Nucleic acid delivery composition and nucleic-acid-containing composition

Abstract

Provided is a nucleic acid delivery composition with which it is possible to highly efficiently deliver a nucleic acid drug to central nervous system tissues by nasal administration and to form composite particles that are exceptional in physical property stability and uniformity.　A nucleic acid delivery composition according to the present invention is used in order to deliver a nucleic acid to central nervous system tissues or the trigeminal nerve, the nucleic acid delivery composition containing (I) a block copolymer having (a) a polyethylene glycol segment and (b) a polycaprolactone segment, and (II) a peptide composed of 8-20 amino acid residues modified with a fatty acid. In the block copolymer (I), the average molecular weight Ma of the polyethylene glycol segment (a) is 1,500-2,500 or 3,500-6,500, and the average molecular weight Mb of the polycaprolactone segment (b) is 1.5-3 times the average molecular weight Ma of the polyethylene glycol segment (a). The nucleic acid delivery composition is delivered to the central nervous system tissues or the trigeminal nerve by nasal administration.

Description

Compositions for nucleic acid delivery and nucleic acid-containing compositions

[0001] The present invention relates to a nucleic acid delivery composition and a nucleic acid-containing composition used to deliver nucleic acids to target tissues, and in particular to a nucleic acid delivery composition and a nucleic acid-containing composition that can deliver nucleic acids to central nervous system tissues such as the brain and spinal cord or the trigeminal nerve by nasal administration.

[0002] Research is underway into nucleic acid drugs that can knock down CNS disease-related molecules at the mRNA level for central nervous system (CNS) diseases such as neurodegenerative diseases, brain tumors, cerebrovascular disorders, and brain infections, for which no effective therapeutic drugs have been available until now. Currently, nucleic acid drugs developed for CNS diseases include nusinersen (a therapeutic agent for spinal muscular atrophy in patients with SMN gene mutations) and tofersen (a therapeutic agent for amyotrophic lateral sclerosis in patients with SOD1 gene mutations). However, these drugs are all administered intrathecally, which is highly invasive and places a heavy burden on patients.

[0003] Meanwhile, the nose-to-brain pathway via nasal administration has attracted attention as a new drug delivery route to the central nervous system. This pathway allows drugs to reach the central nervous system directly from the nasal mucosa via neural circuits (the olfactory nerve and the trigeminal nerve). Therefore, it is expected to provide both efficient drug delivery to the central nervous system without passing through the blood-brain barrier and reduced side effects on peripheral organs, which are non-target tissues. The present inventors have conducted research into drug delivery technologies via the nose-to-brain pathway, and as one of the results of their research, they have discovered and reported that a compound obtained by binding a specific membrane-permeable peptide to the other end of a block copolymer in which a polyethylene glycol segment and a hydrophobic polyester segment are linked (the end not linked to the polyethylene glycol segment) forms nanoparticles with a micellar structure and functions as a drug delivery composition that delivers drugs to the spinal cord via nasal administration (Patent Document 1).

[0004] Patent No. 7211603

[0005] However, the nanoparticles formed by the drug delivery composition reported in Patent Document 1 have problems such as a wide particle size distribution, poor uniformity, and poor physical stability, with the average particle size, zeta potential, etc. easily changing over time.

[0006] Therefore, the present invention has been made in consideration of the above-mentioned points, and its object is to provide a nucleic acid delivery composition that can deliver nucleic acid drugs to central nervous system tissue with high efficiency by nasal administration and can form complex particles that are also excellent in physical stability and uniformity.

[0007] Another object of the present invention is to provide a composition containing easily obtainable ingredients that does not require the complicated synthetic reaction for binding a block copolymer to a peptide, because the compound used in the drug delivery composition reported in Patent Document 1 is obtained by binding a block copolymer to a specific membrane-permeable peptide, and the synthetic reaction and purification processes are time-consuming (costly).

[0008] The present inventors have conducted extensive research into combinations of block copolymers with specific functional peptides, aiming to achieve highly efficient delivery of nucleic acid drugs to central nervous system tissues via nasal administration. As a result, they have discovered a nucleic acid delivery composition that can deliver nucleic acid drugs to central nervous system tissues with high efficiency and that forms complex particles with excellent physical stability and uniformity. Based on this finding, they have completed the present invention.

[0009] In order to solve the above-mentioned problems, the nucleic acid delivery composition of the present invention is a nucleic acid delivery composition used to deliver a nucleic acid to central nervous system tissue or the trigeminal nerve, and contains (I) a block copolymer having (a) a polyethylene glycol segment and (b) a polycaprolactone segment, and (II) a peptide consisting of 8 to 20 amino acid residues modified with a fatty acid, wherein in this block copolymer (I), the (a) polyethylene glycol segment has an average molecular weight Ma of 1,500 to 2,500 or 3,500 to 6,500, and the (b) polycaprolactone segment has an average molecular weight Mb that is 1.5 to 3 times the average molecular weight Ma of the above-mentioned (a) polyethylene glycol segment, and is delivered to central nervous system tissue or the trigeminal nerve by nasal administration.

[0010] The nucleic acid delivery composition of the present invention comprises (I) a block copolymer having (a) a polyethylene glycol segment and (b) a polycaprolactone segment, and (II) a peptide consisting of 8 to 20 amino acid residues modified with a fatty acid. By adjusting the average molecular weight Ma of the (a) polyethylene glycol segment and the average molecular weight Mb of the (b) polycaprolactone segment constituting the block copolymer (I) within a predetermined range, a nucleic acid delivery composition capable of highly efficient delivery of nucleic acid drugs to central nervous system tissue or the trigeminal nerve via nasal administration can be obtained. Note that, in this specification, the numerical range of "lower limit to upper limit" refers to a numerical range "between the lower limit and the upper limit" unless otherwise specified. Furthermore, the "average molecular weight" in this specification refers to the peak-top molecular weight (weight average) measured by GPC using a polyethylene glycol standard (Sigma-Aldrich) as the standard.

[0011] Furthermore, in the nucleic acid delivery composition of the present invention, the average molecular weight Mb of the polycaprolactone segment (b) in the block copolymer (I) described above is preferably 3,500 to 6,500 or 8,000 to 12,000. This allows the selection of a particularly suitable range for the average molecular weight of the polycaprolactone segment (b).

[0012] Furthermore, the nucleic acid delivery composition of the present invention preferably has a molar ratio of the block copolymer (I) to the peptide (II) of (I):(II) = 0.25:1 to 2:1, thereby providing a nucleic acid delivery composition that forms complex particles with particularly excellent physical stability and uniformity.

[0013] In addition, in the nucleic acid delivery composition of the present invention, the block copolymer (I) is preferably methoxypolyethylene glycol-poly(ε-caprolactone), thereby allowing the selection of a particularly suitable block copolymer as the block copolymer (I).

[0014] In addition, in the nucleic acid delivery composition of the present invention, it is also preferred that the peptide (II) is a peptide consisting of cysteine, histidine, and arginine residues, and the fatty acid modifying this peptide is stearic acid, thereby selecting a particularly suitable peptide as the peptide (II).

[0015] In addition, the nucleic acid delivery composition of the present invention preferably comprises nanoparticles formed by the block copolymer (I) and the peptide (II).

[0016] Furthermore, the nucleic acid delivery composition of the present invention forms nanoparticles, and the nanoparticles preferably have an average particle size of 50 nm or less, a polydispersity index (PDI) of 0.3 or less, and a zeta potential of 1 to 20 mV. This allows the formation of complex particles with excellent physical stability and uniformity, and provides a nucleic acid delivery composition that can deliver nucleic acids to target tissues with high efficiency.

[0017] Furthermore, the nucleic acid-containing composition of the present invention is the above-mentioned nucleic acid delivery composition containing a nucleic acid, whereby the nucleic acid is stably retained in the nucleic acid delivery composition, thereby obtaining a nucleic acid-containing composition that can efficiently deliver the nucleic acid to a target tissue.

[0018] Furthermore, it is also preferable that the nucleic acid-containing composition of the present invention has a ratio (N / P ratio) of the total number of moles (N) of cationic groups derived from the peptide (II) above to the total number of moles (P) of phosphate groups derived from the nucleic acid, of 3 to 30. This allows the formation of complex particles with excellent physical stability and uniformity, and provides a nucleic acid-containing composition that can deliver nucleic acids to target tissues with high efficiency.

[0019] Furthermore, the nucleic acid-containing composition of the present invention preferably contains, as the nucleic acid, an antisense oligonucleic acid, siRNA, or mRNA for treating a disease in central nervous system tissue. Since the nucleic acid-containing composition of the present invention is delivered intranasally to central nervous system tissue, the antisense oligonucleic acid, siRNA, or mRNA for treating a disease in central nervous system tissue can be delivered directly to the target tissue.

[0020] Furthermore, the pharmaceutical of the present invention contains as an active ingredient a nucleic acid-containing composition containing the above-mentioned nucleic acid for treating diseases of central nervous system tissue. This allows for the delivery of a therapeutic nucleic acid drug to central nervous system tissue, which is a target tissue, by nasal administration. Because the pharmaceutical of the present invention is delivered to central nervous system tissue via the nasal route, minimally invasive treatment can be performed while reducing side effects on non-target tissues.

[0021] According to the present invention, it is possible to provide a nucleic acid delivery composition and a nucleic acid-containing composition that have the following excellent effects: (1) Nucleic acids such as nucleic acid drugs can be delivered to central nervous system (CNS) tissues or the trigeminal nerve. (2) Because nucleic acid drugs can be delivered to target tissues by nasal administration, non-invasive treatment can be achieved and side effects on non-target tissues can be reduced.

[0022] 1 is a graph showing (a) the average particle size, (b) zeta potential, and (c) PDI (polydispersity index) of nanoparticles in the nucleic acid delivery composition prepared in Example 1.

[0023] FIG. 1 is a graph showing (a) the average particle size, (b) zeta potential, and (c) PDI (polydispersity index) of nanoparticles in the nucleic acid delivery composition prepared in Example 1 by varying the molar ratio of (I) block copolymer to (II) peptide.

[0024] FIG. 1 is a graph showing the physical properties over time of the nucleic acid delivery composition prepared using the components of Tests 1-3 and 1-5 as the (I) block copolymer in Example 1.

[0025] FIG. 1 is a graph showing the physical properties over time of the nucleic acid delivery composition prepared using the components of Tests 1-5 as the (I) block copolymer in Example 1.

[0026] FIG. 1 is a graph showing the particle size distribution of nanoparticles in the nucleic acid (Malat-1-targeting siRNA)-containing composition prepared in Example 2. 1 is a graph showing the target gene knockdown effect in various central nervous system tissues by nasal administration of a composition containing a nucleic acid (Malat-1-targeting siRNA) in Example 3. 2 is a graph showing the (a) average particle size, (b) zeta potential, and (c) PDI (polydispersity index) of nanoparticles in a composition containing a Malat-1-targeting antisense oligonucleic acid prepared in Example 4. 3 is a graph showing the target gene knockdown effect in various central nervous system tissues by nasal administration of a composition containing a Malat-1-targeting antisense oligonucleic acid in Example 5. 4 is a graph showing the (a) average particle size, (b) zeta potential, and (c) PDI (polydispersity index) of nanoparticles in a composition containing a Malat-1-targeting antisense oligonucleic acid prepared by a microfluidic device method in Example 6. 5 is a graph showing the target gene knockdown effect in various central nervous system tissues by nasal administration of a composition containing a Malat-1-targeting antisense oligonucleic acid prepared by a microfluidic device method in Example 7.In Example 8, (a) is a graph showing the average particle size, PDI, and zeta potential of nanoparticles in an hSOD1-targeted antisense oligonucleic acid-containing composition prepared using the components of Test 8-1 as the (I) block copolymer, (b) is a graph showing the average particle size, PDI, and zeta potential of nanoparticles in an hSOD1-targeted antisense oligonucleic acid-containing composition prepared using the components of Test 8-2 as the (I) block copolymer, (c) is an electron microscope photograph showing nanoparticles in an hSOD1-targeted antisense oligonucleic acid-containing composition prepared using the components of Test 8-1 as the (I) block copolymer, and (d) is an electron microscope photograph showing nanoparticles in an hSOD1-targeted antisense oligonucleic acid-containing composition prepared using the components of Test 8-2 as the (I) block copolymer. In Example 9, (a) is a graph showing the knockdown effect of target genes in various central nervous system tissues by nasal administration of an hSOD1-targeted antisense oligonucleic acid-containing composition to ALS model mice. 1 is a graph showing the concentration distribution of hSOD1-targeted antisense oligonucleic acid in various central nervous system tissues and the trigeminal nerve following nasal administration of a composition containing an hSOD1-targeted antisense oligonucleic acid to cynomolgus monkeys in Example 10. FIG. 2 is a graph showing the knockdown effect of target genes in various central nervous system tissues following nasal administration of a composition containing an hSOD1-targeted antisense oligonucleic acid to cynomolgus monkeys in Example 10.

[0023] [Regarding the Nucleic Acid Delivery Composition] The nucleic acid delivery composition of the present invention is described in detail below. The nucleic acid delivery composition of the present invention is a composition used for delivering a nucleic acid to central nervous system tissue or the trigeminal nerve, and is composed of at least (I) a block copolymer having (a) a polyethylene glycol segment and (b) a polycaprolactone segment, and (II) a peptide consisting of 8 to 20 amino acid residues modified with a fatty acid. In the block copolymer (I), the (a) polyethylene glycol segment has an average molecular weight Ma of 1,500 to 2,500 or 3,500 to 6,500, and the (b) polycaprolactone segment has an average molecular weight Mb that is 1.5 to 3 times the average molecular weight Ma of the (a) polyethylene glycol segment.

[0024] The target tissue of the nucleic acid delivery composition of the present invention is central nervous system tissue or the trigeminal nerve. Furthermore, the nucleic acid delivery composition of the present invention is administered intranasally and delivered via the so-called nose-to-brain pathway. Central nervous system (CNS) tissues include the spinal cord and brain, and are not particularly limited to the spinal cord, which may include the cervical, thoracic, lumbar, and sacral spinal cord, and the brain, which may include the cerebrum, diencephalon, midbrain, pons, cerebellum, and medulla oblongata.

[0025] [(I) Block Copolymer] Next, each of the components constituting the nucleic acid delivery composition of the present invention will be described. The nucleic acid delivery composition of the present invention contains (I) a block copolymer having (a) a polyethylene glycol segment and (b) a polycaprolactone segment.

[0026] The (a) polyethylene glycol segment constituting the block copolymer (I) contains an ethyleneoxy group (—CH ２ CH ２The (a) polyethylene glycol segment is a segment containing a polyethylene glycol chain having a repeating structure of (O-) units. One end of the (a) polyethylene glycol segment is linked directly or via a bonding group to the (b) polycaprolactone segment described below. The other end may be a hydroxy group at the polyethylene glycol terminal, or may be any terminal group obtained by modifying the terminal hydroxy group. Specific examples of the other terminal group include, but are not limited to, an alkoxy group having 1 to 12 carbon atoms, an alkenyloxy group having 1 to 12 carbon atoms which may have a substituent, and an aralkyloxy group having 7 to 20 carbon atoms which may have a substituent. Of these, an alkoxy group having 1 to 6 carbon atoms which may have a substituent is preferred, an alkoxy group having 1 to 3 carbon atoms which may have no substituent is more preferred, and a methoxy group is particularly preferred.

[0027] Furthermore, the (a) polyethylene glycol segment may have a targeting molecule via the above-mentioned terminal group. Examples of targeting molecules include sugars, lipids, peptides, proteins, and derivatives thereof, as well as folic acid. Among these, sugars (e.g., glucose, sucrose, etc.) are used from the viewpoint of highly specific and efficient delivery to the brain. Furthermore, receptor ligands, antibodies, and peptide or protein fragments thereof are used from the viewpoint of highly specific and efficient delivery to the organ by interacting with various proteins on the surface of nerve cells in the spinal cord.

[0028] The (b) polycaprolactone segment constituting the block copolymer (I) is a homopolymer of ε-caprolactone, a segment made of poly(ε-caprolactone), and a hydrophobic polyester. In the block copolymer (I), the (a) polyethylene glycol segment and the (b) polycaprolactone segment described above may be linked directly or indirectly via a linking group, but are preferably linked directly. The direct linking mode between the (a) polyethylene glycol segment and the (b) polycaprolactone segment is preferably an ester bond formed between a terminal hydroxy group of the (a) polyethylene glycol segment and a terminal carboxy group of the (b) polycaprolactone segment. When the (a) polyethylene glycol segment and the (b) polycaprolactone segment are indirectly linked, the linking group is not particularly limited as long as it is a group that chemically links the two polymer segments, and may be a linking group formed from a functional group capable of bonding to the terminal group of the (a) polyethylene glycol segment and the terminal group of the (b) polycaprolactone segment. Preferably, it is an alkylene group having 1 to 6 carbon atoms. The bonding mode of this bonding group to the (a) polyethylene glycol segment is preferably an ether bond via a terminal oxygen atom of the poly(oxyethylene) group, and the bonding mode of this bonding group to the (b) polycaprolactone segment is preferably an amide bond or an ester bond.

[0029] It is important that the block copolymer (I) of the present invention is configured such that the average molecular weight Mb of the (b) polycaprolactone segment is greater than the average molecular weight Ma of the (a) polyethylene glycol segment. Specifically, the average molecular weight Ma of the (a) polyethylene glycol segment is 1,500 to 2,500 or 3,500 to 6,500, and the average molecular weight Mb of the (b) polycaprolactone segment is 1.5 to 3 times, and preferably 2 to 2.5 times, the average molecular weight Ma of the (a) polyethylene glycol segment. This results in a nucleic acid delivery composition that can deliver nucleic acid drugs to central nervous system tissue or the trigeminal nerve with high efficiency via nasal administration. More specifically, the average molecular weight Ma of the (a) polyethylene glycol segment is preferably 1,500 to 2,500 or 3,500 to 6,500, and more preferably 1,800 to 2,200 or 4,000 to 6,000. The average molecular weight Mb of the (b) polycaprolactone segment is adjusted to be 1.5 to 3 times, and preferably 2 to 2.5 times, the average molecular weight Ma of the (a) polyethylene glycol segment, and is preferably 3,500 to 6,500 or 8,000 to 12,000, and more preferably 4,000 to 6,000 or 9,000 to 11,000.

[0030] Specific examples of block copolymers include polyethylene glycol-poly(ε-caprolactone) copolymer and monomethoxypolyethylene glycol-poly(ε-caprolactone) copolymer, with monomethoxypolyethylene glycol-poly(ε-caprolactone) copolymer being particularly preferred. Of these, particularly preferred are monomethoxypolyethylene glycol-poly(ε-caprolactone) copolymers in which the polyethylene glycol has an average molecular weight of 1,800 to 2,200 and the poly(ε-caprolactone) has an average molecular weight of 4,000 to 6,000, and monomethoxypolyethylene glycol-poly(ε-caprolactone) copolymers in which the polyethylene glycol has an average molecular weight of 4,000 to 6,000 and the poly(ε-caprolactone) has an average molecular weight of 9,000 to 11,000.

[0031] [Regarding (II) Peptides] The nucleic acid delivery composition of the present invention also contains (II) a peptide consisting of 8 to 20 amino acid residues modified with a fatty acid. The bond type of the peptide (II) is a peptide bond, and it may be an α-linked or β-linked bond, or a mixture thereof. The amino acid residues constituting the peptide (II) may be natural or unnatural amino acids, and either L- or D-isomers may be used without particular limitation. The peptide (II) is modified with a fatty acid, and the fatty acid may be saturated or unsaturated fatty acids having 8 to 24 carbon atoms. Among these, saturated higher fatty acids (e.g., capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, etc.) or unsaturated higher fatty acids (e.g., oleic acid, elaidic acid, linoleic acid, linolenic acid, eleostearic acid, etc.) are preferred, saturated fatty acids having 16 to 20 carbon atoms are more preferred, and stearic acid is particularly preferred. The fatty acid can be bound to the N-terminal amino group or the C-terminal carboxyl group of the peptide directly or via a linking group, and is more preferably bound to the N-terminal amino group of the peptide directly or via a linking group. Examples of the linking group used to bind the peptide and the fatty acid include -CO-, -O-CO-, -NH-CO-, etc., and -CO- is particularly preferred.

[0032] The number of amino acid residues in the peptide (II) is 8 to 20, preferably 8 to 15, more preferably 8 to 12, and particularly preferably 9 to 11. The amino acid residues constituting the peptide (II) preferably include at least arginine. Arginine is a basic amino acid that is positively charged under neutral conditions and can form a complex with negatively charged nucleic acids through electrostatic interaction. Furthermore, arginine contains a guanidine unit, which enhances intracellular uptake through interaction with cell membranes or organelle membranes such as endosomes, contributing to improved nucleic acid delivery efficiency. For this reason, it is preferable for the peptide to contain at least arginine as an amino acid residue, and the number of arginine residues is preferably 30 to 70%, more preferably 40 to 60%, of the total number of peptide residues. Furthermore, sulfur-based amino acids such as cysteine ​​form intermolecular disulfide bonds, thereby improving the stability of nucleic acid delivery compositions or nucleic acid-containing compositions. Therefore, cysteine ​​is also preferably used as an amino acid residue constituting the peptide (II). Furthermore, because basic amino acids such as histidine can be protonated in an acidic biological environment to form salts, thereby improving the efficiency of nucleic acid delivery, it is preferable to use histidine as the amino acid residue constituting the peptide of (II). For these reasons, the amino acid residues constituting the peptide preferably contain arginine, cysteine, and histidine, and the total number of these three types of amino acid residues is preferably 50 to 100%, more preferably 75 to 100%, of the total number of all peptide residues.

[0033] As the peptide (II), STR(stearic acid)-CHHRRRRHHC (SEQ ID NO: 1) from the N-terminus is particularly preferred. Furthermore, a peptide obtained by modifying a known peptide as a membrane-permeable peptide (CPP) with a fatty acid may also be used. Other examples of membrane-permeable peptides include TAT peptide, oligoarginine such as octaarginine, and (PRP) ｎ , penetratin, etc.

[0034] In the nucleic acid delivery composition of the present invention, the molar ratio of the block copolymer (I) to the peptide (II) is preferably (I):(II) = 0.25:1 to 2:1, more preferably (I):(II) = 0.4:1 to 1.5:1, and particularly preferably (I):(II) = 0.5:1 to 1.25:1. By adjusting the ratio within this range, a nucleic acid delivery composition can be obtained that exhibits favorable physical properties (average particle size and zeta potential) and forms nanoparticles that are excellent in physical property stability and uniformity.

[0035] The nucleic acid delivery composition of the present invention forms nanoparticles by forming a complex between a block copolymer (I) and a peptide (II). These nanoparticles are thought to be formed by the hydrophobic interaction of the polycaprolactone segment (b) of the block copolymer (I) and the modified fatty acid group of the peptide (II) to form a micellar structure (micelle particle). However, the nanoparticles are not limited to micellar particles and may be nanoparticles with other structures. The average particle size of the nanoparticles formed by the nucleic acid delivery composition of the present invention is preferably 50 nm or less, more preferably 40 nm or less, and particularly preferably 30 nm or less. By achieving an average particle size within this range, the delivery of nucleic acids to target tissues such as the central nervous system tissue or trigeminal nerve and the distribution efficiency in the target tissue can be improved. Note that, in this specification, the average particle size refers to the average particle size measured by dynamic light scattering and can be measured using a light scattering particle size measurement device (e.g., a Zetasizer Ultra manufactured by Malvern Panalytical). The light scattering particle size measuring device can measure the cumulant average particle size and the mass average particle size, and preferably the cumulant average particle size measured by Zetasizer Ultra manufactured by Malvern Panalytical is used.

[0036] Furthermore, the polydispersity index (PDI), measured by dynamic light scattering and determined by cumulant analysis, is an index for evaluating the width (breadth) of particle size distribution. The polydispersity index of the nanoparticles formed by the nucleic acid delivery composition of the present invention is preferably 0.3 or less, more preferably 0.2 or less, and particularly preferably 0.15 or less. By setting the PDI of the nanoparticles within this range, a nucleic acid delivery composition can be obtained that forms nanoparticles with excellent physical property stability and uniformity.

[0037] Furthermore, the zeta potential of the nanoparticles formed by the nucleic acid delivery composition of the present invention is preferably 1 to 30 mV, more preferably 1 to 20 mV, and particularly preferably 5 to 15 mV. By setting the zeta potential of the nanoparticles within this range, it is possible to improve the delivery of nucleic acids to target tissues, such as central nervous system tissues or trigeminal nerves, and the distribution efficiency in the target tissues.

[0038] The nucleic acid delivery composition of the present invention can also contain components other than the above-mentioned components (I) and (II) as needed. Specifically, for example, pharmaceutically acceptable additives commonly used to prepare nasal nucleic acid-containing compositions may be included. Examples of additives that can be used include excipients, bulking agents, fillers, binders, wetting agents, lubricants, surfactants, disintegrants, solvents, solubilizers, dispersants, buffers, stabilizers, suspending agents, solubilizers, preservatives, antiseptics, flavoring agents, soothing agents, isotonic agents, thickeners, dyes, and fragrances. Additives may be used alone or in combination of two or more in any ratio. Details such as the type and amount of these other components can be appropriately determined by those skilled in the art depending on the purpose, application, and method of use of the nucleic acid delivery composition or nucleic acid-containing composition of the present invention.

[0039] Next, a method for producing the nucleic acid delivery composition of the present invention will be described. The nucleic acid delivery composition of the present invention is not particularly limited, but can be obtained by separately preparing a block copolymer solution (I) obtained by dissolving or dispersing the block copolymer in a water-soluble organic solvent and a peptide solution (II) obtained by dissolving or dispersing the peptide in an aqueous solution, mixing and stirring the block copolymer solution and the peptide solution, and then removing the organic solvent. The organic solvent used to prepare the block copolymer solution may be alcohols such as ethanol or methanol, or ethers such as tetrahydrofuran and 1,4-dioxane, acetone, or acetonitrile. The aqueous solution used to prepare the peptide solution may be water itself, a buffer solution (e.g., PBS, HEPES), physiological saline, or an aqueous glucose solution. When mixing the block copolymer solution and the peptide solution, any mixing method known in the art for producing nucleic acid delivery compositions and nucleic acid-containing compositions may be used. Methods for removing the organic solvent from the mixed solution include methods using an ultrafiltration membrane (e.g., dialysis or methods using a centrifugal ultrafiltration device) and solvent evaporation, with the method using an ultrafiltration membrane being preferred. The pH of the solutions of each component and the mixed solution thereof can be adjusted appropriately within a range that does not inhibit the nanoparticle-forming ability. The pH is preferably 5 to 9, more preferably 6.5 to 8.0, and even more preferably 7.0 to 8.0. The pH can be easily adjusted by using a buffer solution as the solvent. The salt concentration of the solutions of each component and the buffer solution of the mixed solution thereof can be adjusted appropriately as long as it does not inhibit the particle-forming ability.

[0040] Furthermore, the nucleic acid delivery composition of the present invention can also be prepared using a microfluidic device method, in which multiple solutions are mixed in a mixing channel with a special channel structure. In particular, a dispersion containing the nucleic acid delivery composition of the present invention can be easily obtained by using an iLiNP microfluidic chip (manufactured by Lilac Pharma) made of quartz glass. Specifically, a dispersion containing nanoparticles of the nucleic acid delivery composition can be produced by mixing a block copolymer solution and a peptide solution in the iLiNP microfluidic chip. The average particle size of the resulting nanoparticles of the nucleic acid delivery composition can be controlled by varying the polymer concentration in the block copolymer solution, the peptide concentration in the peptide solution, and the total flow rate (TFR) and flow rate ratio (FRR) of the block copolymer solution and the peptide solution. The dispersion prepared by this method contains nanoparticles of the nucleic acid delivery composition and the solvents used in each solution. However, the solvents can be removed and exchanged by processes such as dialysis or ultrafiltration, thereby concentrating the dispersion.

[0041] [Regarding the Nucleic Acid-Containing Composition] The nucleic acid-containing composition according to the present invention is a composition comprising the above-described nucleic acid delivery composition and a nucleic acid. The nucleic acid-containing composition can be administered intranasally to deliver nucleic acid molecules to central nervous system tissue or the trigeminal nerve via the nose-to-brain pathway and introduce them into cells. By using the present invention, a physiologically active nucleic acid can be delivered to the trigeminal nerve or central nervous system tissue while avoiding various nucleic acid-degrading factors in the body, and the nucleic acid can be introduced into cells and released into the cells by escaping from the endosome, allowing the nucleic acid to exert its functions.

[0042] In the present invention, the nucleic acid contained in the nucleic acid-containing composition is not particularly limited, and examples thereof include DNA, RNA, other natural nucleic acids, and modified nucleic acids thereof. Furthermore, the nucleic acid may be a single-stranded nucleic acid or a double-stranded nucleic acid. Examples of the nucleic acid include antisense nucleic acids, siRNA, miRNA, mRNA, shRNA, pre-miRNA, pri-miRNA, plasmid DNA, decoy nucleic acids, ribozymes, DNA aptamers, RNA aptamers, DNA enzymes, and various inhibitory genes. Among these, nucleic acids for treating central nervous system tissues are preferably used in the present invention. In particular, RNA (siRNA or miRNA) or antisense nucleic acids that have the effect of inhibiting the expression of target genes using RNA interference are preferred, with siRNA or antisense nucleic acids being more preferred.

[0043] The content ratio of peptide (II) to nucleic acid in the nucleic acid-containing composition of the present invention is expressed by the N / P ratio (the value obtained by dividing the value of N by the value of P), which is defined as the ratio of the total number of moles of cationic groups derived from the peptide (II) (N) to the total number of moles of phosphate groups (anionic groups) derived from the nucleic acid (P). In the nucleic acid-containing composition of the present invention, the N / P ratio is preferably 1 to 50, more preferably 3 to 30, and even more preferably 5 to 20. By setting the N / P ratio within the above-mentioned range, it is possible to achieve both improved physical stability and improved intracellular introduction rate.

[0044] In the present invention, when nucleic acid-containing compositions are prepared by adding nucleic acid to a nucleic acid delivery composition containing a block copolymer (I) and a peptide (II), it is preferable that a complex is formed by the interaction of these components. Because the peptide (II) has a modified fatty acid group, the anionic-charged nucleic acid and the cationic-charged peptide (II) associate through electrostatic interaction, and further, the (b) polycaprolactone segment of the block copolymer (I) associates with the fatty acid group of the peptide (II) through hydrophobic interaction, thereby forming nanoparticles with a micellar structure. The physical properties of the nanoparticles formed from the nucleic acid-containing composition are similar to those of the nucleic acid delivery composition described above, and the average particle size of the nanoparticles is preferably 50 nm or less, more preferably 40 nm or less, and particularly preferably 30 nm or less. Furthermore, the polydispersity index (PDI) is preferably 0.3 or less, more preferably 0.2 or less, and particularly preferably 0.15 or less. The zeta potential of the nanoparticles is preferably 1 to 30 mV, more preferably 1 to 20 mV, and particularly preferably 5 to 15 mV.

[0045] The method for preparing the nucleic acid-containing composition of the present invention can be carried out, for example, by mixing solutions in which the block copolymer (I), the peptide (II), and the nucleic acid are individually dissolved or dispersed. From the viewpoint of being able to produce nucleic acid-containing compositions that form nanoparticles with a particle diameter of approximately 50 nm or less with high reproducibility, a nucleic acid delivery composition dispersed in water can be prepared by the above-described method, and then mixing the composition with an aqueous solution of nucleic acid to obtain a dispersion in which the nucleic acid-containing composition is dispersed in water. The resulting nucleic acid-containing composition solution can be further subjected to appropriate operations such as dilution, stirring, ultrasonic irradiation, dialysis, and concentration.

[0046] In addition to the above-mentioned components, the nucleic acid-containing composition of the present invention can also contain other drugs than nucleic acids, pharmacologically acceptable carriers for conventional formulations, and additives that are acceptable as additives for pharmaceuticals.

[0047] The pharmaceutical of the present invention contains, as an active ingredient, a nucleic acid-containing composition containing the above-described nucleic acid for treating central nervous system tissue. This allows use in the prevention, amelioration, or treatment of pathologies, symptoms, or diseases involving damage or dysfunction of central nervous system tissue. Treatment and amelioration of pathologies herein also include prevention and prognosis of diseases or conditions of central nervous system tissue. Diseases of central nervous system tissue include, for example, amyotrophic lateral sclerosis (ALS), spinocerebellar degeneration, spinal muscular atrophy, primary lateral sclerosis, spinal-bulbar muscular atrophy, chronic pain, spinal cord injury, spinal cord tumor, brain tumor, cerebrovascular disorder, Parkinson's disease, viral infection, Alzheimer's disease, dementia, and the like, or symptoms or diseases related thereto. Pharmaceuticals containing the nucleic acid-containing composition of the present invention as an active ingredient can be used to treat or ameliorate one or more of these pathologies, symptoms, or diseases, or to suppress or prevent symptoms or conditions.

[0048] The administration route of a pharmaceutical containing the nucleic acid-containing composition of the present invention is intranasal administration. Nasal administration allows the nucleic acid pharmaceutical contained in the nucleic acid delivery composition to be efficiently delivered to central nervous system tissue or the trigeminal nerve. Therefore, the nucleic acid-containing composition and pharmaceutical of the present invention can be in any dosage form suitable for intranasal administration. Examples of dosage forms suitable for intranasal administration include liquid formulations, aerosol formulations, and powder formulations. The dosage or effective intake of a pharmaceutical containing the nucleic acid-containing composition of the present invention as an active ingredient is not particularly limited, as it varies depending on the type and content of the nucleic acid contained in the nucleic acid delivery composition, the desired therapeutic effect, the administration method, the subject, and the dosage form.

[0049] The present invention will be described in detail below with reference to examples and comparative examples, but the present invention is not particularly limited to these examples. The components of each nucleic acid delivery composition and nucleic acid-containing composition prepared in the following examples and comparative examples are shown below.

[0050] (I) Block copolymers: Methoxypolyethylene glycol-poly(ε-caprolactone), {molecular weight of polyethylene glycol segment by HNMR: 1,800 to 2,200, molecular weight of polycaprolactone segment by HNMR: 4,000 to 6,000 (hereinafter also referred to as "mPEG2k-PCL5k")}, manufactured by Sigma-Aldrich (product number 900648, Methoxy poly(ethylene glycol)-block-poly(ε-caprolactone), 2k-5k) Methoxypolyethylene glycol-poly(ε-caprolactone), {molecular weight of polyethylene glycol segment by HNMR: 4,000 to 6,000, molecular weight of polycaprolactone segment by HNMR: 9,000 to 11,000 (hereinafter also referred to as "mPEG5k-PCL10k")}, manufactured by Sigma-Aldrich (product number 900672, Methoxy poly(ethylene glycol)-block-poly(ε-caprolactone), 5k-10k) Methoxypolyethylene glycol-poly(ε-caprolactone), {molecular weight of polyethylene glycol segment by HNMR: 1,800 to 2,200, molecular weight of polycaprolactone segment by HNMR: 1,800 to 2,200 (hereinafter also referred to as "mPEG2k-PCL2k")}, manufactured by Sigma-Aldrich (product number 900649, Methoxy poly(ethylene glycol)-block-poly(ε-caprolactone), 2k-2k) (II) Peptide: A peptide consisting of the amino acid sequence: CHHRRRRHHC (SEQ ID NO: 1), in which the N-terminal amino group is modified with stearic acid (hereinafter referred to as "STR-CH ２ R ４ H ２ C), manufactured by Bex Co., Ltd. Amino acid sequence: stearic acid unmodified peptide consisting of CHHRRRRHHC (SEQ ID NO: 2) (hereinafter referred to as "HO-CH ２ R ４ H ２ Also known as "C"), manufactured by Becks Co., Ltd.

[0051] Example 1 1. Preparation of Nucleic Acid Delivery Composition In this example, a nucleic acid delivery composition was prepared by the mixing / stirring-ultrafiltration method. The (I) block copolymer and (II) peptide constituting the nucleic acid delivery composition were combined as shown in Table 1 below, Tests 1-1 to 1-6, and multiple nucleic acid delivery compositions were prepared with different molar ratios of the (I) block copolymer and (II) peptide.

[0052]

[0053] The preparation method was as follows. First, (I) a block copolymer solution was prepared by dissolving the block copolymer in tetrahydrofuran (THF). On the other hand, (II) a peptide solution was prepared by dissolving the peptide in HEPES buffer (10 mM, pH 7.4). Furthermore, (±) dithiothreonol (DTT) was dissolved in HEPES buffer (10 mM, pH 7.4) to prepare a 100 mM DTT / HEPES solution.

[0054] Using each solution prepared as described above, composite particles of (I) block copolymer and (II) peptide were prepared. Preparation was carried out under conditions of 20°C. First, a DTT / HEPES solution was added to the peptide solution so that 61.7 nmol of DTT was present per 1 nmol of (II) peptide, and HEPES buffer (10 mM, pH 7.4) was added until the total solution volume reached 150 μL. This peptide solution was stirred and mixed using a vortex mixer and then allowed to stand for 15 minutes. After standing, 60 μL of block copolymer solution, each adjusted to a concentration such that the molar ratio of (I) block copolymer to 1 mol of (II) peptide was in the range of 0.25 mol to 2 mol, was added dropwise, and the mixture was stirred and mixed using a vortex mixer, followed by standing for 15 minutes. Next, 290 μL of HEPES buffer (10 mM, pH 7.4) was placed into the pretreated centrifugal filter unit (Amicon Ultra, membrane NMWL3,000-0.5 mL, manufactured by Merck) and the mixture of the block copolymer solution and peptide solution was added dropwise. The mixture was then centrifuged at 20°C and 4,700 G using a refrigerated high-speed centrifuge (model number: MDX-310, manufactured by Tomy Seiko Co., Ltd.) to perform ultrafiltration. The solution was diluted with HEPES buffer (10 mM, pH 7.4) and centrifuged until the concentration of residual THF in the solution was 0.1% or less, and the solution in the filter unit was concentrated to 100 μL. The entire solution in the filter unit was recovered, yielding a nucleic acid delivery composition in which complex particles of (I) block copolymer and (II) peptide were dispersed in the solution. The average particle size (cumulant average particle size measured by dynamic light scattering), polydispersity index (PDI), and zeta potential (particle charge) of the nucleic acid delivery composition were measured using a light scattering analyzer (Zetasizer Ultra, Malvern Panalytical). The measurement temperature was 25°C. The results are shown in Figures 1 and 2. The prepared nucleic acid delivery composition was transferred to a glass vial and refrigerated at 4°C. The physical properties of the complex particles of the nucleic acid delivery composition were measured over time to evaluate the storage stability of the nucleic acid delivery composition. The results are shown in Figure 3.

[0055] Figure 1 shows the physical properties of each nucleic acid delivery composition obtained by combining the (I) block copolymer and the (II) peptide in Tests 1-1 to 1-6. The molar ratio of the (I) block copolymer to the (II) peptide for each nucleic acid delivery composition shown in the graph was (I) block copolymer:(II) peptide = 1:1 for mPEG2k-PCL2k and mPEG2k-PCL5k, and (I) block copolymer:(II) peptide = 0.5:1 for mPEG5K-PCL10K. The number n was 3.

[0056] As shown in Figure 1(a), nanoparticles with an average particle size of less than 40 nm were formed in all test nucleic acid delivery compositions. On the other hand, as shown in Figure 1(b), for all polymer molecular weights, the nucleic acid delivery composition prepared with the stearic acid-modified basic peptide exhibited a positive zeta potential derived from the basic peptide, whereas the composition prepared with the unmodified stearic acid peptide exhibited a near-neutral zeta potential. This suggests that the use of stearic acid-modified peptides results in the formation of nanoparticles formed by polymer-peptide complexes, whereas the use of unmodified peptides results in the formation of nanoparticles formed by the polymer-peptide complexes. Furthermore, as shown in Figure 1(c), the polydispersity index (PDI) values ​​decreased in the order mPEG2k-PCL2k > mPEG2k-PCL5k > mPEG5K-PCL10K, indicating the formation of nanoparticles with a uniform particle size.

[0057] Figure 2 shows the physical properties of each nucleic acid delivery composition prepared in Tests 1-1, 1-3, and 1-5 using a combination of (I) block copolymer and (II) stearic acid-modified peptide, with varying molar ratios of (I) block copolymer and (II) peptide. As shown in Figure 2(a), the average particle size of the complex particles for each nucleic acid delivery composition varied depending on the type and molar ratio of the (I) block copolymer. Specifically, the complex particles of the nucleic acid delivery composition formed with mPEG2k-PCL2k (Test 1-1) had a larger average particle size than the other particles, and even at a molar ratio of (I) block copolymer / (II) peptide of 2.0, they had an average particle size of approximately 100 nm. On the other hand, the complex particles of the nucleic acid delivery composition formed with mPEG2k-PCL5k (Test 1-3) consistently maintained an average particle size of 50 nm or less when the molar ratio of (I) block copolymer / (II) peptide was 0.5 or higher. Furthermore, the complex particles of the nucleic acid delivery composition formed with mPEG5k-PCL10k (Tests 1-5) consistently exhibited an average particle size of 50 nm or less at any molar ratio of (I) block copolymer / (II) peptide.

[0058] On the other hand, as shown in Figure 2(b), the zeta potential of the complex particles of each nucleic acid delivery composition tended to decrease with increasing (I) block copolymer / (II) peptide molar ratio, regardless of the molecular weight of the polymer used. The absolute charge order was mPEG2k-PCL2k > mPEG2k-PCL5k > mPEG5k-PCL10k. In particular, the zeta potential of the complex particles of the nucleic acid delivery composition formed with mPEG2k-PCL5k and mPEG5k-PCL10k significantly decreased with increasing (I) block copolymer / (II) peptide molar ratio. Furthermore, as shown in Figure 2(c), the polydispersity index (PDI) of the complex particles of each nucleic acid delivery composition was high (0.3 or higher) for particles formed with mPEG2k-PCL2k, an index of particle size uniformity, and the manufacturing reproducibility was poor. On the other hand, for particles of the nucleic acid delivery composition using mPEG2k-PCL5k, the PDI decreased with increasing molar ratio of (I) block copolymer / (II) peptide, reaching a minimum of 0.2 or less. In particular, particles of the nucleic acid delivery composition using mPEG5k-PCL10k showed very high uniformity, with a PDI of approximately 0.1.

[0059] 3 shows the physical properties over time of long-term storage of nanoparticles of each nucleic acid delivery composition prepared by combining the (I) block copolymer and the (II) peptide in Tests 1-3 and 1-5. Note that the molar ratio of the (I) block copolymer to the (II) peptide in each nucleic acid delivery composition shown in the graph is (I) block copolymer:(II) peptide = 1:1 for mPEG2k-PCL5k (Test 1-3) and (I) block copolymer:(II) peptide = 0.5:1 for mPEG5k-PCL10k (Test 1-5).

[0060] As shown in the graphs in Figure 3(a), the composite particles of the nucleic acid delivery composition containing mPEG2k-PCL5k and mPEG5k-PCL10k components maintained good average particle size, zeta potential, and PDI over a three-week period, demonstrating high physical property stability. Furthermore, as shown in Figure 3(b), the nanoparticles of the nucleic acid delivery composition containing mPEG5k-PCL10k components exhibited stable average particle size, zeta potential, and PDI over a three-month period. These results demonstrate that the nucleic acid delivery compositions containing mPEG5k-PCL10k and mPEG2k-PCL5k components exhibit excellent physical property stability and uniformity. This is not only important for pharmaceutical distribution but also for in vivo administration, suggesting that these nucleic acid delivery compositions can stably deliver nucleic acids to central nervous system tissues even when administered intranasally.

[0061] Example 2 2. Preparation of nucleic acid (siRNA)-containing composition In this example, two types of nucleic acid-containing compositions containing siRNA as the nucleic acid were prepared. The (I) block copolymer, (II) peptide, and nucleic acid that constitute each nucleic acid-containing composition are shown in Tables 2 and 3 below. In this example, siRNA (manufactured by Bioneer) designed to target the Malat-1 (metastasis associated lung adenocarcinoma transcript 1) gene, which is constitutively expressed in the brain, was used as the nucleic acid. The content ratio of nucleic acid in the nucleic acid-containing composition, "N / P ratio," was calculated using the following formula: N / P ratio = number of cationic groups in nucleic acid delivery composition / number of anionic groups in nucleic acid = (N in (II) peptide that constitutes nucleic acid delivery composition) ＋ (number of moles of peptide in nucleic acid) / (number of moles of PO in nucleic acid) ４ － × number of moles of nucleic acid × number of strands)

[0062]

[0063]

[0064] A 1 mM nucleic acid solution of nucleic acid (siRNA) was prepared using nuclease-free water and stored at -80°C. Using the same procedure and method as in Example 1, a nucleic acid delivery composition was prepared from (I) block copolymer and (II) peptide, resulting in a dispersion of complex particles of the nucleic acid delivery composition dispersed in the solution. HEPES buffer (10 mM, pH 7.4) was added to the nucleic acid solution to an equal volume to the dispersion. An equal amount of this nucleic acid solution was added dropwise to the dispersion of complex particles of the nucleic acid delivery composition, followed by gentle vortexing and allowing to stand for 30 minutes to obtain a nucleic acid-containing composition in which complex particles of the nucleic acid, (I) block copolymer, and (II) peptide were dispersed in the solution. The physical properties of the complex particles in the resulting nucleic acid-containing composition, including the average particle size (cumulant average particle size measured by dynamic light scattering), polydispersity index (PDI), and zeta potential (particle charge), were measured using a light scattering analyzer (Zetasizer Ultra, manufactured by Malvern Panalytical). The measurement temperature was 25° C. The morphology of the complex particles in the nucleic acid-containing composition was also observed using a transmission electron microscope (TEM). The results are shown in Table 4 below and FIG. 4.

[0065]

[0066] According to the results of Figure 4 and Table 4, the nucleic acid-containing composition prepared using mPEG2k-PCL2k (Test 2-1) as the (I) block copolymer not only had a large average particle size of 60 nm, but also had a broad particle size distribution with two peaks, as shown in the graph of Figure 4. Consequently, the PDI was also high, at 0.482. On the other hand, the nucleic acid-containing composition prepared using mPEG2k-PCL5k (Test 2-2) as the (I) block copolymer had a small average particle size of 30 nm and a narrow particle size distribution with a single peak. Consequently, the PDI was also low, at 0.190. These results demonstrate that the nucleic acid-containing composition prepared using mPEG2k-PCL5k exhibits very good physical properties, similar to the nucleic acid delivery composition before the nucleic acid was incorporated.

[0067] Example 3 3. Examination of knockdown effect by nasal administration of nucleic acid (siRNA)-containing composition In this example, the nucleic acid (Malat-1-targeting siRNA)-containing compositions of Test 2-1 and Test 2-2 prepared in Example 2 above were intranasally administered to mice, and the expression level of the Malat-1 gene was confirmed to verify whether the Malat-1 gene, which is constitutively expressed in the brain, was knocked down by the siRNA, thereby verifying the nucleic acid delivery effect.

[0068] Animal experiments were conducted in accordance with a protocol approved under the University of Shizuoka Animal Experimentation Regulations. Five- to six-week-old ICR mice (Japan SLC) were used for administration of the siRNA-containing composition. ICR mice were anesthetized by inhalation with isoflurane using an SN-487 laboratory animal anesthesia device. The anesthetic concentration was 4% for induction and 2% for maintenance. The mice were placed supine, and under inhalation anesthesia, the Malat-1-targeting siRNA-containing composition prepared in Test 2-1 or Test 2-2 was administered intranasally using an anesthesia mask for nasal administration (model number: SN-487-70-09, manufactured by Shinano Seisakusho). Nasal administration was performed by administering 2 μL alternately into the left and right nostrils at 1-minute intervals, for a total of 20 μL, as a single administration. The dose of Malat-1-targeting siRNA was 2 nmol (26.5 μg) / 20 μL. The concentrations and N / P ratios of the administered Malat-1 targeting siRNA-containing compositions are as follows: Malat-1 targeting siRNA: 0.1 nmol / μL (I) Concentration of block copolymer (mPEG2k-PCL2k or mPEG2k-PCL5k): 31.5 nmol / μL (II) Peptide (STR-CH ２ R ４ H ２ Concentration of C): 31.5 nmol / μL ・N / P ratio: 30

[0069] Forty-eight hours after the completion of intranasal administration, mice were euthanized with 5% anesthesia, and the brain, spinal cord, and peripheral tissues were removed. Each removed tissue was washed with 0.9% saline. The brain was fractionated into the olfactory bulb, cerebral cortex, hippocampus, cerebellum, and brainstem (combined midbrain, pons, and medulla oblongata). RNA extraction reagent (RNAiso, Takara Bio Inc.) was added to each removed and fractionated region, and the mixture was homogenized to extract crude total RNA. DNase (product number: 313-03161, Nippon Gene Co., Ltd.) was added to this crude total RNA, and purification was performed by DNase treatment. Reverse transcriptase (ReverTra Ace (registered trademark), Toyobo Co., Ltd.) was added to the purified total RNA, and reverse transcription reaction was performed using a thermal cycler under conditions of 42°C for 40 seconds and then 99°C for 5 seconds to prepare cDNA.

[0070] The resulting cDNA was subjected to qPCR using the SYBR® Green assay. To 2 μL of the prepared cDNA, qPCR reaction solution (SYBR green, manufactured by Promega Corporation), the mMALAT-1 primer set of SEQ ID NOS: 5 and 6 shown in Table 4 below, and nuclease-free water were added, mixed by pipetting, spun down, and then qPCR was performed using a real-time PCR analysis system (CFX connect, manufactured by Bio-Rad Laboratories). qPCR was performed by heating at 95°C for 30 seconds to activate the polymerase, followed by 40 cycles of [95°C for 30 seconds, 58°C for 10 seconds, and 72°C for 20 seconds]. Meanwhile, human glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a housekeeping gene, was selected as an internal standard, and qPCR was performed using the primer set of SEQ ID NOS: 7 and 8 shown in Table 5 below, instead of the mMALAT-1 primer set described above.

[0071]

[0072] Using the Ct values ​​obtained from the qPCR results, the ΔCt values ​​were calculated using the following formula: ΔCt value = Ct value of target gene (Malat-1) - Ct value of reference gene (GAPDH) The ΔΔCt value was calculated by subtracting the ΔCt value of the calibrator sample from the ΔCT value of each sample. The calculated ΔΔCt values ​​were used to calculate the relative expression level of the target gene compared to the untreated group. The results are shown in Figure 5.

[0073] As shown in the graphs in Figure 5, compared to the nucleic acid-containing composition prepared with mPEG2k-PCL2k (Test 2-1), the nucleic acid-containing composition formed from mPEG2k-PCL5k (Test 2-2) with an average particle size of 50 nm or less and a PDI of 0.2 or less exhibited significant target gene knockdown activity throughout the brain and spinal cord. This demonstrates that intranasal administration of the nucleic acid-containing composition formed from mPEG2k-PCL5k (Test 2-2) delivered Malat-1-targeting siRNA to central nervous system tissues and exerted sequence-specific siRNA silencing activity throughout the central nervous system tissues.

[0074] Example 4 4. Preparation of Antisense Oligonucleic Acid-Containing Composition (1) / Preparation by Mixing and Stirring-Ultrafiltration In this example, multiple nucleic acid-containing compositions containing antisense oligonucleic acid (hereinafter also referred to as "ASO") as the nucleic acid were prepared by varying the N / P ratio. The (I) block copolymer, (II) peptide, and nucleic acid constituting the nucleic acid-containing composition are shown in Tables 6 and 7 below. In this example, an antisense oligonucleic acid (manufactured by Gene Design) designed to target the Malat-1 gene was used as the nucleic acid.

[0075]

[0076]

[0077] A 1 mM nucleic acid solution of nucleic acid (ASO) was prepared using nuclease-free water and stored at -80°C. Using the same procedure and method as in Example 1, a nucleic acid delivery composition was prepared from (I) block copolymer and (II) peptide, resulting in a dispersion in which complex particles of the nucleic acid delivery composition were dispersed in the solution. HEPES buffer (10 mM, pH 7.4) was added to the nucleic acid solution to an equal volume to the dispersion. An equal amount of this nucleic acid solution was added dropwise to the dispersion in which complex particles of the nucleic acid delivery composition were dispersed, followed by gentle vortexing and allowing to stand for 30 minutes, resulting in a nucleic acid-containing composition in which complex particles of the nucleic acid, (I) block copolymer, and (II) peptide were dispersed in the solution. Furthermore, by adjusting the concentration of the nucleic acid solution added dropwise to the dispersion in which complex particles of the nucleic acid delivery composition were dispersed, nucleic acid-containing compositions with N / P ratios of 3 to 30 were obtained. The average particle size (cumulant average particle size measured by dynamic light scattering), polydispersity index (PDI), and zeta potential (particle charge) of the complex particles in each of the resulting nucleic acid-containing compositions were measured using a light scattering analyzer (Zetasizer Ultra, manufactured by Malvern Panalytical) (n=4). The measurement temperature was 25°C. The morphology of the nanoparticles in the dispersion was also observed using a transmission electron microscope (TEM). The results are shown in Figure 6.

[0078] The physical properties of the nucleic acid-containing composition prepared in this example are shown in the graph in Figure 6. The physical properties of the complex particles of the nucleic acid delivery composition before the incorporation of nucleic acid were an average particle size of 27.3 nm, a PDI of 0.0467, and a zeta potential of 8.66 mV. After the incorporation of nucleic acid (ASO), the average particle size increased slightly as the amount of nucleic acid contained in the composition increased (the N / P ratio decreased), but the average particle size remained below 50 nm even at an N / P ratio of 3. On the other hand, the zeta potential decreased as the amount of nucleic acid contained in the composition increased (the N / P ratio decreased), and a negative charge was observed at an N / P ratio of 3. Therefore, from the perspective of zeta potential, an N / P ratio of greater than 3 is preferred, 4 or greater is more preferred, and 5 or greater is particularly preferred. The PDI values ​​were small at all N / P ratios, demonstrating high uniformity.

[0079] Example 5 5. Examination of knockdown effect by nasal administration of antisense oligonucleic acid-containing composition (1) In this example, the Malat-1-targeting antisense oligonucleic acid-containing composition of Test 4 prepared in Example 4 above was intranasally administered to mice, and the nucleic acid delivery effect was verified by confirming the expression level of the Malat-1 gene to determine whether the Malat-1 gene, which is constitutively expressed in the brain, was knocked down by this ASO.

[0080] Five- to six-week-old ICR mice (Japan SLC) were used for administration of the Malat-1-targeting ASO-containing composition. The ICR mice were anesthetized with isoflurane using an SN-487 laboratory animal anesthesia device. The anesthesia concentration was 4% for induction and 2% for maintenance. The mice were placed supine, and under inhalation anesthesia, the Malat-1-targeting ASO-containing composition prepared in Test 4 was administered intranasally using an anesthesia mask for nasal administration (model number: SN-487-70-09, manufactured by Shinano Seisakusho). Each intranasal administration consisted of 2 μL administered alternately into the left and right nostrils at 1-minute intervals, for a total of 20 μL. The dose of Malat-1-targeting ASO per administration was 2.36 nmol (12.5 μg) / 20 μL. This was administered intranasally twice daily (the second administration was administered 6 hours after the first administration) every other day for three days. The concentrations and N / P ratios of the administered Malat-1 targeting ASO-containing compositions are as follows: Malat-1 targeting antisense oligonucleotide: 0.118 nmol / μL (I) Concentration of block copolymer (PEG5k-PCL10k): 2.36 nmol / μL (II) Peptide (STR-CH ２ R ４ H ２ Concentration of C): 4.72 nmol / μL ・N / P ratio: 10

[0081] One week after the initial intranasal administration, mice were euthanized with 5% anesthesia, and the brain, spinal cord, and peripheral tissues were removed. Each removed tissue was washed with 0.9% saline. The brain was fractionated into the olfactory bulb, hippocampus, midbrain, and brainstem (the combined pons and medulla oblongata). An RNA extraction reagent (RNAiso, manufactured by Takara Bio Inc.) was added to each removed and fractionated region, and the mixture was homogenized to extract crude total RNA. DNase (product number: 313-03161, manufactured by Nippon Gene Co., Ltd.) was added to this crude total RNA, and purification was performed by DNase treatment. Reverse transcriptase (Rever tra Ace (registered trademark), manufactured by Toyobo Co., Ltd.) was added to the purified total RNA, and a reverse transcription reaction was performed using a thermal cycler under conditions of 42°C for 40 seconds, followed by 99°C for 5 seconds, to prepare cDNA.

[0082] The resulting cDNA was subjected to qPCR using the TaqMan assay. 2 μL of the prepared cDNA was mixed with a qPCR reaction solution (TaqMan Fast Advanced Master Mix, Thermo Fisher Scientific), TaqMan Gene Expression Assay (FAM) Malat-1 (Assay ID: Mm01227912_s1, Thermo Fisher Scientific), TaqMan Gene Expression Assay (VIC) β-Actin (Assay ID: Mm02619580_g1, Thermo Fisher Scientific), and nuclease-free water. The mixture was mixed by pipetting and spun down, after which qPCR was performed using a real-time PCR analysis system (CFX connect, Bio-Rad Laboratories). qPCR was performed by incubating at 50°C for 2 minutes, followed by heating at 95°C for 20 seconds to activate the polymerase, and then repeating 40 sets of [95°C for 1 second → 60°C for 20 seconds].

[0083] Using the Ct values ​​obtained from the qPCR results, the ΔCt values ​​were calculated using the following formula: ΔCt value = Ct value of target gene (Malat-1) - Ct value of reference gene (β-actin) The ΔΔCt value was calculated by subtracting the ΔCt value of the calibrator sample from the ΔCT value of each sample. The calculated ΔΔCt values ​​were used to calculate the relative expression level of the target gene compared to the untreated group. The results are shown in Figure 7.

[0084] As shown in the graph in Figure 7, an ASO-containing composition formed from mPEG5k-PCL10k and having physical properties such as an average particle size of 40 nm or less, a PDI of 0.1 or less, and a zeta potential of 5-10 mV demonstrated target gene knockdown activity throughout the brain and spinal cord. This demonstrates that nasal administration of an ASO-containing composition formed from mPEG5k-PCL10k delivered Malat-1-targeting ASO to central nervous system tissue, resulting in sequence-specific silencing of the ASO throughout central nervous system tissue.

[0085] Example 6 6. Preparation of Antisense Oligonucleic Acid-Containing Composition (2) / Microfluidic Device Method In this example, multiple nucleic acid-containing compositions containing antisense oligonucleic acids (ASOs) were prepared by the microfluidic device method, with varying N / P ratios. The (I) block copolymer, (II) peptide, and nucleic acid constituting the nucleic acid-containing compositions were the same as those used in Examples 4 and 5 (see Tables 6 and 7).

[0086] In this example, an ASO-containing composition was prepared by a microfluidic device method using iLiNP (manufactured by Lilac Pharma), which is capable of controlling the stirring efficiency of each solution within the device. Sample preparation was carried out under conditions of 20°C. The total flow rate (TFR) of the solutions flowing through the microfluidic device was set to 250 μL / min. The flow rate (FRR) of the peptide solution before mixing relative to the flow rate of the block copolymer solution before mixing was set to 9. The concentrations of the block copolymer solution before mixing and the peptide solution before mixing were calculated using the following formulas: - Concentration of block copolymer solution before mixing = Final block copolymer concentration × (FRR + 1) - Concentration of peptide solution before mixing = (Final block copolymer concentration × (FRR + 1)) / (FRR × molar ratio of block copolymer / peptide)

[0087] THF was added to a weighed amount of block copolymer to achieve the calculated concentration of the pre-mixing block copolymer solution, and the mixture was completely dissolved using a vortex mixer. To the pre-mixing peptide solution (dissolved in 10 mM HEPES buffer, pH 7.4), 61.7 mol / L of DTT was added per 1 mol / L of peptide, and the mixture was mixed using a vortex mixer to prepare a peptide solution. The block copolymer solution and peptide solution were each loaded into a glass microsyringe. A complex particle dispersion of the nucleic acid delivery composition was prepared by extruding the solution into a glass nanoparticle manufacturing device iLiNP using a high-precision syringe pump (PUMP 11 ELITE, Harvard) under the above-mentioned conditions. The resulting complex particle dispersion of the nucleic acid delivery composition was dialyzed for 120 minutes using a dialysis tube with a molecular weight cutoff of 12,000 to 14,000 (Spectra / Por (registered trademark) 4, 132697, manufactured by REPLIGEN) by filling the external solution with a 500-fold amount of HEPES buffer solution (10 mM, pH 7.4) relative to the sample, while stirring the external solution at 60 rpm with a stirrer.

[0088] On the other hand, a 1 mM nucleic acid solution of nucleic acid (ASO) was prepared using nuclease-free water and stored at -80°C. A nucleic acid delivery composition was prepared from (I) block copolymer and (II) peptide using a microfluidic device method, resulting in a dispersion in which complex particles of the nucleic acid delivery composition were dispersed in the solution. HEPES buffer (10 mM, pH 7.4) was added to the nucleic acid solution to an equal volume to the dispersion. An equal amount of this nucleic acid solution was added dropwise to the dispersion in which complex particles of the nucleic acid delivery composition were dispersed, followed by gentle vortexing and allowing to stand for 30 minutes, resulting in a nucleic acid-containing composition in which complex particles of the nucleic acid, (I) block copolymer, and (II) peptide were dispersed in the solution. Furthermore, by adjusting the concentration of the nucleic acid solution added dropwise to the dispersion in which complex particles of the nucleic acid delivery composition were dispersed, nucleic acid-containing compositions with N / P ratios of 3 to 30 were obtained. The average particle size (cumulant average particle size measured by dynamic light scattering), polydispersity index (PDI), and zeta potential (particle charge) of the complex particles in each of the resulting nucleic acid-containing compositions were measured using a light scattering analyzer (Zetasizer Ultra, manufactured by Malvern Panalytical) (n=4). The measurement temperature was 25°C. The morphology of the nanoparticles in the dispersion was also observed using a transmission electron microscope (TEM). The results are shown in Figure 8.

[0089] The physical properties of the nucleic acid-containing composition prepared in this example are shown in the graph in Figure 8. The physical properties of the complex particles of the nucleic acid delivery composition before the incorporation of nucleic acid were an average particle size of 31.3 nm, a PDI of 0.0494, and a zeta potential of 9.11 mV. After the incorporation of nucleic acid (ASO), the average particle size increased slightly as the amount of nucleic acid contained in the composition increased (the N / P ratio decreased), but the average particle size remained below 40 nm even at an N / P ratio of 3. On the other hand, the zeta potential decreased as the amount of nucleic acid contained in the composition increased (the N / P ratio decreased), and a negative charge was observed at an N / P ratio of 3. Therefore, from the perspective of zeta potential, an N / P ratio of 5 or more was found to be preferable. The PDI value was small, below 0.1, at all N / P ratios, demonstrating high uniformity. Furthermore, there was no significant difference in physical properties between the nucleic acid-containing composition prepared by mixing and stirring and ultrafiltration in Example 4 and the nucleic acid-containing composition prepared by the microfluidic device method in this Example, and it was found that the nucleic acid delivery composition and nucleic acid-containing composition of the present invention can also be prepared by the microfluidic device method.

[0090] Example 7 7. Examination of knockdown effect by nasal administration of antisense oligonucleic acid-containing composition (2) In this example, a Malat-1-targeted antisense oligonucleic acid-containing composition prepared by the microfluidic device method in Example 6 above was intranasally administered to mice, and the nucleic acid delivery effect was verified by checking the expression level of the Malat-1 gene and whether the Malat-1 gene was knocked down by this ASO. The ASO-containing composition was administered using the same method and procedure as in Example 5. In this example, the concentration and N / P of each component of the administered Malat-1-targeted ASO-containing composition are shown below. Malat-1-targeted ASO: 0.118 nmol / μL (I) Concentration of block copolymer (PEG5k-PCL10k): 1.18 nmol / μL or 2.36 nmol / μL (II) Peptide (STR-CH ２ R ４ H ２ C) Concentration: 2.36 nmol / μL or 4.72 nmol / μL N / P ratio: 5 or 10

[0091] One week after the initial intranasal administration, the mice were euthanized with 5% anesthesia, and the brain, spinal cord, and peripheral tissues were removed. Each removed tissue was washed with 0.9% saline. The brain was fractionated into the olfactory bulb, cerebral cortex, hippocampus, cerebellum, midbrain, and brainstem (the combined pons and medulla oblongata). Total RNA was extracted from each removed and fractionated region using the same method as in Example 5, and cDNA was prepared by reverse transcription. qPCR was performed on the obtained cDNA using TaqMan assay to calculate the expression ratio of the target gene relative to the untreated group. The results are shown in Figure 9.

[0092] As shown in the graph in Figure 9, ASO-containing compositions formed from mPEG5k-PCL10k and having physical properties of an average particle size of 40 nm or less and a PDI of 0.1 or less demonstrated target gene knockdown activity throughout the brain and spinal cord. Among these, the ASO-containing composition with an N / P ratio of 10 exhibited higher knockdown activity in many central nervous system tissues. This is presumably because the zeta potential of the composition with an N / P ratio of 10 was in the range of 5-10 mV compared to the composition with an N / P ratio of 5. These results demonstrate that intranasal administration of an ASO-containing composition formed from mPEG5k-PCL10k delivered Malat-1-targeting ASO to central nervous system tissues, resulting in sequence-specific ASO silencing throughout the central nervous system.

[0093] Example 8 8. Preparation of Antisense Oligonucleic Acid-Containing Composition (3) In this example, multiple nucleic acid-containing compositions containing antisense oligonucleic acids targeting human superoxide dismutase 1 (hSOD1) as the nucleic acid were prepared with varying N / P ratios. The (I) block copolymer, (II) peptide, and nucleic acid constituting the nucleic acid-containing composition are shown in Tables 8 and 9 below. The hSOD1-targeting antisense oligonucleic acid used in this example was an antisense oligonucleic acid (manufactured by Cosmobio) having a sequence similar to that of QALSODY (Tofersen), a drug for treating amyotrophic lateral sclerosis (ALS).

[0094]

[0095]

[0096] A 1 mM nucleic acid solution of nucleic acid (ASO) was prepared using nuclease-free water and stored at -80°C. Using the same procedure and method as in Example 1, a nucleic acid delivery composition was prepared from (I) block copolymer and (II) peptide, resulting in a dispersion in which complex particles of the nucleic acid delivery composition were dispersed in the solution. HEPES buffer (10 mM, pH 7.4) was added to the nucleic acid solution to an equal volume to the dispersion. An equal amount of this nucleic acid solution was added dropwise to the dispersion in which complex particles of the nucleic acid delivery composition were dispersed, followed by gentle vortexing and allowing to stand for 30 minutes, resulting in a nucleic acid-containing composition in which complex particles of the nucleic acid, (I) block copolymer, and (II) peptide were dispersed in the solution. Furthermore, by adjusting the concentration of the nucleic acid solution added dropwise to the dispersion in which complex particles of the nucleic acid delivery composition were dispersed, nucleic acid-containing compositions with N / P ratios of 5 to 30 were obtained. The average particle size (cumulant average particle size measured by dynamic light scattering), polydispersity index (PDI), and zeta potential (particle charge) of the complex particles in each of the resulting nucleic acid-containing compositions were measured using a light scattering analyzer (Zetasizer Ultra, manufactured by Malvern Panalytical). The measurement temperature was 25°C. The morphology of the nanoparticles in the dispersion was also observed using a transmission electron microscope (TEM).

[0097] The physical properties of the ASO-containing compositions prepared in this example are shown in the graphs (a) and (b) of Figure 10. (I) The ASO-containing compositions prepared using mPEG2k-PCL5k (Test 8-1) and mPEG5k-PCL10k (Test 8-2) as block copolymers had average particle sizes of 50 nm or less and PDIs of 0.3 or less at all N / P ratios, indicating the formation of highly uniform particles. On the other hand, the zeta electron charge of the ASO-containing composition prepared using mPEG2k-PCL5k (Test 8-1) was near zero at an N / P ratio of 5, whereas the nucleic acid-containing composition prepared using mPEG5k-PCL10k (Test 8-2) exhibited a stable positive charge over the range of N / P ratios tested. 10(c) and 10(d) are transmission electron microscope photographs showing nanoparticles in a dispersion of an ASO-containing composition, both of which were observed for a composition with an N / P ratio of 5. The photographs show that uniform particles of approximately the same size, approximately 30 to 40 nm, are formed for both mPEG2k-PCL5k (Test 8-1) and mPEG5k-PCL10k (Test 8-2).

[0098] Example 9 9. Examination of knockdown effect by nasal administration of antisense oligonucleic acid-containing composition (3) In this example, the hSOD1-targeting antisense oligonucleic acid-containing compositions of Test 8-1 and Test 8-2 prepared in Example 8 above were intranasally administered to amyotrophic lateral sclerosis (ALS) model mice, and the nucleic acid delivery effect was verified by confirming whether the hSOD1 gene was knocked down by this ASO and the expression level of the hSOD1 gene.

[0099] The hSOD1-targeted ASO-containing composition was administered to 15-week-old male ALS model mice (human mutant SOD1-G93A transgenic mice, B6SJL-Tg(SOD1*G93A)1Gur / J, Jackson Laboratories). The hSOD1-targeted ASO-containing composition was administered intranasally to the ALS model mice using the same method as in Example 5 above. Each intranasal administration consisted of 2 μL administered alternately into the left and right nostrils at 1-minute intervals, for a total of 20 μL. The dose of hSOD1-targeted ASO per administration was 10 nmol (71.3 μg) / 20 μL. This was administered intranasally twice a day (the second administration was 6 hours after the first administration) every other day for three days. The concentrations of each component and the N / P ratio of the administered hSOD1-targeted ASO-containing composition are shown below. hSOD1 target ASO concentration: 0.5 nmol / μL (I) Block copolymer (PEG2k-PCL5k) concentration: 12.5 nmol / μL (I) Block copolymer (PEG5k-PCL10k) concentration: 6.25 nmol / μL (II) Peptide (STR-CH ２ R ４ H ２ Concentration of C): 12.5 nmol / μL ・N / P ratio: 5

[0100] One week after the initial intranasal administration, the mice were euthanized with 5% anesthesia, and the brainstem (the combined midbrain, pons, and medulla oblongata), spinal cord, and peripheral tissues were removed. Each of the removed tissues was washed with 0.9% saline. The spinal cord was fractionated into the upper spinal cord (cervical to thoracic spinal cord region) and the lower spinal cord (lumbar to sacral spinal cord region). Total RNA was extracted from each of the removed and fractionated regions using the same method as in Example 5, and cDNA was prepared by reverse transcription.

[0101] The resulting cDNA was subjected to qPCR using the SYBR® Green assay. To 2 μL of the prepared cDNA, qPCR reaction solution (SYBR green, manufactured by Promega Corporation), the SOD1 primer set of SEQ ID NOS: 11 and 12 shown in Table 9 below, and nuclease-free water were added, mixed by pipetting, spun down, and then qPCR was performed using a real-time PCR analysis system (CFX connect, manufactured by Bio-Rad Laboratories). qPCR was performed by heating at 95°C for 30 seconds to activate the polymerase, followed by 40 cycles of [95°C for 30 seconds → 58°C for 10 seconds → 72°C for 20 seconds]. Meanwhile, human glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a housekeeping gene, was selected as an internal standard, and qPCR was performed using the primer set of SEQ ID NOS: 7 and 8 shown in Table 10 below, instead of the SOD1 primer set described above.

[0102]

[0103] Using the Ct values ​​obtained from the qPCR results, ΔCt and ΔΔCt values ​​were calculated in the same manner as in Example 5, and the relative expression level of the target gene compared to the untreated group was calculated. The results are shown in Figure 11. As shown in the graph in Figure 11, both the ASO-containing composition prepared with mPEG2k-PCL5k (Test 8-1) and the ASO-containing composition prepared with mPEG5k-PCL10k (Test 8-2) as the (I) block copolymer demonstrated target gene knockdown activity throughout the brain and spinal cord. Furthermore, the ASO-containing composition prepared with mPEG5k-PCL10k (Test 8-2) demonstrated higher knockdown activity in the brainstem and lower spinal cord. This is presumably because the ASO-containing composition prepared with mPEG5k-PCL10k (Test 8-2) had a positive zeta potential at the N / P ratio of 5 used in the test. These results demonstrate that by intranasally administering an ASO-containing composition formed from mPEG2k-PCL5k and mPEG5k-PCL10k to ALS model mice, hSOD1-targeting ASO was delivered to central nervous system tissue, and the ASO exerted a sequence-specific silencing effect throughout the central nervous system tissue.

[0104] Example 10 10. Investigation of nucleic acid delivery distribution by nasal administration of antisense oligonucleic acid-containing composition In this example, the hSOD1-targeting antisense oligonucleic acid-containing composition shown in Table 11 below, which was prepared using the same materials and procedures as in Example 8 above, was intranasally administered to cynomolgus monkeys, and the concentration of the hSOD1-targeting antisense oligonucleic acid delivered to central nervous system tissue and the expression level of the hSOD1 gene were confirmed, thereby verifying the nucleic acid delivery effect of the composition of the present invention by intranasal administration.

[0105]

[0106] Cynomolgus monkeys weighing 4-7 kg (Shin-Nihon Kagaku Co., Ltd.) were used to administer the hSOD1-targeted ASO-containing composition. For nasal administration, atropine sulfate was administered intramuscularly to the cynomolgus monkeys to suppress airway mucus secretion, and propofol was administered intravenously to induce anesthesia. The trachea was intubated, and inhalation anesthesia was administered using a mixture of nitrogen gas, oxygen gas (2:1), and isoflurane. The cynomolgus monkeys were placed supine, and an endoscope was inserted into the nasal cavity and secured at the middle turbinate. A catheter was advanced to the vicinity of the cribriform plate of the ethmoid bone. A nanoparticle dispersion of the hSOD1-targeted ASO-containing composition was instilled to terminate administration, and the administration was then completed. The monkeys were maintained under anesthesia until the 30-minute incubation period was over. Administration was performed three times over 24 hours (the second administration was administered approximately 6 hours after the first administration, and the third administration was administered 24 hours after the first administration). The total dose of hSOD1-targeting ASO was 19 to 21 mg per mouse. The concentrations and N / P ratios of the administered hSOD1-targeting ASO-containing compositions are shown below. hSOD1-targeting ASO concentration: 6.55 μmol (46.7 mg) / mL (I) Block copolymer (PEG5k-PCL10k) concentration: 6.25 μmol / μL (II) Peptide (STR-CH ２ R ４ H ２ Concentration of C): 98.25 μmol / mL N / P ratio: 3

[0107] The brain, spinal cord, and trigeminal nerve were collected as follows: After euthanasia under anesthesia 4 hours or 72 hours after the completion of the third intranasal administration, the olfactory bulb, cerebral cortex, striatum, cerebellum, midbrain, pons, medulla oblongata, thoracic spinal cord, lumbar spinal cord, and trigeminal nerve were collected, promptly frozen in liquid nitrogen, and stored frozen in an ultra-low temperature freezer at -70°C or below.

[0108] Cerebrospinal fluid (CSF) was collected as follows. 8, 24, or 72 hours after the third nasal administration, cerebrospinal fluid (CSF) was collected by lumbar puncture under combined anesthesia with intramuscular administration of ketamine hydrochloride or ketamine and medetomidine hydrochloride. Approximately 0.2 mL of CSF was collected. The collected CSF was immediately cooled on ice and centrifuged (4°C, 1700 × g, 5 minutes) to remove blood cell components. Approximately 0.1 mL of the supernatant was dispensed into sample tubes and stored frozen in an ultra-low temperature freezer at -70°C or below.

[0109] [Measurement of the concentration of hSOD1-targeted antisense oligonucleotides delivered to central nervous system tissues] A nucleic acid extraction reagent (RNAiso, manufactured by Takara Bio Inc.) was added to each collected tissue, homogenized, and then centrifuged to collect the supernatant to obtain tissue lysates. Using these tissue lysate samples and cerebrospinal fluid samples, the hSOD1-targeted ASO content in each sample was measured by hybridization ELISA. 50 μL of a template probe (5 nM) with a sequence complementary to the hSOD1-targeted ASO was added to a PCR plate and mixed with the sample solution or standard solution. The PCR plate was sealed, and hybridization was performed in a thermal cycler at 95°C for 5 minutes, followed by 42°C for 1.5 hours. After blocking with blocking buffer (SuperBlock Blocking Buffer, Thermo Fisher Scientific), 40 μL of each sample solution and standard solution after hybridization was added to the assay plate and incubated at 37°C for 60 minutes. On the other hand, detection probe (100 μM, 4.08 μL), T4 Ligase (400 U / μL, 0.6 μL), 10x reaction buffer (300 μL), and ddH２ The assay plate was washed six times with washing buffer, and then 50 μL of the detection probe solution was added to each well. The plate was incubated at 25°C for 60 minutes for ligation, and then stored at 4°C overnight. S1 nuclease (75 U / μL, 1.005 μL), 10x nuclease buffer (75 μL), and ddH ２ 0 (675 μL) was mixed to form solution A. 10× nuclease buffer (608 μL) and ddH ２ The S1 nuclease solution was prepared by mixing the ligated plate with 5467 μL of solution A and adding 675 μL of solution A. After the ligation was completed, the assay plate was washed six times, and then 50 μL of S1 nuclease solution was added to each plate. The plate was sealed and incubated at 37°C for 30 minutes. Next, anti-digoxigenin-AP, Fab fragment (3 μL), blocking buffer (600 μL), and ddH ２ A labeled antibody solution was prepared by mixing 0 (5397 μL). The nuclease-treated plate was washed six times, and 50 μL of the labeled antibody solution was added to each well. The mixture was then incubated at 25°C for 30 minutes. Next, the incubated plate was washed six times, and a substrate solution (QuantaBlu Fluorogenic Peroxidase Substrate Kits, Thermo Fisher Scientific) was added and incubated at 25°C for 15 minutes. After the reaction, 90 μL of the solution from each well of the assay plate was transferred to a measurement plate, and the fluorescence intensity was measured at an excitation wavelength of 325 nm and a detection wavelength of 420 nm using a microplate reader (SpectraMax iD3, Molecular Devices). The hSOD1 target ASO concentration in each sample was calculated from the calibration curve information based on the standard solution. The results are shown in Figure 12.

[0110] In the nose-to-brain route via nasal administration, the drug reaches the central nervous system directly via the olfactory nerve and trigeminal nerve from the nasal mucosa. This is supported by the graphs in Figure 12, which show that the hSOD1-targeted ASO concentration in the olfactory bulb and trigeminal nerve is extremely high 4 hours after nasal administration, and that the hSOD1-targeted ASO is also distributed in other tissues at concentrations on the order of nanograms per tissue. Meanwhile, the hSOD1-targeted ASO concentration in the cerebrospinal fluid was highest 24 hours after nasal administration, which is thought to indicate that the ASO delivered to each central nervous system tissue was excreted via the cerebrospinal fluid. This demonstrates that nasal administration of an ASO-containing composition formed from the nucleic acid delivery composition of the present invention results in highly efficient delivery of the hSOD1-targeted ASO to central nervous system tissues, distribution at high concentrations throughout the central nervous system tissues, and subsequent excretion via the cerebrospinal fluid.

[0111] [Measurement of hSOD1 gene expression levels] Total RNA was extracted from each excised and fractionated site using the same method as in Example 5, and cDNA was prepared by reverse transcription. The resulting cDNA was subjected to qPCR using the TaqMan assay to calculate the expression ratio of the target gene relative to the untreated group. The primers and probes used in the TaqMan assay were TaqMan Gene Expression Assay (FAM) SOD1 (Assay ID: Mf04363557_m1, Thermo Fisher Scientific) and TaqMan Gene Expression Assay (VIC) β-Actin (Assay ID: Mf04354341_g1, Thermo Fisher Scientific). The results are shown in Figure 13.

[0112] As shown in the graph in Figure 13, the hSOD1-targeting ASO-containing composition prepared in this example demonstrated target gene knockdown activity throughout the brain and spinal cord. This demonstrates that nasal administration of an ASO-containing composition formed from the nucleic acid delivery composition of the present invention delivered hSOD1-targeting ASO to central nervous system tissues, resulting in sequence-specific silencing of the ASO throughout central nervous system tissues.

[0113] The present invention is not limited to the above-described embodiments or examples, and its technical scope also includes various modified designs within the scope that does not deviate from the gist of the invention described in the claims.

[0114] The present invention provides a nucleic acid delivery composition and a nucleic acid-containing composition that can deliver nucleic acids to central nervous system tissues such as the brain and spinal cord by nasal administration. The nucleic acid delivery composition and nucleic acid-containing composition of the present invention can be used, for example, as a pharmaceutical for treating diseases of central nervous system tissues, and are therefore widely useful in industries such as the pharmaceutical and medical fields.

Claims

1. A nucleic acid delivery composition used for delivering a nucleic acid to central nervous system tissue or trigeminal nerve, comprising: (I) a block copolymer having (a) a polyethylene glycol segment and (b) a polycaprolactone segment; and (II) a peptide consisting of 8 to 20 amino acid residues modified with a fatty acid, wherein in the block copolymer (I), the (a) polyethylene glycol segment has an average molecular weight Ma of 1,500 to 2,500 or 3,500 to 6,500, and the (b) polycaprolactone segment has an average molecular weight Mb of 1.5 to 3 times the average molecular weight Ma of the (a) polyethylene glycol segment, and wherein the nucleic acid delivery composition is delivered to central nervous system tissue or trigeminal nerve by intranasal administration.

2. The nucleic acid delivery composition described in claim 1, characterized in that the average molecular weight Mb of the (b) polycaprolactone segment in the (I) block copolymer is 3,500 to 6,500 or 8,000 to 12,000.

3. The nucleic acid delivery composition according to claim 1, characterized in that the blending ratio of the block copolymer (I) to the peptide (II) is, in molar ratio, (I):(II) = 0.25:1 to 2:

1.

4. The nucleic acid delivery composition according to claim 1, characterized in that the block copolymer (I) is methoxypolyethylene glycol-poly(ε-caprolactone).

5. The nucleic acid delivery composition according to claim 1, characterized in that the peptide (II) is a peptide consisting of cysteine ​​residues, histidine residues and arginine residues, and the fatty acid that modifies the peptide is stearic acid.

6. The nucleic acid delivery composition according to claim 1, characterized in that the block copolymer (I) and the peptide (II) form nanoparticles.

7. The nucleic acid delivery composition according to claim 6, characterized in that the nanoparticles have an average particle size of 50 nm or less, a polydispersity index (PDI) of 0.3 or less, and a zeta potential of 1 to 20 mV.

8. A nucleic acid-containing composition, further comprising the nucleic acid delivery composition according to any one of claims 1 to 7.

9. The nucleic acid-containing composition according to claim 8, characterized in that the ratio (N / P ratio) of the total number of moles (N) of cationic groups derived from the peptide (II) to the total number of moles (P) of phosphate groups derived from the nucleic acid is 3 to 30.

10. The nucleic acid-containing composition according to claim 8, wherein the nucleic acid is an antisense oligonucleic acid, siRNA or mRNA for treating a disease of central nervous system tissue.

11. A medicine comprising the nucleic acid-containing composition according to claim 8 as an active ingredient.

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