Regulatory t cells manipulated to overexpress CX3CR1

Abstract

The present invention relates to uses of regulatory T cells manipulated to overexpress CX3CR1. The regulatory T cells induced to overexpress CX3CR1 according to the present invention reduce microglial activation in an LPS-induced neuroinflammation animal model and suppress immune inflammatory responses of activated microglia by inhibiting the expression of pro-inflammatory markers and cytokines in the cortical and hippocampal regions of the brain, and thus can be used for reducing microglial activation or for treating neuroinflammatory diseases or neurodegenerative diseases caused by hyperactivation of microglia in the brain.

Description

Regulatory T cells engineered to overexpress CX3CR1

[0001] The present invention relates to the use of regulatory T cells engineered to overexpress CX3CR1.

[0002] Neurodegenerative diseases are conditions characterized by the degeneration of mental function caused by the progressive structural and functional loss of neurons. These diseases involve the progressive degeneration of nerve cells in specific parts of the nervous system, accompanied by symptoms such as dementia, extrapyramidal abnormalities, cerebellar abnormalities, sensory disorders, and motor disorders; furthermore, abnormalities may occur in multiple areas simultaneously, resulting in complex symptoms. Diagnosis is based on the clinical presentation of the patient, but it is characterized by difficulty due to the diverse nature of symptoms and the frequent occurrence of common clinical manifestations across different diseases. These neurodegenerative diseases often manifest gradually and frequently develop alongside aging. Once onset, the disease progresses continuously over years or decades until death, and fundamental treatment is difficult, placing a significant social burden. While genetic factors based on family history play a role in the onset, acquired factors are also known to contribute significantly. Degenerative neurological diseases are broadly classified according to their clinical symptoms into progressive dementia (Alzheimer's disease, etc.), neurological abnormalities (Pick's disease, etc.), postural and motor abnormalities (Parkinson's disease, etc.), progressive ataxia, muscle atrophy and weakness, and sensory and motor disorders. Among these, Alzheimer's dementia, which has the highest prevalence of 6.54% in those aged 65 and older, accounts for 71.3% of all dementia cases, and cytotoxicity caused by beta-amyloid plaques (β-plaque), neuroinflammation, and neurofibrillary tangles is receiving attention as a direct cause of the disease.

[0003] Microglia are cells that perform primary immune functions in the central nervous system (CNS). They maintain a shape with long, slender branches and thin cell bodies, but when toxins are introduced from the outside or generated internally, they transform into an activated form with thick, short branches and round cell bodies to protect neurons from these toxins. Unlike normal microglia, activated microglia actively engage in phagocytosis and cell proliferation, and produce inflammatory mediators by expressing genes such as cytokines like TNF-α, IL-1β, and IL-6, chemokines, iNOS (inducible nitric oxide synthase), and COX-2 (cyclooxygenase-2). While microglia activation serves to remove damaged cells and protect neurons from invading bacteria or viruses, substances such as nitric oxide produced by excessively expressed iNOS, prostaglandins produced by COX-2, and TNF-α exhibit toxicity to neurons; consequently, microglia activation exacerbates neuronal damage. Furthermore, substances released by dying neurons trigger microglia activation, leading to a continuous vicious cycle of neurodegeneration. In fact, microglia activation has been reported to be associated with various neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), Creutzfeldt-Jakob disease (CJD), and multiple sclerosis. Substances that activate microglia include the bacterial endotoxin lipopolysaccharide (LPS), interferon-γ, beta-amyloid, and gangliosides. Signaling pathways involved in the activation of microglia include MAPK, PKC, ROS, and NF-kB.The mitogen-activated protein (MAP) kinase family consists of key proteins acting as intracellular signaling mediators. In the human body, these proteins are activated in response to various extracellular signals, such as inflammatory responses, apoptosis, cell differentiation, and growth. They activate transcription factors to regulate the transcription of necessary genes. The promoters of the iNOS, TNF-α, and COX-2 genes expressed in activated microglia share a common binding site for NF-kB, and the expression of these genes is regulated by the activation of NF-kB. It is known that beta-amyloid and LPS activate NF-kB in microglia, as do gangliosides and thrombin. The activation of NF-kB by these activating agents occurs within 15 minutes, promoting the production of inflammatory cytokines. Although the relationship between microglia activation and neurodegenerative diseases has not yet been fully elucidated, it is generally accepted that microglia activation is involved in the onset and progression of these diseases. Therefore, inhibiting microglia activation could be an effective treatment to mitigate the progression of neurodegenerative diseases.

[0004] The object of the present invention is to provide a recombinant vector for CX3CR1 overexpression.

[0005] In addition, the object of the present invention is to provide a regulatory T cell that overexpresses the CX3CR1 protein.

[0006] In addition, the objective of the present invention is to provide a cell therapeutic composition for the prevention or treatment of neuroinflammatory diseases.

[0007] In addition, the objective of the present invention is to provide a pharmaceutical composition for the prevention or treatment of neuroinflammatory diseases.

[0008] In addition, the objective of the present invention is to provide a method for producing regulatory T cells that overexpress the CX3CR1 protein.

[0009] In addition, the objective of the present invention is to provide a method for preventing or treating neuroinflammatory diseases.

[0010] To achieve the above objective, the present invention provides a recombinant vector comprising a gene encoding CX3CR1.

[0011] In addition, the present invention provides regulatory T cells that overexpress the CX3CR1 protein by transduction with the recombinant vector.

[0012] In addition, the present invention provides a cell therapy composition for the prevention or treatment of neuroinflammatory diseases comprising the regulatory T cells as an active ingredient.

[0013] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of neuroinflammatory diseases comprising the vector or regulatory T cell as an active ingredient.

[0014] In addition, the present invention provides a method for producing regulatory T cells that overexpress the CX3CR1 protein.

[0015] In addition, the present invention provides a method for preventing or treating neuroinflammatory diseases, comprising the step of administering the cell therapeutic composition or the pharmaceutical composition to an individual.

[0016] According to the present invention, regulatory T cells induced to overexpress CX3CR1 of the present invention reduce the activation of microglia in an LPS-induced neuroinflammatory animal model and suppress the immune inflammatory response of activated microglia by suppressing the expression of inflammatory markers and cytokines in the cortex and hippocampus regions of the brain; therefore, the present invention can be used for reducing microglia activation or for treating neuroinflammatory diseases or neurodegenerative diseases caused by the overactivation of microglia in the brain.

[0017] FIGS. 1 to 4 are CX3CR1 + This is a diagram confirming the effect of Tregs administration on improving memory function in a neuroinflammatory mouse model:

[0018] Fig. 1: Schematic diagram of a workflow showing the timeline of experiments and tests;

[0019] Fig. 2: Identification of WT Tregs using flow cytometry and retrovirus-transduced Tregs (CX3CR1 - Flag - and CX3CR1 + Flag + Separation of )

[0020] Fig. 3: Passive avoidance test results; and

[0021] Fig. 4: Y-maze test results.

[0022] FIGS. 5 to 8 are CX3CR1 + This figure confirms the effects of Tregs on homing and the expression of activated microglia:

[0023] Fig. 5: CX3CR1 in the paramedian sagittal section stained with Flag tags + Flag + Cell distribution;

[0024] Fig. 6: CX3CR1 in the hippocampus region + Flag + High-resolution analysis of cells;

[0025] Fig. 7: Immunofluorescence image of Iba1 in the hippocampal DG region; and

[0026] Fig. 8: Quantification of Iba1 immunofluorescence in the hippocampal DG region.

[0027] FIGS. 9 to 13 are CX3CR1 + This is a diagram confirming the gene expression regulatory effects of pro-inflammatory markers by Tregs using qRT-PCR:

[0028] Fig. 9: CD86;

[0029] Fig. 10: NOS2;

[0030] Fig. 11: IL12a;

[0031] Fig. 12: IL1b; and

[0032] Fig. 13: IL23.

[0033] Figures 14 and 15 are CX3CR1 + This is a diagram confirming the regulatory effect of Tregs on the expression of proteins in the inflammatory pathway:

[0034] Fig. 14: Western blot analysis results of NOS2 and COX2 proteins; and

[0035] Fig. 15: Differences in expression in the cortex and hippocampus of the mouse brain.

[0036] FIGS. 16 to 18 are CX3CR1 + This is a study confirming the inhibitory effect of Tregs on pro-inflammatory cytokine expression:

[0037] Fig. 16: TNF-α;

[0038] Fig. 17: IL-6; and

[0039] Fig. 18: IL-1β.

[0040] Hereinafter, the present invention will be described in detail with reference to the attached drawings for embodiments of the present invention. However, the following embodiments are presented as examples of the present invention, and if it is determined that a detailed description of a technology or configuration well known to those skilled in the art may unnecessarily obscure the essence of the present invention, such detailed description may be omitted, and the present invention is not limited thereby. The present invention is capable of various modifications and applications within the scope of the claims set forth below and the equivalents interpreted therefrom.

[0041] Furthermore, the terminology used in this specification is used to appropriately describe preferred embodiments of the present invention, and may vary depending on the intent of the user or operator, or the conventions of the field to which the present invention belongs. Accordingly, the definitions of these terms should be based on the content throughout this specification. Throughout the specification, when a part is described as "comprising" a certain component, unless specifically stated otherwise, this means that it does not exclude other components but may include additional components.

[0042]

[0043] In one aspect, the present invention relates to a recombinant vector comprising a gene encoding CX3CR1 (fractalkine receptor).

[0044] In one embodiment, the recombinant vector may be a vector for overexpressing CX3CR1 (fractalkine receptor) in Treg.

[0045] In one embodiment, the recombinant vector may be a virus vector and may be a retrovirus.

[0046] In one embodiment, the recombinant vector may additionally include a marker, and the marker may be a tag gene, an antibiotic resistance gene, a selection marker gene, a gene encoding beta-glucuronidase (β-glucuronidase encoding gene), chloramphenicol acetyltransferase, luciferase, or a gene encoding a fluorescent protein (fluorescent protein encoding gene).

[0047] In one embodiment, the tag may be a His tag, a Myc(c-myc) tag, a FLAG tag, a HA tag, or a T7 tag.

[0048] In one embodiment, the fluorescent protein may be a green fluorescent protein (GFP), an enhanced green fluorescent protein (EGFP), a yellow fluorescent protein (YFP), a red fluorescent protein (RFP), an orange fluorescent protein (OFP), a cyan fluorescent protein (CFP), a blue fluorescent protein (BFP), a far-red fluorescent protein, or a tetracysteine ​​motif.

[0049] In one embodiment, the selection marker gene may be selected from the group consisting of neomycin phosphotransferase, hygromycin phosphotransferase, puromycin, histidinol dehydrogenase, guanine phosphotransferase, and zeocin.

[0050] As used in the present invention, the term “recombinant vector” refers to a DNA construct containing a nucleic acid molecule operably linked to a suitable regulatory sequence capable of carrying out the expression of the nucleic acid molecule in a suitable host. Such regulatory sequences include a promoter for carrying out transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence regulating the termination of transcription and translation. The vector may be a plasmid, a phage particle, a virus, or simply a potential genome insert. When converted into a suitable host, the vector may replicate and function independently of the host genome, or, in some cases, be integrated into the genome itself. In this specification, “plasmid,” “expression plasmid,” “virus,” and “vector” are often used interchangeably.

[0051] As used in the present invention, the term “viral vector” includes retroviruses, adenoviruses, parvoviruses (e.g., adeno-associated viruses), coronaviruses, negative-strand RNA viruses, such as orthomyxoviruses (e.g., influenza viruses), rhabdoviruses (e.g., rabies and vesicular stomatitis viruses), paramyxoviruses (e.g., measles and sendai viruses), positive-strand RNA viruses, such as piconaviruses and alphaviruses, and double-strand DNA viruses, such as adenoviruses, herpesviruses (e.g., herpes simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxviruses (e.g., cowpox, fowlpox, and canary smallpox viruses). Other viruses include, for example, Norwak virus, togavirus, flavivirus, reovirus, papovavirus, hepadnavirus, and hepatitis virus. Examples of retroviruses include avian leukemia-sarcoma, mammalian type C, type B, and type D viruses, the HTLV-BLV group, lentiviruses, and spumaviruses (Coffin, JM, Retroviridae: The Virus and their replication, In Fundamental Virology, Third Edition, BN Fields et al., Eds., LippincottRaven Publishers, Philadelphia, 1996).

[0052] As used in the present invention, the term "expression" refers to the process in which a polypeptide is generated based on the nucleic acid sequence of a gene. In this sense, gene expression includes not only the processes of transcription and translation, but also post-transcriptional and post-translational processes that may affect the biological activity of the gene or gene product. These processes include, but are not limited to, RNA synthesis, processing, and transport, as well as polypeptide synthesis, transport, and post-translational modification of the polypeptide.

[0053] The term "overexpression" as used in this invention refers to the significant up-regulation of the expression of a specific gene into mRNA or protein by intracellular gene transcription or translation.

[0054] In the present invention, the term “introduction” in relation to the insertion of a nucleic acid sequence into a cell means “transfection,” or “transformation,” or “transduction,” and includes the introduction of a nucleic acid sequence into a eukaryotic or prokaryotic cell, wherein the nucleic acid molecule may be introduced into the cell’s genome (e.g., chromosomes, plasmids, chloroplasts, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).

[0055] Microglia, which act as macrophages in the brain, are important cells that regulate immune responses within the central nervous system. Their activation plays a crucial role in maintaining CNS homeostasis by removing foreign substances caused by drugs or toxins and secreting nerve growth factors. However, exposure to harmful stressors—such as signals from damaged neurons, the accumulation of abnormally modified proteins due to external stimuli, or the invasion of pathogens—can lead to excessive microglia activity, causing neuronal damage and potentially resulting in neurodegenerative diseases. In other words, unlike normal microglia, overly activated microglia actively engage in phagocytosis, proliferate, and generate inflammatory mediators by expressing pro-inflammatory cytokines and inflammation-related genes.

[0056] While the activation of microglia exhibits a beneficial effect of removing damaged cells and protecting neurons from invading bacteria or viruses, the activation of astrocytes, the production of nitric oxide (NO), and the increase in TNF-α cytokines are toxic to neurons and lead to neuronal death. Consequently, the activation of microglia exacerbates neuronal damage and becomes a cause of neurodegenerative diseases. Therefore, methods to inhibit excessive microglia activation can serve as a treatment for neurodegenerative diseases.

[0057] In one aspect, the present invention relates to a regulatory T cell (Treg) that overexpresses a CX3CR1 protein by transfection with a recombinant vector containing a gene encoding the CX3CR1 (fractalkine receptor) of the present invention.

[0058] In one embodiment, the regulatory T cell is CD4 + or CD25 + It could be a cell.

[0059] In one aspect, the present invention relates to a cell therapy composition for the prevention or treatment of neuroinflammatory diseases, comprising the regulatory T cells of the present invention as an active ingredient.

[0060] In one embodiment, the composition can inhibit neuroinflammation of the cerebral cortex or hippocampus induced by the activation of microglia.

[0061] In one embodiment, the composition can inhibit the expression of CD86, NOS2, IL12a, IL1b, or IL23 genes in the cerebral cortex or hippocampus.

[0062] In one embodiment, the composition can inhibit the expression of NOS2, COX2, TNF-α, or IL-6 in the cerebral cortex or hippocampus.

[0063] In one embodiment, the composition can inhibit the hyperactivation of microglia in the cerebral cortex or hippocampus.

[0064] The above cell therapeutic agent may be administered to the human body through any general route as long as it can reach the target tissue, and may be administered parenterally or orally; in the case of oral administration, the cells may be administered encapsulated in an implant material, etc.

[0065] In one aspect, the present invention relates to a pharmaceutical composition for the prevention or treatment of neuroinflammatory diseases, comprising the recombinant vector of the present invention or the regulatory T cell of the present invention as an active ingredient.

[0066] In one embodiment, the neuroinflammatory disease may be a neuroinflammatory disease in which microglia are activated.

[0067] In one embodiment, the composition can inhibit the activation of microglia.

[0068] In one embodiment, the neuroinflammation may be brain neuroinflammation.

[0069] In one embodiment, the neuroinflammation may be a neurodegenerative disease, and the neurodegenerative disease may be any one selected from the group consisting of Alzheimer's disease, Parkinson's disease, Creutzfeldt-Jakob disease (CJD), Hallerforten-Spatz disease, Huntington's disease, multiple system atrophy, dementia, pretemporal dementia, amyotrophic lateral sclerosis, spinal muscular atrophy, spinocerebellar atrophy (SCA), meningoencephalitis, bacterial meningoencephalitis, viral meningoencephalitis, CNS autoimmune disorder, multiple sclerosis (MS), and acute ischemic injury, and the neurodegenerative disease may be a neurodegenerative disease in which microglia activation is increased.

[0070] In one embodiment, the composition can improve cognitive function and can inhibit the expression of CD86, NOS2, IL12a, IL1b, or IL23 genes, and can inhibit the expression of NOS2, COX2, TNF-α, or IL-6.

[0071] In the present invention, the term "prevention" refers to any act of suppressing or delaying the occurrence, spread, and recurrence of neuroinflammatory diseases or neurodegenerative diseases by administering a pharmaceutical composition according to the present invention, and the term "treatment" refers to any act of improving or beneficially altering the symptoms of neuroinflammatory diseases or neurodegenerative diseases by administering a component of the composition of the present invention. A person skilled in the art to which the present invention pertains would be able to determine the precise criteria for diseases to which the composition of the present invention is effective, and to judge the degree of improvement, enhancement, and treatment, by referring to materials provided by organizations such as the Korean Medical Association.

[0072] In the present invention, the term "therapeutically effective amount" used in combination with the active ingredient refers to an amount effective for preventing or treating neuroinflammatory or neurodegenerative diseases, and the therapeutically effective amount of the composition of the present invention may vary depending on various factors, such as the method of administration, the target site, and the patient's condition. Therefore, when used in humans, the dosage should be determined as an appropriate amount by considering both safety and efficacy. It is also possible to estimate the amount used in humans from the effective amount determined through animal experiments. These matters to be considered when determining the effective amount are described, for example, in Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed. (2001), Pergamon Press; and EW Martin ed., Remington's Pharmaceutical Sciences, 18th ed. (1990), Mack Publishing Co.

[0073] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. As used in the present invention, the term "pharmaceuticalally effective amount" refers to an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment and that does not cause side effects. The effective dose level may be determined based on factors including the patient's health condition, the type of neuroinflammatory or neurodegenerative disease, the etiology or severity of the neuroinflammatory or neurodegenerative disease, the drug's activity, sensitivity to the drug, the method of administration, the time of administration, the route of administration and elimination rate, the duration of treatment, drugs used in combination or concurrently, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered as a single or multiple doses. Considering all of the above factors, it is important to administer an amount that obtains maximum effect with a minimum amount without side effects, and this can be easily determined by a person skilled in the art.

[0074] The pharmaceutical composition of the present invention may include a carrier, a diluent, an excipient, or a combination of two or more of these commonly used in biological preparations. As used in the present invention, the term "pharmaceutical acceptable" means exhibiting properties that are not toxic to cells or humans exposed to the composition. The carrier is not particularly limited as long as it is suitable for in vivo delivery of the composition, and may be used, for example, compounds listed in Merck Index, 13th ed., Merck & Co. Inc., saline solution, sterile water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and mixtures of one or more of these components, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added as needed. Additionally, diluents, dispersants, surfactants, binders, and lubricants may be added to formulate the composition into primary formulations such as aqueous solutions, suspensions, and emulsions, as well as pills, capsules, granules, or tablets. Furthermore, it can be preferably formulated according to each disease or component using appropriate methods in the field or methods disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).

[0075] In one embodiment, the pharmaceutical composition may be one or more formulations selected from the group comprising oral formulations, topical preparations, suppositories, sterile injectable solutions, and sprays, and an oral or injectable formulation is more preferred.

[0076] As used in the present invention, the term "administration" means providing a specific substance to an individual or patient by any appropriate method. Depending on the intended method, it may be administered parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or locally as an injectable formulation) or orally. The dosage varies depending on the patient's body weight, age, gender, health status, diet, time of administration, method of administration, excretion rate, and severity of the disease. Liquid formulations for oral administration of the composition of the present invention include suspensions, liquid formulations, emulsions, syrups, etc. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients, such as humectants, sweeteners, flavorings, and preservatives, may be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, suppositories, etc. The pharmaceutical composition of the present invention may also be administered by any device capable of delivering the active substance to target cells. Preferred modes of administration and formulations include intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, drip infusions, etc. Injectables can be prepared using aqueous solvents such as physiological saline solution and Ringer's solution, vegetable oils, higher fatty acid esters (e.g., ethyl oleate), alcohols (e.g., ethanol, benzyl alcohol, propylene glycol, glycerin, etc.), non-aqueous solvents, and may include pharmaceutical carriers such as stabilizers to prevent deterioration (e.g., ascorbic acid, sodium bisulfite, sodium pyrosulfite, BHA, tocopherol, EDTA, etc.), emulsifiers, buffers to adjust pH, and preservatives to inhibit microbial growth (e.g., phenylmercury nitrate, thimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, etc.).

[0077] The term "individual" as used in the present invention refers to any animal, including humans, monkeys, cattle, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits, or guinea pigs, that has developed or may develop the aforementioned neuroinflammatory disease or neurodegenerative disease, and the said diseases can be effectively prevented or treated by administering the pharmaceutical composition of the present invention to the individual. The pharmaceutical composition of the present invention may be administered in conjunction with existing therapeutic agents.

[0078] The pharmaceutical composition of the present invention may further include pharmaceutically acceptable additives, wherein the pharmaceutically acceptable additives may include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose, mannitol, malt syrup, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, sucrose, dextrose, sorbitol, and talc. The pharmaceutically acceptable additive according to the present invention is preferably included in an amount of 0.1 to 90 parts by weight with respect to the composition, but is not limited thereto.

[0079] In one aspect, the present invention relates to a method for producing regulatory T cells overexpressing a CX3CR1 protein, comprising the steps of: a) transfecting a plasmid containing a gene encoding CX3CR1 (fractalkine receptor) into a retroviral vector; b) isolating regulatory T cells from splenocytes; and c) transducing the retroviral vector into the regulatory T cells.

[0080] In one aspect, the present invention relates to a method for preventing or treating cancer comprising the step of administering a cell therapeutic composition or a pharmaceutical composition of the present invention to an individual.

[0081] The term "individual" as used in this invention refers to a subject requiring a method for the prevention, control, or treatment of a disease, and may be used without limitation and includes humans, dogs, monkeys, cats, rodents, e.g., mice, genetically modified mice, etc. More specifically, it refers to mammals such as humans or non-human primates, mice, rats, dogs, cats, horses, and cattle.

[0082] The present invention will be explained in more detail through the following examples. However, the following examples are intended only to illustrate the content of the present invention and do not limit the present invention.

[0083]

[0084] Example 1. Production of regulatory T cells (Tregs) engineered to overexpress CX3CR1

[0085] To construct the retroviral vector, 5 × 10⁶ were prepared with 2 mL of DMDM ​​(Dulbecco's Modified Dulbecco's medium) one day before transfection. 5platA cells / well were seeded into 6-well plates, and platA cells (Retroviral Packaging Cell Line) were transfected by treating them for 48 h with Optimem (Gibco, Grand Island, NY, USA), which contained 2 μg of the packaging vector pCL-Eco (Novus, Centennial, CO, USA) conjugated with a plasmid encoding m5p-CX3CR1-2A-Flag and 1.6% Lipofectamine 3000 (Invitrogen, Waltham, MA, USA). To generate Tregs (Regulatory T cells), splenocytes were extracted from C57BL / 6 mice (7–8 weeks old), homogenized, and mechanically lysed. The obtained splenocytes were filtered through a 40-μm cell strainer, and according to the manufacturer's protocol (CD4 + CD25 + CD4 using MACS (magnetic-activated cell sorting) according to the regulatory T cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) + CD25 + Treg(s) were isolated. Subsequently, the filtered solution of the supernatant of the transfected platA cells, IL-2, and protamine sulfate (Sigma Aldrich; Saint Louis, MO, USA) at a final concentration of 6 mg / mL were mixed with the isolated CD4 + CD25 + A retrovirus overexpressing CX3CR1 (fractalkine receptor) by treating Tregs with CD4 + CD25 +Tregs were transduced. The transduced Tregs were stirred at 1800 rpm at 32 ℃ for 2 hours, and after one day, CX3CR1 was sorted using an S3e cell sorter (Bio-Rad; Hercules, CA, USA). - Flag - Treg and CX3CR1 + Flag + Treg was classified (Fig. 2). Classified CX3CR1 - Flag - Treg and CX3CR1 + Flag + Treg was cultured for 3 days in complete medium supplemented with IL-2 (100 UI / mL).

[0086]

[0087] Example 2. CX3CR1 in a neuroinflammation animal model + The cognitive function enhancement effect of Treg

[0088] To determine whether adoptive transfer therapy using Tregs designed to selectively migrate in the forebrain, as prepared in Example 1 above, affects neuroinflammation, CX3CR1 (fractalkine receptor 1)-transgenic Tregs (Regulatory T cells) (CX3CR1 - or CX3CR1 + ) was administered, and the improvement in memory and cognitive function impairments of neuroinflammatory diseases induced by LPS administration was confirmed using Y-maze tests (Fig. 1). Specifically, 11 to 12-week-old male C57BL / 6J mice were divided into (I) a saline group (control), (II) an LPS (500 μg / kg) administration group (LPS), and (III) an LPS + Treg administration group (cx3cr1 - Flag - ; cx3cr1 -) and (IV) LPS + Treg administration group (cx3cr1 + Flag + ; cx3cr1 + They were divided into 4 groups (5 mice per group), the control group was administered saline (0.9% NaCl) every test day, the LPS group was administered LPS intraperitoneally (ip) at a dose of 500 μg / kg in saline for 7 consecutive days for 1 week, and the LPS + Treg group was administered LPS for 1 week, followed by Treg (cx3cr1 - or cx3cr1 + ; 1 × 10 5 Treg cells / mouse iv) were administered intravenously via the tail. Eleven days after administration of Treg, spatial working memory, learning ability, and memory were evaluated using the Y-maze test and passive avoidance test, and the mice were euthanized. For the Y-maze test, mice were placed in a Y-maze consisting of three identical arms spaced 120° apart and allowed to navigate for 5 minutes, after which their spatial working memory was investigated. Additionally, for the passive avoidance test, a training regimen was conducted for 2 days in a door-separated light chamber and a dark chamber (20 × 20 × 30 cm). On the training day, when a mouse entered the dark chamber, the door was closed and an electric shock (0.35 mA, 2 s) was applied to its paw, and the mouse was removed 30 seconds after the shock was administered. On the day of the test, the mice were placed in the light chamber, and the waiting time until they entered the dark chamber was recorded.

[0089] As a result of the passive avoidance test, the LPS (500 μg / kg, ip) administration group and cx3cr1 -Mice in the Treg administration group (86.25 ± 7.40 s and 94.2 ± 9.06 s, respectively) remained in the bright chamber for a shorter time compared to control mice (263.2 ± 40.81 s), and cx3cr1 + Mice in the Treg administration group (278 ± 42.52 s) were found to remain in the bright chamber for a longer period than control mice (Fig. 3). Additionally, Y-maze test results showed that short-term spatial memory dysfunction was induced in the LPS administration group compared to the control group, but cx3cr1 + In the Treg administration group, the rate of spontaneous alteration behavior improved (Fig. 4), cx3cr1 + We confirmed that Treg treatment improved spatial working memory function in LPS-induced neuroinflammatory mice.

[0090]

[0091] Example 3. CX3CR1 in a neuroinflammation animal model + Inhibitory effect of Treg on activated microglia expression

[0092] Since it is known that activated microglia induce neurodegeneration mediated by neuroinflammation upon LPS administration, CX3CR1 + CX3CR1 in Treg homing and expression of activated microglia + The effects of Tregs were investigated. First, to visualize retrovirus-induced Tregs in mouse brain subregions, CX3CR1, which was confirmed to migrate to the brain, was examined using a confocal laser scanning microscope with antibodies against the Flag (DYKDDDDK) tag. +Paramedian sagittal sections of Tregs were analyzed by immunofluorescence staining. Additionally, Iba1 (ionized calcium-binding adapter protein 1) was analyzed by immunofluorescence staining to measure microglia activation and neuronal loss in the mouse cortex or hippocampus. For immunofluorescence staining, mice were perfused with standard saline and then perfused with 4% PFA (paraformaldehyde) (in 0.1 M sodium phosphate buffer, pH 7.4). Cerebral tissue was removed, incubated overnight in fixatives, and then stored in 30% sucrose solution. After transferring the brain to a 30% sucrose solution, it was cut into 30-μm thick sections using a cryostat microtome (Leica CM 1850; Leica Microsystems, Wetzlar, Germany) and incubated overnight at 37°C. It was washed three times for 5 minutes each with PBS (pH 7.4) and permeabilized by heating at 65°C for 20 minutes in 10 mM sodium citrate buffer (pH 6.0). Brain sections were blocked with 1% BSA solution and treated with a 1:100 dilution of either the antibody against the DYKDDDDK tag (Flag-tag) (Cell Signaling Technology, Danvers, MA, USA) or the antibody against Iba1 (ionized calcium-binding adapter protein 1) (anti-Iba1 antibody; Millipore Corp, Billerica, MA, USA) as the primary antibody, and incubated overnight at 4°C. Subsequently, the sections were incubated with an Alexa Fluor 488 or Alexa Fluor 594 conjugated IgG secondary antibody at room temperature for 2 hours and washed three times with PBS for 10 minutes each.Washed sections were stained with DAPI staining solution, and fluorescence images were captured using an LSM 800 confocal laser-scanning microscope (Carl Zeiss, Oberkochen, Germany). Then, fluorescence intensity was measured and quantified using ImageJ software (http: / rsb.info.nih.gov / ij / ).

[0093] As a result, the relative increase in Flag-tag expression was most pronounced in the hippocampus (Figs. 5 and 6), and it was found that the expression of Iba1 (ionized calcium-binding adapter protein 1) increased in mice of the LPS-administered group compared to the control group, and CX3CR1 + In the Treg administration group, the expression of Iba1 in the DG region of the hippocampus was found to be significantly reduced compared to the LPS administration group (Figs. 7 and 8).

[0094]

[0095] Example 4. CX3CR1 in a neuroinflammation animal model + Inhibitory effect of Treg on LPS-induced pro-inflammatory markers

[0096] CX3CR1 of the present invention +To determine whether Tregs suppress the expression of activated microglia in an animal model of neuroinflammation, the expression of pro-inflammatory markers CD86, NOS2, IL12a, IL1b, and IL23 genes in the cortex and hippocampus of the animal model of neuroinflammation was evaluated by RT-qPCR. Specifically, total RNA was isolated from mouse brain tissues (cortex and hippocampus) of each group using the easy-BLUE RNA extraction kit (iNtRON Biotechnology, Seongnam, Korea, #17061), and cDNA was synthesized using Cyclescript reverse transcriptase (Bioneer, Daejeon, Korea). Subsequently, samples for RT-qPCR were prepared using the SensiFAST SYBR no-Rox kit (Bioline, London, UK), and RT-qPCR was performed using the CFX Connect System (Bio-Rad) under cycling conditions of 95°C for 10 s, 55°C for 10 s, and 72°C for 10 s.Target mRNA expression was analyzed using the ΔΔCt method, mouse β-actin was used as an endogenous control, and the primers used for RT-qPCR were: Cd86: forward, 5'-GACCGTTGTGTGTGTTCTGG-3' and reverse, 5'-GATGAGCAGCATCACAAGGA-3'; NOS2: forward, 5'-CAGCTGGGCTGTACAAACCTT-3' and reverse, 5'-CATTGGAAGTGAAGCGTTTCG-3'; Il23: forward, 5'-CCTTCTCCGTTCCAAGATCCT-3' and reverse, 5'-ACTAAGGGCTCAGTCAGAGTTGCT-3'; Il1b: forward, 5'-AAGCCTCGTGCTGTCGGACC-3' and reverse, 5'-TGAGGCCCAAGGCCACAGG-3'; IL12a: forward, 5'-AAGCTCTGCATCCTGCTTCAC-3' and reverse, 5'-GATAGCCCATCACCCTGTTGA-3'; and β-Actin: forward, 5'-GTGCTATGTTGCTCTAGACTTCG-3' and reverse, 5'-ATGCCACAGGATTCCATACC-3'.

[0097] Analysis results showed that the expression of pro-inflammatory genes increased in the cortex and hippocampus by LPS administration was Treg (CX3CR1 - It was reduced by ) administration, and this is CX3CR1 + It was shown to be reduced more significantly in the Treg administration group (Figs. 9 to 13), CX3CR1 + Treg is CX3CR1 - It can be seen that LPS-treatment significantly suppresses the expression of pro-inflammatory mRNA in the inflamed cortex and hippocampus compared to Treg.

[0098]

[0099] Example 5. CX3CR1 in a neuroinflammation animal model + Inhibitory effect of Tregs on LPS-induced neuroinflammation-related markers

[0100] CX3CR1 of the present invention +To determine whether Tregs alter the expression of neuroinflammation-related markers induced by LPS administration in the cortex and hippocampus in an animal model of neuroinflammation, the expression of NOS-2 and COX2 in the cerebral cortex and hippocampus was confirmed by Western blot analysis. Specifically, brain tissue from mice of each group was disrupted with PRO-PREP (iNtRON Biotechnology) at 4°C for 20 minutes, samples were extracted using 80% formic acid, and treated with a glass Dounce homogenizer. After adjusting the protein concentration of each sample using the Bradford assay, 30 μg of protein was subjected to 10% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and then transferred to a nitrocellulose membrane. The membranes were blocked with 5% skim milk at room temperature for 30 minutes and incubated overnight at 4°C with anti-CD86 antibody (1:1000; Cell Signaling Technology), anti-NOS2 antibody (1:2000; Santa Cruz Biotechnology, Dallas, TX, USA), or anti-β-actin antibody (1:5000; Santa Cruz Biotechnology), respectively. After washing the membranes with a TBS (Tris-buffered saline) solution containing 25 mM Tris-Cl, 150 mM NaCl, and 0.05% Tween-20, they were reacted with HRP (horseradish peroxidase)-conjugated secondary antibodies (anti-rabbit or anti-mouse IgG) for 2 hours. Immunoreactivity was visualized using a western blot detection reagent kit (Thermo, Waltham, MA, USA), and antibody binding was measured using a chemiluminescence detection system.Each band was quantified by performing densitometric analysis using ImageJ software.

[0101] As a result, LPS administration increased the expression of NOS2 and COX2 in the cortex and hippocampus (Fig. 14), which was reduced by the administration of Tregs, in particular, CX3CR1 - CX3CR1 over Treg + It was found that the reduction was even greater when Treg was administered (Fig. 15).

[0102]

[0103] Example 5. CX3CR1 in a neuroinflammation animal model + Inhibitory effect of Treg on LPS-induced inflammatory cytokines

[0104] The levels of TNF-α, IL-6, and IL-1β increased by treatment with LPS, a pro-inflammatory activator of microglia, are CX3CR1 of the present invention + To determine whether it is reduced by Treg administration, protein extracts were collected from the brain tissue of mice in each group according to the manufacturer's protocol (R&D Systems, Minneapolis, MN, USA) and an ELISA analysis was performed.

[0105] As a result, the levels of TNF-α, IL-6, and IL-1β in the group administered LPS increased significantly compared to the control group, and the expression levels of TNF-α and IL-6 were CX3CR1 - Treg administration group and CX3CR1 + Although it was significantly reduced in the Treg administration group, the expression level of IL-1β did not change significantly (Figs. 16 to 18). Through this, LPS induces the expression of the pro-inflammatory cytokines TNF-α and IL-6 in both the cerebral cortex and hippocampus, and CX3CR1 + It was confirmed that Treg administration inhibits this.

Claims

1. A recombinant vector containing a gene encoding CX3CR1 (fractalkine receptor).

2. In claim 1, a recombinant vector that is a retrovirus.

3. The recombinant vector according to claim 1, further comprising a marker.

4. In claim 3, the marker is a recombinant vector that is a tag gene, an antibiotic resistance gene, a selection marker gene, a gene encoding beta-glucuronidase (β-glucuronidase encoding gene), chloramphenicol acetyltransferase, luciferase, or a gene encoding fluorescent protein (fluorescent protein encoding gene).

5. Regulatory T cells (Treg) that overexpress the CX3CR1 protein by transduction with the recombinant vector of claim 1.

6. In Paragraph 5, CD4 + or CD25 + Regulatory T cells.

7. A cell therapy composition for the prevention or treatment of neuroinflammatory diseases, comprising the regulatory T cells of claim 5 as an active ingredient.

8. A cell therapy composition for the prevention or treatment of neuroinflammatory diseases, wherein the composition of claim 7 inhibits neuroinflammation of the cerebral cortex or hippocampus induced by the activation of microglia.

9. A cell therapy composition for the prevention or treatment of neuroinflammatory diseases, wherein the composition of claim 7 inhibits the expression of CD86, NOS2, IL12a, IL1b, or IL23 genes in the cerebral cortex or hippocampus.

10. A cell therapeutic composition for the prevention or treatment of neuroinflammatory diseases, wherein the composition of claim 7 inhibits the expression of NOS2 or COX2 in the cerebral cortex or hippocampus.

11. A cell therapeutic composition for the prevention or treatment of neuroinflammatory diseases, wherein the composition of claim 7 inhibits the expression of TNF-α or IL-6 in the cerebral cortex or hippocampus.

12. A pharmaceutical composition for the prevention or treatment of neuroinflammatory diseases, comprising the recombinant vector of claim 1 or the regulatory T cell of claim 5 as an active ingredient.

13. A pharmaceutical composition for the prevention or treatment of a neuroinflammatory disease, wherein the neuroinflammatory disease is one in which microglia are activated, as described in claim 12.

14. A pharmaceutical composition for the prevention or treatment of neuroinflammatory diseases, which inhibits the activation of microglia in claim 12.

15. A pharmaceutical composition for the prevention or treatment of neuroinflammatory diseases that improves cognitive function, according to claim 12.

16. A pharmaceutical composition for the prevention or treatment of neuroinflammatory diseases, wherein the composition of claim 12 inhibits the expression of CD86, NOS2, IL12a, IL1b, or IL23 genes.

17. A pharmaceutical composition for the prevention or treatment of neuroinflammatory diseases, wherein the composition inhibits the expression of NOS2 or COX2 according to claim 12.

18. A pharmaceutical composition for the prevention or treatment of neuroinflammatory diseases, wherein the composition inhibits the expression of TNF-α or IL-6, as described in claim 12. 19.a) Transfecting a plasmid containing a gene encoding CX3CR1 (fractalkine receptor) into a retroviral vector; b) a step of isolating regulatory T cells from splenocytes; and c) A method for producing regulatory T cells overexpressing CX3CR1 protein, comprising the step of transducing a retroviral vector into regulatory T cells.

20. A method for preventing or treating a neuroinflammatory disease comprising the step of administering the cell therapeutic composition of claim 7 or the pharmaceutical composition of claim 12 to an individual.

Images