Nucleic acid introduction aid, nucleic acid introduction agent, and nucleic acid introduction method

Abstract

Provided is a nucleic acid introduction aid having a novel composition. The nucleic acid introduction aid comprises an anti-inflammatory agent and / or an antioxidant.

Description

Nucleic Acid Introduction Aid, Nucleic Acid Introduction Agent, and Nucleic Acid Introduction Method

[0001] The present invention relates to a nucleic acid introduction aid, a nucleic acid introduction agent, and a nucleic acid introduction method.

[0002] Techniques for introducing genes such as nucleic acids into cells are known. Examples of methods for introducing nucleic acids into cells include the lipofection method, the electroporation method, the calcium phosphate method, the microinjection method, and the viral vector method (see Patent Document 1 below). In these methods, nucleic acid introduction aids and nucleic acid introduction agents are used when introducing nucleic acids into cells.

[0003] Japanese Patent Application Laid-Open No. 2017-127200

[0004] Regarding the composition of nucleic acid introduction aids and nucleic acid introduction agents used when introducing nucleic acids into cells, there is still room for improvement. Therefore, nucleic acid introduction aids and nucleic acid introduction agents having novel compositions, as well as nucleic acid introduction methods using them, are desired.

[0005] The nucleic acid introduction aid according to one aspect contains at least one of an anti-inflammatory agent and an antioxidant.

[0006] The nucleic acid introduction agent according to one aspect contains the above nucleic acid introduction aid and a nucleic acid or a vector containing a nucleic acid.

[0007] The nucleic acid introduction method according to one aspect includes a step of introducing a nucleic acid into the cell by bringing both a nucleic acid or a vector containing a nucleic acid and the above nucleic acid introduction aid into contact with the cell, or by bringing the above nucleic acid introduction agent into contact with the cell.

[0008] The cell according to one aspect is a cell containing a nucleic acid introduced by the above nucleic acid introduction method.

[0009] The product according to one aspect is a product produced from the above cell.

[0010] Figure 1 is a micrograph of adipose tissue after nucleic acid introduction in Experimental Example 1. Figure 2 is a micrograph of adipose tissue after nucleic acid introduction in Experimental Example 2. Figure 3 is a micrograph of adipose tissue after nucleic acid introduction in Experimental Example 3. Figure 4 is a micrograph of adipose tissue after nucleic acid introduction in Experimental Example 4. Figure 5 is a micrograph of adipose tissue after nucleic acid introduction in Experimental Example 5. Figure 6 is a micrograph of adipose tissue after nucleic acid introduction in Experimental Example 6. Figure 7 is a micrograph of adipose tissue after nucleic acid introduction in Experimental Example 7 for experiment numbers D02, D03, G01, and experiment numbers other than G02. Figure 8 is a micrograph of adipose tissue after nucleic acid introduction in Experimental Example 8. Figure 9 is a micrograph of adipose tissue after nucleic acid introduction in Experimental Example 9. Figure 10 is a micrograph of adipose tissue after nucleic acid introduction in Experimental Example 10. Figure 11 is a micrograph of adipose tissue after nucleic acid introduction in Experimental Example 11. Figure 12 shows micrographs of adipose tissue samples after nucleic acid introduction in Experiments D02, D03, G01, and G02 of Experimental Example 7. Figure 13 shows micrographs of adipose tissue samples after treatment with reactive oxygen species detection reagent in Experimental Example 12. Figure 14 shows micrographs of adipose tissue samples after nucleic acid introduction in Experimental Example 13. Figure 15 shows micrographs of mouse tissue after nucleic acid introduction in Experimental Example 14.

[0011] Embodiments will be described below with reference to the drawings. In this specification, "nucleic acid" includes nucleotides, oligonucleotides, and polynucleotides, and may be DNA, RNA, or DNA-RNA hybrids. These nucleic acids may have a double-stranded or single-stranded form. These nucleic acid molecules may have a cyclic or linear form. These nucleic acid molecules may be artificially synthesized or of biological origin.

[0012] In this specification, the term "gene" includes not only structural genes that define the primary structure of proteins, tRNA, rRNA, etc., but also regions on nucleic acids that have specific regulatory functions, such as promoters or operators, unless otherwise specified. Therefore, unless otherwise specified, the term "gene" refers to regulatory regions, coding regions, exons, and introns without distinction. Furthermore, nucleic acid molecules such as siRNA, miRNA, and ncRNA that interfere with gene expression are also included in the definition of "gene."

[0013] [Method for introducing nucleic acids] The nucleic acid introduction method according to one embodiment relates to a method for introducing nucleic acids into cells. The nucleic acids to be introduced include nucleotides, oligonucleotides, and polynucleotides, and may be DNA, RNA, or DNA-RNA hybrids. The nucleic acids to be introduced may also include modified nucleic acids. The nucleic acid introduction method may be a so-called gene introduction method.

[0014] The nucleic acid delivery method includes the step of introducing nucleic acids into cells by bringing both the nucleic acid delivery aid and the nucleic acid (described later) or the nucleic acid delivery aid (described later) into contact with the cells. The nucleic acid delivery aid may contain the nucleic acid to be introduced or a vector having a region of the nucleic acid to be introduced. On the other hand, the nucleic acid delivery aid may be an aid that does not contain the nucleic acid to be introduced or a vector having a region of the nucleic acid to be introduced. In this case, the nucleic acid delivery aid may be used together with the nucleic acid to be introduced or a vector having a region of the nucleic acid to be introduced.

[0015] The nucleic acid to be introduced may be prepared in a form mounted on a desired vector, or it may be prepared in a form not mounted on a vector (directly in the solvent). The vector is not particularly limited, but may contain at least one selected from the group consisting of plasmids, minicircles, closed-end DNA, retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, aneroviruses, and herpesviruses.

[0016] The step of introducing nucleic acids into cells can be carried out by various known methods, except for the composition of nucleic acid delivery aids and / or nucleic acid delivery agents. Methods for introducing nucleic acids into cells include electrical and / or magnetic perforation, ultrasound, plasma, microinjection, calcium phosphate, lipofection, polyfection, lipid nanoparticles, polymer nanoparticles, inorganic nanoparticles, and virus-like particle methods.

[0017] The step of introducing nucleic acids into cells may involve culturing the cells for a desired period of time after or during contact with the nucleic acid delivery aid and / or by applying physical stimulation to the cells.

[0018] The physical stimuli applied to the cells may be, for example, electrical and / or magnetic stimuli, specifically the application of electrical or magnetic pulses. The conditions for the electrical or magnetic pulses can be set as appropriate.

[0019] The target cells to which nucleic acids are introduced may be living cells or cells from outside the body. In other words, the nucleic acid introduction method may be performed in vivo or in vitro.

[0020] When nucleic acid delivery is performed in vivo, the nucleic acid, nucleic acid delivery aid, and / or nucleic acid delivery agent may be injected into the body, for example, by syringe, catheter, or intravenous infusion, or inhaled by an inhaler. Alternatively, the nucleic acid delivery aid or nucleic acid delivery agent may be administered orally separately as a compounded tablet, capsule, or powder.

[0021] When the nucleic acid delivery method is performed in vitro, the nucleic acid, nucleic acid delivery aid, and / or nucleic acid delivery agent may be injected directly into the cells, spheroids, or tissue fragments, for example, by a syringe. Alternatively, the nucleic acid, nucleic acid delivery aid, and / or nucleic acid delivery agent may be brought into contact with the cells, spheroids, or tissue fragments by adding them to the culture medium in which the cells, spheroids, or tissue fragments are cultured in a medium containing the nucleic acid, nucleic acid delivery aid, and / or nucleic acid delivery agent.

[0022] The cells into which nucleic acids are introduced are not particularly limited. The cells may be, for example, animal cells. The animal cells may preferably be mammalian cells, and more preferably human cells.

[0023] The cells into which nucleic acids are introduced may be in the form of isolated cells, spheroids, or tissues. The cells into which nucleic acids are introduced may also be cells from an animal, such as a human body. The type of cell is not particularly limited, but may include, for example, adipocytes, muscle cells, cardiomyocytes, chondrocytes, epithelial cells, sensory cells, stem cells, immune cells, fibroblasts, nerve cells, retinal cells, vascular cells, cancer cells, etc.

[0024] [Cells and Products] A cell according to one embodiment is a cell containing nucleic acid introduced by the nucleic acid introduction method described above. The type of cell is not particularly limited.

[0025] The product according to one embodiment is a product produced from the above-described cells. That is, the product may be produced from cells into which nucleic acids have been introduced by the above-described nucleic acid introduction method. In particular, when the nucleic acid introduction method is performed in a culture vessel (in vitro), the product produced from the cells can be used as a drug or the like.

[0026] Such products may be, for example, proteins or RNA molecules, or genes encoded by nucleic acids introduced into cells, or RNA molecules. RNA molecules include siRNA, miRNA, ncRNA, etc.

[0027] [Nucleic Acid Delivery Aid] A nucleic acid delivery aid according to one embodiment includes at least one of an anti-inflammatory agent and an antioxidant. The nucleic acid delivery aid may contain an anti-inflammatory agent but not an antioxidant, or it may contain an antioxidant but not an anti-inflammatory agent.

[0028] The inventors of this invention have found that the efficiency of nucleic acid delivery to target cells can be improved by using at least one of an anti-inflammatory agent and an antioxidant as a nucleic acid delivery aid during nucleic acid delivery to target cells.

[0029] The nucleic acid delivery aid may include, for example, at least an anti-inflammatory agent. In this case, the anti-inflammatory agent may include at least one of a histamine receptor inhibitor, a muscarinic receptor inhibitor, and a steroid. In this case, the histamine receptor inhibitor is preferably at least one of a histamine H1 receptor inhibitor, a histamine H2 receptor inhibitor, a histamine H3 receptor inhibitor, and a histamine H4 receptor inhibitor. More preferably, the histamine receptor inhibitor is a histamine H2 receptor inhibitor or a histamine H4 receptor inhibitor.

[0030] Preferably, the histamine receptor inhibitor may be an antagonist to the histamine receptors on the target cells. Preferably, the muscarinic receptor inhibitor may be an antagonist to the muscarinic receptors on the target cells.

[0031] Nucleic acid delivery vectors can sometimes trigger biological defense responses or inflammatory responses in target cells to which nucleic acids are being introduced, which can reduce the efficiency of nucleic acid delivery. If the nucleic acid delivery aid contains an anti-inflammatory agent, the inflammatory response associated with vector delivery may be suppressed, potentially improving the efficiency of nucleic acid delivery to target cells.

[0032] Histamine and muscarinic receptors can trigger the generation of reactive oxygen species in some cells. These reactive oxygen species may damage nucleic acids introduced into cells, potentially reducing their delivery efficiency. Therefore, including histamine receptor inhibitors and / or muscarinic receptor inhibitors in nucleic acid delivery aids may improve the efficiency of nucleic acid delivery to target cells.

[0033] Steroids are thought to potentially improve nucleic acid gene expression through their anti-inflammatory effects.

[0034] Furthermore, nucleic acid delivery aids may contain at least an antioxidant. During the delivery of nucleic acids into cells, reactive oxygen species may be generated as a biological defense response. These reactive oxygen species can damage the nucleic acids delivered to the cells. Therefore, including antioxidants in nucleic acid delivery aids may improve the efficiency of nucleic acid delivery.

[0035] Histamine H1 receptor inhibitors include brompheniramine, chlorpheniramine, diphenhydramine, doxylamine, chlorcyclidine, dexbrompheniramine, dexchlorpheniramine, metapyrylene, phenindamine, pheniramine, phenyltroxamine, pyriramine, tenyldiamine, tondilamine, triprolidine, acribastine, astemizole, bepotastine, bilastine, cetirizine, desloratadine, ebastine, fexofenadine, ketotifen, levocetirizine, loratadine, mizolastine, quifenadine, rupatadine, terfenadine, azelastine, levocabastine, olopatadine, and mequita It may contain at least one selected from the group consisting of din, homochlorcyclidine, alimazine, mepyramine, novelelastine, hydroxyzine, meclizine, buclicidine, triprolidine, triperenamine, mebuhydroline, pimethixen, tadiphylline, oxatomide, emedastine, niaprazine, oxomemazine, chloropyramine, carebastine, fenspirid, deptropin, dasemazine, icotidine, mirtazapine, esmirtazapine, paliperidone, cetastine, antazoline, alinastine, dosurepin, pivaxidine, epinastine, temelastine, dorispan, carbinoxamine, and cremisole.

[0036] More preferably, the histamine H1 receptor inhibitor comprises at least one selected from the group consisting of brompheniramine, doxylamine, chlorcyclidine, pheniramine, pyriramine, tenyldiamine, tondilamine, triprolidine, cetirizine, desloratadine, fexofenadine, ketotifen, olopatadine, mequitazine, homochlorcyclidine, alimazine, novellastine, hydroxyzine, meclizine, triprolidine, triperenamin, pimethixen, tadiphylline, emedastine, niaprazine, oxomemazine, chloropyramine, fenspiride, dasemazine, icotidine, mirtazapine, esmirtazapine, pivaxidine, and dolispan. These histamine H1 receptor inhibitors have the advantage of good water solubility and the advantage of being safe to administer in humans.

[0037] Histamine H2 receptor inhibitors may include at least one selected from the group consisting of cimetidine, ranitidine, famotidine, nizatidine, roxatidine, lafutidine, thiotidine, nipelotidine, sfotidine, methiaamide, donetidine, ramoxotidine, zaltidine, labortidine, zolantidine, dalcotidine, mefentidine, brimamide, oxymethidine, ethintidine, and icotidine.

[0038] Histamine H3 receptor inhibitors may include at least one selected from the group consisting of pitrisant, proxyphan, conesin, ildabisant, thiperamide, babisant, clobemprit, siproxyphan, enalisanto, idofenpropit, sipralisanto, and samelisanto.

[0039] The histamine H4 receptor inhibitor may include at least one selected from the group consisting of adriforant, isuforant, mequitazine, treforant, pimozide, ceriforant, thioperamide, JNJ39758979, JNJ7777120, JNJ10191584, ZPL389, VUF6002, A987306, and A943931.

[0040] Muscarinic receptor inhibitors include throspium, dalifenacin, atropine, tolterodine, oxybutynin, solifenacin, fesoterodine, propiverine, oxyphenonium, benzatropine, trihexyphenidyl, procyclidine, profenamine, hyoscyamine, anisotropine, pirenzepine, scopolamine, propantheline, dicyclomine, tropicamide, biperiden, quinidine, methantheline, cyclopentolate, glycopyrrolium, orphenadrine, dexetimide, chlorphenoxamine, benactidine, and buty Ruscopolamine, Lebefenacin, Othironium, Emepronium, Bornaprine, Propiomazine, Olanzapine, Methixene, Oxyphencycline, Promazine, Tridihexetyl, Homatropin, Benzquinamide, Promethazine, Diphenidol, Methotrimeprazine, Tiotropium, Ipratropium, Nicardipine, Quetiapine, Ziprasidone, Clozapine, Pipecuronium, Pancuronium, Disopyramide, Chlorprothixene, Doxepin, Amitriptyline, Desipramine, Imipramine, Nortriptyline, Paro Xetine, cyproheptadine, mepenzolate, isopropamide, tramadol, methoscopolamine, acridinium, umecridinium, hexocycline, dosulepin, imidafenacin, bizotifen, tondiamine, cridinium, cyclimine, buclidine, doxylamine, amoxapine, escitalopram, flupentixol, galamine, rocuronium, doxacrium, metacholine, dimethindene, diphenhydramine, chlorpromazine, rapacronium, camillofin, difemeline, dihexyberine, mebeberine It comprises at least one selected from the group consisting of piperidolate, rosiverine, trimebutine, benzylonium, bevonium, difemanil, fenpiberinium, pentienate, pipenzolate, porzin, prifinium, thiemonium, thimepidium, fenglutalimid, oxytropium, tropatepine, etibenzatropin, batephenterol, piroxazine, clemastine, diphenylpyraline, homochlorcyclidine, alimazine, mequitazine, tarafenacin, zamifenacin, terenzepine, and lidocaine.

[0041] More preferably, the muscarinic receptor inhibitor comprises at least one selected from the group consisting of tolterodine, solifenacin, benzatropine, procyclidine, profenamine, pirenzepine, biperiden, orphenadrine, lebefenacin, propiomazine, methixene, promazine, promethazine, quetiapine, clozapine, chlorprothixene, doxepin, desipramine, imipramine, nortriptyline, paroxetine, cyproheptadine, isopropamide, tramadol, hexocycline, imidafenacin, pizotifen, galamine, diphenhydramine, chlorpromazine, alimazine, mequitazine, terenzepine, and lidocaine. These muscarinic receptor inhibitors have the advantage of good water solubility and the advantage of being safe to administer in humans.

[0042] Steroids include fluticasone, diflorasone, alclometasone, medrison, amcinonide, beclometasone, betamethasone, desoxymetasone, urobetasol, mometasone, crocortolone, loteprednol, rimexolone, prednicarbate, butesonide, desonide, cortisone, difluprednate, halcinonide, clobetasolone, fluocortin, fluperolon, formocortar, halomethasone, fluchlorolon, fluocinolone, triamicinolone, flumetasone, hydrocortisone, fludrocortide, prednisolone, It comprises at least one selected from the group consisting of methylprednisolone, fluocinonide, dexamethasone, flupredniden, fluocortone, difluocortone, clobetazone, flunisolide, ciclesonide, thixocortol, deflazacort, fludrocortisone, trilostane, paramethasone, aldosterone, meprednisone, cortibazole, prednilidene, cloprednol, prednisone, fluprednisolone, melengethol, levocetoconazole, vamorolone and its carboxylic acid esters, phosphate esters, and acetonides.

[0043] More preferably, the steroid agent contains at least one selected from the group consisting of betamethasone, dexamethasone, deflazacort, paramethasone, cloprednol, prednisolone, prednisone, fluprednisolone and its phosphate ester.

[0044] The antioxidant contains at least one selected from the group consisting of propofol, fospropofol, probucol, ibakaftole, sonrychromanol, succinobucol, ferulic acid, vanillic acid, 2,6-di-tert-butylphenol, α-tocopherol, tocopherolsolan, tocotrienol, ascorbic acid, allopurinol, levamipide, melatonin, carvedilol, nicarabine, kojic acid, uric acid, ergothioneine, carnitine, pentoxifylline, theophylline, theobromine, paraxanthine, caffeine, DOPA, N-oleyl dopamine, levodopa, methyldopa, melevodopa, droxidopa, dopaxamine, norepinephrine, epinephrine, adrenalone, levonordefrin, theoadrenaline, hydroxythiosol, masoprocol, dobutamine, isoprenaline, albutamine, protokylol, isoproterenol, dichloroisoproterenol, rimiterol, acteoside, quercetin, resveratrol, rosmarinic acid, catechin, epicatechin, ellagic acid, rutin, isofraxidin, chlorogenic acid, caffeic acid, N-acetylcysteine, omeprazole, esomeprazole, pramipexole, atorvastatin, metronidazole, fluvastatin, propylthiouracil, glutathione, β-carotene, nicotinamide, curcumin, luteolin, apigenin, kaempferol, vitamin K, lycopene, zeaxanthin, astaxanthin, canthaxanthin, lipoic acid, and donepezil.

[0045] More preferably, the antioxidant contains at least one selected from the group consisting of fospropofol, sonylchromanol, tocopherolsolan, ascorbic acid, allopurinol, nicarabine, carnitine, pentoxifylline, theophylline, DOPA, levodopa, methyldopa, melevodopa, droxidopa, norepinephrine, epinephrine, adrenalone, levonordefrin, theoadrenaline, isoprenaline, isoproterenol, rimiterol, omeprazole, esomeprazole, pramipexole, atorvastatin, metronidazole, fluvastatin, and donepezil. These antioxidants have the merit of good water solubility and the merit of being safely administered to humans.

[0046] Preferably, the nucleic acid introduction adjuvant contains at least two of a histamine receptor inhibitor, a muscarinic receptor inhibitor, a steroid agent, and an antioxidant. Hereinafter, it should be noted that the "histamine receptor inhibitor", "muscarinic receptor inhibitor", "steroid agent", and "antioxidant" can be selected from the agents listed above in plural numbers. In this case, the histamine receptor inhibitor may be at least one of a histamine H1 receptor inhibitor, a histamine H2 receptor inhibitor, a histamine H3 receptor inhibitor, and a histamine H4 receptor inhibitor.

[0047] The inventor of the present application newly found that it is possible to improve the introduction efficiency of nucleic acids by combining at least two selected from a histamine receptor inhibitor, a muscarinic receptor inhibitor, and a steroid agent. Specifically, the nucleic acid introduction adjuvant may contain a steroid agent and a histamine receptor inhibitor. Instead of this, the nucleic acid introduction adjuvant may contain a steroid agent and a muscarinic receptor inhibitor. Also, the nucleic acid introduction adjuvant may contain a histamine receptor inhibitor and a muscarinic receptor inhibitor.

[0048] From the viewpoint of improving the introduction efficiency of nucleic acids, more preferably, the nucleic acid introduction adjuvant contains at least two of a histamine receptor H1 inhibitor, a histamine receptor H2 inhibitor, a histamine receptor H3 inhibitor, a histamine receptor H4 inhibitor, a muscarinic receptor inhibitor, and a steroid agent.

[0049] From the viewpoint of improving the efficiency of nucleic acid delivery, it is even more preferable that the nucleic acid delivery aid be a combination of a histamine receptor H4 inhibitor or a muscarinic receptor inhibitor and a steroid, or a combination of a histamine receptor inhibitor and a muscarinic receptor inhibitor. In the combination of a histamine receptor inhibitor and a muscarinic receptor inhibitor, the histamine receptor inhibitor may be a histamine receptor H1 inhibitor, a histamine receptor H2 inhibitor, a histamine receptor H3 inhibitor, or a histamine receptor H4 inhibitor. By combining these agents, it is possible to significantly improve the efficiency of nucleic acid delivery.

[0050] Furthermore, the inventors of this application have novelly discovered that nucleic acids can be introduced with minimal individual variation using at least two combinations selected from histamine receptor inhibitors, muscarinic receptor inhibitors, and antioxidants (high robustness).

[0051] Furthermore, the inventors of this application have novelly discovered that the cells to which nucleic acids are readily introduced can be altered by combining at least two selected from histamine receptor inhibitors, muscarinic receptor inhibitors, and antioxidants. This makes it possible to provide a nucleic acid introduction method with cell selectivity. In other words, the combination of at least two selected from histamine receptor inhibitors, muscarinic receptor inhibitors, and antioxidants should be changed depending on the type of cell to which the nucleic acid should be introduced.

[0052] More preferably, the nucleic acid delivery aid comprises at least two of the following: a histamine receptor H1 inhibitor, a histamine receptor H2 inhibitor, a histamine receptor H3 inhibitor, a histamine receptor H4 inhibitor, a muscarinic receptor inhibitor, and an antioxidant. Even more preferably, the nucleic acid delivery aid comprises at least two of the following: a histamine receptor H4 inhibitor, a muscarinic receptor inhibitor, and an antioxidant.

[0053] A combination of at least two selected from histamine receptor inhibitors, muscarinic receptor inhibitors, and antioxidants may be selected as appropriate.

[0054] In addition to or instead of the above embodiments, the nucleic acid delivery aid preferably contains both an anti-inflammatory agent and an antioxidant. Specifically, the nucleic acid delivery aid may contain at least one selected from a histamine receptor inhibitor, a muscarinic receptor inhibitor, and a steroid, and an antioxidant. More preferably, the nucleic acid delivery aid may contain at least one of a histamine receptor inhibitor and a muscarinic receptor inhibitor, and an antioxidant. In this case, the histamine receptor inhibitor is at least one of a histamine H1 receptor inhibitor, a histamine H2 receptor inhibitor, a histamine H3 receptor inhibitor, and a histamine H4 receptor inhibitor. Alternatively, the histamine receptor inhibitor is more preferably a histamine H4 receptor inhibitor.

[0055] It is known that reactive oxygen species (ROS) are generated within cells as a biological defense response when foreign pathogens such as viruses invade cells. Similarly, it is expected that ROS will be generated when nucleic acids are introduced into cells. Since ROS are known to damage nucleic acids, there is a possibility that the introduced nucleic acids may not be able to fully express their effects within the cell due to ROS. Even when antioxidants alone cannot sufficiently suppress ROS, combining them with anti-inflammatory agents can effectively suppress the production of ROS. The inventors have newly discovered that the efficiency of nucleic acid introduction is significantly improved by using antioxidants in combination with anti-inflammatory agents. Even more surprisingly, they have found that by using antioxidants in combination with anti-inflammatory agents, nucleic acids can be introduced with little dependence on individual differences in the sample.

[0056] Furthermore, the inventors discovered that by changing the type and combination of antioxidants or anti-inflammatory agents, the type of cell to which nucleic acids are primarily introduced can be altered. In this case, the anti-inflammatory agent used in combination with the antioxidant may be, for example, a muscarinic receptor inhibitor and / or a histamine receptor inhibitor. The reason why the cell selectivity changes depending on the combination of antioxidants and anti-inflammatory agents is presumed to reflect the fact that the mechanism of reactive oxygen species generation and the reactive oxygen species control mechanisms to which each subtype of histamine receptor or muscarinic receptor contribute differ from cell to cell.

[0057] In a more preferred embodiment, the nucleic acid delivery aid may contain an antioxidant and a muscarinic receptor inhibitor. By including both an antioxidant and a muscarinic receptor inhibitor, the generation of reactive oxygen species associated with nucleic acid delivery is thought to be sufficiently controlled by the synergistic effect of the two agents.

[0058] In another preferred embodiment, the nucleic acid delivery aid may include an antioxidant and a histamine receptor inhibitor. In this case, the histamine receptor inhibitor is at least one of a histamine H1 receptor inhibitor, a histamine H2 receptor inhibitor, a histamine H3 receptor inhibitor, and a histamine H4 receptor inhibitor. Alternatively, the histamine receptor inhibitor is a histamine H4 receptor inhibitor. When the nucleic acid delivery aid includes both an antioxidant and a histamine receptor inhibitor, it is believed that the generation of reactive oxygen species associated with nucleic acid delivery is sufficiently controlled by the synergistic effect of both agents.

[0059] Instead of the embodiments described above, the nucleic acid delivery aid may include a histamine receptor inhibitor and a muscarinic receptor inhibitor. Here, the histamine receptor inhibitor may be at least one of a histamine H1 receptor inhibitor, a histamine H2 receptor inhibitor, a histamine H3 receptor inhibitor, and a histamine H4 receptor inhibitor. Preferably, the histamine receptor inhibitor may be a histamine H2 receptor inhibitor or a histamine H4 receptor inhibitor. The use of such an acid delivery aid may improve the efficiency of nucleic acid delivery.

[0060] Instead of the embodiments described above, the nucleic acid delivery aid may include an agent that has both histamine receptor inhibitory and muscarinic receptor inhibitory effects. That is, the nucleic acid delivery aid does not need to include multiple types of agents as long as it includes one agent that has both histamine receptor inhibitory and muscarinic receptor inhibitory effects. Mequitazine is one example of such an agent.

[0061] However, if the nucleic acid delivery aid contains mequitazine, it may also contain other agents. For example, the nucleic acid delivery aid may contain mequitazine and an antioxidant.

[0062] Preferred combinations of nucleic acid delivery aids for specifically introducing vectors such as plasmids, minicircles, closed-end DNA, and mRNA into adipocytes by electrical and / or magnetic perforation (electromagnetic pulses) are combinations of antioxidants and muscarinic inhibitors, or combinations of antioxidants and muscarinic inhibitors having histamine inhibitory activity. More preferably, the combination of nucleic acid delivery aids is: tocofersolan / mequitazine, tocofersolan / benzatropine, tocofersolan / promethazine, tocofersolan / chlorprothixene, tocofersolan / doxepin, tocofersolan / cyproheptadine, tocofersolan / alimemazine, sonlichromatol / mequitazine, sonlichromatol / benzatropine, sonlichromatol / promethazine, sonlichromatol / chlorprothixene, sonlichromatol / doxepin, sonlichromatol / cyproheptadine, sonlichromatol / alimemazine, propofol / mequitazine, propofol / lidocaine, propofol / imidafenacin, propofol The combinations of propofol / lebefenacin, propofol / tolterodine, propofol / benzatropine, propofol / terenzepine, propofol / promethazine, propofol / cyproheptadine, propofol / procyclidine, propofol / profenamine, propofol / chlorprothixene, propofol / doxepin, propofol / isopropamide, propofol / alimethimazine, propofol / biperiden, propofol / methixene, propofol / hexocycline, propofol / pizotifen, fospropofol / mequitazine, fospropofol / lidocaine, fospropofol / imidafenacin,Fospropofol / lebefenacin combination, fospropofol / tolterodine combination, fospropofol / benzatropine combination, fospropofol / terenzepine combination, fospropofol / promethazine combination, fospropofol / cyproheptadine combination, fospropofol / procyclidine combination, fospropofol / profenamine combination, fospropofol / chlorprothixene combination, fospropofol / doxepin combination, fosprop These combinations include fospropofol / isopropamide, fospropofol / alimemazine, fospropofol / biperiden, fospropofol / methixene, fospropofol / hexocycline, fospropofol / pizotifen, pentoxifylline / lidocaine, pentoxifylline / lebefenacin, pentoxifylline / tolterodine, pentoxifylline / benzatropine, or pentoxifylline / terenzepine.

[0063] The nucleic acid delivery aid described above may be provided in powder or solid form, or in a form dissolved or mixed in a solution. Furthermore, if possible, the nucleic acid delivery aid may be provided in a lyophilized state.

[0064] Furthermore, the nucleic acid delivery aid only needs to contain the above-mentioned anti-inflammatory agent and / or antioxidant as an active ingredient. In addition to the above-mentioned anti-inflammatory agent and / or antioxidant as an active ingredient, the nucleic acid delivery aid may also contain other additives if necessary.

[0065] As mentioned above, nucleic acid delivery aids more preferably contain a combination of several agents selected from antioxidants and / or anti-inflammatory agents. In this case, the nucleic acid delivery aid may contain a mixture of the several agents. Alternatively, the nucleic acid delivery aid may be a kit containing the several agents without mixing them. Furthermore, the nucleic acid delivery aid may contain only one of the above-mentioned combination of several agents, in which case that one agent may be provided for use in combination with the other agent of the above-mentioned combination of several agents.

[0066] [Nucleic Acid Delivery Agent] A nucleic acid delivery agent according to one embodiment includes a nucleic acid delivery aid having the composition described above, and a nucleic acid or a vector carrying a nucleic acid. The nucleic acid delivery aid includes at least one of an anti-inflammatory agent and an antioxidant, as described above. The specific types and combinations of the anti-inflammatory agent and antioxidant are as described above. The nucleic acid delivery agent only needs to contain a nucleic acid delivery aid, i.e., the above-mentioned anti-inflammatory agent and / or antioxidant, as an active ingredient.

[0067] The nucleic acid delivery agent may be in the form of a solution comprising a nucleic acid delivery aid having the composition described above, and a nucleic acid or a vector carrying a nucleic acid. The nucleic acid delivery aid may be provided, for example, as an administration solution or a concentrated administration stock. Here, the administration solution refers to the solution itself to be administered into cells, spheroids, tissue fragments, or the tissues or blood vessels of a living organism. On the other hand, the concentrated administration stock is a concentrated version of the administration solution. The concentrated administration stock is diluted to become an administration solution, and the diluted administration solution is administered into cells, spheroids, tissue fragments, or the tissues or blood vessels of a living organism.

[0068] When the nucleic acid delivery agent is in solution form, the solvent is not particularly limited, but may be water or an aqueous solution consisting substantially of water, or it may be an isotonic solution. The solvent of the administration solution may be an isotonic solution containing, in terms of concentration, 2-5% (w / v) glucose, 1-5% (w / v) mannitol, 100-200 mM sodium chloride, 1 mM magnesium chloride, etc. Furthermore, the administration solution as a nucleic acid delivery agent may also contain buffering salts such as sodium phosphate or tris hydrochloride, surfactants such as polysorbate 20 or poloxamer 188, or solubilizers such as sulfobutyl ether β-cyclodextrin or hydroxy β-cyclodextrin.

[0069] Alternatively, the nucleic acid delivery agent may comprise a nucleic acid delivery aid having the above-described composition and a nucleic acid or a vector carrying a nucleic acid, and may be provided in powder or solid form or in a freeze-dried state. Alternatively, the nucleic acid delivery agent may be provided as an administration solution in the form of an emulsion obtained by mixing the above-described nucleic acid delivery aid and vector with an oil or fat and emulsifying it. In this case, the oil or fat may be, for example, soybean oil, lecithin, glycerin, etc.

[0070] The concentration of nucleic acid delivery aids in nucleic acid delivery agents is not particularly limited. When the nucleic acid delivery agent is in the form of an administration solution, the concentrations of antioxidants and anti-inflammatory agents in the administration solution may be, for example, in the range of 0.001 to 1000 μM each.

[0071] The nucleic acid contained in the nucleic acid delivery agent may be the nucleic acid to be introduced into the cell directly added. Alternatively, the nucleic acid delivery agent may contain a vector carrying the nucleic acid to be introduced into the cell.

[0072] The vector is not particularly limited, but may contain at least one selected from the group consisting of closed-end DNA such as plasmids, minicircles, and doggybone DNA, retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, aneroviruses, and herpesviruses. Any known method can be used to package the target nucleic acid into the vector.

[0073] The nucleic acid delivery aid and / or nucleic acid delivery agent described above can be used as a gene expression promoting reagent and / or gene therapy agent. That is, a gene expression promoting reagent and / or gene therapy agent according to one embodiment may include the nucleic acid delivery aid described above.

[0074] <Experimental Examples> (Experimental Example 1) As samples, 50-100 mg of human subcutaneous adipose tissue fragments and a nucleic acid delivery agent were prepared. The composition of the nucleic acid delivery agent differed for each experiment number A01 to A06, as described below. In experiments A01 to A06, a sample (SF03) collected from the same subject was used. Here, the symbols assigned to the samples (SF01, SF02, SF03, SF04, SF05, SF06, SF07, SF08, SF09, SF10, SF11, SF12) represent human subcutaneous adipose tissue samples shown in Table 16.

[0075] The nucleic acid delivery agent (administration solution) used in experiments A01 to A06 contained plasmid DNA (Aldevron, pALD-Nanoplasmid-CAG-EGFP) containing the reporter gene EGFP gene as the nucleic acid, and was an aqueous solution containing a nucleic acid delivery aid (added as a stock solution as shown in Table 15; the same applies hereafter) and glucose. The glucose concentration in the administration solution, which is the nucleic acid delivery agent, was 5% (w / v), and the plasmid DNA concentration was 200 μg / mL (the same applies hereafter).

[0076] The composition of the nucleic acid delivery aids is shown in Table 1 below. The nucleic acid delivery aid used in experiment number A01 contains a histamine H1 receptor inhibitor as an anti-inflammatory agent. The nucleic acid delivery aid used in experiment number A02 contains a histamine H4 receptor inhibitor as an anti-inflammatory agent. The nucleic acid delivery aids used in experiments number A03 and A04 contain a muscarinic receptor inhibitor as an anti-inflammatory agent. The nucleic acid delivery aids used in experiments number A05 and A06 contain an agent that has both histamine H1 and H4 receptor inhibitory and muscarinic receptor inhibitory effects as an anti-inflammatory agent.

[0077] In Tables 1-14, the symbols "a1", "a2", "a3", "a4", "a5", "a6", "a7", "a8", "a9", "a10", "h1", "h2", "h3", "h4", "m1", "m2", "m3", "m4", "m5", and "m6" indicate the drugs shown in Table 15 below. In Tables 1-14, the numbers in parentheses following these symbols indicate the concentration (μM: micromolar) of the drug in the administration solution, which is a nucleic acid delivery agent. In Tables 1-14, the symbol "-" means that the drug in question is not contained.

[0078] (Table 1)

[0079] In each experiment with the specified experimental number, 5 μL of the nucleic acid delivery agent solution was injected into the sample using a 30-gauge injection needle (Terumo, 1-4909-06). Next, adipose tissue fragments were held between forceps-type electrodes (Neppageen, CUY650P10) via a nonwoven fabric moistened with culture medium, and three electrical pulses were irradiated using an electroporator (Neppageen, NEPA21 Type II) at intervals of 1 to 2 minutes (electroporation). The conditions for each electrical pulse were as follows: • Pulse intensity: 30V • Pulse width: 4 ms • Pulse interval: 10 ms • Number of pulses: 20 pulses • Pulse decay: 2% • Pulse polarity: + / -

[0080] After irradiation with electrical pulses, adipose tissue samples were cultured. The adipose tissue samples were cultured using the culture apparatus described in International Publication No. 2024 / 162464, under the conditions described in International Publication No. 2024 / 162464. The adipose tissue samples were cultured in the culture apparatus while pressed into the culture medium using a mesh with a mesh opening of 55 μm. The culture medium was changed every 2-3 days. After culturing the adipose tissue samples for one week, the samples were imaged in fluorescence mode using a fluorescence microscope (Keyence, BZ-9000) (magnification 4x, exposure time 1 / 20 second). This allowed for the confirmation of the presence and degree of EGFP gene expression.

[0081] Figure 1 shows a micrograph of adipose tissue sample taken with a fluorescence microscope in Experimental Example 1. In the micrographs shown in Figures 1-15, the white areas correspond to the green fluorescence in the original micrograph. Therefore, the white areas in the micrographs represent gene expression, with whiter areas indicating stronger expression.

[0082] Comparing experiment number A01 with experiment numbers A02-A04, experiment number A01 showed an enhanced expression of the EGFP gene. Comparing experiment numbers A05 and A06 with experiment numbers A02-A04, experiment numbers A05 and A06 showed a significantly enhanced expression of the EGFP gene compared to experiment numbers A02-A04. Comparing experiment number A01 with experiment number A05 or A06, experiment numbers A05 or A06 showed an even more significant enhanced expression of the EGFP gene compared to experiment number A01. Therefore, nucleic acid agents containing histamine H1 receptor inhibitors alone showed a moderate gene expression enhancement effect. Furthermore, nucleic acid agents containing anti-inflammatory agents that combine histamine H1, H4 receptor inhibition and muscarinic receptor inhibition showed higher nucleic acid delivery efficiency than nucleic acid agents without anti-inflammatory agents, or nucleic acid agents containing only histamine H1 or H4 receptor inhibitors, or only muscarinic inhibitors.

[0083] Therefore, it can be seen that the synergistic effect of both histamine H1 or H4 receptor inhibition and muscarinic receptor inhibition significantly increases the efficiency of nucleic acid delivery.

[0084] (Experimental Example 2) As samples, 50-100 mg of human subcutaneous adipose tissue fragments and a nucleic acid delivery agent were prepared. The composition of the nucleic acid delivery agent (administration solution) differed for each experiment number B01-B04, as described below. In experiments B01-B04, samples taken from the same subject (SF03) were used.

[0085] The nucleic acid delivery agent used in experiments B01 to B03 was an aqueous solution containing plasmid DNA (Aldevron, pALD-Nanoplasmid-CAG-EGFP) containing the reporter gene EGFP gene as the nucleic acid, a nucleic acid delivery aid, and glucose. The concentrations of glucose and nucleic acid in the administration solution of the nucleic acid delivery agent were the same as in Experimental Example 1.

[0086] The composition of the nucleic acid delivery aids is shown in Table 2 below. The nucleic acid delivery aids used in experiments B01, B02, and B03 contain antioxidants. Specifically, the nucleic acid delivery aids used in experiments B01, B02, and B03 contain pentoxifylline, propofol, and 3-methyl-1-phenyl-5-pyrazolone, respectively.

[0087] The nucleic acid delivery agent used in experiment number B04 (reference example) is an aqueous solution containing plasmid DNA (Aldevron, pALD-Nanoplasmid-CAG-EGFP) containing the reporter gene EGFP gene and glucose. The concentrations of glucose and plasmid DNA in the administration solution of this nucleic acid delivery agent are the same as those in experiments B01 to B03. In other words, the nucleic acid delivery agent used in experiment number B04 substantially does not contain anti-inflammatory agents or antioxidants.

[0088] (Table 2)

[0089] The gene transfer method in Experimental Example 2 was carried out under the same conditions and methods as in Experimental Example 1, except for the person who collected the sample and the composition of the nucleic acid transfer agent. Furthermore, the conditions for culturing the adipose tissue fragments and imaging them under a microscope were also the same as in Experimental Example 1.

[0090] Figure 2 shows micrographs of adipose tissue samples imaged with a fluorescence microscope in Experimental Example 2. Comparing Experimental Numbers B01, B02, and B03 with Experimental Number B04, genes are expressed more strongly and specifically in adipocytes in Experimental Numbers B01, B02, and B03 than in Experimental Number B04. Therefore, it can be seen that nucleic acid delivery agents containing antioxidants have a higher nucleic acid delivery efficiency than nucleic acid delivery agents without antioxidants.

[0091] (Experimental Example 3) As samples, 50-100 mg of human adipose tissue fragments and a nucleic acid delivery agent were prepared. The composition of the nucleic acid delivery agent differed for each experiment number C01-C12, as described below. In all experiment numbers, samples taken from the same subject (SF02) were used.

[0092] The nucleic acid delivery agent (administration solution) used in experiments C01 to C12 was an aqueous solution containing plasmid DNA (Aldevron, pALD-Nanoplasmid-CAG-EGFP) containing the reporter gene EGFP gene as the nucleic acid, a nucleic acid delivery aid, and glucose. The concentrations of glucose and plasmid DNA in the administration solution, which is the nucleic acid delivery agent, were the same as in Experimental Example 1.

[0093] The composition of the nucleic acid delivery aids is shown in Table 3 below. The nucleic acid delivery aids used in experiments C01-C03 and C09 contain both a steroid and a muscarinic receptor inhibitor as anti-inflammatory agents. The nucleic acid delivery aids used in experiments C04-C06 and C12 contain a muscarinic receptor inhibitor as an anti-inflammatory agent. The nucleic acid delivery aid used in experiment C07 contains both a steroid and a histamine receptor H1 inhibitor as anti-inflammatory agents. The nucleic acid delivery aid used in experiment C08 contains both a steroid and a histamine receptor H4 inhibitor as anti-inflammatory agents. The nucleic acid delivery aid used in experiment C10 contains a steroid and an agent that has both histamine receptor (H1 receptor and H4 receptor) inhibitory and muscarinic receptor inhibitory effects as anti-inflammatory agents. The nucleic acid delivery aid used in experiment C11 contains a steroid as an anti-inflammatory agent.

[0094] (Table 3)

[0095] The gene transfer method in Experimental Example 3 was carried out under the same conditions and methods as in Experimental Example 1, except for the person who collected the sample and the composition of the nucleic acid transfer agent.

[0096] Figure 3 shows micrographs of adipose tissue samples imaged with a fluorescence microscope in Experimental Example 3. Comparing Experiments C01-C03 and C09 with Experiments C04-C06 and C12, genes are expressed significantly more strongly in Experiments C01-C03 and C09 than in Experiments C04-C06 and C12. Therefore, nucleic acid delivery agents containing both steroids and muscarinic receptor inhibitors are significantly more efficient at delivering nucleic acids than nucleic acid delivery agents containing only muscarinic receptor inhibitors as anti-inflammatory agents. It can also be inferred that this effect may be observed regardless of the specific types of steroids and muscarinic receptor inhibitors used.

[0097] Furthermore, comparing experiment numbers C09 and C10 with experiment numbers C11 and C12, gene expression was significantly stronger in experiment numbers C09 and C10 than in experiment numbers C11 and C12. Therefore, it is thought that steroids can significantly enhance the effect of nucleic acid delivery through muscarinic receptor inhibition. In other words, nucleic acid delivery agents containing both a muscarinic receptor inhibitor and a steroid are considered to be more suitable as anti-inflammatory agents than nucleic acid delivery agents containing only a muscarinic receptor inhibitor in terms of nucleic acid delivery efficiency.

[0098] Comparing experiment number C07 with experiment number C11, experiment number C07 shows stronger gene expression than experiment number C11. Furthermore, comparing experiment number C08 with experiment number C10, experiment number C08 shows gene expression as strong as that of experiment number C10. Therefore, it is thought that steroids can significantly enhance the effect of nucleic acid introduction through histamine H4 receptor inhibition. In other words, nucleic acid introduction agents containing both an agent with histamine H4 receptor inhibitory and / or muscarinic receptor inhibitory effects and a steroid are considered to be just as suitable in terms of nucleic acid introduction efficiency as nucleic acid introduction agents containing both an agent with muscarinic receptor inhibitory effects and a steroid.

[0099] (Experimental Example 4) As samples, 50-100 mg of human subcutaneous adipose tissue fragments and a nucleic acid delivery agent were prepared. The composition of the nucleic acid delivery agent differed for each experimental number D01-D05, as described below. In each experimental number, samples were taken from the same subject (SF01).

[0100] The nucleic acid delivery agent (administration solution) used in experiments D01 to D05 was an aqueous solution containing plasmid DNA (Aldevron, pALD-Nanoplasmid-CAG-EGFP) containing the reporter gene EGFP gene as the nucleic acid, a nucleic acid delivery aid, and glucose. The concentrations of glucose and plasmid DNA in the administration solution, which is the nucleic acid delivery agent, were the same as in Experimental Example 1.

[0101] The composition of the nucleic acid delivery aids is shown in Table 4 below. The nucleic acid delivery aid used in experiment number D01 contains a muscarinic receptor inhibitor as an anti-inflammatory agent. The nucleic acid delivery aids used in experiments D02 and D03 contain both a histamine receptor H2 inhibitor and a muscarinic receptor inhibitor as anti-inflammatory agents. The nucleic acid delivery aid used in experiment number D04 contains both a histamine receptor H1 inhibitor and a histamine receptor H2 inhibitor as anti-inflammatory agents. The nucleic acid delivery aid used in experiment number D05 contains both a histamine receptor H3 inhibitor and a histamine receptor H2 inhibitor as anti-inflammatory agents.

[0102] (Table 4)

[0103] The gene transfer method in Experimental Example 4 was carried out under the same conditions and methods as in Experimental Example 1, except for the person who collected the sample and the composition of the nucleic acid transfer agent. Furthermore, the conditions for culturing the adipose tissue fragments and imaging them under a microscope were also the same conditions and methods as in Experimental Example 1.

[0104] Figure 4 shows micrographs of adipose tissue samples taken with a fluorescence microscope in Experimental Example 4. Comparing Experiment No. D01 with Experiment Nos. D02 and D03, genes are expressed more specifically and significantly in adipocytes in Experiment Nos. D02 and D03 than in Experiment No. D01. Therefore, it can be seen that nucleic acid delivery agents containing two types of anti-inflammatory agents, specifically histamine receptor inhibitors and muscarinic receptor inhibitors, have significantly higher nucleic acid delivery efficiency than nucleic acid delivery agents containing only muscarinic receptor inhibitors as anti-inflammatory agents. It can also be inferred that this effect may be achieved regardless of the specific type of muscarinic receptor inhibitor used.

[0105] Comparing experiment numbers D04 and D05 with experiment number D01, the gene expression enhancement effect in experiment numbers D04 and D05 was slightly higher than in experiment number D01. Furthermore, comparing experiment numbers D02 and D03 with experiment numbers D04 and D05, the gene expression enhancement effect in experiment numbers D02 and D03 was significantly higher than in experiment numbers D04 and D05. Therefore, it can be seen that the combination of a histamine receptor H2 inhibitor and a muscarinic receptor inhibitor is more important for improving the efficiency of nucleic acid delivery.

[0106] (Experimental Example 5) As samples, 50-100 mg of human subcutaneous adipose tissue fragments and a nucleic acid delivery agent were prepared. The composition of the nucleic acid delivery agent differed for each experiment number, as described below. In all experiment numbers, samples were taken from the same subject (SF03).

[0107] The nucleic acid delivery agent (administration solution) used in experiments E01, E02, B02, E03, E06, E04, E05, and B01 was an aqueous solution containing plasmid DNA (Aldevron, pALD-Nanoplasmid-CAG-EGFP) containing the reporter gene EGFP gene as the nucleic acid, a nucleic acid delivery aid, and glucose. The concentrations of glucose and plasmid DNA in the administration solution, which is the nucleic acid delivery agent, were the same as in Experimental Example 1.

[0108] The composition of the nucleic acid delivery aids is shown in Table 5 below. The nucleic acid delivery aids used in experiments E01, E02, E04, and E05 contain both anti-inflammatory agents and antioxidants. Specifically, the nucleic acid delivery aids used in experiments E01, E02, and E05 contain both muscarinic receptor inhibitors and antioxidants. The nucleic acid delivery aid used in experiment E04 contains an antioxidant and an agent that has both histamine receptor (H1 receptor and H4 receptor) inhibitory and muscarinic receptor inhibitory effects. The nucleic acid delivery aids used in experiments B01 and B02 contain only antioxidants. The nucleic acid delivery aids used in experiments E03 and E06 contain only muscarinic receptor inhibitors.

[0109] (Table 5)

[0110] The gene transfer method in Experimental Example 5 was carried out under the same conditions and methods as in Experimental Example 1, except for the person who collected the sample and the composition of the nucleic acid transfer agent. Furthermore, the conditions for culturing the adipose tissue fragments and imaging them under a microscope were also the same conditions and methods as in Experimental Example 1.

[0111] Figure 5 shows micrographs of adipose tissue samples imaged with a fluorescence microscope in Experimental Example 5. Comparing Experiments E01 and E02 with Experiments E03 and E06, genes are expressed more specifically and significantly in adipocytes in Experiments E01 and E02 than in Experiments E03 and E06. Furthermore, comparing Experiments E01 and E02 with Experiment B02, genes are expressed more specifically and significantly in adipocytes in Experiments E01 and E02 than in Experiment B02. In addition, comparing Experiment E05 with Experiment B01, stronger gene expression is specifically observed in adipocytes in Experiment E05 than in Experiment B01. Therefore, it can be seen that nucleic acid delivery agents containing both anti-inflammatory agents and antioxidants, specifically muscarinic receptor inhibitors and antioxidants, have significantly higher nucleic acid delivery efficiency than nucleic acid delivery agents containing only muscarinic receptor inhibitors or only antioxidants.

[0112] Furthermore, since strong gene expression was specifically observed in adipocytes in experiment number E04, anti-inflammatory agents possessing both muscarinic receptor inhibition and histamine receptor inhibition also exhibit a synergistic effect with antioxidants, similar to the above.

[0113] (Experimental Example 6) As samples, 50-100 mg of human subcutaneous adipose tissue fragments and a nucleic acid delivery agent were prepared. The composition of the nucleic acid delivery agent differed for each experiment number F01-F09, as described below. In each experiment number, samples taken from the same subject (SF06) were used.

[0114] The nucleic acid delivery agent (administration solution) used in experiments F01 to F08 was an aqueous solution containing plasmid DNA (200 ug / mL, Aldevron, pALD-Nanoplasmid-CAG-EGFP) containing the reporter gene EGFP gene as the nucleic acid, a nucleic acid delivery aid, and glucose. The concentrations of glucose and plasmid DNA in the administration solution, which is the nucleic acid delivery agent, were the same as in Experimental Example 1.

[0115] The composition of the nucleic acid delivery aids is shown in Table 6 below. The nucleic acid delivery aids used in experiments F01 to F04 contain both anti-inflammatory agents and antioxidants. Various combinations of anti-inflammatory agents and antioxidants were tested in experiments F01 to F04. On the other hand, the nucleic acid delivery aids used in experiments F05, F06, F07, and F08 each contain only one type of agent, either an anti-inflammatory agent or an antioxidant.

[0116] (Table 6)

[0117] The gene transfer method in Experimental Example 6 was carried out under the same conditions and methods as in Experimental Example 1, except for the person who collected the sample and the composition of the nucleic acid transfer agent. Furthermore, the conditions for culturing the adipose tissue fragments and imaging them under a microscope were also the same conditions and methods as in Experimental Example 1.

[0118] Figure 6 shows micrographs of adipose tissue samples imaged with a fluorescence microscope in Experimental Example 6. Comparing Experimental Numbers F01, F02, and F04 with Experimental Numbers F05, F06, and F08, genes are expressed significantly more strongly in Experimental Numbers F01, F02, and F04 than in Experimental Numbers F05, F06, and F08. Therefore, it can be seen that nucleic acid delivery agents containing both anti-inflammatory agents and antioxidants have significantly higher nucleic acid delivery efficiency than nucleic acid delivery agents containing only one of the anti-inflammatory or antioxidant agents. Comparing Experimental Number F03 with Experimental Number F07, although to a lesser degree than in the above example, an improvement in nucleic acid delivery is observed in Experimental Number F03 compared to Experimental Number F07.

[0119] (Experimental Example 7) As samples, 50-100 mg of human subcutaneous adipose tissue fragments and a nucleic acid delivery agent were prepared. The nucleic acid delivery agent (administration solution) used in each experiment number shown in Table 7 below was an aqueous solution containing plasmid DNA (Aldevron, pALD-Nanoplasmid-CAG-EGFP) containing the reporter gene EGFP gene as the nucleic acid, a nucleic acid delivery aid, and glucose. The concentrations of glucose and plasmid DNA in the administration solution of the nucleic acid delivery agent were the same as in Experimental Example 1.

[0120] In Experiment Example 7, the sample may differ for each experiment number. That is, samples collected from different individuals are used. In Table 7, the same sample number indicates that the sample was collected from the same individual. Conversely, different sample numbers in Table 7 indicate that the sample was collected from different individuals.

[0121] The composition of the nucleic acid delivery aid is shown in Table 7. Here, among the experiment numbers listed in Table 7, those with the same experiment number as the previously described example indicate that they are the same experiment.

[0122] In Table 7, the nucleic acid delivery aids used in experiment numbers other than D02, D03, G01, and G02 include both anti-inflammatory agents and antioxidants, specifically both muscarinic receptor inhibitors and antioxidants. The nucleic acid delivery aids used in experiment numbers F01 and G09 include antioxidants and agents that have both histamine receptor (H1 receptor and H4 receptor) inhibitory and muscarinic receptor inhibitory effects. On the other hand, the nucleic acid delivery aids used in experiment numbers D02, D03, G01, and G02 include muscarinic receptor inhibitors and histamine H2 receptor inhibitors.

[0123] (Table 7)

[0124] The gene transfer method in Experimental Example 7 was carried out under the same conditions and methods as in Experimental Example 1, except for the person who collected the sample and the composition of the nucleic acid transfer agent. Furthermore, the conditions for culturing the adipose tissue fragments and imaging them under a microscope were also the same conditions and methods as in Experimental Example 1.

[0125] Figure 7 shows micrographs of adipose tissue samples imaged with a fluorescence microscope in Experimental Example 7, excluding Experimental Numbers D02, D03, G01, and G02. Figure 12 shows micrographs of adipose tissue samples after nucleic acid introduction in Experimental Numbers D02, D03, G01, and G02 of Experimental Example 7. In Experimental Numbers D02 and G01, the nucleic acid introduction aids have the same composition. Comparing the micrographs in Experimental Numbers D02 and G01, significant gene expression is observed in the D02 sample. Similarly, in Experimental Numbers D03 and G02, the nucleic acid introduction aids have the same composition. Comparing the micrographs in Experimental Numbers D03 and G02, similar levels of gene expression are observed in both D03 and G02. Furthermore, in samples containing only anti-inflammatory agents as nucleic acid introduction aids (D02, D03, G01, G02), it was confirmed that variability in gene expression can be observed depending on the combination of the two anti-inflammatory agents used.

[0126] In experiments G03, G04, E01, G05, and G06, the nucleic acid delivery aids had the same composition. A comparison of the microscopic images from experiments G03, G04, E01, G05, and G06 shows that gene expression was sufficient, with little dependence on the sample (small individual differences).

[0127] Furthermore, in experiments E07, G07, and G08, the nucleic acid delivery aids had the same composition. A comparison of the microscopic images from experiments E07, G07, and G08 shows that the genes were sufficiently expressed, with little dependence on the sample. Similarly, in experiments F01 and G09, the nucleic acid delivery aids had the same composition. A comparison of the microscopic images from experiments F01 and G09 shows that the genes were sufficiently expressed, with little dependence on the sample. In addition, in experiments E02 and G10, the nucleic acid delivery aids had the same composition. A comparison of the microscopic images from experiments E02 and G10 shows that the genes were sufficiently expressed, with little dependence on the sample.

[0128] Based on the above, it is thought that by including both anti-inflammatory and antioxidant agents in the nucleic acid delivery agent, the effect of sufficient gene expression can be obtained with little dependence on the sample (small individual differences).

[0129] As mentioned above, for nucleic acid delivery aids containing only anti-inflammatory agents (D02, D03, G01, G02), variations in gene expression may be observed depending on the combination of the two anti-inflammatory agents used. However, it should be noted that the invention of this application is not intended to exclude nucleic acid delivery aids containing only anti-inflammatory agents. Even with nucleic acid delivery aids containing only anti-inflammatory agents, gene expression is sufficient in some samples (see, for example, experiment numbers D02 and G02). Therefore, it is considered that nucleic acid delivery aids containing only anti-inflammatory agents also contribute to improving the efficiency of nucleic acid delivery.

[0130] (Experimental Example 8) As samples, 50-100 mg of human adipose tissue fragments and a nucleic acid delivery agent were prepared. In experiments H01 and H02, subcutaneous adipose tissue samples (SF06) collected from the same subject were used.

[0131] The nucleic acid delivery agent (administration solution) used in experiment number H01 is an aqueous solution containing mRNA (TriLink, WOTL84901, CleanCap EGFP) containing the reporter gene EGFP gene as the nucleic acid, a nucleic acid delivery aid, and glucose. The glucose concentration in the administration solution is the same as in Experimental Example 1. The mRNA concentration in the administration solution is 200 μg / mL.

[0132] The composition of the nucleic acid delivery aid is shown in Table 8 below. The nucleic acid delivery aid used in experiment number H02 contains an antioxidant and an anti-inflammatory agent that has both histamine receptor inhibitory and muscarinic receptor inhibitory effects. Specifically, the nucleic acid delivery aid used in experiment number H02 contains an antioxidant and mequitazine.

[0133] The nucleic acid delivery agent (administration solution) used in experiment number H01 (reference example) is an aqueous solution containing mRNA (TriLink, WOTL84901, CleanCap EGFP) containing the reporter gene EGFP gene as the nucleic acid, and glucose. The concentrations of glucose and nucleic acid in the administration solution of the nucleic acid delivery agent are the same as in experiment number H02. In other words, the nucleic acid delivery agent used in experiment number H01 substantially does not contain anti-inflammatory agents or antioxidants.

[0134] (Table 8)

[0135] The gene transfer method in Experimental Example 8 was the same as in Experimental Example 1, except for the person who collected the sample, the composition of the nucleic acid transfer agent, the conditions of the electrical pulses in the electroporation method, and the type of nucleic acid (mRNA). The conditions for culturing the adipose tissue fragments and imaging under a microscope were also the same as in Experimental Example 1. The conditions for each electrical pulse were as follows: • Pulse intensity: 100V • Pulse width: 10ms • Pulse interval: 10ms • Number of pulses: 5 pulses • Pulse decay: 10% • Pulse polarity: + / -

[0136] Figure 8 shows micrographs of adipose tissue samples imaged with a fluorescence microscope in Experimental Example 8. Comparing Experimental Example H01 and Experimental Example H02, the gene expression is much stronger in Experimental Example H02 than in Experimental Example H01. Therefore, it can be seen that nucleic acid delivery agents containing antioxidants / anti-inflammatory agents have a higher nucleic acid delivery efficiency than nucleic acid delivery agents that do not contain antioxidants or anti-inflammatory agents.

[0137] Furthermore, comparing Experimental Example 2 and Experimental Example 8, it can be seen that the efficiency of nucleic acid introduction by antioxidants / anti-inflammatory agents is similarly increased whether the vector packaging the nucleic acid is a plasmid or mRNA. Thus, it is considered that the effect of the present invention on the efficiency of nucleic acid introduction is exerted regardless of the type of vector.

[0138] (Experimental Example 9) As samples, 50-100 mg of human adipose tissue fragments and a nucleic acid delivery agent were prepared. In experiments I01-I04, samples (SF05) collected from the same subject were used.

[0139] The nucleic acid delivery agent (administration solution) used in experiments I02, I03, and I04 was an aqueous solution containing adeno-associated virus (serotype AAV8, Vector Builder, AAV8S(VB010000-9394npt)-C) containing the reporter gene EGFP gene as the nucleic acid, a nucleic acid delivery aid, and glucose. The glucose concentration in the administration solution was the same as in Experimental Example 1. The amount of adeno-associated virus in 5 μL of the administration solution was 2.00 × 10⁶. ９ It is GC.

[0140] The composition of the nucleic acid delivery aids is shown in Table 9 below. The nucleic acid delivery aids used in experiments I02 to I04 included anti-inflammatory agents, or a combination of antioxidants and anti-inflammatory agents.

[0141] The nucleic acid delivery agent (administration solution) used in experiment number I01 (reference example) is an aqueous solution containing glucose and adeno-associated virus (AAV8, Vector Builder AAV8S(VB010000-9394npt)-C), which contains the reporter gene EGFP gene as the nucleic acid. The glucose concentration and the amount of adeno-associated virus in the administration solution of the nucleic acid delivery agent are the same as those in experiments I02, I03, and I04. In other words, the nucleic acid delivery agent used in experiment number I01 substantially does not contain anti-inflammatory agents or antioxidants.

[0142] (Table 9)

[0143] In each experiment with the specified experimental number, 5 μL of nucleic acid delivery agent was injected into the sample using a 30-gauge injection needle (Terumo, 1-4909-06). Subsequently, the adipose tissue fragments were cultured.

[0144] The adipose tissue samples were cultured under the same conditions as in Experimental Example 1, except that the culture period was extended to six weeks. Microscopic imaging was performed at 10x magnification. This allowed us to confirm the presence and degree of EGFP gene expression.

[0145] Figure 9 shows micrographs of adipose tissue samples imaged with a fluorescence microscope in Experimental Example 9. Comparing Experiment No. I01 with Experiment Nos. I02-I04, genes are expressed more strongly in Experiment Nos. I02-I04 than in Experiment No. I01. Therefore, it can be seen that nucleic acid delivery agents containing anti-inflammatory agents or antioxidants, or nucleic acid delivery agents containing both anti-inflammatory agents and nucleic acid delivery agents, have a higher nucleic acid delivery efficiency than nucleic acid delivery agents that do not contain anti-inflammatory agents or antioxidants.

[0146] Furthermore, in Experimental Example 9, nucleic acids were introduced into cells using a viral vector method with AAV, without the use of electroporation. Thus, it is thought that the gene transfer efficiency effect of nucleic acid delivery agents containing at least one of an anti-inflammatory agent and an antioxidant can also be exerted using viral vectors.

[0147] Furthermore, in experiments I02 and I04, the effects of gene expression were specifically observed primarily in dendritic cells. On the other hand, in experiment I03, the effects of gene expression were observed primarily in granule cell-like cells and a small number of stem cell-like cells. Thus, by changing the combination of antioxidants and anti-inflammatory agents, nucleic acids can be selectively introduced into specific cells among various cell types.

[0148] (Experimental Example 10) As samples, 50-100 mg of human adipose tissue fragments and a nucleic acid delivery agent were prepared. In experiments J01-J03, samples (SF08) collected from the same subject were used.

[0149] The nucleic acid delivery agent (administration solution) used in experiments J02 and J03 was an aqueous solution containing an adeno-associated virus (AAV5, Vector Builder, AAV5C(VB010000-9287ffw)-i) containing the reporter gene EGFP gene as the nucleic acid, a nucleic acid delivery aid, and glucose. The glucose concentration and the amount of adeno-associated virus in the administration solution were the same as in Experimental Example 9.

[0150] The composition of the nucleic acid delivery aid is shown in Table 10 below. The nucleic acid delivery aid used in experiments J02 to J03 included a muscarinic receptor inhibitor as an anti-inflammatory agent and an antioxidant.

[0151] The nucleic acid delivery agent used in experiment number J01 (reference example) is an aqueous solution containing glucose and adeno-associated virus (AAV5, Vector Builder, AAV5C(VB010000-9287ffw)-i) encoding the EGFP gene. The glucose concentration and adeno-associated virus amount in the administration solution of this nucleic acid delivery agent are the same as those in experiments J02 and J03. In other words, the nucleic acid delivery agent used in experiment number J01 substantially does not contain anti-inflammatory agents or antioxidants.

[0152] (Table 10)

[0153] In each experiment with the specified experimental number, 5 μL of nucleic acid delivery agent was injected into the sample using a 30-gauge injection needle (Terumo, 1-4909-06). Subsequently, the adipose tissue fragments were cultured.

[0154] The adipose tissue samples were cultured under the same conditions as in Experimental Example 1, except that the culture period was extended to four weeks. Microscopic imaging was performed at 10x magnification. This allowed us to confirm the presence and degree of EGFP gene expression.

[0155] Figure 10 shows micrographs of adipose tissue samples imaged with a fluorescence microscope in Experimental Example 10. Comparing Experimental Number J01 and Experimental Number J02, Experimental Number J02 shows specific gene expression in adipocytes where no gene expression was observed at all in Experimental Number J01. On the other hand, in Experimental Number J03, strong gene expression is specifically observed in nerve-like cells where almost no gene expression was observed in Experimental Number J01. Therefore, by changing the combination of antioxidants and anti-inflammatory agents, nucleic acids can be selectively introduced into specific cells among various cell types.

[0156] (Experimental Example 11) As samples, 50-100 mg of human adipose tissue fragments and a nucleic acid delivery agent were prepared. The composition of the nucleic acid delivery agent differed for each experiment number K01-K04, as described below. In each experiment number, a sample (SF06) collected from the same subject was used.

[0157] The nucleic acid delivery agent (administration solution) used in experiments K02 to K04 was an aqueous solution containing plasmid DNA (Aldevron, pALD-Nanoplasmid-CAG-EGFP) containing the reporter gene EGFP gene as the nucleic acid, a nucleic acid delivery aid, and glucose. The concentrations of glucose and plasmid DNA in the administration solution, which is the nucleic acid delivery agent, were the same as in Experimental Example 1.

[0158] The composition of the nucleic acid delivery aids is shown in Table 11 below. The nucleic acid delivery aids used in experiments K02 to K04 contain both antioxidants and anti-inflammatory agents. The nucleic acid delivery aid used in experiment K02 contains an agent (mequitazine) that exhibits both muscarinic receptor inhibition and histamine receptor inhibition (H1 + H4), and an antioxidant. The nucleic acid delivery aids used in experiments K03 and K04 contain a muscarinic receptor inhibitor and an antioxidant.

[0159] The nucleic acid delivery agent (administration solution) used in experiment number K01 (reference example) is an aqueous solution containing plasmid DNA (Aldevron, pALD-Nanoplasmid-CAG-EGFP) containing the reporter gene EGFP gene as the nucleic acid. The concentrations of glucose and plasmid DNA in the administration solution of the nucleic acid delivery agent are the same as those in experiments K02 to K04. In other words, the nucleic acid delivery agent used in experiment number K01 substantially does not contain anti-inflammatory agents or antioxidants.

[0160] (Table 11)

[0161] The gene transfer method in Experimental Example 11 was the same as in Experimental Example 1, except for the person who collected the sample, the composition of the nucleic acid transfer agent, and the conditions of the electrical pulses for the electroporation method. The conditions of the electrical pulses for each pulse were as follows: • Pulse intensity: 50V • Pulse width: 20ms • Pulse interval: 10ms • Number of pulses: 20 pulses • Pulse decay: 5% • Pulse polarity: + / -

[0162] Figure 11 shows micrographs of adipose tissue samples imaged with a fluorescence microscope in Experimental Example 11. Comparing Experimental Number K01 with Experimental Numbers K02-K04, genes are expressed more strongly in Experimental Numbers K02-K04 than in Experimental Number K01. Therefore, it can be seen that nucleic acid delivery agents containing anti-inflammatory and antioxidant agents have a higher nucleic acid delivery efficiency than nucleic acid delivery agents that do not contain anti-inflammatory or antioxidant agents.

[0163] Furthermore, in experiment number K02, a strong gene expression effect was observed primarily in adipocytes. On the other hand, in experiments K03 and K04, little gene expression was observed in adipocytes, and the gene expression effect was observed only in small cells. In this way, by changing the combination of antioxidants and anti-inflammatory agents, nucleic acids can be selectively introduced into specific cells among various cell types.

[0164] Referring to experiment number K02, it is considered preferable that the nucleic acid delivery agent or nucleic acid delivery aid, especially when selectively introducing nucleic acids into adipocytes, contains an agent that exerts both muscarinic receptor inhibitory and histamine receptor inhibitory effects, as well as an antioxidant.

[0165] (Experimental Example 12) As samples, 50-100 mg of human adipose tissue fragments and a nucleic acid delivery agent were prepared. In experiments L01-L03, samples (SF09) collected from the same subject were used.

[0166] The nucleic acid delivery agent (administration solution) used in experiment number L03 is an aqueous solution containing an adeno-associated virus (AAV5, Vector Builder, AAV5C(VB010000-9287ffw)-i) containing the reporter gene EGFP gene as the nucleic acid, a nucleic acid delivery aid, and glucose. The glucose concentration and the amount of adeno-associated virus in the administration solution, which is the nucleic acid delivery agent, are the same as in experiment example 9.

[0167] The composition of the nucleic acid delivery aid is shown in Table 12 below. The nucleic acid delivery aid used in experiment number L03 contains a muscarinic receptor inhibitor as an anti-inflammatory agent and an antioxidant.

[0168] The administration solution used in experiment number L01 (reference example) was a 5% (w / v) glucose aqueous solution. In other words, the administration solution used in experiment number L01 substantially contained neither nucleic acid nor nucleic acid delivery agent. The nucleic acid delivery agent (administration solution) used in experiment number L02 (reference example) was an aqueous solution containing glucose and adeno-associated virus (AAV5, Vector Builder, AAV5C(VB010000-9287ffw)-i), which encodes the reporter gene EGFP gene, as the nucleic acid. The glucose concentration and the amount of nucleic acid in the administration solution, which is the nucleic acid delivery agent, are the same as in experiment number L03. In other words, the nucleic acid delivery agent used in experiment number L02 substantially contained neither anti-inflammatory agents nor antioxidants.

[0169] (Table 12)

[0170] In each experiment with the specified experimental number, 5 μL of nucleic acid delivery agent was injected into the sample using a 30-gauge injection needle (Terumo, 1-4909-06). Subsequently, the adipose tissue fragments were cultured.

[0171] The adipose tissue samples were cultured under the same conditions as in Experimental Example 1, except that the culture period was 21 hours. After 21 hours, 10 μL of reactive oxygen species detection reagent (ROS Assay Kit -Highly Sensitive DCFH-DA-, Dojin, R252) was injected into the adipose tissue samples. The adipose tissue samples were cultured for another hour, and then imaged under a microscope (magnification 10x, exposure time 1 / 500 sec). This allowed for the confirmation of the presence or absence of reactive oxygen species generation and the degree of their expression.

[0172] Figure 13 shows micrographs of adipose tissue samples imaged with a fluorescence microscope in Experiment 12. In Experiment No. L01, where nucleic acid was not administered, only weak fluorescence was observed. Comparing Experiment No. L01 and Experiment No. L02, very strong fluorescence was observed in the adipocytes in Experiment No. L02. This indicates that significant reactive oxygen species generation occurred upon administration of nucleic acid. On the other hand, comparing Experiment No. L03 and Experiment No. L02, the fluorescence of adipocytes in Experiment No. L03 was significantly lower than in Experiment No. L02 and about the same as in Experiment No. L01. Therefore, it was shown that the generation of reactive oxygen species by nucleic acid was suppressed in Experiment No. L03. Thus, it can be seen that nucleic acid agents containing both anti-inflammatory agents and nucleic acid agents can significantly suppress the generation of reactive oxygen species caused by the introduction of nucleic acids.

[0173] (Experimental Example 13) As samples, 50-100 mg of human subcutaneous adipose tissue fragments and a nucleic acid delivery agent were prepared. The composition of the nucleic acid delivery agent differed for each experiment number M01 to M10, as described below. In experiments M01 and M02, samples (SF10) collected from the same subject were used.

[0174] The nucleic acid delivery agent (administration solution) used in experiments M01 and M02 contained a plasmid (Aldevron, pALD-Nanoplasmid-CAG-EGFP) containing the reporter gene EGFP gene as the nucleic acid, and was an aqueous solution containing a nucleic acid delivery aid (added as a stock solution as shown in Table 15; the same applies hereafter) and glucose. The glucose concentration in the administration solution, which is the nucleic acid delivery agent, was 5% (w / v), and the plasmid DNA concentration was the same as in Experimental Example 1.

[0175] The composition of the nucleic acid delivery aid used in experiment number M02 is shown in Table 13 below. The nucleic acid delivery aid used in experiment number M02 included a muscarinic receptor inhibitor as an anti-inflammatory agent and an antioxidant.

[0176] The nucleic acid delivery agent used in experiment number M01 (reference example) is an aqueous solution containing plasmid DNA (shown in Table 13) containing the reporter gene, the EGFP gene, and glucose. In other words, the nucleic acid delivery agent used in experiment number M01 substantially does not contain anti-inflammatory agents or antioxidants.

[0177] (Table 13)

[0178] Experiments M01 and M02 were conducted in the same manner as in Experiment Example 1, except for the electrical pulse conditions of the electroporator. The electrical pulse conditions were as follows: • Pulse intensity: 20V • Pulse width: 99.9ms • Pulse interval: 10ms • Number of pulses: 1 pulse • Pulse decay: 0% • Pulse polarity: +

[0179] In experiments M01 and M02, samples were cultured after irradiation with electrical pulses. The culture of the samples and the evaluation of the cultured samples (degree of EGFP gene expression) were performed in the same manner as in Experimental Example 1. The degree of EGFP gene expression is shown in Table 13.

[0180] Figure 14 is a micrograph of adipose tissue after nucleic acid introduction in Experimental Example 13. A comparison between Experimental Numbers M01 and M02 shows that using a nucleic acid introduction agent containing antioxidants and anti-inflammatory agents significantly enhances EGFP gene expression.

[0181] (Experimental Example 14) Mice (ICR, 9 weeks old, female) and a nucleic acid delivery agent were prepared. The composition of the nucleic acid delivery agent differed between experiment numbers Q01 and Q02, as described below. The nucleic acid delivery agent (administration solution) used in experiment number Q02 contained a plasmid (Vector Builder, VB010000-9834vem) containing the reporter gene EGFP gene as the nucleic acid, and was an aqueous solution containing a nucleic acid delivery aid (added as a stock solution as shown in Table 15; the same applies below) and glucose. The glucose concentration in the administration solution of the nucleic acid delivery agent was 5% (w / v), and the plasmid DNA concentration was 400 μg / mL (the same applies below).

[0182] The composition of the nucleic acid delivery aid is shown in Table 14 below. The nucleic acid delivery aid used in experiment number Q02 contains a muscarinic inhibitor and an antioxidant as anti-inflammatory agents. In experiment number Q01, the nucleic acid delivery agent (administration solution) does not contain the nucleic acid delivery aid (anti-inflammatory agent and antioxidant).

[0183] (Table 14)

[0184] In each experiment with the specified experimental number, under isoflurane anesthesia, the thighs of mice were depilated with clippers, and then punctured with a syringe (BD; 326666, 29G, 12.7mm) filled with a nucleic acid delivery agent along with a needle-type electrode (Nepagine; CUY568-4-0.5). After injecting 20 μL of the nucleic acid delivery agent, the mice were irradiated three times in a row with two types of electrical pulses consisting of a first pulse and a subsequent second pulse using an electroporator (Nepagine, NEPA21 Type II) (electroporation). The conditions for the electrical pulses are as follows. <First Pulse> - Pulse strength: 50V - Pulse width: 15ms - Pulse interval: 50ms - Number of pulses: 3 pulses - Pulse decay: 10% - Pulse polarity: + <Second Pulse> - Pulse strength: 7V - Pulse width: 50ms - Pulse interval: 50ms - Number of pulses: 3 pulses - Pulse decay: 40% - Pulse polarity: + / -

[0185] After irradiation with electrical pulses, mice were housed for 7 days. After collecting the thigh muscles of the mice, imaging was performed using a fluorescence microscope (Keyence, BZ-9000) in fluorescence mode (magnification 4x, exposure time 1 / 10 second). Figure 15 is a micrograph of the mouse tissue after nucleic acid introduction in Experimental Example 14. Comparing Experimental Number Q01 and Experimental Number Q02, a significant enhancement of EGFP gene expression is observed in Experimental Number Q02. Therefore, a significant gene expression enhancement effect can be observed by using a nucleic acid introduction agent containing a muscarinic receptor inhibitor as an anti-inflammatory agent and an antioxidant. Furthermore, it can be seen that the gene expression enhancement effect by anti-inflammatory agents and / or antioxidants described above can also be observed in the tissues of animals other than humans, such as mammals like mice.

[0186] Table 15 below shows the drugs corresponding to each symbol shown in Tables 1 to 14. (Table 15)

[0187] Furthermore, in each experimental example, stock solutions were prepared by dissolving each drug in the solvent shown in Table 15. As mentioned above, the nucleic acid delivery agent (administration solution) was prepared by mixing the stock solution and glucose solution with the nucleic acid solution. The final concentration of DMSO contained in the nucleic acid delivery agent was kept below 1%. Since DMSO is thought to diffuse into adipose tissue after injection of the nucleic acid delivery agent, the DMSO concentration in adipose tissue is considered to be below 0.1% when calculated from the volume of adipose tissue (50 μL or more).

[0188] Table 16 below details the samples indicated by each sample number for the adipose tissue used in each experimental example. (Table 16)

[0189] As described above, the content of the present invention has been disclosed through embodiments and examples, but the descriptions and drawings that constitute part of this disclosure should not be understood as limiting the invention. Various alternative embodiments, examples, and operational techniques will become apparent to those skilled in the art from this disclosure. Therefore, the technical scope of the present invention is defined solely by the inventive features relating to the claims that are reasonable from the above description.

[0190] This application claims priority under Japanese Patent Application No. 2024-195502, filed on 7 November 2024, the entire contents of said patent application are incorporated herein by reference.

Claims

1. A nucleic acid delivery aid comprising at least one of an anti-inflammatory agent and an antioxidant.

2. The nucleic acid delivery aid according to claim 1, wherein the anti-inflammatory agent comprises at least one of a histamine receptor inhibitor, a muscarinic receptor inhibitor, and a steroid.

3. A nucleic acid delivery aid according to claim 1 or 2, comprising at least two of a histamine receptor inhibitor, a muscarinic receptor inhibitor, a steroid, and an antioxidant.

4. A nucleic acid delivery aid according to any one of claims 1 to 3, comprising at least an antioxidant.

5. A nucleic acid delivery aid according to any one of claims 1 to 4, comprising both an anti-inflammatory agent and an antioxidant.

6. A nucleic acid delivery aid according to any one of claims 1 to 5, comprising at least a muscarinic receptor inhibitor.

7. A nucleic acid delivery aid according to any one of claims 1 to 6, comprising an antioxidant and a muscarinic receptor inhibitor.

8. A nucleic acid delivery aid according to any one of claims 1 to 7, comprising an antioxidant and a histamine receptor inhibitor.

9. A nucleic acid delivery aid according to any one of claims 1 to 8, comprising a steroid and a histamine receptor inhibitor.

10. A nucleic acid delivery aid according to any one of claims 1 to 9, comprising a steroid and a muscarinic receptor inhibitor.

11. A nucleic acid delivery aid according to any one of claims 1 to 10, comprising a histamine receptor inhibitor and a muscarinic receptor inhibitor.

12. The nucleic acid delivery aid according to any one of claims 2, 3, 8, 9, or 11, wherein the histamine receptor inhibitor is at least one of a histamine H1 receptor inhibitor, a histamine H2 receptor inhibitor, a histamine H3 receptor inhibitor, and a histamine H4 receptor inhibitor.

13. A nucleic acid delivery aid according to any one of claims 1 to 12, comprising a histamine H4 receptor inhibitor.

14. A nucleic acid delivery aid according to any one of claims 1 to 13, comprising an agent having both histamine receptor inhibitory and muscarinic receptor inhibitory effects.

15. The nucleic acid delivery aid according to claim 14, wherein the agent is mequitazine.

16. The aforementioned antioxidants include propofol, fospropofol, probucol, ibakhtol, sonolichromanol, succinobucol, ferulic acid, vanillic acid, 2,6-di-tert-butylphenol, α-tocopherol, tocofersolan, tocotrienol, ascorbic acid, allopurinol, rebamipide, melatonin, carvedilol, nicaraben, kojic acid, uric acid, ergothioneine, carnitine, pentoxifylline, theophylline, theobromine, paraxanthine, caffeine, DOPA, N-oleyldopamine, levodopa, methyldopa, melevodopa, droxidopa, dopaxamine, norepinephrine, epinephrine, adrenaline, levonordefrine, theoadrenaline, hydroxythiosol, masopropyl alcohol, dobutamine, isoprenaline A nucleic acid delivery aid according to any one of claims 1 to 15, comprising at least one selected from the group consisting of phosphorus, albumin, protochlor, isoproterenol, dichloroisoproterenol, limiterol, acteoside, quercetin, resveratrol, rosmarinic acid, catechin, epicatechin, ellagic acid, rutin, isofraxidin, chlorogenic acid, caffeic acid, N-acetylcysteine, omeprazole, esomeprazole, pramipexole, atorvastatin, metronidazole, fluvastatin, propylthiouracil, glutathione, β-carotene, nicotinamide, curcumin, luteolin, apigenin, kaempferol, vitamin K, lycopene, zeaxanthin, astaxanthin, canthaxanthin, lipoic acid, and donepezil.

17. The muscarinic receptor inhibitors include throspium, dalifenacin, atropine, tolterodine, oxybutynin, solifenacin, fesoterodine, propiverine, oxyphenonium, benzatropine, trihexyphenidyl, procyclidine, profenamine, hyoscyamine, anisotropine, pirenzepine, scopolamine, propantheline, dicyclomine, tropicamide, biperiden, quinidine, methantheline, cyclopentolate, glycopyrrolium, orphenadrine, dexetimide, chlorphenoxamine, benactidine, and butyl Scopolamine, Lebefenacin, Othironium, Emepronium, Bornaprine, Propiomazine, Olanzapine, Methixene, Oxyphenecycline, Promazine, Tridihexetyl, Homatropin, Benzquinamide, Promethazine, Diphenidol, Methotrimeprazine, Tiotropium, Ipratropium, Nicardipine, Quetiapine, Ziprasidone, Clozapine, Pipecuronium, Pancuronium, Disopyramide, Chlorprothixene, Doxepin, Amitriptyline, Desipramine, Imipramine, Nortriptyline, Paroxetine Cyproheptadine, mepenzolate, isopropamide, tramadol, methoscopolamine, acridinium, umecridinium, hexocycline, dosulepin, imidafenacin, bizotifen, tondiamine, cridinium, cyclimine, buclidine, doxylamine, amoxapine, escitalopram, flupentixol, galamine, rocuronium, doxacrium, methacholine, dimethindene, diphenhydramine, chlorpromazine, rapacronium, camylofin, difemeline, dihexyberine, mebeberine, piperidolate Claims 2, 3, 6, 7, comprising at least one selected from the group consisting of rosiverine, trimebutine, benzylonium, bevonium, difemanil, fenpiberinium, pentienate, pipenzolate, porzin, prifinium, thiemonium, thimepidium, fenglutalimid, oxytropium, tropatepine, etibenzatropin, batephenterol, biloxazine, clemastine, diphenylpyraline, homochlorcyclidine, alimazine, mequitazine, tarafenacin, zamifenacin, terenzepine, and lidocaine.A nucleic acid delivery aid as described in 10 or 11.

18. The steroids mentioned above are fluticasone, diflorasone, alclometasone, medrison, amcinonide, beclometasone, betamethasone, desoximetasone, urobetasol, mometasone, crocortolone, loteprednol, rimexolone, prednicarbate, butesonide, desonide, cortisone, difluprednate, halcinonide, clobetasolone, fluocortin, fluperolon, formocortal, halomethasone, fluchlorolon, fluocinolone, triamicinolone, flumetasone, hydrocortisone, fludroxycortide, and prednisolone. A nucleic acid delivery aid according to claim 2, 3, 9, or 10, comprising at least one selected from the group consisting of lon, methylprednisolone, fluocinonide, dexamethasone, flupredniden, fluocortol, difluocortol, clobetazone, flunisolide, ciclesonide, thixocortol, deflazacort, fludrocortisone, trilostane, paramethasone, aldosterone, meprednisone, cortibazole, prednilidene, cloprednol, prednisone, fluprednisolone, melengethol, levocetoconazole, and vamorolone.

19. The histamine H1 receptor inhibitors include brompheniramine, chlorpheniramine, diphenhydramine, doxylamine, chlorcyclidine, dexbrompheniramine, dexchlorpheniramine, metapyrylene, phenyndamine, pheniramine, phenyltroxamine, pyriramine, tenyldiamine, tondilamine, triprolidine, acribastine, astemizole, bepotastine, bilastine, cetirizine, desloratadine, ebastine, fexofenadine, ketotifen, levocetirizine, loratadine, mizolastine, quifenadine, rupatadine, terfenadine, azelastine, levocabastine, olopatadine, mequitazine, homo A nucleic acid delivery aid according to claim 12, comprising at least one selected from the group consisting of chlorcyclidine, alimazine, mepyramine, novelelastine, hydroxyzine, meclizine, buclidene, triprolidine, triperenamine, mebuhydroline, pimethixen, tadiphylline, oxatomide, emedastine, niaprazine, oxomemazine, chloropyramine, carebastine, fenspirid, deptropin, dasemazine, icotidine, mirtazapine, esmirtazapine, paliperidone, cetastine, antazoline, alinastine, dosurepin, pivaxidin, epinastine, temerastine, dorispan, carbinoxamine, and cremisole.

20. The nucleic acid delivery aid according to claim 12, wherein the histamine H2 receptor inhibitor comprises at least one selected from the group consisting of cimetidine, ranitidine, famotidine, nizatidine, roxatidine, lafutidine, thiotidine, nipelotidine, sfotidine, methiaamide, donetidine, ramoxotidine, zartidine, labortidine, zolantidine, dalcotidine, mefentidine, brimamide, oxymethidine, ethintidine, and icotidine.

21. The nucleic acid delivery aid according to claim 12, wherein the histamine H3 receptor inhibitor comprises at least one selected from the group consisting of pitrisant, proxyphan, conesin, ildabisant, thioperamide, babisant, clobemprit, siproxyphan, enalisanto, idofenpropit, sipralisanto, and samelisanto.

22. The nucleic acid delivery aid according to claim 12 or 13, wherein the histamine H4 receptor inhibitor comprises at least one selected from the group consisting of adriforant, isuforant, mequitazine, treforant, pimozide, ceriforant, thioperamide, JNJ39758979, JNJ7777120, JNJ10191584, ZPL389, VUF6002, A987306, and A943931.

23. A nucleic acid delivery agent comprising a nucleic acid delivery aid according to any one of claims 1 to 22, and a nucleic acid or a vector containing a nucleic acid.

24. A method for introducing nucleic acids into cells, comprising the step of contacting cells with both a nucleic acid or a vector containing a nucleic acid and a nucleic acid delivery aid according to any one of claims 1 to 22, or contacting cells with the nucleic acid delivery agent according to claim 23.

25. The nucleic acid delivery method according to claim 24, wherein the step of introducing nucleic acids comprises performing electrical and / or magnetic perforation on the cells.

26. A gene therapy agent comprising a nucleic acid delivery agent according to claim 23, or a nucleic acid delivery aid according to any one of claims 1 to 22.

27. A gene expression promoting reagent comprising a nucleic acid delivery agent according to claim 23, or a nucleic acid delivery aid according to any one of claims 1 to 22.

28. Cells or tissues containing nucleic acids introduced by the nucleic acid introduction method according to claim 24 or 25.

29. A product produced from the cells or tissue described in claim 28.

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