred at room temperature for 20 hours. The precipitated solid was collected by filtration and dried under reduced pressure to obtain the target product (1.02 g, yield 89%). 1H-NMR(400MHz,DMSO-d6,22℃)δ:7.84(brs,1H),7.68-7.66(m,1H),6.98-6.96(m,1H),4.18-4.17(m,1H),3.96-3.90(m,1H),2.97(brs,2H), 1.59-1.11(m,9H),1.37(s,9H),0.71-0.67(m,6H), 13C-NMR(100MHz,DMSO-d6,22℃)δ:17 2.1,171.0,155.3,78.0,52.9,52.0,40.5,38.3,31.9,28.6,28.1,24.1,22.9,22.4,21.4

[0137] (Example 10-2) Synthesis of leucinyl polylysine hydrochloride N-(N-tert-butoxycarbonyl-leucinyl)polylysine (0.40 g) was added to 2 M hydrogen chloride in isopropanol (4.0 g, Fujifilm Wako Pure Chemical Industries, Ltd.) and stirred under reflux for 4 hours. After cooling to room temperature, the precipitate was collected by filtration and dried under reduced pressure to obtain the target product (0.30 g, 92% yield). The pKa value of the obtained leucinyl polylysine hydrochloride was 7.43. H-NMR (400 MHz, DMSO-d6, 22°C) δ: 8.79-8.77 (m, 1H), 8.42 (brs, 3H), 8.19 (brs, 1H), 4.24-4.22 (m, 1H), 3.86 (brs, 1H), 3.02 (brs, 2H). 1.71-1.57(m,5H),1.37-1.24(m,4H)0.89-0.87(m,6H), 13C-NMR(100MHz,DMSO-d6,21℃)δ:170.9(C),168.6(C),52.9(C H),50.9(CH),40.2(CH2),38.4(CH2),31.7(CH2),28.7(CH2),23.6(CH),22.7(CH2,CH3),22.3(CH3),13C-NMR(100MHz,D MSO-d6,80℃)δ:170.5(C),168.1(C),53.0(CH),51.1(CH),39.7(CH2),38.1(CH2),31.2(CH2),28.2(CH2),23.3(CH),22 .4(CH2),22.2(CH3),21.9(CH3), FT-IR(ATR method):3239,3052,2955,2934,2870,1652,1548,1369,1264,1237,1185,1133cm -1

[0138] (Example 10-3) The same procedure as in Comparative Example 3-3 was repeated except that chitosan_H52 was replaced with leucinyl polylysine hydrochloride prepared in Example 10-2.

[0139] (Example 10-4) Preparation of the complex The same procedure as in Comparative Example 3-3 was repeated except that chitosan_H52 was replaced with leucinyl polylysine hydrochloride prepared in Example 10-2.

[0140] (Example 10-5) Measurement of the amount of double-stranded RNA eluted from the complex The composite obtained in Example 10-4 was measured in the same manner as in Comparative Example 3-4.

[0141] (Example 10-6) Preparation of compound feed The same procedure as in Comparative Example 6-1 was repeated, except that chitosan_H76 was replaced with leucinyl polylysine hydrochloride prepared in Example 10-2.

[0142] (Example 10-7) Rearing of Vannamei shrimp using formulated feed and extraction of total RNA The compound feed prepared in Example 10-6 was used, and the same procedure as in Comparative Example 4-2 was followed.

[0143] (Example 10-8) Measurement of mRNA expression level and evaluation of efficacy The total RNA obtained in Example 10-7 was used, and the same procedure as in Comparative Example 1-3 was followed.

[0144] Example 11 (Example 11-1) Preparation of compound feed The same procedure as in Example 3-1 was repeated, except that chitosan_H52 was replaced with leucinyl polylysine hydrochloride prepared in Example 10-2.

[0145] (Example 11-2) Rearing of Vannamei shrimp using compound feed and extraction of total RNA The compound feed prepared in Example 11-1 was used, and the same procedure as in Comparative Example 4-2 was followed.

[0146] (Example 11-3) Measurement of mRNA expression level and evaluation of efficacy The total RNA obtained in Example 11-2 was used, and the same procedure as in Comparative Example 1-3 was followed.

[0147] 5. Test results of comparative examples and examples The test results of the above Comparative Examples and Examples are explained below. Table 6 shows the measurement results of the amount of double-stranded RNA (SEQ ID NO: 1) eluted from the complex of each Test Example in 0.5% artificial seawater and 3.0% artificial seawater. Table 7 shows the dsRNA dose, Rab7 protein mRNA expression level, and significant differences from the control experiment in Comparative Examples 1 to 6 and Examples 1 to 11. The results of Comparative Examples 1 to 3 and Examples 1 to 11 in Table 7 are also shown in Figures 3 and 4.

[0148] [Table 6]

[0149] As shown in Table 6 above, the complex of Comparative Example 1 eluted large amounts of RNA into water with a salt concentration of 0.0% by mass and artificial seawater with a salt concentration of 0.5% by mass, whereas the amounts of RNA eluted were less than 0.2 μg / mL for all of the complexes of Examples 4, 7, and 10. Regarding the elution of RNA into artificial seawater with a salt concentration of 3.0% by mass, the complex of Comparative Example 3 eluted 42.9 μg / mL, whereas the complexes of Examples 4 and 7 eluted less than 0.2 μg / mL, and the complex of Example 10 eluted 1.3 μg / mL. On the other hand, the amounts of RNA eluted into water with a salt concentration of 0.0% by mass and artificial seawater with a salt concentration of 0.5% by mass for the complex of Example 1 were lower than those for the complex of Comparative Example 3, but the amount eluted into artificial seawater with a salt concentration of 3.0% by mass was conversely higher. From the above results, it was confirmed that in the basic polymer, modification of the basic amino acid lysine with guanidine, phenylalanine, or leucine further suppresses the elution of RNA from the complex, and that guanidine and phenylalanine modifications are particularly preferable.

[0150] [Table 7]

[0151] As shown in Table 7, mixing double-stranded RNA (SEQ ID NO: 1) with polylysine enabled the expression of RNA interference through oral administration. Furthermore, by using its derivative, a significant interference effect was observed even when a low dose of dsRNA (12.5 ppm) was administered to shrimp.

[0152] 6. Examination of the content ratio (RNA / basic polymer) of basic polymer (wt%) and double-stranded RNA (wt%) in the complex 25 wt% ε-polylysine (JNC) was diluted with sodium acetate buffer adjusted to pH 4.6 to prepare a 0.2 wt% polylysine solution. A predetermined amount of 0.2 wt% polylysine solution (1 μL, 10 μL, 20 μL, 200 μL, or 600 μL) was added to a microtube, and the total volume was adjusted to 960 μL with sodium acetate buffer adjusted to pH 4.6. 40 μL of double-stranded RNA (SEQ ID NO: 1) solution (1 μg / μL) was added and mixed by vortexing. After allowing to stand at room temperature for 1 hour, the presence or absence of double-stranded RNA elution was verified by electrophoresis. The disappearance of the double-stranded RNA band confirmed the formation of the complex (Table 8).

[0153] [Table 8]

[0154] As shown in Table 8 above, when the content ratio (RNA / basic polymer) of the basic polymer (wt%) to the double-stranded RNA (wt%) in the complex was 0.5, 1, 10, or 30, no elution of the double-stranded RNA from the complex occurred.

[0155] 7. pKa of basic polymers The pKa of each basic polymer was calculated using the following method and procedure. 25 mg of basic polymer was dissolved in 0.01 mol / L hydrochloric acid, and then 3.0 mol / L sodium chloride solution was added to adjust the ion concentration to 0.1. Ultrapure water was added to the solution to a volume of 30 mL, and the solution temperature was adjusted to 25 ± 1°C. Using a self-propelled titrator (Metronome Titrando 905) and a pH electrode (Metronome Ecotrode Plus), the titration volume and pH were recorded while adding 0.1 mol / L sodium hydroxide solution. The degree of dissociation (α) of the basic polymer at each titration volume of 0.1 mol / L sodium hydroxide solution was calculated using the titration volume of 0.1 mol / L sodium hydroxide solution required to neutralize the hydrochloric acid and the titration volume of 0.1 mol / L sodium hydroxide solution required to neutralize the ammonium groups. Using the Henderson-Hasselbalch equation as a reference, a linear regression equation was calculated for the relationship between the recorded pH and the calculated degree of dissociation (α), and the intercept of the linear regression equation was taken as the pKa of the basic polymer (Table 9).

[0156]

number

[0157] In the above formula, A: Each titration volume of 0.1 mol / L sodium hydroxide solution [mL] B: Titration volume of 0.1 mol / L sodium hydroxide solution required to neutralize hydrochloric acid [mL] C: Titration volume of 0.1 mol / L sodium hydroxide solution required to neutralize the ammonium group [mL]

[0158] The Henderson-Hasselbalch equation is as follows:

[0159]

number

[0160] [Table 9]

[0161] 8. Average molecular weight of polylysine A 25% polylysine solution (80 mg, manufactured by JNC) was diluted with 10 mL of mobile phase, and the number-average molecular weight (Mn) and weight-average molecular weight (Mw) were calculated by comparing the retention times of gel permeation chromatography (GFC) using pullulan as a standard. The GFC conditions were as follows. The average molecular weight of chitosan_H76 was measured using the same procedure, except that polylysine was replaced with chitosan_H76.

[0162] GFC conditions: Column: GMPW XL (7.8mm*30cm, 13μm, Tosoh Corporation x 2) Mobile phase: 0.5 mol / L acetic acid - 0.5 mol / L sodium acetate aqueous solution Flow rate: 0.5mL / min Column temperature: 40℃ Detector: Differential refractive index detector (RI) Injection volume: 30μL (8mg / mL)

[0163] The retention times of the standard and polylysine are shown below.

[0164] [Table 10]

[0165] The nucleic acid sequences of SEQ ID NOs: 2 to 9 listed in the sequence listing are shown below.

[0166] [Table 11] [Industrial Applicability]

[0167] The RNA delivery composition of the present invention contains RNA effective against viral infections in aquatic animals such as crustaceans, and a basic polymer, thereby preventing RNA elution in breeding water, suppressing its degradation in gastric juice, and promoting uptake in the small intestine, allowing it to be sufficiently taken up into the body even by oral administration. As a result, the RNA delivery composition of the present invention can be suitably used for the prevention and / or treatment of viral infections. Furthermore, raising aquatic animals using the above-described RNA delivery composition of the present invention can effectively prevent and / or treat viral infections in aquatic animals, thereby greatly contributing to solving the problem of viral infections in aquaculture.

Claims

1. The nucleic acid comprises a basic polymer and RNA having 20 or more bases, The composition for RNA delivery, wherein the basic polymer is a polymer having a repeating unit containing a basic amino acid and has a pKa of 7.0 or higher.

2. The composition for RNA delivery according to claim 1 , wherein the basic amino acid is a lysine derivative.

3. The composition for RNA delivery according to claim 1 or 2, wherein the basic polymer contains a compound represented by the following formula (I) or a salt thereof: 【Chemistry 4】 (wherein n is an integer of 6 to 75; X is hydrogen, —C(═NH)—NH ２ or C(═O)—CHR—NH ２ and R is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted alkyl ether, substituted or unsubstituted alkylthioether, substituted or unsubstituted hydroxyalkyl, substituted or unsubstituted hydroxyaryl, substituted or unsubstituted sulfanylalkyl, substituted or unsubstituted sulfanylaryl, substituted or unsubstituted alkylthioalkyl, substituted or unsubstituted alkylthioaryl, substituted or unsubstituted aminoalkyl, substituted or unsubstituted aminoaryl, substituted or unsubstituted aminocarbonylalkyl, substituted or unsubstituted aminocarbonylaryl, substituted or unsubstituted guanidylalkyl, or substituted or unsubstituted guanidylaryl, provided that multiple Xs may be the same or different.

4. The composition for RNA delivery according to any one of claims 1 to 3, wherein the Mn of the basic polymer is 1,000 to 50,000.

5. The composition for RNA delivery according to claim 1 , wherein the RNA is double-stranded RNA (dsRNA).

6. The RNA delivery composition according to any one of claims 1 to 3, wherein the content ratio of the basic polymer (wt%) to the RNA having 20 or more bases (wt%) in the RNA delivery composition (RNA / basic polymer) is 0.5 to 50.

7. The composition for RNA delivery according to any one of claims 1 to 6, which is in the form of a pharmaceutical or feed.

8. The composition for RNA delivery according to claim 7, which is in the form of a medicine or feed for aquatic animals.

9. The composition for RNA delivery according to claim 8, wherein the aquatic animal is a crustacean.

10. mixing an aqueous solution of a basic polymer and an aqueous solution of RNA having 20 or more bases; A method for producing a composition for RNA delivery, wherein the basic polymer is a polymer having a repeating unit containing a basic amino acid and has a pKa of 7.0 or higher.

11. A method for raising an aquatic animal, comprising a step of feeding the aquatic animal the composition for RNA delivery according to claim 8 or 9.

12. An aquatic animal raised by the method of claim 11.

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