KR-20260067201-A - Tregs engineered to overexpress CX3CR1

Abstract

The present invention relates to the use of regulatory T cells engineered to overexpress CX3CR1. Since the regulatory T cells induced to overexpress CX3CR1 of the present invention reduced microglia activation in an LPS-induced neuroinflammatory animal model and suppressed 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, they can be used for reducing microglia activation or for treating neuroinflammatory diseases or neurodegenerative diseases caused by the overactivation of microglia in the brain.

Inventors

• 배현수
• 양주원
• 박선영
• 양혜진
• 엄기일

Assignees

• 경희대학교 산학협력단

Dates

Publication Date

20260512

Application Date

20241105

Claims (19)

1. A recombinant vector containing a gene encoding CX3CR1 (fractalkine receptor).
2. In claim 1, a recombinant vector that is a retrovirus.
3. A recombinant vector comprising a marker additionally in claim 1.
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, regulatory T cells that are CD4 + or CD25 + 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. In claim 7, a cell therapy composition for the prevention or treatment of neuroinflammatory diseases, which 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, in claim 7, it 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 inhibits the expression of NOS2 or COX2 in the cerebral cortex or hippocampus, as described in claim 7.
11. A cell therapeutic composition for the prevention or treatment of neuroinflammatory diseases, wherein, in claim 7, it 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 microglia are activated, in accordance with claim 12.
14. A pharmaceutical composition for the prevention or treatment of neuroinflammatory diseases, which inhibits the activation of microglia in claim 12.
15. In claim 12, a pharmaceutical composition for the prevention or treatment of neuroinflammatory diseases that improves cognitive function.
16. A pharmaceutical composition for the prevention or treatment of neuroinflammatory diseases, wherein the expression of CD86, NOS2, IL12a, IL1b, or IL23 genes is inhibited.
17. A pharmaceutical composition for the prevention or treatment of neuroinflammatory diseases, which inhibits the expression of NOS2 or COX2 in claim 12.
18. A pharmaceutical composition for the prevention or treatment of neuroinflammatory diseases, which inhibits the expression of TNF-α or IL-6, according to claim 12.
19. a) a step of 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.

Description

Regulatory T cells engineered to overexpress CX3CR1 The present invention relates to the use of regulatory T cells engineered to overexpress CX3CR1. 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. 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 microglia activation include MAPK, PKC, ROS, and NF-kB. The mitogen-activated protein (MAP) kinase family are key proteins acting as intracellular signaling mediators; they are activated in response to extracellular signals such as various inflammatory responses, apoptosis, cell differentiation, and growth in the human body, thereby activating transcription factors to regulate the transcription of necessary genes. The promoters of iNOS, TNF-α, and COX-2 genes expressed in activated microglia share common binding sites for NF-kB, and the expression of these genes is regulated by NF-kB activation. It is known that beta-amyloid and LPS activate NF-kB in microglia, as do gangliosides and thrombin. NF-kB activation by these activators 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. Figure 1 shows the effect of CX3CR1 + Tregs administration on improving memory function in a neuroinflammatory mouse model: A: Schematic diagram of the workflow showing the timeline of experiments and tests; B: Identification of WT Tregs using flow cytometry and isolation of retrovirus-transduced Tregs (CX3CR1 - Flag- and