Phosphorodiamidate morpholino oligonucleotide (PMO) complex comprising PMO and polyanion

Abstract

The purpose of the present invention is to provide a novel means for delivering a phosphorodiamidate morpholino oligonucleotide (PMO). Provided is a phosphorodiamidate morpholino oligonucleotide (PMO) complex which comprises a PMO and a polyanion.

Description

A PMO complex comprising a phosphorodiamidate morpholino oligonucleotide (PMO) and a polyanion.

[0001] The present invention relates to a PMO complex comprising a phosphorodiamidate morpholino oligonucleotide (PMO) and a polyanion.

[0002] Phosphorodiamidate morpholino oligomers (PMOs) are artificial nucleic acids that are resistant to nucleases and are increasingly being used as nucleic acid medicines. Examples of nucleic acid medicines that utilize PMOs include viltolarsen (trade name Viltepso (registered trademark), Non-Patent Document 1), a drug for treating Duchenne muscular dystrophy. While DNA and RNA have a five-membered ring (deoxyribose or ribose) in the sugar moiety and a phosphodiester bond, PMOs have a six-membered morpholine structure and an electrically neutral phosphorodiamidate bond.

[0003] Thus, PMOs are nucleic acids that can be administered to the body. However, when PMOs are administered intravenously alone, for example, they are rapidly excreted from the blood via the kidneys, and therefore relatively large doses are required to achieve systemic efficacy. Therefore, the development of new methods for delivering PMOs is important.

[0004] Viltepso® Intravenous Infusion 250 mg Package Insert, November 2021 (4th Edition), February 2021 (revised), Prescription Drug Viltolarsen Intravenous Infusion for Duchenne Muscular Dystrophy

[0005] The object of the present invention is to provide a new means useful for the delivery of phosphorodiamidate morpholino oligonucleotides (PMOs).

[0006] As a result of extensive research, the present inventors have found that a PMO complex containing a PMO and a polyanion can be easily produced by combining a PMO with a polyanion. They have also found that the particle size and / or zeta potential of the PMO complex can be altered by changing the mixing ratio of the PMO and the polyanion. The present invention was completed through further research based on these findings, and the present disclosure encompasses, for example, the following representative inventions. Item 1. A PMO complex containing a phosphorodiamidate morpholino oligonucleotide (PMO) and a polyanion. Item 2. The PMO complex according to Item 1, wherein the polyanion is at least one selected from the group consisting of polyamino acids and polysaccharides. Item 3. The PMO complex according to Item 1 or 2, wherein the polyanion is at least one selected from the group consisting of polyglutamic acid, polyaspartic acid, chondroitin sulfate C, heparin, hyaluronic acid, alginic acid, carboxymethylcellulose, carrageenan, and salts thereof. Item 4. Item 5. The PMO complex according to any one of Items 1 to 3, wherein the polyanion is at least one selected from the group consisting of polyglutamic acid, polyaspartic acid, chondroitin sulfate, and salts thereof. Item 6. The PMO complex according to any one of Items 1 to 4, wherein the PMO is present in an amount of 0.05 to 300 parts by mass per part by mass of the polyanion. Item 7. The PMO complex according to Item 5, wherein the PMO is present in an amount of 4 to 300 parts by mass per part by mass of the polyanion. Item 8. The PMO complex according to Item 5, wherein the PMO is present in an amount of 10 to 250 parts by mass per part by mass of the polyanion. Item 9. The PMO complex according to Item 5, wherein the PMO is present in an amount of 20 to 250 parts by mass per part by mass of the polyanion. Item 10. Item 10. The PMO complex according to Item 5, wherein the PMO is present in an amount of 25 to 250 parts by mass per part by mass of polyanion. Item 11. The PMO complex according to Item 5, wherein the PMO is present in an amount of 30 to 250 parts by mass per part by mass of polyanion. Item 12. The PMO complex according to Item 5, wherein the PMO is present in an amount of 40 to 250 parts by mass per part by mass of polyanion. Item 13. The PMO complex according to Item 5, wherein the PMO is present in an amount of 40 to 100 parts by mass per part by mass of polyanion.Item 14. The PMO complex according to any one of Items 1 to 13, having a zeta potential of -50 mV or more and less than 0 mV. Item 15. The PMO complex according to any one of Items 1 to 14, having a zeta potential of -30 mV or more and -5 mV or less. Item 16. The PMO complex according to any one of Items 1 to 15, having a zeta potential of -30 mV or more and -10 mV or less. Item 17. The PMO complex according to any one of Items 1 to 13, having a zeta potential greater than 0 mV and 50 mV or less. Item 18. The PMO complex according to any one of Items 1 to 13 and 17, having a zeta potential of 10 mV or more and 30 mV or less. Item 19. The PMO complex according to any one of Items 1 to 13 and 17 to 18, having a zeta potential of 10 mV or more and 20 mV or less. Item 20. Item 21. The PMO complex according to any one of Items 1 to 19, having an average particle size of 30 to 2000 nm. Item 22. The PMO complex according to any one of Items 1 to 5 and 20 to 21, wherein the polyanion is at least one selected from the group consisting of polyglutamic acid and salts thereof, the amount of PMO is 0.05 to 1 part by mass per part by mass of the polyanion, and the zeta potential is -50 mV or more and less than 0 mV. Item 23. The PMO complex according to any one of Items 1 to 5 and 20 to 21, wherein the polyanion is at least one selected from the group consisting of polyaspartic acid and salts thereof, the amount of PMO is 0.05 to 0.2 part by mass per part by mass of the polyanion, and the zeta potential is -50 mV or more and less than 0 mV. Item 24. Item 24. The PMO complex according to any one of Items 1 to 5 and 20 to 21, wherein the polyanion is at least one selected from the group consisting of chondroitin sulfate and salts thereof, the amount of PMO is 0.1 to 3 parts by mass per part by mass of the polyanion, and the zeta potential is -50 mV or more and less than 0 mV. Item 25. The PMO complex according to any one of Items 1 to 5 and 20 to 21, wherein the polyanion is at least one selected from the group consisting of polyglutamic acid and salts thereof, the amount of PMO is 30 to 300 parts by mass per part by mass of the polyanion, and the zeta potential is greater than 0 mV and less than 50 mV.Item 26. The PMO complex according to any one of Items 1 to 5 and 20 to 21, wherein the polyanion is at least one selected from the group consisting of polyaspartic acid and salts thereof, the amount of PMO is 15 to 30 parts by mass per part by mass of the polyanion, and the zeta potential is greater than 0 mV and not greater than 50 mV. Item 27. The PMO complex according to any one of Items 1 to 5 and 20 to 21, wherein the polyanion is at least one selected from the group consisting of chondroitin sulfate and salts thereof, the amount of PMO is 40 to 100 parts by mass per part by mass of the polyanion, and the zeta potential is greater than 0 mV and not greater than 50 mV.

[0007] A PMO complex containing a PMO and a polyanion can be provided. By changing the mixing ratio of the PMO and the polyanion, the particle size and / or zeta potential of the PMO complex can be changed.

[0008] 1 is a diagram showing examples of a PMO represented by SEQ ID NO: 1 and a PMO represented by SEQ ID NO: 2. It is a diagram showing the PMO / PGA ratio, average particle size, polydispersity index, and zeta potential of the produced PMO complex. It is a diagram showing the relationship between the average particle size and zeta potential of the PMO complex, created based on FIG. 2. It is a diagram showing the relationship between the average particle size and PMO / PGA ratio ... zeta potential and PMO / PGA ratio of the PMO complex, created based on FIG. 2. It is a diagram showing the PMO / PGA ratio, average particle size, polydispersity index, and zeta potential of the produced PMO complex. It is a diagram showing the relationship between the average particle size and zeta potential of the PMO complex, created based on Table 5. It is a diagram showing the relationship between the average particle size and PMO / PAA ratio of the PMO complex, created based on Table 5. It is a diagram showing the relationship between the zeta potential and PMO / PAA ratio of the PMO complex, created based on Table 5. It is a diagram showing the relationship between the average particle size and zeta potential of the PMO complex, created based on Table 7. FIG. 11 is a diagram showing the relationship between the average particle size of the PMO complex and the PMO / CSC ratio, prepared based on Table 7. FIG. 12 is a diagram showing the relationship between the zeta potential of the PMO complex and the PMO / CSC ratio, prepared based on Table 7. FIG. 4 is a diagram showing moving averages. FIG. 8 is a diagram showing a polynomial approximation curve. FIG. 11 is a diagram showing a polynomial approximation curve.

[0009] Hereinafter, embodiments included in the present disclosure will be described in more detail. In this disclosure, "comprise" also includes the meanings of "substantially consist of" and "consist of." This disclosure relates to artificial nucleic acid complexes. Below, a PMO complex will be described as an example of an artificial nucleic acid complex.

[0010] PMO Complex The present disclosure encompasses a PMO complex comprising a phosphorodiamidate morpholino oligonucleotide (PMO) and a polyanion. In the present disclosure, the complex may be referred to as the "PMO complex of the present disclosure."

[0011] Phosphorodiamidate Morpholino Oligonucleotide (PMO) PMO is a known artificial nucleic acid and is a compound that has attracted attention for its use as an antisense oligonucleotide. While RNA has a five-membered ring (ribose) and a phosphodiester bond in the sugar moiety, PMO has a six-membered morpholine structure and an electrically neutral phosphorodiamidate bond. PMO and methods for producing them are publicly known (see, for example, WO 2012 / 029986, etc.). Therefore, the PMO in this application is not limited as long as it is a publicly known PMO, and can be produced according to publicly known procedures.

[0012] In the present disclosure, a preferred PMO is an oligomer having a group represented by the following general formula (1) as a constituent unit.

[0013]

[0014] In formula (1), R １ and R ２ are the same or different and represent a hydrogen atom, an alkyl group, an alkoxy group, a cycloalkyl group or an aryl group, and Base represents a nucleic acid base.

[0015] Preferred examples of the alkyl group include linear or branched alkyl groups having 1 to 6 carbon atoms. Specific examples of the alkyl group include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, tert-pentyl, n-hexyl, and isohexyl. The alkyl group may have a substituent, and examples of the substituent include halogen atoms, alkoxy groups, cyano groups, and nitro groups. For example, these may be the same or different and may have 1 to 3 substituents. Examples of halogen atoms include fluorine atoms, chlorine atoms, bromine atoms, and iodine atoms. Examples of the alkoxy group include linear or branched alkoxy groups having 1 to 6 carbon atoms. Specific examples of the alkoxy group include a methoxy group, an ethoxy group, an n-propoxy group, an isopropoxy group, an n-butoxy group, an isobutoxy group, a sec-butoxy group, a tert-butoxy group, an n-pentyloxy group, an isopentyloxy group, an n-hexyloxy group, an isohexyloxy group, etc. Of these, an alkoxy group having 1 to 3 carbon atoms is more preferred.

[0016] Examples of the alkoxy group include linear or branched alkoxy groups having 1 to 6 carbon atoms. Specific examples of the alkoxy group include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, n-pentyloxy, isopentyloxy, n-hexyloxy, and isohexyloxy. Of these, alkoxy groups having 1 to 3 carbon atoms are more preferred.

[0017] Preferred examples of the cycloalkyl group include cycloalkyl groups having 5 to 12 carbon atoms. Specific examples of the cycloalkyl group include a cyclopentyl group, a cyclohexyl group, a cycloheptyl group, a cyclooctyl group, a cyclodecyl group, and a cyclododecyl group.

[0018] The aryl group is preferably an aryl group having 6 to 10 carbon atoms. Specific examples of the aryl group include a phenyl group, an α-naphthyl group, and a β-naphthyl group. Of these, a phenyl group is more preferable. The aryl group may have a substituent, and examples of the substituent include an alkyl group, a halogen atom, an alkoxy group, a cyano group, and a nitro group. For example, the aryl group may have 1 to 3 of these, which may be the same or different, as a substituent. The alkyl group, halogen, and alkoxy group are as described above.

[0019] In formula (1), R １ and R ２ are the same or different, and are preferably exemplified by an alkyl group, more preferably an alkyl group having no substituent. １ and R ２ are the same or different, and more preferably, methyl group, ethyl group, etc. are exemplified, and particularly preferably, methyl group is exemplified.

[0020] Examples of nucleic acid bases constituting PMOs include adenine, guanine, hypoxanthine, cytosine, thymine, uracil, and modified bases thereof. Examples of such modified bases include pseudouracil, 3-methyluracil, dihydrouracil, 5-alkylcytosine (e.g., 5-methylcytosine), 5-alkyluracil (e.g., 5-ethyluracil), 5-halouracil (5-bromouracil), 6-azapyrimidine, 6-alkylpyrimidine (6-methyluracil), 2-thiouracil, 4-thiouracil, 4-acetylcytosine, and 5-(carboxyhydroxymethyl)uracil. Examples of amino acids include, but are not limited to, uracil, 5'-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, 1-methyladenine, 1-methylhypoxanthine, 2,2-dimethylguanine, 3-methylcytosine, 2-methyladenine, 2-methylguanine, N6-methyladenine, 7-methylguanine, 5-methoxyaminomethyl-2-thiouracil, 5-methylaminomethyluracil, 5-methylcarbonylmethyluracil, 5-methyloxyuracil, 5-methyl-2-thiouracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid, 2-thiocytosine, purine, 2,6-diaminopurine, 2-aminopurine, isoguanine, indole, imidazole, and xanthine. In the present disclosure, preferred examples of nucleic acid bases constituting PMOs include adenine, guanine, hypoxanthine, cytosine, thymine, uracil, etc., more preferred examples include adenine, guanine, cytosine, thymine, uracil, etc., and even more preferred examples include adenine, guanine, cytosine, thymine, etc.

[0021] Although not limiting the present disclosure, a preferred embodiment of PMO is exemplified by a PMO represented by the following general formula (1a):

[0022]

[0023] In formula (1a), R １ , R ２ and Base are as defined above. In formula (1a), m is an integer ranging from 1 to 99.

[0024] In formula (1a), m is preferably an integer in the range of 11 to 49, more preferably 11 to 39, even more preferably 18 to 29, and particularly preferably 20 to 24.

[0025] The base length of the PMO is not limited as long as the effects of the present disclosure can be obtained. Examples of the base length of the PMO include preferably 12 to 40 bases, more preferably 19 to 30 bases, and even more preferably 21 to 25 bases. PMOs such as viltolarsen (21 bases long) described in Non-Patent Document 1 are actually used as nucleic acid drugs. Thus, the base length of the PMO can be appropriately determined depending on the purpose.

[0026] The base sequence of the PMO may also be appropriately determined depending on the purpose. Examples of the base sequence of the PMO include the sequence represented by SEQ ID NO: 1 and the sequence represented by SEQ ID NO: 2. The PMO represented by SEQ ID NO: 1 and the PMO represented by SEQ ID NO: 2 are shown in Figure 1. The former has exon 53 skipping activity of the human dystrophin gene, and the latter has exon 23 skipping activity of the mouse dystrophin gene.

[0027] The PMO may be used alone or in combination of two or more.

[0028] Examples of polyanions include polyamino acids and polysaccharides. Examples of polyamino acids include polyglutamic acid (γ-PGA), polyaspartic acid, and salts thereof. The salts may be any pharmaceutically acceptable salt, such as alkali metal salts (e.g., sodium salts and potassium salts) and alkaline earth metal salts (e.g., calcium salts). More preferred examples of polyamino acids include polyglutamic acid, polyaspartic acid, and sodium salts thereof, with sodium salts of polyglutamic acid being even more preferred. Examples of polysaccharides include chondroitin sulfate (e.g., chondroitin sulfate C), heparin, hyaluronic acid, alginic acid (10 g / l aqueous solution, viscosity at 20°C: 80 to 120 mPa·s), carboxymethylcellulose (2% aqueous solution, viscosity at 25°C: 500 to 900 mPa·s), carrageenan, and salts thereof. The salts used herein may be any pharmaceutically acceptable salt, and examples thereof include alkali metal salts such as sodium salts and potassium salts, alkaline earth metal salts such as calcium salts, etc. More preferred examples of polysaccharides include chondroitin sulfate C, heparin, alginic acid, carrageenan, and sodium salts thereof, and even more preferred examples include chondroitin sulfate C, heparin, alginic acid, and sodium salts thereof.

[0029] The polyglutamic acid and polyaspartic acid may be L-, D-, or LD-isomers. The polyglutamic acid or its salts are not limited as long as the effects of the present disclosure are obtained; however, preferred examples of the average molecular weight are 500,000 to 700,000, and more preferably 580,000 to 600,000. The polyaspartic acid or its salts are not limited as long as the effects of the present disclosure are obtained; however, preferred examples of the average molecular weight are 8,000 to 9,500, and more preferably 8,900. Here, the average molecular weight refers to the weight-average molecular weight and can be measured by gel permeation chromatography (GPC). Polyglutamic acid or its salts are commercially available; for example, the product under the trade name Poly-γ-Sodium Glutamate (average molecular weight 400,000) manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. is an example. Polyaspartic acid or its salts are commercially available, for example, under the trade name Poly-(α,β)-DL-aspartic acid sodium salt-mol wt 2,000-11,000, manufactured by Sigma-Aldrich (average molecular weight 2,000-11,000).

[0030] Furthermore, chondroitin sulfate or a salt thereof is not limited as long as the effects of the present disclosure can be obtained. Chondroitin sulfate or a salt thereof is commercially available, and an example thereof is manufactured by Nacalai Tesque, Inc. (product code 08815-84, chondroitin sulfate C sodium, CAS number 12678-07-8). Heparin or a salt thereof is not limited as long as the effects of the present disclosure can be obtained. A preferred pH is 6 to 8, and a more preferred pH is 6.5 to 7.5 (25°C). For example, heparin or a salt thereof is commercially available, and an example thereof is manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. (product code 081-00136, heparin sodium, CAS number 9041-08-1). Hyaluronic acid ...12 to 40 m ３ / kg, more preferably 15 to 30 m ３ / kg is an example. The intrinsic viscosity of hyaluronic acid or a salt thereof can be measured using a capillary viscometer such as an Ubbelohde viscometer in accordance with the method for measuring liquid viscosity of Japanese Industrial Standard JIS Z8803. For example, hyaluronic acid or a salt thereof is available from Nacalai Tesque, Inc. (product code 18237-41, sodium hyaluronate, CAS number 9067-32-7). Alginic acid, carboxymethylcellulose, carrageenan, and salts thereof are also commercially available. For example, sodium alginate is available from Fujifilm Wako Pure Chemical Industries, Ltd. (product code 194-13321). Sodium carboxymethylcellulose is available from Tokyo Chemical Industry Co., Ltd. (product code C0045). Carrageenan is available from Fujifilm Wako Pure Chemical Industries, Ltd. (product code 039-09691).

[0031] The polyanions may be used singly or in combination of two or more.

[0032] In the PMO complex of the present disclosure, the contents of PMO and polyanion are not particularly limited and may be appropriately determined taking into consideration the target particle size of the PMO complex, etc. For example, the mass ratio of PMO to 1 part by mass of polyanion (hereinafter sometimes referred to as the "PMO / polyanion ratio") is preferably 0.05 to 300, more preferably 0.1 to 300, even more preferably 0.2 to 300, and particularly preferably 0.25 to 250, for example.

[0033] From the viewpoint of further reducing the particle size of the PMO complex, the PMO / polyanion ratio in the PMO complex is preferably 0.05 to 3 or 4 to 300, more preferably 0.05 to 1 or 4 to 300, and even more preferably 0.1 to 1 or 4 to 250. From this viewpoint, when the ratio is 3 or less, more preferably 0.1 to 3, even more preferably 0.2 to 3, and particularly preferably 0.25 to 3. When the ratio is 1 or less, more preferably 0.05 to 1, 0.2 to 1, even more preferably 0.25 to 1, particularly preferably 0.3 to 1, especially more preferably 0.4 to 1, and especially more preferably 0.4 to 0.75. Furthermore, when the ratio is 4 or more, examples of the ratio include more preferably 10 to 250, even more preferably 20 to 250, particularly preferably 25 to 250, particularly more preferably 30 to 250, particularly more preferably 40 to 250, and particularly still more preferably 40 to 100.

[0034] The average particle size of the PMO complex of the present disclosure is not limited and may be determined depending on the purpose. Examples of average particle sizes include a preferred average particle size of 2000 nm or less, more preferably 30 to 1500 nm, and even more preferably 30 to 1000 nm. Since smaller substances are generally easier to deliver, a smaller average particle size is preferred from the perspective of increasing the delivery efficiency of PMO. From this perspective, examples of average particle sizes of the PMO complex of the present disclosure include a preferred average particle size of 30 to 450 nm, more preferably 30 to 400 nm, even more preferably 30 to 350 nm, particularly preferably 30 to 300 nm, and even more preferably 30 to 200 nm. In the present disclosure, the average particle size is measured and determined by dynamic light scattering using a nanoparticle measuring device (ZetaSizer Nano ZS-90, Malvern). Specifically, as shown in the test examples described below, the temperature (solution temperature) is set to 25°C, the dispersion medium viscosity and dispersion medium refractive index are set according to the dispersion medium used, and the cumulant diameter is measured.

[0035] The charge of the PMO complex of the present disclosure is not limited and may be negative, neutral, or positive. Conventionally, carriers such as various particles, including lipid nanoparticles (LNPs) and liposomes, have been known as drug delivery systems (DDS) for delivering nucleic acids and the like. The PMO complex may be used in combination with such a carrier, and in such cases, the charge of the PMO complex may be determined according to the characteristics of the carrier to be combined.

[0036] While not limiting the present disclosure, LNPs will be used as an example of such carriers. LNPs have recently been used in nucleic acid drugs such as Onpattro® and COVID-19 vaccines. The particle size of LNPs is generally approximately 10 to 1,000 nm. LNPs contain ionized lipids as one of their constituent components. The ionized lipids have a tertiary amine at the end of their molecular structure and are electrically neutral in blood (pH 7.4) and positively charged in cellular endosomes (pH 5 to 6). Therefore, in the process of encapsulating a target substance in LNPs, it is considered desirable for the target substance to be negatively charged, taking into account the interaction with the positively charged ionized lipid. For this reason, when the PMO complex of the present disclosure is encapsulated in a positively charged carrier, such as LNP, and used for delivery, the PMO complex is preferably negatively charged.

[0037] From the viewpoint of obtaining a negatively charged PMO complex, the PMO / polyanion ratio in the PMO complex of the present disclosure is preferably 0.05 to 3, and more preferably 0.05 to 2. The ratio is more preferably 0.05 to 1, even more preferably 0.1 to 1, and particularly preferably 0.2 to 1, 0.25 to 1, 0.3 to 1, 0.4 to 1, or 0.4 to 0.75.

[0038] In the present disclosure, a negative charge refers to a zeta potential of less than 0 mV, a neutral charge refers to a zeta potential of 0 mV, and a positive charge refers to a zeta potential greater than 0 mV. In the case of a negative charge, preferably, the charge is less than 0 mV and greater than or equal to -50 mV, more preferably between -5 and -50 mV, even more preferably between -10 and -50 mV or between -5 and -30 mV, and particularly preferably between -10 and -30 mV. In the case of a positive charge, preferably, the charge is greater than 0 mV and less than or equal to 50 mV, more preferably between 10 and 30 mV, and even more preferably between 10 and 20 mV. In the present disclosure, the charge is measured and determined by dynamic light scattering using a nanoparticle measuring device (ZetaSizer Nano ZS-90, Malvern). Specifically, as shown in the test examples described below, the viscosity, refractive index, and dielectric constant of the dispersion medium are set and determined at 25°C (solution temperature) according to the dispersion medium used.

[0039] Furthermore, from the viewpoint of obtaining a PMO complex having a relatively small average particle size (for example, preferably 450 nm or less, more preferably 300 nm or less) and a negative charge, the PMO / polyanion ratio in the PMO complex of the present disclosure is preferably 0.05 to 3, and more preferably 0.05 to 2. Furthermore, the ratio is more preferably 0.05 to 1, even more preferably 0.1 to 1, and particularly preferably 0.2 to 1, 0.25 to 1, 0.3 to 1, 0.4 to 1, or 0.4 to 0.75.

[0040] Although not limiting the present disclosure, as an example of a preferred embodiment of the PMO complex of the present disclosure, when polyglutamic acid and / or a salt thereof (hereinafter sometimes referred to as polyglutamic acids) is used as the polyanion, from the viewpoint of obtaining a PMO complex with a relatively small average particle size, the PMO / polyglutamic acid ratio is preferably 0.05 to 1 or 30 to 300. Furthermore, the PMO / polyglutamic acid ratio is more preferably 0.1 to 1, and even more preferably within the range of 0.2 to 1, 0.25 to 1, 0.3 to 1, 0.4 to 1, or 0.4 to 0.75, or more preferably within the range of 40 to 300, and even more preferably within the range of 40 to 250. In this case, from the viewpoint of obtaining a PMO complex having a relatively small average particle size and a negative charge, the PMO / polyglutamic acid ratio is preferably, for example, 0.05 to 1, more preferably 0.1 to 1, and even more preferably 0.2 to 1, 0.25 to 1, 0.3 to 1, 0.4 to 1, or 0.4 to 0.75. In this case, from the viewpoint of obtaining a PMO complex having a relatively small average particle size and a positive charge, the PMO / polyglutamic acid ratio is preferably, for example, 30 to 300, more preferably 40 to 300, and even more preferably 40 to 250. Furthermore, when the polyanion is a polyglutamic acid, from the viewpoint of obtaining a PMO complex having a relatively small average particle size and a negative charge, the zeta potential of the PMO complex is exemplified as being less than 0 mV and not less than −50 mV, more preferably −5 to −50 mV, even more preferably −10 to −50 mV or −5 to −30 mV, and particularly preferably −10 to −30 mV. In this case, from the viewpoint of obtaining a PMO complex having a relatively small average particle size and a positive charge, the zeta potential of the PMO complex is exemplified as being more than 0 mV and not more than 50 mV, more preferably 10 to 30 mV, and particularly preferably 10 to 20 mV.

[0041] Furthermore, although not limiting the present disclosure, an example of a more preferred embodiment in this case is a PMO complex having a PMO / polyglutamic acid ratio of 0.05 to 1 and a zeta potential of −50 mV or more and less than 0 mV, or a PMO / polyglutamic acid ratio of 30 to 300 and a zeta potential of more than 0 mV and not more than 50 mV. In these cases, the average particle size of the PMO complex is more preferably 30 to 450 nm, and even more preferably 30 to 300 nm.

[0042] While not limiting the present disclosure, as an example of a preferred embodiment of the PMO complex of the present disclosure, when polyaspartic acid and / or a salt thereof (hereinafter, sometimes referred to as polyaspartic acids) is used as the polyanion, from the viewpoint of obtaining a PMO complex having a relatively small average particle size, the PMO / polyaspartic acid ratio is preferably 0.05 to 0.2 or 15 to 30, more preferably 0.05 to 0.15, even more preferably 0.05 to 0.1, or more preferably 15 to 25, and even more preferably 15 to 20. In this case, from the viewpoint of obtaining a PMO complex having a relatively small average particle size and a negative charge, the PMO / polyaspartic acid ratio is preferably 0.05 to 0.2, more preferably 0.05 to 0.15, and even more preferably 0.05 to 0.1. In this case, from the viewpoint of obtaining a PMO complex having a relatively small average particle size and a positive charge, the PMO / polyaspartic acid ratio is preferably 15 to 30, more preferably 15 to 25, and even more preferably 15 to 20. Furthermore, when the polyanion is a polyaspartic acid, from the viewpoint of obtaining a PMO complex having a relatively small average particle size and a negative charge, the zeta potential of the PMO complex is preferably less than 0 mV and not less than −50 mV, more preferably −5 to −50 mV, even more preferably −10 to −50 mV or −5 to −30 mV, and particularly preferably −10 to −30 mV. In this case, from the viewpoint of obtaining a PMO complex having a relatively small average particle size and a positive charge, the zeta potential of the PMO complex is preferably greater than 0 mV and not more than 50 mV, more preferably 10 to 30 mV, and particularly preferably 10 to 20 mV.

[0043] Furthermore, although not limiting the present disclosure, an example of a more preferred embodiment in this case is a PMO complex having a PMO / polyaspartic acid ratio of 0.05 to 0.2 and a zeta potential of −50 mV or more and less than 0 mV, or a PMO / polyaspartic acid ratio of 15 to 30 and a zeta potential of more than 0 mV and not more than 50 mV. In these cases, the average particle size of the PMO complex is more preferably 30 to 450 nm, and even more preferably 30 to 300 nm.

[0044] While not limiting the present disclosure, as an example of a preferred embodiment of the PMO complex of the present disclosure, when chondroitin sulfate and / or a salt thereof (hereinafter sometimes referred to as chondroitin sulfates) is used as the polyanion, from the viewpoint of obtaining a PMO complex having a relatively small average particle size, the PMO / chondroitin sulfate ratio is preferably 0.1 to 3 or 40 to 100, more preferably 0.1 to 2, even more preferably 0.2 to 1, or more preferably 60 to 100, and even more preferably 80 to 100. In this case, from the viewpoint of obtaining a PMO complex having a relatively small average particle size and a negative charge, the PMO / chondroitin sulfate ratio is preferably 0.1 to 3.0, more preferably 0.1 to 2, and even more preferably 0.2 to 1. In this case, from the viewpoint of obtaining a PMO complex having a relatively small average particle size and a positive charge, the PMO / chondroitin sulfate ratio is preferably 40 to 100, more preferably 60 to 100, and even more preferably 80 to 100. Furthermore, when the polyanion is chondroitin sulfate, from the viewpoint of obtaining a PMO complex having a relatively small average particle size and a negative charge, the zeta potential of the PMO complex is exemplified as less than 0 mV and not less than -50 mV, more preferably -5 to -50 mV, even more preferably -10 to -50 mV or -5 to -30 mV, and particularly preferably -10 to -30 mV. In this case, from the viewpoint of obtaining a PMO complex having a relatively small average particle size and a positive charge, the zeta potential of the PMO complex is exemplified as a value greater than 0 mV and not more than 50 mV, more preferably 10 to 30 mV, and particularly preferably 10 to 20 mV.

[0045] Furthermore, although not limiting the present disclosure, an example of a more preferred embodiment in this case is a PMO complex having a PMO / chondroitin sulfate ratio of 0.1 to 3 and a zeta potential of −50 mV or more and less than 0 mV, or a PMO / chondroitin sulfate ratio of 40 to 100 and a zeta potential of more than 0 mV and not more than 50 mV. In these cases, the average particle size of the PMO complex is more preferably 30 to 450 nm, and even more preferably 30 to 300 nm.

[0046] Method for Producing PMO Complexes There are no limitations on the method for producing the PMO complexes of the present disclosure, as long as these complexes can be produced. As a simple example, the PMO complexes of the present disclosure can be produced by causing a PMO and a polyanion to coexist in an acidic solution.

[0047] The pH of the acidic solution is not limited as long as a complex of PMO and polyanion is obtained, but is exemplified as a pH of 2.8 to 5 at 15 to 25°C (solution temperature), more preferably a pH of 3 to 3.8. The pH is measured using a pH meter (product name F-72, manufactured by Horiba, Ltd.). The pH is measured in the same manner below.

[0048] As a more specific and simple example of the production method, for example, the following method can be mentioned: (Production Method 1) An acidic solution, PMO, and polyanion are mixed and stirred. (Production Method 2) An acidic solution (solution 2a) and PMO are mixed and stirred to obtain a PMO solution. An acidic solution (solution 2b) and polyanion are mixed and stirred to obtain a polyanion solution. The PMO solution and polyanion solution are mixed and stirred.

[0049] In (Production Example 1) and (Production Example 2), the temperature of each solution and water is not limited as long as the PMO complex of the present disclosure can be obtained, and is typically, for example, 15 to 25° C. In (Production Example 2), Solution 2a and Solution 2b may be the same solution or different solutions, and preferably, they are the same solution.

[0050] In (Production Example 1) and (Production Example 2), the acidic solution is not limited as long as it can produce the PMO complex of the present disclosure. Preferred examples of the acidic solution include citrate buffer (a mixture of water, citric acid monohydrate, and trisodium citrate dihydrate (for example, a buffer prepared by dissolving 351 mg of citric acid monohydrate and 97 mg of trisodium citrate dihydrate in water and making up to 100 mL with water)), buffer solutions such as phosphate buffer, hydrochloric acid, etc. These may be used alone or in combination of two or more.

[0051] The contents of PMO and polyanion in the solution are not particularly limited. As described above, it is sufficient that the PMO and polyanion are contained at the PMO / polyanion ratio corresponding to the desired PMO complex of the present disclosure. While not limiting the present disclosure, the PMO content in the final solution (i.e., in the acidic solution after mixing of the PMO and polyanion) is preferably 0.05 to 5 mg / mL, more preferably 0.1 to 2.5 mg / mL, even more preferably 0.1 to 1.5 mg / mL, and particularly preferably 0.2 to 1 mg / mL. Furthermore, the polyanion content in the solution is preferably 0.001 to 2.5 mg / mL, more preferably 0.02 to 2 mg / mL, even more preferably 0.1 to 2 mg / mL, and particularly preferably 0.5 to 1 mg / mL. A preferred example is having the PMO and polyanion coexist in the solution so that both of these ranges are satisfied. Furthermore, the PMO complex thus produced may be further mixed with any component (for example, a liquid such as ethanol) within a range that does not impair the effects of the present disclosure.

[0052] In this way, the present disclosure can provide a PMO complex. Furthermore, as described above, in the past, for example, in DDS, target substances have often been encapsulated in carriers (e.g., LNPs) and used for delivery. According to the present disclosure, the average particle size and charge of the PMO complex of the present disclosure can be changed by changing the mixing ratio of PMO and polyanion. Therefore, the present disclosure can easily provide a PMO complex with a desired average particle size and / or charge, depending on the characteristics of the carrier and the characteristics of other components to be combined.

[0053] Thus, according to the present disclosure, the average particle size and / or charge can be changed depending on the characteristics of the carrier or the like to be combined. Furthermore, the PMO complex of the present disclosure can be used for PMO delivery via administration routes such as intravenous, subcutaneous, intramuscular, intrathecal, and intravitreal, in the same way as conventional nucleic acid drugs. The PMO complex of the present invention can be used in any subject requiring the PMO complex, including humans and non-human mammals. The subject can be of any age or gender.

[0054] Furthermore, from this, it can be said that the PMO complex of the present disclosure is useful for PMO delivery. Therefore, the PMO complex of the present disclosure can be used to produce a PMO delivery composition. The composition is not limited as long as it contains the PMO complex of the present disclosure, and may also contain, in addition to the PMO complex, the solution used in producing the PMO complex, etc. Furthermore, any other components may be contained within a range that does not impair the effects of the present disclosure. These components may be used alone or in combination of two or more. Furthermore, the composition may be used in the form of a kit (PMO delivery kit) together with instructions for use, etc. The instructions may include a webpage URL or a readable code, etc., and instructions for use, etc. may be accessible via the URL or readable code, etc. Therefore, the present disclosure can also be said to provide a method for administering an effective amount of the PMO complex of the present disclosure to a subject in need of the PMO complex. In this case, the PMO complex may be encapsulated in a carrier (e.g., LNP) as needed. The effective amount may be determined appropriately depending on the age and sex of the subject, the desired effect, etc.

[0055] Although the present disclosure describes a PMO complex as an example of an artificial nucleic acid complex, embodiments are not limited thereto. For example, an artificial nucleic acid complex may include an artificial nucleic acid other than a PMO as the artificial nucleic acid and the above-described polyanion. Examples of artificial nucleic acids include methylphosphonate (MP) and methoxypropylphosphonate (MOP).

[0056] Furthermore, although the present disclosure has exemplified the combination of a PMO complex with a DDS carrier (such as an LNP or a liposome), the embodiment is not limited thereto, and the artificial nucleic acid complex may be combined with any carrier. Furthermore, as an example of such a combination, an artificial nucleic acid complex such as a negatively charged PMO complex may be combined with a cationic liposome and / or a non-cationic liposome, and an artificial nucleic acid complex such as a positively charged PMO complex may be combined with a non-cationic liposome.

[0057] Hereinafter, the embodiments of the present disclosure will be described more specifically with reference to examples, but the embodiments of the present disclosure are not limited to the following examples.

[0058] Test Example 1: Examination of the effect of the PMO / polyanion concentration ratio on the particle size of the PMO complex In Test Example 1, the effect on the particle size of the PMO complex was examined by changing the PMO concentration while fixing the polyanion concentration.

[0059] 1. Preparation of PMO Complex and Test Method for Particle Size The PMO represented by SEQ ID NO: 1 (hereinafter referred to as PMO-NS-1, Figure 1) was used as the PMO. Sodium gamma-polyglutamate (hereinafter referred to as PGA) was used as the polyanion. The weight-average molecular weight of the PGA used in this test example was 600,000 (product name: poly-sodium gamma-glutamate, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.).

[0060] First, 351 mg of citric acid monohydrate and 97 mg of trisodium citrate dihydrate were dissolved in water (water for injection), and the resulting solution was diluted to 100 mL with water for injection (citric acid buffer, pH 3.1). A citrate buffer solution of PMO-NS-1 (a solution obtained by adding PMO-NS-1 to citrate buffer and dissolving it) and a citrate buffer solution of PGA (a solution obtained by adding PGA to citrate buffer and dissolving it) were prepared, and the respective solutions were stirred and mixed to the final concentrations listed in Table 1. In Test Example 1, the citrate buffer solution of PGA was fixed at 0.5 mg / mL, and the citrate buffer solutions of PMO-NS-1 were set to 0.2 mg / mL (Example 1) and 0.5 mg / mL (Example 2). The average particle size and polydispersity index of the obtained PMO complex were measured and determined by dynamic light scattering using a nanoparticle measuring device (ZetaSizer Nano ZS-90, Malvern). During the measurement, the temperature was set to 25°C, the dispersion medium viscosity was 0.8956 mPa.s, and the dispersion medium refractive index was 1.331. Here, the average particle size refers to the average particle size calculated by the cumulant method. The pH was measured using a pH meter (product name F-72, manufactured by Horiba, Ltd.). The same applies hereinafter unless otherwise specified.

[0061] 2. Test Results The average particle size and polydispersity index of the PMO / PGA composites prepared by the above-mentioned methods are shown in Table 1. In Examples 1 and 2, uniform composite particles with an average particle size of 220.5 nm or less were obtained.

[0062]

[0063] Test Example 2: Investigation of the effect of the PMO / PGA ratio on the surface charge of the PMO complex In Test Example 2, the PMO concentration was fixed at a level at which no aggregation occurred visually, and the polyanion concentration was varied to investigate the effect on the surface charge of the PMO complex.

[0064] 1. Preparation of PMO Complex and Test Method for Particle Size and Zeta Potential A citrate buffer solution of PMO-NS-1 and a citrate buffer solution of PGA were prepared in the same manner as in Test Example 1, and the respective solutions were stirred and mixed to the final concentrations listed in Table 2. The average particle size and zeta potential of the obtained PMO complex were measured and determined using a nanoparticle measuring device (ZetaSizer Nano ZS-90, Malvern). The average particle size was measured and determined using the same settings as in Test Example 1. When measuring the zeta potential, the temperature was set to 25°C, the dispersion medium viscosity was 0.8956 mPa s, the dispersion medium refractive index was 1.331, and the dispersion medium dielectric constant was 78.5.

[0065] 2. Test Results The measurement results of the average particle size and zeta potential of the composite particles prepared by the above method are shown in Table 2. PMO composites were successfully produced in all of Examples 3 to 6. Furthermore, PMO composites with positive zeta potentials were produced in Examples 3 and 4, and PMO composites with negative zeta potentials were produced in Examples 5 and 6. This demonstrates that PMO composites can be produced by combining PMO with polyanions, and that by appropriately changing the mixing ratio of PMO to polyanions, composites with the desired average particle size and zeta potential can be produced. Furthermore, in Examples 5 and 6, uniform composite particles with an average particle size of approximately 200 nm or less were obtained, and the zeta potentials were negative, while in Examples 3 and 4, relatively large particles were obtained, and the zeta potentials were positive. These results confirmed that negatively charged nanoparticles could be obtained when the PMO / PGA concentration ratio was 1 or less.

[0066]

[0067] Further, several PMO complexes were produced in the same manner as in Test Examples 1 and 2. Figure 2 shows a table summarizing the PMO / PGA ratio, average particle size, polydispersity index, and zeta potential of the PMO complex and the PMO complexes of Examples 1 to 6. Figure 3 shows the relationship between the average particle size and zeta potential of the PMO complex, prepared based on Figure 2. Figure 4 shows the relationship between the average particle size and PMO / PGA ratio of the PMO complex, prepared based on Figure 2. Figure 5 shows the relationship between the zeta potential and PMO / PGA ratio of the PMO complex, prepared based on Figure 2.

[0068] As shown in Figure 2, a PMO complex can be produced by combining PMO and a polyanion. Furthermore, Figures 2 to 5 show that by appropriately changing the mixing ratio of PMO and polyanion, a complex with the desired average particle size and zeta potential can be produced. In particular, it was found that a PMO / PGA ratio of 1 or less or 10 or more, and even 1 or less or 25 or more, can produce a PMO complex with a relatively small average particle size. Furthermore, it was found that the average particle size tends to be affected by the concentration of PMO and / or PGA in the solution used. Therefore, it was found that the desired PMO complex can be produced by appropriately changing the concentration in consideration of this tendency. Furthermore, it was found that a PMO / PGA ratio of 1 or less can produce a PMO complex with a negative charge.

[0069] Test Example 3: Investigation of PMO concentration in the production of PMO complex In Test Example 3, the influence of PMO concentration and PGA concentration on the particle size of the PMO complex was investigated.

[0070] 1. Preparation of PMO Complex and Test Method for Particle Size and Zeta Potential A citrate buffer solution of PMO-NS-1 and a citrate buffer solution of PGA were prepared in the same manner as in Test Example 1, and the respective solutions were mixed by stirring to the final concentrations shown in Table 3. The particle size and zeta potential of the obtained PMO complex were measured and determined using a nanoparticle measuring device (ZetaSizer Nano ZS-90, Malvern) in the same manner as in Test Example 2.

[0071] 2. Test results The results are shown in Table 3. In all cases, PMO complexes with relatively small particle sizes were obtained, and it was shown that negatively charged PMO complexes were obtained when the PMO / PGA concentration ratio was in the range of 0.25 to 1.

[0072]

[0073] Test Example 4: Evaluation of particle size of PMO complex after mixing with ethanol In Test Example 4, the effect of mixing ethanol with a PMO complex suspension on the particle size of the PMO complex was examined.

[0074] 1. Preparation of PMO Complex and Test Method for Particle Size: A citrate buffer solution of PMO-NS-1 and a citrate buffer solution of PGA were prepared in the same manner as in Examples 11 to 14 shown in Test Example 3 above, and the respective solutions were stirred and mixed to the final concentrations listed in Table 4. The resulting PMO complex suspension and ethanol were mixed in equal amounts and stirred. The stirred solution was used as a sample solution, and the average particle size was measured and determined using a nanoparticle measuring device (ZetaSizer Nano ZS-90, Malvern). During particle size measurement, the temperature was set to 25°C, the dispersion medium viscosity to 2.6901 mPa.s, and the dispersion medium refractive index to 1.356.

[0075] 2. Test Results The results are shown in Table 4. It was shown that particles prepared using the same formulations as in Examples 11 to 14 of Test Example 3 above had an average particle size of about 200 nm or less even when mixed with an equal amount of ethanol.

[0076]

[0077] Further, several PMO complexes were produced in the same manner as in Test Examples 3 and 4. The PMO / PGA ratio, average particle size, and polydispersity index of the PMO complexes and the PMO complexes of Examples 19 to 22 are summarized in a table in Figure 6. The average particle size, polydispersity index, and zeta potential of the solution before ethanol mixing were measured in the same manner as in Test Examples 1 to 3. The average particle size and polydispersity index of the solution after ethanol mixing were measured under the same conditions as in Test Example 4. Figure 6 also demonstrates that PMO complexes can be produced by combining PMO and polyanions. In particular, it was found that negatively charged PMO complexes can be obtained when the PMO / PGA ratio is 1 or less.

[0078] As can be seen from the above test examples, the present disclosure enables the production of PMO complexes. Furthermore, it was found that the desired PMO complexes can be easily produced by appropriately changing the PMO concentration, PGA concentration, and the mixing ratio of PMO and PGA, taking into consideration the target average particle size, zeta potential, etc.

[0079] Test Example 5: Preparation of PMO Complex Using Polyaspartic Acid In Test Example 5, sodium polyaspartate (hereinafter referred to as PAA) was used as the polyanion, and the effects of the PMO concentration and PAA concentration on the particle size and zeta potential of the PMO complex were investigated.

[0080] 1. Preparation of PMO Complex and Test Method for Particle Size and Zeta Potential The same PMO as used in Test Example 1 was used. The weight-average molecular weight of the PAA used in this Test Example was 8,900 (product name: Poly-(α,β)-DL-aspartic acid sodium solt-mol wt 2,000-11,000, manufactured by Sigma-Aldrich). A citrate buffer solution of PMO-NS-1 was prepared in the same manner as in Test Example 1, and a citrate buffer solution of PAA was prepared in the same manner as in Test Example 1, except that PGA was replaced with PAA. The respective solutions were stirred and mixed to the final concentrations listed in Table 5. The particle size and zeta potential of the obtained PMO complex were measured and determined using a nanoparticle measuring device (ZetaSizer Nano ZS-90, Malvern) in the same manner as in Test Example 2.

[0081] 2. Test Results The results are shown in Table 5. PMO complexes were produced in all of Examples 23 to 29. Furthermore, when the PMO / PAA concentration ratio was 0.1 or less (Examples 25 to 27) or 20 (Example 28), particles with an average particle size of 300 nm or less were obtained. Furthermore, PMO complexes with negative zeta potentials were produced in Examples 23 to 27, and PMO complexes with positive zeta potentials were produced in Examples 28 and 29. This also demonstrates that PMO complexes can be produced by combining PMO and polyanions, and that complexes with the desired average particle size and zeta potential can be produced by appropriately changing the mixing ratio of PMO and polyanions.

[0082]

[0083] The relationship between the average particle size and zeta potential of the PMO complex prepared based on Table 5 is shown in Figure 7. The relationship between the average particle size and PMO / PAA ratio of the PMO complex prepared based on Table 5 is shown in Figure 8. The relationship between the zeta potential and PMO / PAA ratio of the PMO complex prepared based on Table 5 is shown in Figure 9.

[0084] Test Example 6: Evaluation of particle size of PMO complex after mixing with ethanol In Test Example 6, the effect of mixing ethanol with a PMO / PAA complex suspension on the particle size of the complex was examined.

[0085] 1. Preparation of PMO Complex and Test Method for Particle Size: A citrate buffer solution of PMO-NS-1 and a citrate buffer solution of PAA were prepared in the same manner as in Examples 25 and 28 shown in Test Example 5 above, and the respective solutions were stirred and mixed to the final concentrations listed in Table 6. The resulting PMO complex suspension and ethanol were mixed in equal amounts and stirred. The stirred solution was used as a sample solution, and the average particle size was measured and determined using a nanoparticle measuring device (ZetaSizer Nano ZS-90, Malvern). During particle size measurement, the temperature was set to 25°C, the dispersion medium viscosity to 2.6901 mPa.s, and the dispersion medium refractive index to 1.356.

[0086] 2. Test results The results are shown in Table 6. It was shown that when the produced particles were mixed with an equal amount of ethanol, the average particle size was approximately 200 nm or less.

[0087]

[0088] Test Example 7: Preparation of PMO Complex Using Chondroitin Sulfate In Test Example 7, chondroitin sulfate C sodium salt (hereinafter referred to as CSC) was used as the polyanion, and the effects of the PMO concentration, CSC concentration, etc. on the particle size and zeta potential of the PMO complex were investigated.

[0089] 1. Preparation of PMO Complex and Test Method for Particle Size and Zeta Potential The same PMO as used in Test Example 1 was used. The trade name of the CSC used in this Test Example is chondroitin sulfate C sodium salt (manufactured by Nacalai Tesque, Inc., Code: 08815-84). A citrate buffer solution of PMO-NS-1 was prepared in the same manner as in Test Example 1, and a citrate buffer solution of CSC was prepared in the same manner as in Test Example 1, except that PGA was replaced with CSC. The respective solutions were stirred and mixed to the final concentrations listed in Table 7. The particle size and zeta potential of the obtained PMO complex were measured and determined using a nanoparticle measuring device (ZetaSizer Nano ZS-90, Malvern) in the same manner as in Test Example 2.

[0090] 2. Test Results The results are shown in Table 7. PMO complexes were produced in all of Examples 32 to 41. For example, negatively charged particles with an average particle size of 200 nm or less were obtained when the PMO / CSC concentration ratio was in the range of 0.1 to 3. This also demonstrates that PMO complexes can be produced by combining PMO and polyanions, and that complexes with the desired average particle size and zeta potential can be produced by appropriately changing the mixing ratio of PMO and polyanions.

[0091]

[0092] The relationship between the average particle size and the zeta potential of the PMO complex prepared based on Table 7 is shown in Figure 10. The relationship between the average particle size and the PMO / CSC ratio of the PMO complex prepared based on Table 7 is shown in Figure 11. The relationship between the zeta potential and the PMO / CSC ratio of the PMO complex prepared based on Table 7 is shown in Figure 12.

[0093] Test Example 8: Evaluation of particle size of PMO complex after mixing with ethanol In Test Example 8, the effect of mixing ethanol with a PMO / CSC complex suspension on the particle size of the complex was investigated.

[0094] 1. Preparation of PMO Complex and Test Method for Particle Size: A citrate buffer solution of PMO-NS-1 and a citrate buffer solution of CSC were prepared in the same manner as in Examples 36, 38, and 39 shown in Test Example 7 above, and the respective solutions were stirred and mixed to the final concentrations listed in Table 8. The resulting PMO complex suspension and ethanol were mixed in equal amounts and stirred. The stirred solution was used as a sample solution, and the average particle size was measured and determined using a nanoparticle measuring device (ZetaSizer Nano ZS-90, Malvern). During particle size measurement, the temperature was set to 25°C, the dispersion medium viscosity to 2.6901 mPa.s, and the dispersion medium refractive index to 1.356.

[0095] 2. Test results The results are shown in Table 8. It was shown that when the produced particles were mixed with an equal amount of ethanol, the average particle size was approximately 200 nm or less.

[0096]

[0097] 13 to 15 are diagrams in which moving averages or approximation curves have been added to Figures 4, 8, and 11. From the results of each of the above test examples, it can be said that in each graph, a rough guideline for the zeta potential of 0 mV exists near the peak of the moving average (Figure 13) or approximation curve (Figures 14 and 15), and it was observed that the zeta potential tended to be less than 0 mV in the left side of the peak, and more than 0 mV in the right side of the peak.

[0098] This also demonstrates that the present disclosure enables the production of PMO complexes. Furthermore, it has been demonstrated that the desired PMO complexes can be easily produced by appropriately changing the mixing ratio of PMO and polyanion, etc., taking into consideration the target average particle size, zeta potential, etc.

Claims

1. A phosphorodiamidate morpholino oligonucleotide (PMO) complex comprising a PMO and a polyanion.

2. The PMO complex of claim 1, wherein the polyanion is at least one selected from the group consisting of polyamino acids and polysaccharides.

3. The PMO complex according to claim 1, wherein the PMO is present in an amount of 0.05 to 300 parts by mass per part by mass of the polyanion.

4. The PMO complex according to claim 3, wherein the PMO is 0.05 to 3 parts by mass per part by mass of polyanion.

5. The PMO complex according to claim 3, wherein the PMO is present in an amount of 4 to 300 parts by mass per part by mass of polyanion.

6. The PMO complex according to any one of claims 1 to 5, having a zeta potential of -50 mV or more and less than 0 mV.

7. The PMO complex according to any one of claims 1 to 5, having a zeta potential of more than 0 mV and not more than 50 mV.

8. The PMO complex according to any one of claims 1 to 5, having an average particle size of 30 to 450 nm.

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