CN117004632A - Chimeric antigen receptor modified glial cells and uses thereof - Google Patents
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Abstract
The present invention relates to chimeric antigen receptor-modified glial cells and uses thereof. In particular, the present invention relates to chimeric antigen receptors comprising an anti-amyloid oligomer binding domain, a transmembrane domain and an intracellular signaling domain, nucleic acid molecules encoding the same, compositions comprising the same, and uses thereof in the treatment and/or prevention of neurodegenerative diseases. The glial cell modified by the chimeric antigen receptor can effectively identify and swallow amyloid oligomers, reduce the release of inflammation and pro-inflammatory cytokines in the brain, and provide a brand-new treatment strategy for the field of neurodegenerative diseases.
Description
Technical Field
The present invention relates to chimeric antigen receptor-modified glial cells and uses thereof. In particular, the present invention relates to chimeric antigen receptors comprising an anti-amyloid oligomer binding domain, a transmembrane domain and an intracellular signaling domain, nucleic acid molecules encoding the same, compositions comprising the same, and uses thereof in the treatment and/or prevention of neurodegenerative diseases.
Background
Neurodegenerative diseases are a general term for diseases caused by degenerative degeneration and loss of chronic progressive nerve cells, and include Alzheimer's Disease (AD), parkinson's Disease (PD), huntington's Disease (HD), amyotrophic lateral sclerosis, etc., and are characterized by neuronal structure and dysfunction, neuronal loss, cognition and behavior abnormalities. Targets currently used to treat neurodegenerative diseases include: neurotransmitters, amyloid, intestinal flora, neuroinflammation, mitochondrial disability, oxidative stress, and intracellular signaling pathways, and the like. While these therapeutic strategies have shown some therapeutic effect in preclinical studies, most have no significant therapeutic effect at the clinical trial stage, and so it is of particular importance to develop therapies against disease pathogenesis.
AD, commonly known as senile dementia, is a neurodegenerative disease characterized mainly by progressive memory impairment, impaired cognitive function, personality changes, language disorders, etc. AD pathogenesis is mainly the abnormal aggregation of aβ and hyperphosphorylated tau protein to form toxic oligomers, leading to senile plaques and neurofibrillary tangles in the brain, leading to neuronal death. Currently, the therapeutic strategies under investigation for AD mainly include natural compounds, immunotherapy, gene therapy, stem cell therapy, and intestinal flora regulation. However, specific therapeutic drugs targeting the etiology have not been developed so far.
PD is a dyskinesia disease with an incidence of about 0.3%. The main pathological feature of PD is the death of Dopamine (DA) capable neurons and the appearance of Lewy bodies (Lewy bodies) in substantia nigra neurons formed by abnormal aggregation of alpha-synuclein. Abnormal aggregation of α -synuclein can disrupt synaptic vesicle integrity, interfere with dopamine metabolism, impair mitochondrial function, interfere with intracellular transport, and ultimately trigger dopaminergic neuronal death.
HD is a neurodegenerative disorder characterized by chorea-like symptoms, manifested as PD-like dyskinesias and ataxia, and AD-like dementia. HD is caused by the aggregation of a large number of polyglutamine-containing mHTT proteins in nerve cells, resulting in dysfunction and apoptosis of nerve cells in the cortex and striatum.
The cause of neurodegenerative diseases is complex, and many neurodegenerative diseases are characterized by abnormal aggregation of amyloid, such as beta amyloid (Abeta), microtubule-associated protein (tau), alpha synuclein (alpha synuclein) and mutant huntingtin (mHTT), etc., and oligomers formed by aggregation of protein monomers are key causative factors for the development of AD, PD, HD, etc., and are effective targets for diagnosing and treating the diseases. Among all aggregate forms, the most cytotoxic of the oligomers is directly related to the occurrence and development of the disease. Oligomers and fibers of different amyloid proteins deposit at different sites in the brain, damaging nerve cells in the corresponding areas, leading to cognitive and behavioral disorders. These amyloid proteins, although of differing primary sequences, aggregate to form similar spatial stereoisomers and possess similar toxic mechanisms. Therefore, a therapeutic strategy targeting amyloid toxic oligomers is of great importance for the diagnosis and treatment of a variety of neurodegenerative diseases.
In recent years, more and more researchers have focused their eyes on glial cells. Glial cells are another major class of cells in the nervous system, apart from neurons, mainly comprising: microglia, astrocytes, oligodendrocytes, oligodendrocyte Precursor Cells (OPC), and the like. In the central nervous system, glial cells play a variety of important physiological functions, such as clearing foreign bodies, participating in the formation of myelin sheath around axons, regulating normal physiological functions of synapses and synaptic regeneration, maintaining the integrity of the Blood Brain Barrier (BBB) and homeostasis of ions and neurotransmitters in the brain, etc.
Microglial cells are widely distributed in the brain and spinal cord, are the most important innate immune cells in the central nervous system, play roles in monitoring immunity in the brain and endogenous immune defenses, and play an important role in the occurrence and development of neurodegenerative diseases. In AD, microglia have a critical role in the clearance of aβ in the brain. The Abeta oligomer can interact with certain microglial cell surface receptors, on one hand, the Abeta oligomer induces the microglial cells to phagocytose and clear Abeta, and protects neurons from Abeta, on the other hand, a large amount of Abeta oligomer can excessively activate the microglial cells, promote the release of inflammatory factors and lead to neuroinflammation. At the same time, the effect of overactivated microglia phagocytosis of aβ is inhibited, further causing deposition of aβ. Aβ oligomers can also cause abnormal phagocytosis of synapses by microglia, by activating complement-associated clearance pathways, resulting in excessive loss of synapses and reduced cognitive ability. In addition, aβ oligomers can cause autophagic dysfunction of microglia, with reduced ability to degrade aβ. Therefore, microglial cells play a role of a double sword in the pathological process of AD, maintain the normal activation state of microglial cells, and acceleration of phagocytosis and clearance of Abeta toxic oligomers is a key to AD treatment. Current studies suggest that activated microglia can be divided into two states, M1 and M2. Microglia in M2 state can release anti-inflammatory factors (such as IL-4, IL-10 and the like), engulf damaged nerve cell fragments, promote tissue repair and regeneration of neurons, but when microglia are overactivated, the microglia can turn to M1 state, release a large amount of inflammatory factors such as IL-1 beta, IL-6, TNF-alpha, ROS, NO and the like, and have toxic effects on nerve cells, so that the nerve cells are apoptotic. Therefore, maintaining the normal M2 state of microglial cells and reducing the release of neuroinflammatory factors are of great significance for maintaining the healthy state of the central nervous system, and have great value for AD drug development.
The scavenger receptor SR-A is an important receptor that mediates microglial phagocytosis and clearance of Abeta. Studies have shown that SR-A promotes phagocytosis and clearance of Abeta while down-regulating expression of inflammation-associated genes. In the brain of AD patients, SR-a expression levels decrease and microglial cells have a significantly reduced ability to phagocytose and clear aβ oligomers, which gradually accumulate, resulting in neuronal apoptosis. The prior art has reported a cyclic heptapeptide XD4 capable of activating SR-A, which promotes binding of Abeta to SR-A receptor and enhances the ability of microglia to clear Abeta oligomers. Similarly, up-regulating the expression of SR-A can promote the phagocytosis and clearance of microglia to Abeta and reduce the release of neuroinflammatory factors, and is an effective AD gene therapy means. Astrocytes, a type of glial cell with the largest volume in the brain, are part of the BBB and play an important role in controlling the outward Zhou Zhuaiyun of aβ in the brain and the transport of peripheral aβ into the brain. In the brain of AD patients, astrocyte dysfunction leads to a decrease in aβ in the brain outward Zhou Zhuaiyun, leading to accumulation of aβ in the brain and formation of aβ plaques. In addition, reactive astrocytes are able to promote the activity of beta-secretase and gamma-secretase, resulting in overproduction of aβ. Aβ pathology in turn triggers astrocyte activation and dysfunction. Studies have shown that astrocyte dysfunction is also closely related to tau hyperphosphorylation and NFT formation. Tau protein in neurons may spread into astrocytes, resulting in loss of normal physiological function of astrocytes, further exacerbating AD conditions. In pathological conditions, aβ stimulates astrocytes to produce excessive reactive oxygen species (Reactive oxygen species, ROS). Although low levels of ROS have some protective effect on neurons. However, excessive ROS can induce nitrosation stress in neurons, leading to loss of normal function and death of neurons. The level of ROS released by astrocytes is closely related to the level of amyloid in the brain. Excessive levels of aβ and tau in the brain directly lead to damaging effects of reactive astrocytes on neurons. Inhibit astrocyte activation and reduce the release of inflammatory factors, and is a common means for modifying astrocyte functions at present.
MerTK receptors are predominantly expressed on M2 astrocytes and macrophages, and when activated mediate phagocytosis while inhibiting the release of pro-inflammatory cytokines. The MerTK structure is similar to the CAR structure in conventional CAR-T cells, including antigen recognition, transmembrane and intracellular signaling regions, and is an ideal receptor for CAR engineering.
Oligodendrocytes and oligodendrocyte precursor cells are a large number of glial cells in the brain. Oligodendrocytes express MHC-I molecules and immune receptors, have an immune regulation function, can regulate the functional state of microglia by expressing various cytokines and chemokines, can express various neurotrophic factors, and play a supporting role on neurons. Oligodendrocyte precursor cells are widely present in the central nervous system and can proliferate and differentiate in the demyelinated area to form oligodendrocytes, which repair damage caused by demyelination.
In the brain of AD patients, oligodendrocytes are easily stimulated by aβ oligomers, and demyelination occurs, resulting in impaired neuronal function and neuronal death. In addition, oligodendrocytes are also highly sensitive to the environment of inflammation in the brain caused by aβ. Oligodendrocytes are rich in microtubules, and both stabilization of microtubules and myelination require tau protein involvement, whereas tauopathies severely affect myelination and axonal transport. Thus, in AD, the status of oligodendrocytes is closely related to aβ and tau pathology.
Chimeric antigen receptor T cell therapy (Chimeric antigen receptor T-cell immunotherapy, CAR-T) is a precise targeted therapy for the treatment of tumors. The chimeric antigen receptor in CAR-T therapy is an artificially designed chimeric receptor, mainly comprising a recognition domain for known tumor antigen binding, hinge and transmembrane regions, an intracellular signaling domain of a T cell receptor and a co-stimulatory domain triggering T cell activation. After the chimeric antigen receptor is combined with the corresponding antigen, the activation of inflammatory pathways in immune cells is mediated, a large amount of cell killing factors and pro-inflammatory factors are released, and tumor cells are killed.
The antigen binding domain is the extracellular portion of the CAR, can mediate killing of the corresponding target cells by CAR-T cells, and cytokines, single chain antibodies, ligands, and the like can all be used as antigen binding domains of the CAR, with single chain antibodies being the most common.
The hinge region and transmembrane domain of the CAR are bridges connecting the extracellular antigen binding domain and the intracellular signaling domain. The hinge region provides sufficient flexibility to overcome steric hindrance of antigen and scFv binding. The transmembrane domain can anchor the CAR on the T cell membrane, typically derived from a type I protein. Different transmembrane domains affect the stability and function of CARs. Different hinge regions and transmembrane domains may be selected according to different requirements.
The intracellular signaling domain typically consists of one activation domain and one or more co-stimulatory domains. Most CARs activate CAR-T cells through cd3ζ. However, the signal mediated by cd3ζ alone is insufficient to induce reactive proliferation of T cells, resulting in limited persistence and activity of T cells in vivo. Adding a co-stimulatory domain may be effective in solving this problem. In addition to traditional CARs, researchers have tried to modify other receptors, such as Megf10, TLR4, TLR2, merTK, fcγ, etc., discussing the therapeutic advantages of multiple receptor modifications in different diseases, and hopefully expanding the scope of application of CAR technology.
In view of the fact that glial cells play a role of a double sword in the occurrence and development of neurodegenerative diseases, modifying the functions of the glial cells, increasing the beneficial functions of the glial cells, reducing the harmful functions of the glial cells, maintaining the normal activation state of the glial cells and accelerating the clearance of Abeta oligomers are potential therapeutic strategies. However, no drugs based on such therapeutic strategies are currently in clinical use. There are two main reasons, firstly, the existing glial cell function modification strategies are mainly to inhibit the inflammatory state of glial cells through nonsteroidal anti-inflammatory drugs or small molecule drugs, however, these strategies reduce the clearing capacity of the glial cells to Abeta oligomers while reducing the inflammation in the brain, so that AD cannot be effectively treated. How to solve the contradiction between the phagocytosis of Abeta oligomer by glial cells and the release of inflammatory factors, and the release of a large amount of pro-inflammatory factors is not caused while the phagocytosis of Abeta oligomer by glial cells is realized, which is the key point of a glial cell function modification strategy. Second, there is a lack of effective glial cell-targeted drug delivery systems. Some drugs used for microglial remodeling, such as Nec-1s, have a short half-life in vivo, are easily cleared in the blood circulation, and due to the BBB, only a very small amount of the drug can enter the brain. These drugs cannot target glial cells precisely after entering the brain, resulting in higher side effects. Therefore, enhancing the ability of drugs to penetrate the BBB, increasing the bioavailability of drugs within the central nervous system, is key to the successful development of such drugs.
The liposome nano-carrier is a high-efficiency gene drug delivery system and is widely applied to the research of diseases such as AD and the like. The liposome is used as a non-viral vector, and can overcome the defects of high immunogenicity, high toxicity, limited number of carried genes and the like on the viral vector. The phospholipid bilayer membrane can promote the transport of various biological membranes including BBB, and can exist in tissues for a long time so that the target medicine or gene can be fully infiltrated into the target object. Simple "non-targeted" liposomes can enter the brain through the olfactory nerve pathway. Targeted liposomes can then cross the BBB by active transport mechanisms.
The liposome can be functionally modified or modified in various ways, and small molecular compounds, biological macromolecules, imaging probes and the like can be attached to the outside of the liposome or encapsulated in a water core or a lipid bilayer, for example, a stealth liposome can be prepared by modifying with polyethylene glycol (PEG), so that the solubility of the liposome is increased, the interaction with plasma proteins is resisted, and the liposome is prevented from being phagocytized by a reticuloendothelial system. In addition, these PEG liposomes can facilitate BBB transport. At present, various novel liposome gene vectors, such as magnetic nanoliposome gene vectors, have been reported.
Up to now, there is still an urgent need in the art for a drug effective in treating and/or preventing neurodegenerative diseases, and at the same time, there is no report in the art that chimeric antigen receptor technology is applied to modification of glial cells and thus treatment and/or prevention of neurodegenerative diseases such as AD is achieved.
Disclosure of Invention
One aspect of the invention relates to a nucleic acid molecule comprising a polynucleotide sequence encoding a chimeric antigen receptor comprising: 1) A binding domain for an anti-amyloid oligomer; 2) A transmembrane domain; and 3) an intracellular signaling domain.
In some embodiments, the amyloid oligomer is selected from the group consisting of beta amyloid oligomer, microtubule-associated protein tau oligomer, alpha-synuclein oligomer, huntingtin oligomer, pancreatic amyloid oligomer, SOD1 oligomer, and TDP-43 oligomer.
In some embodiments, the amyloid oligomer is a beta amyloid oligomer, preferably the beta amyloid oligomer is aβo42 or aβo40.
In some embodiments, the binding domain of the anti-amyloid oligomer is a single chain variable region fragment scFV, preferably the binding domain of the anti-amyloid oligomer is a binding domain of an anti-beta-amyloid oligomer, more preferably the binding domain of the anti-beta-amyloid oligomer is a scFV, an a Du Nashan antibody (aducanamab) or a kriging mab (Crenezumab) comprising heavy chain CDRs 1-3 and light chain CDRs 1-3 of a single chain antibody W20, wherein the amino acid sequence of the single chain antibody W20 is as set forth in SEQ ID NO:1, the amino acid sequences of the heavy chain CDR1-3 and the light chain CDR1-3 of the single chain antibody W20 are respectively shown in SEQ ID NO:2-7, more preferably, the binding domain of the anti-beta amyloid oligomer is single chain antibody W20.
In some embodiments, the intracellular signaling domain is an intracellular signaling domain of an anti-inflammatory receptor, preferably the intracellular signaling domain of an anti-inflammatory receptor is an intracellular signaling domain selected from the group consisting of a class a scavenger receptor (SR-a), merTK, tyro3, ax1, itgB5, BAI1, ELMO, MRC1, stabilins, ADGRB1, TIMs, and αvβ3/αvβ5 integrins.
In some embodiments, the chimeric antigen receptor further comprises one or more selected from the group consisting of: a hinge region and a co-stimulatory domain that triggers glial cell activation.
In some embodiments, the hinge region is derived from the group consisting of MerTK receptor FNIII domain, CD8 a, CD28, igG1 and IgG 4.
In some embodiments, the transmembrane domain is derived from a transmembrane domain consisting of SR-A, merTK, axl, tyro3, tim1, tim4, tim3, fcR, BAI1, CD4, DAP12, MRC1, CD8 a, CD3, ICOS and CD 28. In a further embodiment, the transmembrane domain is a transmembrane domain derived from SR-A or MerTK.
In some embodiments, the binding domain of the anti-amyloid-beta oligomer is single chain antibody W20, the chimeric antigen receptor further intracellular signaling domain is an intracellular signaling domain of a class a scavenger receptor or MerTK, and the transmembrane domain is a transmembrane domain derived from SRA, merTK.
In some embodiments, the nucleic acid molecule comprises an operably linked glial cell-specific promoter, preferably the glial cell-specific promoter is a microglial cell-specific promoter, an astrocyte and/or an oligodendrocyte-specific promoter, more preferably the glial cell-specific promoter is a promoter selected from the group consisting of gfa2, GFAP104, gfabc 1D, ALDH L1, cst3, CX30, CX3CR1, IBa-1, pdgfra, oligo 2 and NG 2. In further embodiments, the glial cell-specific promoter is a gfa, CX3CR1 or oligo 2 promoter.
In some embodiments, the binding domain of the anti-amyloid oligomer co-fusion expresses an SR-A, merTK receptor, a glycosylated end product receptor, a G protein-coupled receptor, a CC-type receptor, a CXC-type receptor, a C-receptor, a CX 3C-receptor, or a lamp2a receptor.
In some embodiments, the glial cell is selected from the group consisting of a microglial cell, an astrocyte, an oligodendrocyte, and an oligodendrocyte precursor cell. In further embodiments, the glial cell is a microglial cell or an astrocyte.
In another aspect, the invention relates to a vector comprising a nucleic acid molecule as described above.
In some embodiments, the vector is a plasmid, a retroviral vector, an adenoviral vector, an adeno-associated vector, or a lentiviral vector, preferably the vector is plasmid pGFP-N1, retroviral vector pRetrox, adenoviral vector pDC315, pDC311, adeno-associated viral vector pAAV, or lentiviral vector pCDH.
Another aspect of the invention relates to a cell comprising a nucleic acid molecule or vector as described above.
In some embodiments, the cell is a glial cell, preferably the glial cell is selected from the group consisting of microglial cell, astrocyte, oligodendrocyte, and oligodendrocyte precursor cell. In further embodiments, the glial cell is a microglial cell or an astrocyte.
Another aspect of the invention relates to a chimeric antigen receptor encoded by a nucleic acid molecule as described above, expressed by a vector as described above or expressed by a cell as described above.
Another aspect of the invention relates to a pharmaceutical composition comprising a nucleic acid molecule as described above, a vector as described above, a cell as described above and/or a chimeric antigen receptor as described above, and a pharmaceutically acceptable excipient.
In some embodiments, the pharmaceutical composition is a nanoparticle prepared using liposomes, preferably the liposomes comprise one or more cationic lipids and/or one or more non-cationic lipids.
In some embodiments, the liposome is selected from cKK-E12, OF-02, C12-200, MC3, DLinDMA, DLinkC2DMA, ICE, HGT5000, HGT5001, HGT4003, DODAC, DDAB, DMRIE, DOSPA, DOGS, DODAP, DODMA and DMDMA, DODAC, DLenDMA, DMRIE, CLinDMA, cpLinDMA, DMOBA, DOcarbDAP, DLinDAP, DLincarbDAP, DLinCDAP, DLinSSDMA, KLin-K-DMA, DLin-K-XTC2-DMA, N1GL, N2GL, V1GL, ccBene, ML7, ribocationic lipid or combinations thereof, preferably the one or more non-cationic lipids are selected from DSPC, DOPC, DPPC, DOPG, DPPG, DOPE, POPC, POPE, DOPE-mal, DPPE, DMPE, DSPE, phosphatidylserine, sphingolipids, cerebrosides, gangliosides, 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, SOPE or combinations thereof, preferably the liposome is selected from POPC, DDAB, DSPE, DOPC, DOTAP or combinations thereof; more preferably, the liposome is selected from the following combinations: 1): POPC, DDAB, DSPE; (2): DSPE, DOPC, DOTAP; more preferably, the DSPE is PEG modified DSPE, preferably, the DSPE is DSPE-PEG2000.
In some embodiments, the nanoparticle prepared using the liposome is conjugated to a glial cell specific targeting peptide. In some embodiments, the targeting peptide is selected from the group consisting of polypeptides of AS1, MG1, V9, NGR, and the like.
In some embodiments, the pharmaceutical composition is in the form of nasal drops, intravenous injection, intravenous infusion, subcutaneous injection, or the pharmaceutical composition is administered directly to the brain.
Another aspect of the invention relates to the use of a nucleic acid molecule as described above, a vector as described above, a cell as described above, a chimeric antigen receptor as described above or a pharmaceutical composition as described above for the manufacture of a medicament for promoting cellular clearance of amyloid oligomers in a subject, treating and/or preventing inflammatory diseases, reducing senile plaques in the brain, treating and/or preventing neurodegenerative diseases, inhibiting gliosis, increasing brain synapse levels.
Another aspect of the invention relates to a method of promoting cellular clearance of amyloid oligomers, treating and/or preventing inflammatory diseases, reducing senile plaques in the brain, treating and/or preventing neurodegenerative diseases, inhibiting gliosis, increasing brain synapse levels in a subject in need thereof comprising administering to a subject in need thereof a nucleic acid molecule as described above, a vector as described above, a cell as described above, a chimeric antigen receptor as described above or a pharmaceutical composition as described above.
The invention also relates to a nucleic acid molecule as described above, a vector as described above, a cell as described above, a chimeric antigen receptor as described above or a pharmaceutical composition as described above for use in promoting cellular clearance of amyloid oligomers in a subject, treating and/or preventing an inflammatory disease, reducing senile plaques in the brain, treating and/or neurodegenerative diseases, inhibiting glioblastoma, increasing brain synaptic levels.
In some embodiments, the neurodegenerative disease is selected from the group consisting of alzheimer's disease, parkinson's disease, huntington's disease, amyotrophic lateral sclerosis, or spinocerebellar ataxia. In further embodiments, the neurodegenerative disease is alzheimer's disease, parkinson's disease, or huntington's disease.
In other words, in view of the fact that glial cells have the function of 'double sword' in the pathological change process of neurodegenerative diseases such as AD, the invention can not only phagocytose Abeta Os, protect neurons, but also induce inflammation.
Specifically, in order to regulate the normal functions of microglia, the invention delivers the Abeta oligomer specific single-chain antibody W20 and a scavenger receptor SR-A fusion gene to the brain through a liposome gene delivery system, so that the W20 and the SR-A are fused and expressed on the surface of microglia, the microglia in the brain are remodeled into microglia (Chimeric Antigen Receptor Microglia, CAR-M) of a chimeric antigen receptor, the expressed W20 is directly targeted to Abeta oligomers in the brain, and then the linked SR-A is activated, phagocytic clearance of the Abeta oligomers is promoted, and release of inflammatory factors is reduced, thereby providing a new and feasible strategy for treating AD and other neurodegenerative diseases.
Single chain antibody W20 (the amino acid sequence of which is shown as SEQ ID NO:1, the amino acid sequences of heavy chain CDR1-3 are shown as SEQ ID NO:2-4, and the amino acid sequences of light chain CDR1-3 are shown as SEQ ID NO: 5-7) for specifically recognizing amyloid oligomer is screened out by phage display technology. The antibody has strong affinity, good stability, high safety, good oligomer specificity, no combination with monomer and fiber forms, and can inhibit aggregation and cytotoxicity of amyloid such as Abeta. The single chain antibody W20 is different from the intact antibody, and can avoid the inflammatory reaction and excessive phagocytosis of synapses of the antibody mediated by the Fc fragment, thereby avoiding the occurrence of adverse reactions.
Class A scavenger receptors (SR-A, the nucleotide sequence of which is shown as SEQ ID NO: 8) on microglia participate in Abeta phagocytosis, SR-A down-regulation can reduce the phagocytic function of microglia on Abeta and induce Abeta accumulation to trigger AD and other neurodegenerative diseases, while up-regulation of SR-A expression can reduce Abeta level in brain and reduce the generation of inflammatory factors in microglia by inhibiting the activation of NF- κB signal channels. Since neuroinflammation is closely related to the pathology of neurodegenerative diseases such as AD, up-regulating SR-a without inducing inflammatory factor production, and increasing the clearance of aβ are an ideal strategy for treating neurodegenerative diseases such as AD. The invention enables the antibody W20 and SR-A to be co-fused and expressed on the surface of microglial cells, and converts the microglial cells into microglial cell CAR-M of chimeric antigen receptor. Meanwhile, the liposome gene vector is subjected to PLGA modification, so that the liposome gene vector has longer blood half-life and higher brain intake, and is subjected to targeted microglial cell administration through nose and brain, thereby solving the most main problem faced by microglial cell function modification. Namely, the invention designs and constructs a W20-SRA gene expression vector, prepares liposome vector delivery particles containing genes, characterizes the morphology, particle size and stability of the liposome in vitro, verifies plasmid transfection capacity and protein expression condition through a cell immunofluorescence experiment, and verifies inflammatory factor level and polarization state of CAR-M cells incubated by the Abeta oligomer through ELISA, QPCR, western-blot and other methods under the conditions of CAR-M recognition, phagocytosis and Abeta oligomer elimination. Meanwhile, W20-SRA liposome nano particles are delivered into the brain of an AD transgenic mouse in a nasal administration mode, transfection capacity of microglial cells in the brain of the mouse and functional characteristics of CAR-M are detected, cognitive behavior functions of the mouse are detected by using new things cognition, a Y maze and a water maze, pathological changes in the brain of the mouse are detected by using histoimmunochemistry, ELISA, western-blot, QPCR and the like, and treatment effects of the CAR-M on the AD mouse are examined.
The experimental result of the invention shows that the prepared liposome gene delivery nanoparticle can transfect microglial cells such as BV2 cells, and convert the BV2 cells into CAR-BV2 cells, so that the surface of the CAR-BV2 cells is fused and expressed with SRA-W20. The SRA-W20 liposome nano particles have good dispersibility, uniform size and high biological safety, and the particle size is about 91.3+/-5.0 nm. SRA-W20 was successfully fusion expressed on the surface of CAR-BV2 cells. After W20 is specifically combined with Abeta Os, the fusion expressed SR-A can be activated, the lysosome pathway and autophagy system of cells are activated, the phagocytosis and degradation of Abeta Os are promoted, the inflammatory reaction of cells is reduced, the cells are promoted to be converted from M1 type to M2 type, the normal phagocytosis function is not influenced, the excessive phagocytosis of synapses is not caused, and the neuroprotection is realized. Meanwhile, the nasal administration mode can bypass the blood brain barrier, so that the medicine directly enters the brain through the olfactory bulb, and the liposome encapsulation can protect genes from degradation and promote transmucosal transport of the genes, so that the gene vector more effectively enters the brain, and has good application prospect in AD treatment. Namely, the CAR-M of the invention effectively improves the cognitive function of the AD mice and remarkably reduces the pathological changes in the brains of the AD mice. The main conclusion is as follows:
(1) CAR-M gene therapy significantly improved cognitive function and memory in AD mice. The behavioral experiments of the APP/PS1 mice for 28 days in a nasal administration mode prove that the CAR-M therapy can effectively improve the spatial memory capacity and the cognition level of the AD mice.
(2) CAR-M gene therapy significantly reduced the aβ pathology in the brain of AD mice. Compared with AD mice in a solvent control group, the number of senile plaques in the brain of the AD mice treated by the CAR-M is obviously reduced, and the level of soluble and insoluble Abeta 40/42 is also obviously reduced, which proves that the CAR-M can effectively promote the clearance of Abeta.
(3) The CAR-M gene therapy remarkably reduces the neuroinflammation in the brain of the AD mice and promotes the conversion of microglial cells from inflammatory M1 type to anti-inflammatory M2 type. CAR-M therapy significantly reduced the degree of activation of astrocytes and microglia in the brain of AD mice and reduced the levels of inflammatory factors such as TNF- α, IL-1β, IL-6, and the like. Meanwhile, CAR-M promotes the expression of microglial cell M2 related genes Arg-1, IL-10, TGF-beta, CD206 and other proteins, so that microglial cells are converted into anti-inflammatory M2 type directions. Their anti-inflammatory mechanisms include inhibition of activation of NLRP3 inflammatory corpuscles and NF- κB.
(4) CAR-M gene therapy inhibits synaptic loss in AD mice, improving survival of neurons in the mice brain. CAR-M therapy significantly increased the levels of synaptosin and postsynaptic proteins (PSD 95) in the brain of AD mice and improved the survival status of neurons, which is closely related to the improvement of cognitive ability in AD mice.
(5) CAR-M gene therapy reduced oxidative stress levels in AD mice brains. Compared with the AD mice of the solvent control group, the ratio of GSH/GSSG in the brain of the AD mice treated by the CAR-M is obviously improved, and the ROS level is obviously reduced.
(6) The CAR-M gene therapy has high safety and no toxic or side effect. The CAR-M therapy can not promote excessive phagocytosis of synapses by microglia in the brain of the AD mice, and has high safety when being administered in a large dose for a long time, and has no adverse effect on the mental state, the weight, the biochemical index, the nasal mucosa and various internal organs of the mice.
The present invention further demonstrates the chimeric antigen receptor system of the present invention in astrocytes. The present invention designs and prepares W20-MerTK engineered astrocytes (CAR-a). The anti-inflammatory chimeric antigen receptor W20-MerTK expression plasmid specifically expressed by astrocytes is designed and constructed and loaded in liposome nano-particles of targeted astrocytes. The morphology, particle size and cytotoxicity of the lipid complexes were characterized. The expression condition of W20-MerTK in astrocytes, the phagocytic capacity of the modified astrocytes to Abeta oligomer and the release condition of inflammatory factors are characterized by using methods such as cellular immunofluorescence, flow cytometry and the like. The invention also provides a lipid complex for expressing W20-merTK through nasal administration, and detects the expression condition of W20-merTK in astrocytes in the brain of an AD mouse, the clearance capacity of CAR-A on Abeta oligomer in the brain and the release condition of inflammatory factors. The improvement of the memory capacity of the CAR-A on the AD mice is detected through water maze, new thing cognition and Y maze experiments, and the pathological changes in the brains of the AD mice are detected by using methods such as immunohistochemistry, ELISA and the like.
Specifically, the invention replaces an antigen binding region (19 Gly-275 Asn) of MerTK (the nucleotide sequence of which is shown as SEQ ID NO: 9) with a single-chain antibody fragment (W20) specifically bound with the Abeta oligomer, and constructs a novel anti-inflammatory chimeric antigen receptor (W20-MerTK) for recognizing the Abeta oligomer. The W20-MerTK expression plasmid is loaded in liposome nano particles of targeted astrocytes, and the lipid complex has uniform particle size, good dispersibility and no obvious cytotoxicity. The chimeric antigen receptor is expressed completely in astrocytes, so that the astrocytes are programmed and transformed into CAR-A, and when W20-MerTK is combined with the Abeta oligomer, intracellular RhoA, rac1 and Cdc42 signal paths can be activated to guide the CAR-A to phagocytose the Abeta oligomer. Meanwhile, the CAR-A can activate SOCS1/3 signal path when phagocytizing Abeta oligomer, inhibit cytokine receptor and reduce the forward feedback effect of cytokine receptor on cytokine. In addition, the CAR can inhibit the release of inflammatory factors caused by the receptor such as RAGE and TLR phagocytosing Abeta oligomers by inhibiting NF- κB signaling pathway, thereby improving the cell microenvironment. The constructed W20-MerTK plasmid was delivered into the brain by nasal administration using a liposome gene delivery system, and W20-MerTK was successfully expressed on astrocytes in the brain and converted to CAR-A. The CAR-A can effectively remove the Abeta oligomer in the brain, reduce the Abeta plaque quantity and lower the Abeta 40 and Abeta 42 levels in the brain, and can reduce the release of pro-inflammatory cytokines and the inflammatory reaction in the brain when the Abeta oligomer in the brain is removed, the phenotype of microglia cells can be converted into M0 type from MGnD by the treatment of the CAR-A gene, the levels of synaptotin and PSD95 can be obviously improved, the loss of synaptogenesis in the brain of an AD mouse is inhibited, thereby improving the cognitive function of the mouse, and the astrocyte programming modification technology provides a brand-new treatment strategy for the treatment of neurodegenerative diseases such as AD.
Experimental results show that the obtained lipid complex has the particle size of 132+/-19.5 nm, good dispersibility and uniform size, and the W20-merTK can be completely expressed in astrocytes and is not expressed in neurons and microglia. Binding of W20-MerTK to the Abeta oligomer activates RhoA, rac1 and Cdc42 signaling pathways within astrocytes, causing phagocytosis of Abeta oligomer by CAR-A. The CAR-A can inhibit NF- κB passage and release of inflammatory factors when phagocytizing Abeta oligomer, and can also obviously improve intracellular SOCS1/3 protein level, inhibit cytokine receptor and reduce the forward feedback effect of cells on cytokines. When the CAR-A phagocytoses the Abeta oligomer, the activation of microglia and the damage of neurons are reduced by reducing the release of pro-inflammatory cytokines caused by the receptor such as RAGE, TLR and the like when the receptor phagocytoses the Abeta oligomer, so that the cell microenvironment is improved. Following nasal administration of the lipid complex containing the W20-MerTK plasmid, the chimeric antigen receptor successfully engineered the astrocytes in the brain into CAR-a. The CAR-A can effectively remove Abeta oligomers in the brain, reduce the number of Abeta plaques, lower the Abeta 40 and Abeta 42 levels in the brain and the proinflammatory cytokine levels in the brain, and improve the anti-inflammatory cytokine levels. After CAR-a gene therapy, the phenotype of microglial cells is changed from MGnD to M0 and the level of synapses in the brain is significantly increased.
The CAR-M and CAR-a of the invention are different from CAR-T, which returns a large number of in vitro expanded CAR-T cells to the patient for tumor treatment at a significant cost and may induce cytokine storms. The chimeric antigen receptor is introduced into glial cells such as microglial cells and astrocytes, microglial cells and astrocytes are respectively induced in situ in brain to become CAR-M and CAR-A, so that the recognition and phagocytic capacity of the glial cells such as microglial cells and astrocytes to amyloid oligomers are improved, the obvious side effects such as inflammatory reaction and synaptic loss are not caused, and the implementation of CAR-M and CAR-A gene therapy is hopeful to overcome the defects of the existing neurodegenerative disease treatment means and provide a brand-new strategy for the treatment of various nervous system diseases.
Drawings
Fig. 1: plasmid map.
Fig. 2: characterization of liposomes. (A) TEM observe the morphology of NP and SRA-W20-NP, scale bar = 200nm; (B) TEM measuring particle size distribution of NP and SRA-W20-NP; (C) The hydration diameters of the NPs and SRA-W20-NPs were measured by the DLS method.
Fig. 3: expression of CAR-BV2 cell surface W20 and GFP. (A) CAR-BV2 cells were immunostained with W20 antibody (red), observed with confocal microscopy, scale bar = 25 μm; (B) The fraction of GFP expressing positive CAR-BV2 cells was analyzed using a flow cytometer.
Fig. 4: cytotoxicity assay of liposomes. Different concentrations of NP, SRA-W20-NP, and SRA-ns-scFv-NP were added to the medium of BV2, N2a, and U87-mg cells and incubated at 37℃for 48h to determine cell activity as MTT.
Fig. 5: the phagocytic capacity of CAR-BV2 on Abeta Os is improved. (A) After CAR-BV2 cells were transfected with SRA, SRA-W20 and SRA-ns-scFv plasmids, aβos were added and after incubation for 0, 1, 6, 12 and 24 hours, conditions of aβ (aβ antibody staining, red) in CAR-BV2 cells (green) were observed, scale bar = 25 μm; (B) quantitative analysis of the intracellular Aβ levels of CAR-BV 2.
Fig. 6: CAR-BV2 promotes phagocytosis of Abeta Os by SR-A. (A) BV2 cells were transfected with SRA, SRA-W20 and SRA-ns-scFv, then added with fucose (SR-A receptor antagonist) and Abeta Os, and intracellular Abeta levels were detected using cellular immunofluorescence, with a scale = 25 μm; (B) quantitative analysis of the intracellular Aβ levels of CAR-BV 2.
Fig. 7: the clearance capacity of CAR-BV2 to Abeta Os is improved. (A) After 2h incubation with aβos in CAR-BV2 cell culture medium transfected with SRA, SRA-W20 and SRA-ns-scFv, aβ (aβ antibody staining, red) levels in CAR-BV2 cells (green) were detected at 0, 3, 6, 12, 24 and 36h later, scale bar = 25 μm. (B) The intracellular aβ levels of CAR-BV2 were detected at different times by flow-through.
Fig. 8: CAR-BV2 activates the endolysosomal pathway. (A) Adding Abeta Os into a CAR-BV2 cell culture medium transfected with SRA, SRA-W20 and SRA-ns-scFv, and detecting the level of a lysosomal marker LAPM1 by using cell immunofluorescence; (B) quantitative analysis of LAMP1 Signal in CAR-BV 2.
Fig. 9: western-blot detection of intracellular LAMP1 protein levels. (A) Performing SDS-PAGE electrophoresis on the transfected BV2 cell extract, and detecting the level of the BV2 cell extract by using an LAMP1 antibody after transferring the membrane; and (B) quantitative analysis of protein band density.
Fig. 10: qPCR measures mRNA levels of pro-inflammatory and anti-inflammatory factors in CAR-BV2 cells. mRNA levels of IL-6, TNF- α, IL-10, TGF- β, YM-1 and Arg-1 in cells were determined by qPCR by adding 4μ M A βOs to CAR-BV2 cell culture medium transfected with SRA, SRA-W20 and SRA-ns-scFv for 24h co-incubation.
Fig. 11: expression of fusion gene SRA-W20 in mouse brain. Fusion proteins GFP (green) and W20 (anti-W20 antibody staining, red) were observed by confocal microscopy, scale bar = 25 μm.
Fig. 12: the therapeutic effect of CAR-M on AD mice was examined using the Y maze assay. The number of times (a) that mice of each group entered the new arm (B) was determined using the Y maze test 28 days after the mice were treated with the various liposome gene vectors.
Fig. 13: morris water maze assay (MWM) effect of CAR-M on spatial learning and memory function in AD mice. (a) during the training phase, the incubation period in which the mice find the platform; (B-D) after withdrawal of the platform, the incubation period (B) for the mice to reach the platform area, the number of passes through the location of the platform (C) and the target quadrant residence time (D).
Fig. 14: CAR-M treatment reduced aβ plaques in the brains of AD mice. (A) AD mice and WT mice were treated with CAR-M and immunohistochemical staining with 6E10 antibody, scale bar = 100 μm; (B) quantitative analysis of A.beta.plaque staining areas.
Fig. 15: CAR-M reduced aβ levels in the brains of AD mice. AD and WT mice treated with CAR-M were assayed for soluble aβ40 (a), insoluble aβ40 (B), soluble aβ42 (C) and insoluble aβ42 (D) levels in the brain of the mice by ELISA.
Fig. 16: CAR-M reduced aβ oligomer levels in the brains of AD mice. The content of Abeta oligomer in the brain homogenate of the AD mice is detected by ELISA method.
Fig. 17: CAR-M promotes phagocytosis of aβ in vivo. (A) Conditions of phagocytosis of Abeta (red) in Aba-1 positive (yellow) microglia cells in mice treated with various liposome gene vectors; (B) The level of aβ phagocytosis by microglia was quantitatively analyzed, with a scale bar=500 nm.
Fig. 18: CAR-M gene therapy reduced oxidative stress and ROS levels in AD mice brains. GSH (A), GSSG (B) and GSH/GSSG ratios (C), and ROS (D) levels in mouse brain homogenates were determined using the corresponding kits.
Fig. 19: CAR-M gene therapy inhibits loss of synapses in the brain of AD mice. (A) Synaptophysin immunostaining (green) and PSD95 immunostaining (red) in brains of AD and WT mice treated with different liposomes, scale bar = 200 μm; quantitative analysis of (B-D) synaptorin and PSD95 immunostained regions.
Fig. 20: CAR-M gene therapy reduced the extent of glial cell activation in the brain of AD mice. (A) After treatment with different liposomes and PBS, activation of astrocytes and microglial cells in the mouse brain was detected by GFAP immunostaining and Iba-1 immunostaining, scale = 250 μm; (B) quantitative statistics of areas of GFAP and Iba-1 positive staining.
Fig. 21: CAR-M gene therapy promotes conversion of microglial cells to M2 type in the brain of AD mice.
Fig. 22: construction of CAR-A expression plasmids. gfa2: astrocyte-specific promoters; poly (A): a polyadenylation signal; ORI: an origin of replication; kanR: kanamycin resistance gene.
Fig. 23: ICC and Western blot analysis CAR expression intact in astrocytes. (A) CARs were stained using W20 and MerTK antibodies to identify CAR complete expression. Scale bar: 5 μm. (B) Membrane proteins extracted from ns-CAR-A and CAR-A were subjected to Western blot analysis using W20 and MerTK antibodies.
Fig. 24: CARs are specifically expressed in astrocytes. The CAR lipid complexes were transfected with astrocytes (a), neuronal cells (B), microglia cells (C), and the expression of CARs in different cells was examined with a laser confocal microscope. Scale bar: 50 μm.
Fig. 25: CAR-a phagocytosis aβ monomers and oligomers were flow analyzed. Astrocytes were transfected with CAR lipid complexes or ns-CAR lipid complexes, and after co-incubation of the transfected astrocytes with 200nM aβ monomers or aβ oligomers for 6h, the cells were stained with aβ antibodies and the phagocytic capacity of the astrocytes for each type of aβ was analyzed by flow cytometry.
Fig. 26: the CAR reduces the transcription level of inflammatory factors within astrocytes. Abeta oligomers were added to CAR-A, ns-CAR-A or control astrocyte cultures and incubated at 37℃for 24h to detect mRNA levels of pro-inflammatory cytokines IL1 beta, TNF alpha, iNOS, IL6 (A) and anti-inflammatory cytokines IL4, arg1, CD206, TGF beta (B) using qPCR.
Fig. 27: distribution of CAR lipid complexes in mice.
Fig. 28: expression of CAR in astrocytes in mouse brain. Scale bar: 50 μm.
Fig. 29: CAR-a ameliorates spatial learning and memory impairment in AD transgenic mice. WT and AD mice were evaluated for spatial learning and memory capacity by the water maze test following nasal administration of liposomes, CAR lipid complexes or ns-CAR lipid complexes. During the training experiments, the movement trace of the mice (a) and the latency of the mice to find the platform (C) were recorded over time. During the exploration experiment, the mouse motion profile (B), the latency of the mouse to the plateau region (D), the residence time in the target quadrant (E) and the number of plateau crossings (F) were recorded.
Fig. 30: CAR-a ameliorates spatial memory impairment in AD transgenic mice. WT and AD mice spatial memory capacity of WT and AD mice was assessed by Y maze experiments following nasal administration of PBS, CAR lipid complexes or ns-CAR lipid complexes. The mouse movement trace (a), residence time in the new arm (B) and number of entries (C) were recorded.
Fig. 31: the CAR-a in the mouse brain is more capable of phagocytosing aβ oligomers. (A) Representative pictures of astrocyte phagocytic aβ oligomers in the brains of mice from different experimental groups. Scale bar: 5 μm. (B) Abeta oligomers in astrocytes were quantified using Image J software and values were normalized against the AD-PBS group.
Fig. 32: CAR-a reduced the level of aβ oligomers in the brains of AD transgenic mice. After nasal administration of PBS, CAR lipid complexes or ns-CAR lipid complexes to WT and AD mice, the soluble aβ oligomer content in the mice brain homogenates was determined using a kit.
Fig. 33: CAR-a reduced the number of aβ plaques in the brains of AD transgenic mice. After nasal administration of PBS, CAR lipid complex or ns-CAR lipid complex to WT and AD mice, immunostaining was performed using 6E10 antibodies to detect the levels of senile plaques in the brain (a), and statistics were performed on the area of senile plaques using Image J software (B). Scale bar: 200 μm.
Fig. 34: CAR-a reduced levels of aβ40 and aβ42 in the brains of AD transgenic mice. After nasal administration of liposomes, CAR lipid complexes or ns-CAR lipid complexes to WT and AD mice, the content of insoluble aβ40 (a) and insoluble aβ42 (B) in the brain homogenates of the mice was determined using the corresponding detection kit.
Fig. 35: CAR-a reduces pro-inflammatory cytokine mRNA levels in AD transgenic mice brain, increasing anti-inflammatory cytokine mRNA levels. After nasal administration of liposomes, CAR lipid complexes or ns-CAR lipid complexes to WT and AD mice, mRNA levels of pro-inflammatory cytokines (il1β, tnfα, iNOS, IL 6) and anti-inflammatory cytokines IL4, arg1, tgfβ) were determined in each group of mice using qPCR.
Fig. 36: western blot detects GFAP and Iba 1 protein levels in the brains of mice in each group. After nasal administration of liposomes, CAR lipid complexes or ns-CAR lipid complexes to WT and AD mice, western blot experiments were performed using GFAP and Iba 1 antibodies to detect the amount of GFAP and Iba 1 protein in the mice brain (a) and statistics of the banding area by Image J (B).
Fig. 37: CAR-a reduced intra-brain synaptic loss in AD mice. Following nasal administration of liposomes, CAR lipid complexes or ns-CAR lipid complexes by WT and AD mice, the levels of PSD95 (red) and SYN (green) in the mice brain were detected by immunofluorescence, co-localization representing intact synapses (yellow) a scale bar: 5 μm. The area was counted (B) using Image J software.
Detailed Description
Definition of the definition
Amyloid oligomer refers to an aggregated form of two or more amyloid monomer molecules aggregated. Amyloid proteins include beta-amyloid, microtubule-associated protein tau, alpha-synuclein, huntingtin, pancreatic amyloid, superoxide dismutase 1 (SOD 1), TDP-43 proteins, and the like.
As used herein, anti-inflammatory receptors refer to glial cell surface receptors that do not promote the production of inflammatory factors, including class a scavenger receptors (SR-a), MERTK, tyro3, ax1, itgB5, BAI1, ELMO or MRC1, stabilins, ADGRB1, TIMs, αvβ3/αvβ5 integrins. Preferably, the anti-inflammatory receptor is SR-A, MERTK. The abbreviations SRA and SR-A for the class A scavenger receptors are used interchangeably herein.
Chimeric antigen receptor glial cells refer to chimeric antigen receptor-engineered glial cells that comprise an amyloid-oligomer binding domain, a transmembrane domain, and an intracellular signaling domain.
CAR-M refers to chimeric antigen receptor engineered microglia.
CAR-a refers to chimeric antigen receptor engineered astrocytes.
"CAR" or "chimeric antigen receptor" as used interchangeably herein refers to a molecule comprising at least three domains, namely an extracellular domain comprising an antigen binding domain (in the present invention an anti-amyloid oligomer binding domain), a transmembrane domain, and an intracellular domain comprising an intracellular signaling domain.
Thus, when the CAR is expressed on a host cell (particularly an effector cell), the antigen binding domain will be present within or as an extracellular domain. Typically, most or all of the antigen binding domain will be present extracellular to allow the CAR to bind to the target antigen (e.g., at least 90%, 95%, 97%, 99% or 100% of the antigen binding domain will be present extracellular when the CAR is expressed in the host cell, transported to the cell membrane, and presented). In some embodiments, the antigen binding domain is an antibody, particularly a single chain antibody.
In some embodiments, the antigen binding domain is a single chain antibody that specifically targets an amyloid oligomer, which can be engineered from antibodies known in the art that specifically target an amyloid oligomer by genetic manipulation. The person skilled in the art is aware of the methods of modification performed. In some embodiments, the antigen binding domain is a W20 antibody, an a Du Nashan antibody (aducanaumab) or a kerrimab (Crenezumab), preferably in the scFV form thereof.
The transmembrane domain connects the extracellular domain comprising the antigen binding domain (i.e., the anti-amyloid oligomer binding domain of the invention) to the intracellular signaling domain and typically spans the cell membrane of the host cell after CAR expression and membrane targeting. The transmembrane domain may be derived from a protein or portion of a protein having both transmembrane and intracellular regions, such as CD28, and both of these domains or portions thereof may be included in a CAR of the invention. The transmembrane domain may be based on or derived from the transmembrane domain of any transmembrane protein. In some embodiments, it may be or may be derived from transmembrane domains from SRA, merTK, axl, tyro3, tim1, tim4, tim3, fcR, BAI1, DAP12, MRC1, ICOS, CD8a, CD28, CD4, CD3 ζ, CD45, CD9, CD16, CD22, CD33, CD64, CD80, CD86, CD134 (OX 40), CD137 (4-1 BB) and CD 154. In further embodiments, it may be or may originate from a transmembrane domain from SRA, merTK, axl, tyro3, tim1, tim4, tim3, fcR, BAI1, CD4, DAP12, MRC1, CD8a, CD3, ICOS or CD 28.
As used herein, an "intracellular signaling domain" refers to a portion of a CAR protein that is involved in transducing information of the effective binding of a CAR to a target antigen (amyloid oligomer) into the interior of a cell (host cell, e.g., glial cell) to elicit a cellular function (e.g., effector cell function). The intracellular signaling domain of the CAR is present within the host cell after expression of the CAR (i.e., contained within the intracellular domain of the CAR), typically within the cytoplasm of the cell. The domain is capable of activating one or more normal functions of a host cell expressing the CAR. For example, if the host cell is a microglial cell, the intracellular signaling domain can increase its phagocytic capacity for aβ oligomers and by engineering the downstream signaling pathway of the chimeric antigen receptor, it does not produce excessive pro-inflammatory factors. In some embodiments, the intracellular signaling domain is selected from the group consisting of intracellular signaling domains of class A scavenger receptors (SR-A), merTK, tyro3, ax1, itgB5, BAI1, ELMO or MRC1, stabilins, ADGRB1, TIMs, αvβ3/αvβ5 integrins, and the like.
The CAR may also comprise a hinge domain or spacer region (used interchangeably herein) between the anti-amyloid-oligomer binding domain and the transmembrane domain. The hinge domain and/or spacer region may have flexibility that allows it to be oriented in different directions, which may help antigen-binding anti-amyloid-oligomer binding domains. In certain embodiments, the hinge region and/or spacer region can be an immunoglobulin hinge region, and can be a wild-type immunoglobulin hinge region or an altered wild-type immunoglobulin hinge region, such as a truncated hinge region. Other exemplary hinge and/or spacer regions that may be used include hinge and/or spacer regions derived from the extracellular region of a type 1 membrane protein, such as CD8a, CD4, CD28, CD7, igG1 or IgG4, which may be wild-type hinge/spacer regions from these molecules or may be altered. Preferably, the hinge/spacer region is or is derived from the human MerTK receptor FNIII domain, CD8a, CD4, CD28, CD 7. IgD, CH3 and Fc spacers or hinges may also be used in the CARs of the invention. The hinge domain or spacer region as used herein may be at least 10 amino acids in length, for example, at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, or 200 amino acids in length.
In a specific embodiment of the invention, the hinge domain may not be used and the anti-amyloid-oligomer binding domain may be directly linked to the transmembrane domain.
The invention also encompasses vectors comprising the nucleic acids of the invention. The vector may be, for example, an expression vector (e.g., a eukaryotic gene expression vector, particularly for glial cell expression) or a cloning vector. Possible expression vectors include, but are not limited to, plasmids, modified viruses (e.g., replication defective retroviruses, adenoviruses, adeno-associated viruses, and lentiviruses), transposons, or cosmids, provided that the vector is compatible with the host cell used.
Thus, the present invention encompasses recombinant expression vectors comprising the nucleic acid molecules of the invention, as well as regulatory sequences necessary for the transcription and translation of the protein sequences encoded by the nucleic acid molecules of the invention. Suitable regulatory sequences may be derived from a variety of sources including bacterial, fungal, viral, mammalian or insect genes. The selection of the appropriate regulatory sequences depends on the host cell selected as discussed below and can be readily accomplished by one of ordinary skill in the art. In some embodiments, the host cell is a human glial cell as described above. In some embodiments, examples of such regulatory sequences include: transcriptional promoters, enhancers, introns, RNA polymerase binding sequences, TATA boxes, ribosome binding sequences such as SD sequences, including translation initiation signals. In addition, depending on the host cell selected and the vector used, other sequences, such as origins of replication, additional DNA restriction sites, enhancers, and sequences that confer transcriptional inducibility, may be introduced into the expression vector.
An example of a promoter capable of expressing a CAR molecule in a cell (mammalian cell) is a glial cell specific promoter. Glial cell-specific promoters refer to promoters specific for glial cells. Preferably, the glial cell-specific promoter is selected from the group consisting of gfa, GFAP104, gfabac 1D, ALDH L1, cst3, CX30, CX3CR1, IBa1, pdgfra, oligo 2, NG2, CNP, CD68, etc., such as the gfa, GFAP, cst3, GFAP104, gfabac 1D or CX30 promoters for the astrocytes, oligo 2 or CNP promoters for the oligodendrocytes, CD68, CX3CR1 or Iba1 promoters for the microglia.
Recombinant expression vectors can be introduced into host cells to produce transformed host cells. The terms "transforming with," "transfecting with," "transforming," "transduction," and "transfection" are intended to include the introduction of a nucleic acid (e.g., vector) into a cell by one of many possible techniques known in the art. As used herein, the term "transformed host cell" or "transduced host cell" is also intended to include cells that have been transformed with the recombinant expression vectors of the invention. Prokaryotic cells may be transformed with nucleic acids by, for example, electroporation or calcium chloride mediated transformation. For example, the nucleic acid may be introduced into the mammalian cells by conventional techniques, such as calcium phosphate or calcium chloride co-precipitation, DEAE-dextran mediated transfection, lipofection, electroporation, or microinjection. Suitable methods for transforming and transfecting host cells can be found in Sambrook, J., fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual.Cold Spring Harbor Press, new York and other Laboratory textbooks.
It is also understood that the cells of the invention may comprise more than one nucleic acid or vector of the invention. In particular, the cells of the invention may comprise 2, 3, 4 or 5 or more nucleic acids or vectors of the invention, each expressing a different CAR molecule.
As used herein, the term "subject" refers to any mammal, but in particular to humans, domestic animals (e.g., cats, dogs, etc.), horses, mice, rats, primates, e.g., monkeys, cows, pigs, etc.
As discussed herein, degenerate variants of defined nucleic acid or polynucleotide sequences are also contemplated, which result from redundancy of the genetic codons. In particular, codon-optimized nucleotide sequences are contemplated that are optimized for intracellular expression in a particular organism. For example, codon optimization may be developed for polynucleotide sequences expressed in human or murine glial cells and is encompassed by the present invention.
Although the nucleotide sequence defined above is DNA, in alternative embodiments of the invention the nucleotide sequence may be RNA. Thus encompassing the corresponding RNA sequences to the DNA sequences described herein. The skilled artisan will understand how to derive RNA sequences encoding the same protein/polypeptide product into the DNA sequences shown above. "T" should be replaced with "U".
As used herein, the term "nucleic acid sequence" or "nucleic acid molecule" or "polynucleotide", "polynucleotide sequence" or "nucleotide sequence" refers to a sequence of nucleosides or nucleotide monomers comprised of naturally occurring bases, sugars, and inter-sugar (backbone) linkages. The term also includes modified or substituted sequences that contain non-naturally occurring monomers or portions thereof. The nucleic acid, polynucleotide, or nucleotide sequence of the present invention may be a deoxyribonucleic acid sequence (DNA) or a ribonucleic acid sequence (RNA), and may include naturally occurring bases including adenine, guanine, cytosine, thymidine, and uracil. The sequence may also contain modified bases. Examples of such modified bases include aza and deazaadenine, guanine, cytosine, thymidine and uracil; and xanthines and hypoxanthines. The nucleic acid, polynucleotide, or nucleotide sequence may be double-stranded or single-stranded. The nucleic acid, polynucleotide or nucleotide sequence may be fully or partially synthetic or recombinant.
The glial cells of the invention may be in situ glial cells, isolated primary glial cells or established glial cells.
The altered glial cells of the invention have therapeutic utility, particularly for the recognition and endocytosis of amyloid oligomers. At the time of treatment, the gene vector may be delivered into the brain using methods well known in the art.
Prior to therapeutic use of the cells, it may be desirable to subject the cells to an activation or expansion step using methods well known in the art.
The invention further provides, inter alia, a population of cells, wherein at least one cell of the population comprises a nucleic acid or vector of the invention. The cell population may comprise cells comprising different nucleic acids or vectors of the invention. Thus, one cell in a population may comprise a nucleic acid of the invention encoding a first CAR, e.g., W20-SRA, and a second cell in the population may comprise a nucleic acid of the invention encoding a second CAR, e.g., W20-MerTK.
Although the nucleic acids, vectors, or cells of the invention may be effective against a disease when used alone, additional therapeutic agents may be used in combination with the nucleic acids, vectors, or cells of the invention to combat a disease. Thus, in another embodiment of the invention, at least one additional or additional therapeutic agent (e.g., other neurodegenerative disease treatment drug) may be administered to the subject. Thus, the nucleic acids, vectors, CARs, cells or cell populations of the invention and other therapeutic agents (e.g., other neurodegenerative disease treatment agents) can be administered to a subject. Thus, the compositions or pharmaceutical compositions of the invention may comprise other active or therapeutic agents, as well as the nucleic acids, vectors, CARs, cells and/or cell populations of the invention. However, it is to be understood that the nucleic acids, vectors, CARs, cells or cell populations of the invention and other therapeutic agents (e.g., other neurodegenerative disease treatment agents) may be administered separately, e.g., by separate routes of administration. In addition, the nucleic acids, vectors, CARs, cells or cell populations of the invention and at least one other therapeutic agent (e.g., other neurodegenerative disease such drugs) can be administered sequentially or (substantially) simultaneously. They may be administered in the same pharmaceutical formulation or drug, or they may be formulated and administered separately. For sequential administration, the additional therapeutic agent may be administered at least 1 minute, 10 minutes, 1 hour, 6 hours, 12 hours, 1 day, 5 days, 10 days, 2 weeks, 4 weeks, or 6 weeks before or after administration of the nucleic acid/vector/cells.
"pharmaceutically acceptable" includes preparations which are sterile and pyrogen-free. Suitable pharmaceutical carriers, diluents and excipients are well known in the pharmaceutical arts. The carrier must be "acceptable" in the sense of being compatible with the drug and not deleterious to the recipient thereof. Typically, the carrier will be saline or an infusion medium (alternatively referred to as an infusion solution), which will be sterile and pyrogen-free; however, other acceptable carriers may be used.
The pharmaceutical composition of the present invention may be administered in a manner suitable for the disease to be treated (or prevented). The number and frequency of administration will be determined by factors such as the condition of the patient and the type and severity of the patient's disease, although the appropriate dosage may be determined by clinical trials.
The compositions of the present invention may be administered in a single dose or in multiple doses. In particular, the composition may be administered in a single, disposable application.
The nucleic acid molecules or compositions of the invention may be administered by any parenteral route in the form of pharmaceutical preparations containing the active ingredient. Depending on the condition and the patient to be treated and the route of administration, the composition may be administered in different dosages. In any event, the physician will determine the actual dosage that is most appropriate for any individual patient, and it will vary with the age, weight and response of the particular patient.
In human therapy, the nucleic acid molecules or compositions of the invention are typically administered in admixture with a suitable pharmaceutical excipient, diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice. For each of the embodiments, the nucleic acid molecules or compositions of the invention may be administered in a variety of dosage forms. Examples of such dosage forms include, but are not limited to, reconstitutable powders, elixirs, liquids, solutions, suspensions, emulsions, powders, granules, particles, microparticles, dispersible granules, cachets, inhalants, aerosol inhalants, patches, particle inhalants, implants, long-acting implants, injections (including subcutaneous, intramuscular, intravenous, and intradermal, preferably intravenous), infusions, and combinations thereof. In general, the cells of the invention may be administered in a nasal drop buffer, an injection or infusion buffer. Exemplary formulations can be found, for example, in Remington's Pharmaceutical Sciences, 19 th edition, grennaro, a., editions, 1995, which is incorporated herein by reference.
The nucleic acid molecules or compositions of the invention may also be administered parenterally, for example nasally, intravenously, intra-arterially, intraperitoneally, intrathecally, intracranially, topically, intramuscularly, buccally, subcutaneously, transdermally, epidurally, inhaled, intracardially, intraventricular, intraocular, intraspinal, sublingual, transdermal or transmucosally, or they may be administered by infusion techniques. They are preferably used in the form of sterile aqueous solutions which may contain other substances, such as enough salts or glucose to make the solution isotonic with blood. If necessary, the aqueous solution should be suitably buffered (preferably at a pH of 3 to 9). The preparation of a suitable parenteral formulation under sterile conditions can be readily accomplished by standard pharmaceutical techniques well known to those skilled in the art.
The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules, bags and vials.
In some embodiments, the invention relates to the following:
a nucleic acid molecule comprising a polynucleotide sequence encoding a chimeric antigen receptor comprising: 1) A binding domain for an anti-amyloid oligomer; 2) A transmembrane domain; and 3) an intracellular signaling domain.
The nucleic acid molecule of item 1, wherein the amyloid oligomer is selected from the group consisting of beta amyloid oligomer, microtubule-associated protein tau oligomer, alpha-synuclein oligomer, huntingtin oligomer, pancreatic amyloid oligomer, SOD1 oligomer, and TDP-43 oligomer.
The nucleic acid molecule of item 1 or 2, wherein the amyloid oligomer is a beta amyloid oligomer, preferably the beta amyloid oligomer is Abeta O42 or Abeta O40.
The nucleic acid molecule of any one of clauses 1-3, wherein the binding domain of the anti-amyloid oligomer is a single-chain variable region fragment scFV, preferably the binding domain of the anti-amyloid oligomer is a binding domain of an anti-beta-amyloid oligomer, more preferably the binding domain of the anti-beta-amyloid oligomer is a scFV comprising heavy chain CDRs 1-3 and light chain CDRs 1-3 of a single-chain antibody W20, an a Du Nashan antibody (aducauab) or a kriglizumab (Crenezumab), wherein the amino acid sequence of the single-chain antibody W20 is as set forth in SEQ ID NO:1, the amino acid sequences of the heavy chain CDR1-3 and the light chain CDR1-3 of the single chain antibody W20 are respectively shown in SEQ ID NO:2-7, more preferably, the binding domain of the anti-beta amyloid oligomer is single chain antibody W20.
The nucleic acid molecule of any one of items 1-4, wherein the intracellular signaling domain is an intracellular signaling domain of an anti-inflammatory receptor, preferably the intracellular signaling domain of an anti-inflammatory receptor is an intracellular signaling domain selected from the group consisting of a class a scavenger receptor (SR-a), merTK, tyro3, ax1, itgB5, BAI1, ELMO, MRC1, stabilins, ADGRB1, TIMs, and αvβ3/αvβ5 integrins.
The nucleic acid molecule of any one of clauses 1-5, wherein the chimeric antigen receptor further comprises one or more selected from the group consisting of: a hinge region and a co-stimulatory domain that triggers glial cell activation.
The nucleic acid molecule of item 6, wherein the hinge region is derived from the group consisting of MerTK receptor FNIII domain, CD8 a, CD28, igG1 and IgG 4.
The nucleic acid molecule of any one of clauses 1-7, wherein the transmembrane domain is derived from a transmembrane domain consisting of SR-A, merTK, axl, tyro3, tim1, tim4, tim3, fcR, BAI1, CD4, DAP12, MRC1, CD8 a, CD3, ICOS and CD 28.
The nucleic acid molecule of any one of clauses 1-8, wherein the binding domain of the anti-amyloid-beta oligomer is single chain antibody W20, the chimeric antigen receptor further intracellular signaling domain is the intracellular signaling domain of a class a scavenger receptor (SR-a) or MerTK, and the transmembrane domain is a transmembrane domain derived from SR-A, merTK.
The nucleic acid molecule of any one of claims 1-9, wherein the nucleic acid molecule comprises an operably linked glial cell-specific promoter, preferably the glial cell-specific promoter is a microglial-specific promoter, an astrocyte and/or an oligodendrocyte-specific promoter, more preferably the glial cell-specific promoter is a promoter selected from the group consisting of gfa, GFAP104, gfabc 1D, ALDH L1, cst3, CX30, CX3CR1, IBa-1, pdgfra, oligo 2 and NG 2.
The nucleic acid molecule of any one of clauses 1-10, wherein the binding domain of the anti-amyloid oligomer co-fusion expresses an SR-A, merTK receptor, a glycosylated end-product receptor, a G protein-coupled receptor, a class CC receptor, a class CXC receptor, a C receptor, a CX3C receptor, or a lamp2a receptor.
The nucleic acid molecule of any one of clauses 6-11, wherein the glial cell is selected from the group consisting of a microglial cell, an astrocyte, an oligodendrocyte, and an oligodendrocyte precursor cell.
Item 13. A vector comprising the nucleic acid molecule of any one of items 1-12.
The vector according to item 13, wherein the vector is a plasmid, a retrovirus vector, an adenovirus-associated vector or a lentiviral vector, preferably the vector is plasmid pGFP-N1, retrovirus vector pRetrox, adenovirus vector pDC315, pDC311, adenovirus-associated virus vector pAAV or lentiviral vector pCDH.
A cell comprising the nucleic acid molecule of any one of items 1-12 or the vector of item 13 or 14.
The cell of item 15, wherein the cell is a glial cell, preferably the glial cell is selected from the group consisting of microglial cell, astrocyte, oligodendrocyte and oligodendrocyte precursor cell.
A chimeric antigen receptor encoded by the nucleic acid molecule of any one of claims 1-12, expressed by the vector of claim 13 or 14, or expressed by the cell of claim 15 or 16.
A pharmaceutical composition comprising the nucleic acid molecule of any one of items 1-12, the vector of item 13 or 14, the cell of item 15 or 16, and/or the chimeric antigen receptor of item 17, and a pharmaceutically acceptable excipient.
The pharmaceutical composition of item 18, wherein the pharmaceutical composition is a nanoparticle prepared using a liposome, preferably the liposome comprises one or more cationic lipids and/or one or more non-cationic lipids.
The pharmaceutical composition OF item 18 or 19, wherein the liposome is selected from cKK-E12, OF-02, C12-200, MC3, DLinDMA, DLinkC2DMA, ICE, HGT5000, HGT5001, HGT4003, DODAC, DDAB, DMRIE, DOSPA, DOGS, DODAP, DODMA and DMDMA, DODAC, DLenDMA, DMRIE, CLinDMA, cpLinDMA, DMOBA, DOcarbDAP, DLinDAP, DLincarbDAP, DLinCDAP, DLinSSDMA, KLin-K-DMA, DLin-K-XTC2-DMA, N1GL, N2GL, V1GL, ccBene, ML7, a ribocationic lipid, or a combination thereof, preferably the one or more non-cationic lipids is selected from DSPC, DOPC, DPPC, DOPG, DPPG, DOPE, POPC, POPE, DOPE-mal, DPPE, DMPE, DSPE, phosphatidylserine, sphingolipids, cerebrosides, gangliosides, 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, SOPE, or a combination thereof, preferably the liposome is selected from POPC, DDAB, DSPE, DOPC, DOTAP, or a combination thereof; more preferably, the liposome is selected from the following combinations: 1): POPC, DDAB, DSPE; (2): DSPE, DOPC, DOTAP; more preferably, the DSPE is PEG modified DSPE, preferably, the DSPE is DSPE-PEG2000.
The pharmaceutical composition according to item 19 or 20, wherein the nanoparticle prepared using liposomes is conjugated with a glial cell-specific targeting peptide, preferably the targeting peptide is selected from the group consisting of AS1, MG1, V9, NGR polypeptides.
The pharmaceutical composition of any one of items 18-21, wherein the pharmaceutical composition is in the form of nasal drops, intravenous injection, intravenous infusion, subcutaneous injection, or the pharmaceutical composition is administered directly to the brain.
Use of the nucleic acid molecule of any one of items 1-12, the vector of item 13 or 14, the cell of item 15 or 16, the chimeric antigen receptor of item 17, or the pharmaceutical composition of any one of items 18-22 in the manufacture of a medicament for promoting cellular clearance of amyloid oligomers, treating and/or preventing inflammatory diseases, reducing senile plaques in the brain, treating and/or preventing neurodegenerative diseases, inhibiting gliosis, increasing brain synapse levels in a subject.
A method of promoting cellular clearance of amyloid oligomers, treating and/or preventing inflammatory diseases, reducing senile plaques in the brain, treating and/or preventing neurodegenerative diseases, inhibiting gliosis, increasing brain synaptic levels in a subject in need thereof comprising administering to a subject in need thereof the nucleic acid molecule of any one of claims 1-12, the vector of claim 13 or 14, the cell of claim 15 or 16, the chimeric antigen receptor of claim 17, or the pharmaceutical composition of any one of claims 18-22.
The nucleic acid molecule of any one of items 1-12, the vector of item 13 or 14, the cell of item 15 or 16, the chimeric antigen receptor of item 17, or the pharmaceutical composition of any one of items 18-22 for use in promoting cellular clearance of amyloid oligomers, treating and/or preventing inflammatory diseases, reducing senile plaques in the brain, treating and/or neurodegenerative diseases, inhibiting gliosis, increasing brain synaptic levels in a subject.
Item 26. The nucleic acid molecule, vector, cell, chimeric antigen receptor or pharmaceutical composition of item 22, the method of item 23 or item 25, wherein the neurodegenerative disease is selected from the group consisting of alzheimer's disease, parkinson's disease, huntington's disease, amyotrophic lateral sclerosis, or spinocerebellar ataxia.
The technical solution of the present invention will be specifically described below by means of examples, which are descriptive, illustrative and not meant to be limiting. Reagents used in the examples described below, such as those not specifically identified, are readily commercially available from reagent companies such as Sigma Aldrich, merck, and the test methods, such as those not specifically identified, are found in textbooks such as Sambrook, J., fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual Spring Harbor Press, new York.
Examples
Example 1 construction of w20-SRA gene delivery particles and microglial cells of chimeric antigen receptor (CAR-M).
1.1 Experimental materials and methods
1.1.1 Experimental materials
(2, 3-dioleoyl-propyl) -trimethylammonium (DOTAP) is purchased from Avanti Polar Lipids; dioleoyl phosphatidylcholine (DOPC), distearoyl phosphatidylethanolamine-polyethylene glycol-N-hydroxysuccinimide (DSPE-PEG-NHS), N-hydroxysuccinimide (NHS), N-diisopropylethylamine available from Ala-dine; aβ42 peptide, AS1 (CLNSSQPSC) cyclic peptide was purchased from Medium peptide Biochemical Co., ltd; 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), BCA protein quantification kit, DMEM cell culture medium, RPMI-1640 cell culture medium, PS, FBS purchased from Thermo Fisher Scientific; RNA extraction kit, reverse transcription kit, 2X SYBR Green qPCR Mix were purchased from Beijing kang as century Biotech Co., ltd.
The antibodies used were as follows: iba-1 antibody (GeneTex, cat# GTX 101495), GFAP antibody (Cell Signaling Technology, cat# 3670S), PSD95 antibody (Abcam, cat# ab 12093), synaptophysin antibody (Abcam, cat# ab 32127), LAMP1 antibody (Abcam, cat# ab 24170).
1.1.2 laboratory apparatus
The equipment used in the experiment of this example mainly comprises: zetasizer Nano ZS laser particle sizer (Malvern), HT7700 Transmission Electron microscope (Hitachi), LSM780 laser confocal microscope (Zeiss), IX73 fluorescence microscope (Olympus), cytoFLEX LX flow cytometer (Beckman), 7500Fast Real-Time PCR System (Applied Biosystem).
1.1.3 preparation of Main solution
(1) Phosphate Buffer (PBS): 0.62g of sodium dihydrogen phosphate dihydrate, 5.73g of disodium hydrogen phosphate dodecahydrate and 9g of sodium chloride are dissolved in deionized water, the volume is fixed to 1L, and a 0.22 mu m filter membrane is used for filtration sterilization.
(2) 0.1% pbst: 1mL of Tween 20 was dissolved in 1L of PBS.
(3) 5% skim milk: 5g of skim milk was weighed and dissolved in 100mL of 0.1% PBST.
(4) 10 Xrunning buffer: 144g glycine, 30g Tris, 10g sodium dodecyl sulfonate were dissolved in deionized water to a volume of 1L.
(5) 5 x reduced electrophoresis loading buffer: 2.5mL of 1M Tris-HCl (pH 6.8), 1g of sodium dodecyl sulfate, 5mL of glycerol, 0.5mL of beta-mercaptoethanol, 50mg of bromophenol blue were dissolved in deionized water and the volume was fixed to 10mL.
(6) Flow buffer: 2g of bovine serum albumin was dissolved in 100mL PBS.
(7) LB medium: 10g tryptone, 5g yeast extract, 5g sodium chloride were dissolved in deionized water, and the volume was set to 1L and autoclaved at 121℃for 20min.
Remarks: the relevant reagents, equipment and solutions mentioned in examples 2, 3 and 4 but without giving the origin are the same as in example 1.
1.2 construction of plasmid
The pEGFP-N1 vector (product number: P6460 of Beijing Soy Co., ltd.) was digested with Nhe1 and BamH1 endonucleases, and the synthesized target genes (SR-A, SRA-W20, SRA-ns-scFv) were digested with the nucleotide sequences shown in SEQ ID NO: 8. 9 and 10) were ligated into the vector after double cleavage using Nhe1 and BamH1 endonucleases (FIG. 1).
1.3 preparation and characterization of DNA-NP liposome complexes
19.2. Mu. MoL of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine (POPC, available from Allatin), 0.2. Mu. MoL of diformyldimethyl ammonium bromide (DDAB, available from Allatin) and 0.6. Mu. MoL of diformylphosphole-phenylethanolamine (DSPE) -PEG2000 (available from Sigma Aldrich) were dissolved in chloroform and spin-distilled in a rotary evaporator. 1mL of 10mM PBS (pH=7.4) was added for resuspension and sonicated. The plasmid was added and the plasmid was packaged by a series of freeze/thaw cycles. Exo-enzyme III/DNase I was added to remove excess plasmid DNA adsorbed outside the liposome particles and stored at 4℃for use.
A transmission electron microscope (TEM, japanese Hitachi, HT 7700) sample was prepared and observed with TEM, followed by characterization of the dynamic size of the liposomes in aqueous solution using dynamic light scattering instrument DLS. The results showed that the two particles were well monodisperse and uniform in size distribution (panel A of FIG. 2) and diameter of 87.1.+ -. 3.9nm and 91.3.+ -. 5.0nm, respectively (panel B of FIG. 2). Dynamic light scattering measurement NP had a hydration diameter of 92.0.+ -. 2.9nm, SRA-W20-NP had a hydration diameter of 95.1.+ -. 2.5nm (FIG. 2, panel C), and was excellent in dispersibility in water. The NP and SRA-W20-NP solutions were left at 4deg.C for 3 months, and TEM or DLS measurements showed that the particles were still well dispersed, uniform in size distribution, and no significant change in particle size, indicating good stability of the various particles produced (data not shown).
1.4 CAR expression on BV2 cell surface
BV-2 cells (national laboratory cell resource sharing service platform (NICR)) were inoculated into cells containing Opti- -Cell density was 5X 10 in 12 well plates of medium (available from Gibco) and slide 5 Cells/well, added to the prepared CAR lipid complex, cultured for a further 12h, and observed for CAR expression in cells using cellular immunofluorescence. The results indicated that the single chain antibody W20 was expressed on the cell membrane surface and co-localized with GFP fluorescent protein on the cell membrane (FIG. 3, panel A), whereas no W20 signal was observed by BV2 cells transfected with SRA-ns-scFv. Flow cytometry was used (analysis of GFP expression on BV-2 cells and statistics of transfection-positive cell rate. The results indicated that the BV-2 cell rate for GFP expression was much greater than for the control group (FIG. 3, panel B).
To assess the biosafety of the liposomes, different concentrations of NP, SRA-W20-NP, and SRA-ns-scFv-NP were added to three cell cultures of BV2, N2a, and U87-mg (national laboratory cell resource sharing service platform (NICR)), and after 48h, cell activity was determined using the MTT method. The results show that when the concentration of each group of DNA-NP is 1000 mug/mL, the activity of the three types of cells can still reach more than 90% of that of the control cells, and the liposome has smaller cytotoxicity and better biological safety (figure 4).
1.5 Increased phagocytic capacity of CAR-BV2 for Abeta Os
To demonstrate the increased ability of CAR-BV2 cells to phagocytose Abeta Os, the present invention detects Abeta Os levels in BV-2 cells. BV-2 cells transfected with SRA, SRA-W20 and SRA-ns-scFv plasmids were incubated with Abeta Os for 12h, and intracellular Abeta levels were detected by cellular immunofluorescence. The results showed that cells transfected with SRA and SRA-W20 plasmids had significantly improved phagocytosis of aβos compared to untransfected BV-2 cells, with 8-fold and 15-fold increases in the levels of aβos in CAR (SRA) -BV2 cells and CAR (SRA-W20) -BV2 cells, respectively (fig. 5).
1.6 CAR-BV2 promotes phagocytosis of Abeta Os by SR-A
To verify whether CAR-BV2 uptake of aβos was mediated by SR-a, the SR-a receptor antagonist fucose was added to BV2 cells transfected with SRA, SRA-W20 and SRA-ns-scFv, and intracellular aβ levels were detected using cellular immunofluorescence. The results demonstrate that CAR-BV2 cells phagocytosis of aβos was significantly reduced after addition of fucose compared to the control group (fig. 6). It was shown that AβOs was taken up by SR-A receptor mediated endocytosis.
1.7 Increased degradation of Abeta Os by CAR-BV2
In order to verify that the degradation capability of CAR-BV2 cells on Abeta Os changes after the phagocytic capability of Abeta Os is improved, the invention observes and measures the change of the level of Abeta Os phagocytosed into cells with time. The results showed that 0, 3, 6, 12, 24 and 36h after phagocytosis of aβos by the cells, the intracellular aβ levels gradually decreased (fig. 7), indicating that they were degraded by continuous digestion. Further, the results of cellular immunofluorescence and western-blot analysis show that the level of a lysosome marker LAMP1 is remarkably increased in BV2 cells transfected by SRA and SRA-W20 (figures 8 and 9), and the digestion and degradation capability of Abeta in the CAR-BV2 cells is obviously improved.
1.8 CAR-BV2 reduces AβOs-induced inflammatory factor production
AβOs has cytotoxicity, and can promote BV-2 cells to produce inflammatory factors and convert into M1. The invention uses qPCR to detect mRNA of inflammatory factors in BV-2 cells transfected by liposome. The primers used are shown in SEQ ID NO: 11-28. (remarks: the above primers are also used in example 2, 3 or 4)
qPCR results show that after Abeta Os stimulation, mRNA levels of pro-inflammatory cytokines IL-6 and TNF-alpha in BV2 cells are obviously increased, and mRNA levels of anti-inflammatory factors such as Arg-1, TGF-beta, IL-10 and the like are reduced. Whereas in BV2 cells transfected with SR-A as well as SRA-W20, M1 markers TNF- α and IL-6mRNA levels were reduced and M2 markers Arg1, TGF- β and IL-10 levels were elevated, indicating that CAR-M was able to inhibit AβOs-induced inflammatory cytokine production and promote conversion of BV-2 cells from M1 to M2 (FIG. 10).
EXAMPLE 2 in vivo studies of chimeric antigen receptor engineering microglia
2.1 Main Experimental materials and apparatus
In addition to the experimental materials and equipment used in example 1, the following main experimental materials and equipment were used in this example: FXPRO small animal living body imaging system (KODAK), KD-BM paraffin embedding machine (KEDEE), RM2245 paraffin slicing machine (Leica), water maze equipment (China medical science sciences), new thing recognition equipment (China medical science sciences), and Y maze equipment (China medical science sciences). 50% xylene: the equal volume of ethanol and xylene are fully and uniformly mixed. Different percentages of ethanol: the absolute ethyl alcohol with different volumes is fully and evenly mixed with the deionized water. Citric acid antigen retrieval liquid: 3g of trisodium citrate and 0.4g of citric acid are dissolved in deionized water, and the volume is fixed to 1L.
2.2 animal Experimental design
AD mice (purchased from the company fukang biotechnology, ltd, beijing) were divided into 5 groups of 8 mice each, and the dosing strategy was as follows: different liposome particles of DNA-free-NP, SRA-W20-NP, and SRA-ns-scFv-NP were nasal instilled into AD mice and WT mice (available from Beckmann Biotechnology Co., ltd.) each at 10. Mu.L, at 10. Mu.g each dose, once every other day for 28 days. After the last administration, a behavioural test was performed.
2.3 distribution of Liposome Gene vectors in mice
Injecting Cy 7-labeled SRA-W20-NP liposome gene vector into mice in a nasal administration mode, taking organs such as brain, heart, liver, spleen, lung, kidney and the like of the mice after 0.5, 1, 2, 3, 6 and 12 hours, and detecting the distribution of the liposome through a bioluminescence imaging system. The results showed that SRA-W20-NP was distributed in each organ of the mice, and the distribution state was changed with time, and SRA-W20-NP was present in the liver, and Cy7 fluorescent signal was detected in the brain, and peaked at around 3 hours. The results indicate that the liposome gene vector was able to successfully deliver the SRA-W20-NP liposome gene vector to the brain by nasal administration (data not shown).
2.4 expression of fusion Gene W20-SRA in mouse brain
In order to examine the expression condition of fusion gene W20-SRA in the nerve cells in the brain of the mice, the invention observes the fluorescence distribution condition of fusion gene GFP and W20 in the brain of the mice after nasal administration through a laser confocal microscope. The results showed that fusion gene W20-SRA was successfully expressed on microglial cell membranes in the brain (panel A of FIG. 11), whereas control gene SRA-ns-scFv only observed GFP fluorescence signal (panel B of FIG. 11).
2.5 CAR-M therapy improved cognitive impairment in AD mice
AD and WT mice were nasally dosed with CAR-M therapy and after 28 days of treatment, the invention tested the cognitive function of the mice in Y-maze and Morris water maze experiments. The Y-maze and Morris water maze experiments were performed as follows.
2.5.1Y maze
The Y-maze consisted of A, B, C arms, comprising two test phases, spaced 1h apart. The first phase was to let the mice explore the two arms (a, B) of the maze, and the third arm (new arm C) was blocked for 10min. The second phase is to place the mouse on the same starting arm as in the first phase, open the new arm C and let it go freely into and out of all three arms for 5min. The exploration trajectory is recorded with a camera and the number of exploration of the new arm and the time the new arm stays are recorded. The results indicate that PBS and DNA-free-NP treated AD mice do not have a significant preference for the new arm compared to WT mice. AD mice showed a significant increase in both new arm residence time (panel B of fig. 12) and number of searches (panel a of fig. 12) following SRA-W20-NP treatment, indicating improved spatial memory.
2.5.2Morris Water Maze (MWM)
The water maze consists of a water pool with the diameter of 110cm and a platform with the diameter of 10cm, wherein the water pool contains opaque water (22+/-1 ℃), and different symbols are marked on the periphery of the water pool respectively so as to facilitate the positioning and identification of the mice. During the continuous 5 days of training, the platform was placed under water for about 1cm. Mice were placed in a pool from random positions, allowed to swim for 60s to find the platform, and left on the platform for 10s. When the mouse found the platform, the test ended and the time to find the platform was recorded. Mice unable to find the platform were guided onto the platform and latency was recorded as 60s. After each training session, the mice were wiped dry with a dry towel and dried with a heater and returned to the squirrel cage. Training is carried out 2 times a day at intervals of 3-4 hours, and the latency, swimming distance, average swimming speed and exploration mode of each group of mice are recorded. After 24h of the last learning test, the platform was removed, a water entry point was randomly selected, the mice were placed in water, swim for 60s, and the memory retention of the mice was tested without the platform. The parameters of target quadrant retention time, swimming distance, average swimming speed, exploration path, number of platform penetrating times and the like are analyzed and measured by software through monitoring and recording through a camera arranged above the maze.
In the MWM test to assess spatial memory and learning ability, AD mice treated with SRA-NP and SRA-W20-NP reached a significantly shorter plateau time during the training phase compared to AD mice of PBS control group (panel a of fig. 13). In the platform-withdrawal test, AD mice of SRA-W20-NP or SRA-NP treated groups exhibited a pronounced spatially directed swimming behavior, with significantly shorter time required to reach the platform position (panel B of fig. 13), more number of passes (panel C of fig. 13) and longer residence time in the target quadrant (panel D of fig. 13) compared to PBS and DNA-free-NP treated control AD mice. In the training and testing experiments, the swimming speeds of the mice in each group were not significantly different, indicating that the motor functions were not affected. These results indicate that SRA-W20-NP and SRA-NP significantly reduced cognitive dysfunction in AD mice, with SRA-W20-NP providing more significant improvement in cognitive and memory function in AD mice.
2.6 determination of Abeta levels in mice brain
To investigate the effect of CAR-M on aβ plaques in AD mice brains, the present invention examined the levels of 6E10 positive aβ plaques in AD mice brains using immunohistochemistry. Specifically, mice were deeply anesthetized with pentobarbital sodium (50 mg/kg) and perfused with pre-chilled PBS (containing heparin 10U/mL) prior to sacrifice. The brain was dissected and cut into two parts along the sagittal plane, the left hemisphere was fixed overnight at 4% paraformaldehyde, and the next day dehydrated (30% ethanol 1h,50% ethanol 1h,75% ethanol 1h,100% ethanol 1h,50% xylene 1h,100% xylene 1 h), paraffin embedded for 3h. Paraffin-embedded sections were cut 5 μm or 10 μm thick on a microtome. Paraffin sections were placed in an oven at 60 ℃ for 1h and dewaxed in sequence in 100% xylene, 50% xylene, 100% alcohol, 70% alcohol, 50% alcohol and 30% alcohol. Soaking the slices in deionized water for 10min, adding antigen retrieval liquid, boiling for 15min, and cooling at room temperature for standby. Washed 3 times with PBS and blocked for 1h at room temperature in PBST containing 10% sheep serum and 0.3% Triton X-100. Subsequently, sections were incubated with 6E10 (1:100) antibody, washed three times with PBS for 5min each, then with HRP-labeled secondary antibody, washed three times with PBS for 5min each, and observed with confocal microscopy.
Preparation of brain homogenate: the right brain half obtained above was subjected to lysis homogenization in RIPA buffer containing protease inhibitor, and then the tissue was centrifuged at 14000g at 4 ℃ for 30min, and the supernatant containing soluble aβ (RIPA soluble fraction) was collected. The insoluble pellet was resuspended in guanidine hydrochloride buffer (5.0M guanidine hydrochloride, pH 8.0) and centrifuged at 14000g at 4℃for 1h to obtain a supernatant containing insoluble A.beta.s (guanidine soluble fraction). Detection was performed using the IBL company's aβ40ELISA detection kit and aβ42ELISA detection kit.
The results showed that the hippocampal aβ plaque staining area was significantly reduced in mice of the SRA-NP, SRA-W20-NP treated group compared to AD mice of the PBS control group, with the most significant reduction in SRA-W20-NP treated group (fig. 14). The levels of soluble and insoluble A.beta.40/42 in the mouse brain homogenates were further examined using ELISA, and the results showed that both SRA-NP and SRA-W20-NP significantly reduced the levels of soluble and insoluble A.beta.40 and A.beta.42 in the brain (FIG. 15).
2.7 determination of Aβ oligomer level in brain homogenates
To investigate the effect of CAR-M on aβ oligomer levels in AD mice brain, the present invention used W20 antibodies to detect the content of aβ oligomers in mouse brain homogenates by ELISA. The results show that SRA-W20-NP significantly reduced the level of Abeta oligomers in the brains of AD mice (FIG. 16), however, there was no statistical difference in the effect of SRA-NP and SRA-ns-scFv-NP on Abeta oligomer levels compared to PBS or empty liposomes.
2.8 determination of the ability of CAR-M to phagocytose Abeta in the brain of AD mice
To examine the ability of CAR-M to phagocytose aβ in AD mouse brain, the present invention uses immunohistochemistry to detect levels of aβ in gfp+ microglia in the mouse brain. Preparation of Paraffin brain sections and immunohistochemical treatment methods such as 2.6. The primary antibody adopts 6E10 (1:100) and anti-Iba-1 antibody (1:100), and the secondary antibody is corresponding fluorescence labeled anti-IgG antibody. The results indicate that microglia expressing CAR (SRA-W20) in the brain of AD mice have significantly increased levels of phagocytic aβ (panel a of fig. 17). Also, while microglia expressing both CAR (SRA) and CAR (SRA-ns-scFv) had increased aβ phagocytosis, both were significantly lower than the CAR (SRA-W20) group (panel B of fig. 17).
The invention also determines the content of GSH and GSSG in brain homogenate of AD mice and WT mice thereof after various liposome gene vector treatments. The used kits are GSH and GSSG detection kits and ROS detection kits of Biyundian corporation, and the experiment is carried out according to the instruction of the kit. The results show that SRA-W20-NP significantly reduced GSSG levels in the brains of AD mice (panels A and B of FIG. 18) and increased GSH/GSSG ratios (panel C of FIG. 18) relative to the PBS control, whereas neither SRA-NP nor SRA-ns-scFv-NP reached statistical differences. The present invention further examined the ROS content in the mouse brain homogenate, and the results indicate that SRA-W20-NP and SRA-NP were able to significantly reduce ROS levels in the brain of AD mice (FIG. 18, panel D). The above results demonstrate that CAR-M gene therapy is able to reduce oxidative stress in the brain of AD mice.
2.9 CAR-M reduces brain synaptic loss in AD mice
Aβos may cause synaptic dysfunction and loss of synapses in AD brain, leading to a decline in cognitive function. Synaptocins and PSD95 are marker proteins of presynamics and postsynaptic, respectively, and thus, the present invention uses synaptocins and PSD95 antibodies to assess synapse levels in the mouse brain by immunohistochemical methods (FIG. 19). Experimental results indicate that the synaptic levels in the brains of the AD control mice are significantly reduced relative to the WT mice, whereas both SRA-W20-NP and SRA-NP treatments significantly increased the levels of synaptocins and PSD-95 in the brains of the AD mice (FIGS. 19, A, B and C), and the number of synapses is also significantly increased (FIG. 19, panel D). There was no statistical difference in synaptic levels between the SRA-ns-scFv-NP treated group and the PBS control AD mice.
2.10 CAR-M reduces the degree of activation of glial cells in the brain of AD mice
In the development and progression of AD, neuroinflammation caused by glial activation plays a key role. We examined activation of astrocytes and microglia in the mouse brain by GFAP and Iba-1 immunostaining, respectively. The brain GFAP and Iba-1 positive staining area was significantly reduced in AD mice treated with SRA-W20-NP compared to AD mice of PBS and NP control (fig. 20). These results indicate that SRA-W20-NP can significantly reduce the overactivation of astrocytes and microglia in AD brain, reducing neuroinflammation. The therapeutic effect of SRA-W20-NP is better than that of SRA-NP.
2.11 Effect of CAR-M treatment on the microglial phenotype in the brain of AD mice
In order to investigate whether CAR-M can promote the conversion of microglial cells from inflammatory M1 type to anti-inflammatory M2 type, the invention uses qPCR to determine the gene expression conditions of marker genes IL-1 beta, TNF-alpha and marker genes Arg-1, TGF-beta, IL-10 and YM-1 of M1 type microglial cells in the brain of mice after CAR-M treatment. The results show that the SRA-W20-NP treatment can significantly increase the mRNA level of the M2-related marker gene in the brain of the AD mice and reduce the mRNA level of the M1-related gene (FIG. 21). It was shown that CAR-M treatment shifted microglial cells from inflammatory M1 to anti-inflammatory M2 in the brains of AD mice.
Example 3 construction of W20-MerTK gene delivery particles and astrocytes of chimeric antigen receptor (CAR-a).
Similar to microglia, astrocytes were similarly "double edged sword" in the development and progression of AD, and therefore, chimeric antigen receptor technology was also used in this example to functionally engineer astrocytes to confirm whether the technical effects of CAR-M described above are prevalent in glial cells.
Unlike the traditional chimeric antigen receptor used to program CAR-T cells, the chimeric antigen receptor constructed in this example is derived from MerTK receptor, which is expressed primarily on M2-type macrophages and astrocytes, which, upon activation, mediate phagocytosis while inhibiting the release of pro-inflammatory cytokines. The MerTK structure is similar to the CAR structure in traditional CAR-T cells, including antigen recognition, transmembrane and intracellular signal transduction regions, and is an ideal receptor for CAR engineering, and other anti-inflammatory phagocytic receptors, such as Tyro3 and Axl, are also expected to be used for anti-inflammatory CAR engineering by rational design. In this example, the antigen binding region (19 Gly-275 Asn) of merTK was replaced with an Abeta oligomer-specific single-chain antibody (W20), a novel anti-inflammatory chimeric antigen receptor recognizing Abeta oligomer was constructed, and the functional properties of astrocytes transformed with the CAR were studied intensively.
3.1 culture of Primary astrocytes and microglia
Taking 5C 57BL/6J neonates, soaking and sterilizing in 75% alcohol, taking out brains, soaking the brains in HBSS, removing blood meninges, shearing the brain with tissues, placing the brain with the blood meninges removed into a 50mL centrifuge tube, adding 20mL of tissue digestion liquid (DMEM medium containing 0.25% pancreatin and 1mg/mL DNase I), digesting for 20min at 37 ℃, removing undigested tissue blocks of the digested cell suspension through a 70 μm cell screen, centrifuging for 10min at 500g to collect cells, spreading the cells in a T-75 culture flask, and culturing for 5-7 days at 37 ℃. The medium was changed every 2-3 days. After 7 days of cell culture, the cells were taken out and placed in a cell shaker and shaken at 220rpm for 30min, the supernatant was placed in a centrifuge tube and centrifuged at 500g for 10min, and microglial cells were collected. Astrocytes were still attached to the cell culture flask, and the culture was continued with fresh medium added, with medium replacement every 2-3 days.
3.2 culture of Primary neuronal cells
Taking 5C 57BL/6J neonates, soaking and sterilizing in 75% alcohol, taking out brains, soaking the brains in HBSS, peeling blood meninges, peeling hippocampal tissues, shearing the peeled hippocampal tissues with tissues, placing the crushed hippocampal tissues in a 15mL centrifuge tube, adding 5mL of tissue digestion liquid (DMEM medium containing 0.25% pancreatin and 1mg/mL DNase I), digesting for 20min at 37 ℃, removing undigested tissue blocks of the digested cell suspension through a 70 μm cell screen, centrifuging for 10min at 500g to collect cells, spreading the cells in a 12-well plate containing a cell climbing sheet (PDL room temperature coated overnight), and culturing by using a Neurobasal medium (containing B27, L-Glutamax).
3.3 construction of plasmid
pEGFP-N1 vector was double digested with Nhe1 and BamH1 endonucleases, and the nucleotide sequences of the synthesized target genes (MerTK, W20-MERTK, ns-ScFv-merTK (ns-CAR) were shown in SEQ ID NO: 29-31), respectively, were ligated into the vector after double digestion with Nhe1 and BamH1 endonucleases (FIG. 22).
3.4 Synthesis and characterization of DSPE-PEG-AS1
2mg of DSPE-PEG2000-NHS and 1.5mg of AS1 peptide (CLNSSQPSC (C-C ring) were weighed, put in a round bottom flask, 5mL of dimethylformamide (containing 5. Mu.L/mL of triethylamine) was added, stirred at room temperature for 72 hours, and after the reaction solution was put in a dialysis bag (molecular weight cut-off: 1 kDa), dialyzed for 48 hours to remove unreacted AS1 peptide, and then lyophilized and stored at-20℃and the molecular weight of DSPE-PEG2000-AS1 was analyzed using a MALDI-TOF mass spectrometer. Molecular weight data indicated that AS1 was successfully coupled to DSPE-PEG (data not shown).
3.5 preparation of liposomes and lipid complexes
50. Mu. Mol DOTAP, 50. Mu. Mol DOPC and 5. Mu. Mol DSPE-PEG2000-AS1 were dissolved in 5mL chloroform/methanol mixed solution (v: v=2:1). After gentle stirring at room temperature for 2h, the mixture was spin evaporated on a rotary evaporator to give a lipid bilayer membrane, 2mL Tris buffer (10 mm, ph 7.4) was added and sonicated in an ice bath to give unilamellar vesicle liposomes. The resulting liposomes were passed sequentially through 200nm and 100nm polycarbonate membranes using a liposome extruder. To prepare plasmid-liposome complexes (lipid complexes), the present example vortexes the plasmid and liposome mixture (N: p=4:1) and maintained at room temperature for 15-20min. The lipid complexes prepared were then transferred to a cryovial for ten freeze-thaw cycles, and the resulting lipid complexes were passed through 200nm and 100nm membranes in sequence.
Experimental results of dynamic light scattering showed that the hydrated diameter of the empty liposome was 106.+ -. 13.1nm, PDI was 0.16, and the hydrated diameter of the lipid complex was slightly larger than that of the empty liposome, 132.+ -. 19.5nm, and PDI was 0.19 (data not shown). The PDI of both empty liposomes and lipid complexes was less than 0.3, indicating uniform particle size.
3.6 Expression of CARs in astrocytes
To characterize whether CARs can be expressed intact in astrocytes, the present example performs a cellular immunofluorescence assay on astrocytes 24h after transfection of astrocytes with CAR lipid complexes as described in example 1. The results show that the CAR expressed W20 (red fluorescence), 276-994MerTK (yellow fluorescence) and EGFP (green fluorescence) (panel a of fig. 23). Further, membrane proteins of astrocytes were extracted and subjected to Western blot analysis, and the results showed that the CAR receptor contained Mertk and W20 protein fragments, and the molecular weight was about 200kDa, which was consistent with the theoretical molecular weight (fig. 23, panel B).
To characterize CAR expression in various cell types, this example transfected different cell types with CAR lipid complexes, and the results indicated that CAR was specifically expressed in astrocytes and not in neurons and microglia (fig. 24). Since the astrocyte-specific promoter (gfa 2) was used in the CAR expression plasmid, the CAR was expressed only in astrocytes.
3.7 CAR-A phagocytic capacity assay for Abeta
Phagocytosis of Abeta oligomers by CAR-A was analyzed by flow cytometry. Specifically, after fixing cells with 4% paraformaldehyde for 20min, adding 6E10 primary antibody, and incubating for 1h at room temperature; washing with streaming buffer solution for three times, adding PE-marked goat anti-mouse secondary antibody, and incubating for 1h at room temperature; after three washes with streaming buffer, data were collected using a CytoFLEX LX flow cytometer and analyzed with FlowJo X10. The results show that CAR-a has significantly increased ability to phagocytose aβ oligomers compared to control astrocytes, while ns-CAR-a has no significant change in ability to phagocytose aβ oligomers, indicating that W20 mediates CAR recognition of aβ oligomers (fig. 25).
3.8 CAR-A eliminates Abeta oligomers without causing inflammatory reactions
The expression levels of pro-inflammatory cytokines (IL-1. Beta., TNF-. Alpha., iNOS, and IL-6) and anti-inflammatory cytokines (IL-4, arg-1, CD206, and TGF-. Beta.) of CAR-A in phagocytosis of Abeta oligomers were determined using fluorescent quantitative PCR, and the primers were used as described above. The results show that when astrocytes and ns-CAR-a phagocytose aβ oligomers, the pro-inflammatory cytokines are significantly increased and the anti-inflammatory cytokines are significantly reduced. While CAR-a phagocytized aβ oligomers, pro-inflammatory cytokines were not significantly increased, and anti-inflammatory cytokines were not significantly decreased (fig. 26). These results indicate that CAR-a does not elicit an inflammatory response when phagocytosing aβ oligomers.
EXAMPLE 4 therapeutic study of chimeric antigen receptor-programmed astrocytes in AD mice
In AD pathological conditions, astrocytes have reduced phagocytic capacity for aβ and sustained release of inflammatory factors. Regulating and controlling astrocyte function in brain, enhancing the phagocytic capacity of the astrocyte to Abeta, and reducing the release of inflammatory factors, is an ideal strategy for treating AD. Similar to example 2, this example uses methods such as animal in vivo fluorescence imaging and immunohistochemistry to evaluate the metabolism of liposome gene delivery particles in vivo and the expression of chimeric antigen receptor in brain; the influence of CAR-A on the memory capacity of the AD mice is detected through water maze, new thing cognition and Y maze experiments, and the pathological changes in the brain of the AD mice are detected by methods such as immunohistochemistry, ELISA and the like.
4.1 laboratory animals
6 month old APP/PS1 transgenic mice and 6 month old male wild type mice were purchased from Beijing Fukang Biotechnology Co., ltd. The mice are raised in a clean room with the room temperature of 22+/-2 ℃ and the humidity of 45+/-10%, the mice can obtain food and water freely, and all animal experiments are carried out according to the national public health service laboratory animal nursing and use guidelines. Experiments involving mice have been approved by the committee for animal protection and use at the university of bloom.
4.2 experiment
Animal experiment dosing and grouping, morris water maze experiment, Y maze experiment, neology cognitive experiment, collection and treatment of mouse brain tissue, preparation of brain homogenate, immunohistochemistry and immunofluorescence analysis, determination of Abeta 40 and Abeta 42 in mouse brain, determination of transcription level of pro-inflammatory cytokines and anti-inflammatory cytokines related genes in mouse brain by real-time quantitative fluorescence PCR were performed as described in example 2, and the GFAP and Iba-1 protein levels in mouse brains of different experimental groups were determined by Western blot experiment.
4.3 experimental results
4.3.1 lipid complexes are effective in entering the brain
The results show that after nasal administration, the CAR lipid complex is mainly distributed in tissues such as brain, lung, liver and the like, the CY7 signal in the brain reaches the highest at 3h, and the CY7 signal in the brain still exists after 24h (fig. 27), which shows that the CAR lipid complex can enter the brain of the mouse through the nasal cavity. Immunofluorescence results showed better co-localization of the CAR with astrocytes, indicating that the CAR can effectively infect and express astrocytes in the mouse brain (fig. 28).
4.3.2 CAR-A can improve cognitive function of AD transgenic mice
After 6 weeks of treatment of AD mice with CAR lipid complexes, the effect of CAR-a on cognitive function in AD transgenic mice was tested with Morris water maze, Y maze, and neology cognitive experiments.
First, the spatial memory and learning ability of mice was evaluated by Morris water maze test, and in the training period, the time to plateau of AD-PBS (PBS control AD mice), AD-NP (empty vector control AD mice) and AD-ns-CAR (nonspecific scFv-CAR control AD mice) was significantly increased, while the time to plateau of AD mice given with the CAR lipid complex was significantly decreased (A, C graph of FIG. 29), indicating that the CAR lipid complex can effectively improve the spatial memory and learning ability of mice, as compared to wild-type mice (WT-PBS and WT-NP). In the post-withdrawal test, AD mice given the CAR lipid complex were significantly shorter in time to first pass through the target site (figure 29, B, D), and significantly longer in residence time in the target quadrant (figure 29, E), and significantly more times to cross the plateau region (figure 29, F), than AD-PBS, AD-NP, and AD-ns-CAR groups. The above results indicate that the CAR lipid complex is capable of significantly improving cognitive dysfunction in AD transgenic mice.
The effect of CAR-a on AD mice short term memory was further assessed by Y maze experiments. The results showed that the number of times WT mice entered the new arm and the residence time in the new arm were significantly higher than AD-PBS, AD-NP and AD-ns-CAR, whereas the residence time and the number of searches in the new arm were significantly increased for AD mice given the CAR lipid complex (fig. 30), indicating that the CAR lipid complex was able to significantly improve the short term memory capacity of AD transgenic mice.
4.3.3 CAR-A can reduce the level of Abeta oligomers in the brain
Immunofluorescence results showed that the astrocytes in the brains of mice in the AD-PBS, AD-NP and AD-ns-CAR groups had lower levels of Abeta oligomers, indicating that astrocytes in the AD-PBS, AD-NP and AD-ns-CAR groups had a weaker ability to phagocytise Abeta oligomers, whereas the CAR-engineered astrocytes had significantly enhanced phagocytic ability to Abeta oligomers (FIG. 31).
The content of Abeta oligomers in the brains of mice was further determined by ELISA method in this example. The results show a significant decrease in the content of aβ oligomers in the brains of mice from the AD-CAR experimental group compared to the AD-PBS, AD-NP and AD-ns-CAR groups (fig. 32). The above results indicate that CAR-a can phagocytose aβ oligomers in large amounts in AD mouse brain, thereby reducing levels of aβ oligomers in mouse brain.
4.3.4 CAR-A can reduce senile plaque in brain and level of Aβ40/Aβ42
The effect of CAR-a on the level of aβ plaques in the brains of AD mice was evaluated, and the results of immunohistochemistry on aβ plaque staining and aβ plaque area statistics showed a significant decrease in aβ plaque area in the brains of AD-CAR mice compared to AD-PBS, AD-NP and AD-ns-CAR groups (fig. 33).
The levels of insoluble aβ40 and aβ42 in the brain of the mice were further determined by ELISA. The results show that AD mice given CAR lipid complexes have significantly reduced levels of insoluble aβ40 and aβ42 in the brain compared to AD-PBS, AD-NP and AD-ns-CAR groups (fig. 34). CAR-a significantly reduces aβ oligomer levels in the brain by phagocytosing aβ oligomers, while a reduction in aβ oligomer content further reduces aβ plaque formation, thereby reducing total aβ40 and aβ42 levels in the brain.
4.3.5 CAR-A can improve the inflammatory microenvironment in the brain of AD mice
qPCR measures the levels of pro-inflammatory cytokines and anti-inflammatory cytokines in the mouse brain. qPCR experimental results showed that the levels of pro-inflammatory cytokines (IL-1β, TNF- α, iNOS, IL-6) were significantly increased in mice brains of the AD-PBS, AD-NP, and AD-ns-CAR groups relative to WT mice, the levels of anti-inflammatory cytokines (IL-4, arg-1, TGF- β) were significantly decreased, and the levels of pro-inflammatory and anti-inflammatory cytokines were restored to near WT levels in AD mice administered with the CAR lipid complex (FIG. 35).
4.3.6 CAR-A reduces gliosis in AD mice
The Western blot method measured GFAP and Iba-1 protein levels in the mouse brain to evaluate astrocyte and microglial proliferation in the mouse brain, and the results indicate that the CAR lipid complex can effectively reduce glial proliferation in the brain of AD mice (fig. 36).
4.3.7 CAR-A reduces brain synaptic loss in AD mice
Synaptic dysfunction and synaptic loss are one of the major pathological features of AD. The presynaptic proteins Synaptocins (SYN) and postsynaptic proteins 95 (PSD 95) are marker proteins of presynamics and postsynapses, respectively. This example uses immunofluorescence to immunostain presynaptic and postsynaptic proteins in the mouse brain to assess synaptic levels in the mouse brain. The results showed that the brain synaptic levels in mice of the AD-PBS and AD-NP groups were significantly reduced, whereas the brain synaptic levels in mice given the CAR lipid complex were significantly increased, whereas the brain synaptic levels in mice of the AD-ns-CAR control group were not significantly increased, relative to WT mice (fig. 37).
EXAMPLE 5 therapeutic study of chimeric antigen receptor-programmed astrocytes on PD mice
In PD pathological conditions, astrocytes have reduced phagocytic capacity for alpha-synuclein and sustained release of inflammatory factors. Therefore, regulating and controlling astrocyte function in brain, enhancing the phagocytic capacity of alpha-synuclein and reducing the release of inflammatory factors are ideal strategies for treating PD. Similar to example 4, this example uses methods such as cellular immunofluorescence, animal in vivo fluorescence imaging, and immunohistochemistry to evaluate the metabolism of liposomal gene delivery particles in vivo and the expression of chimeric antigen receptors in the brain; the influence of CAR-A on the memory capacity of PD mice was detected by water maze, neology cognition and Y maze experiments, and pathological changes in the brain of PD mice were detected by methods such as immunohistochemistry and ELISA (data not shown).
Equivalent solution
While various embodiments of the invention have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the functions and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the present invention. More broadly, those skilled in the art will readily appreciate that all parameters, materials, and settings described herein are meant to be exemplary and that the actual parameters, materials, and/or settings will depend upon the specific application for which the teachings of the present invention is used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is, therefore, to be understood that the foregoing embodiments and examples are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the invention may be practiced otherwise than as specifically described and claimed. If such features, systems, articles, materials, and/or methods are not mutually conflicting, any combination of two or more such features, systems, articles, materials, and/or methods is included within the scope of the present invention.
The phrase "and/or" as used in the present specification and claims should be understood to mean "either or both" of the elements so combined, i.e., elements that are in some cases combined and in other cases not combined. In addition to the elements specifically identified by the "and/or" clause, other elements may optionally be present, whether related or unrelated to those elements specifically identified unless specifically indicated otherwise. Thus, as a non-limiting example, reference to "a and/or B" when used in conjunction with an open language such as "comprising" can refer in one embodiment to the presence of a without B (optionally including other elements other than B); in another embodiment, meaning that B does not have a (optionally including elements other than a); in yet another embodiment, both a and B (optionally including other elements) are referred to; etc.
As used herein in the specification and claims, "or" should be understood as having the same meaning as "and/or" as defined above. For example, when separating items in a list, "or" and/or "should be understood to be inclusive, i.e., including at least one of the plurality of elements or lists of elements, but also including more than one, and optionally, other items not listed. Only the opposite terms, such as "only one of them" or "exactly one of them", or when used in the claims, "consisting of … …" will refer to exactly one element comprising a plurality or list of elements. Generally, when an exclusive term is provided, such as "either," "one," "only one," or "exactly one," the term "or" as used herein should be understood to mean only an exclusive alternative (i.e., "one or the other but not both"). "consisting essentially of … …" when used in the claims should have its ordinary meaning in the art of patent law.
As used herein in the specification and claims, the phrase "at least one" when referring to a list of one or more elements is understood to mean at least one element selected from any one or more elements of the list of elements, but does not necessarily include at least one of each element specifically listed in the list of elements, and does not exclude any combination of elements in the list of elements. This definition also allows that elements other than the specifically identified elements in the list of elements that the phrase "at least one" refers to may optionally be present, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, in one embodiment, "at least one of a and B" (or equivalently, "at least one of a or B," or equivalently, "at least one of a and/or B") may refer to at least one, optionally including more than one, a, with no B present (and optionally including elements other than B); in another embodiment, at least one, optionally including more than one, B, absent a (and optionally including elements other than a); in yet another embodiment, at least one, optionally including more than one a, and at least one, optionally including more than one B (and optionally including other elements); etc.
In the claims and in the above description, all conjunctions such as "comprising," "including," "carrying," "having," "containing," "involving," "having," etc. are to be construed as open-ended, i.e., to mean including but not limited to. Only the conjunctions "consisting of … …" and "consisting essentially of … …" should be closed or semi-closed conjunctions, respectively.
Use of ordinal terms such as "first," "second," "third," etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements.
Sequence listing
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aagtccggac tcagatctcg agctatgaca gagaatcaga ggctctgccc tcatgaacga 780
gaggatgctg actgcagttc agaatccgtg aaatttgacg cacgttcaat gacagcatcc 840
cttcctcaca gcactaaaaa tggcccctcc gttcaggaga agttgaagtc cttcaaggct 900
gccctcattg ctctctacct ccttgtgttt gcagtactaa tacctgttgt tggaatagtg 960
acagctcagc ttttgaattg ggaaatgaag aactgcttag tttgttcacg taacacaagt 1020
gatacatctc aaggtcctat ggaaaaagaa aataccagta acgtggaaat gagatttaca 1080
attatcatgg cacacatgaa ggacatggag gagagaatcc aaagcatttc aaactcaaaa 1140
gccgacctta tagacacggg acgcttccag aatttcagca tggcaactga ccaaagactt 1200
aatgatattc ttctgcagtt aaattccttg attttgtcag tccaggaaca tgggaattca 1260
ctggatgcaa tctccaagtc cttgcagagt ctgaatatga cactgcttga tgttcaactc 1320
catacagaaa cactgcatgt cagagtccgt gaatctacag caaagcaaca ggaggacatc 1380
agtaaattgg aggaacgtgt gtacaaagta tcagcagaag tccagtctgt gaaagaagaa 1440
caagcgcacg tggaacagga agtaaaacag gaagtgagag tattgaacaa catcaccaac 1500
gacctcagac tgaaggactg ggaacactca cagacactga aaaacatcac cttcattcaa 1560
gggcctcctg gaccccaagg tgaaaaggga gacagagggc ttactggaca aactggtcca 1620
cctggtgctc caggaataag aggtattcca ggtgttaaag gtgatcgggg acaaattggc 1680
ttccctggag gtcgaggaaa cccaggagca ccaggaaagc cagggaggtc gggatctcct 1740
ggacctaaag gacaaaaggg agagaagggg agtgtaggcg gatcaacccc ccttaagaca 1800
gttcgactgg ttggtggtag tggagcccat gagggccgag tggagatctt ccaccaaggc 1860
cagtggggca caatctgtga tgatcgctgg gatatacggg ctggacaagt tgtctgccgg 1920
agtctaggat accaagaagt tctagctgtg cacaagagag ctcactttgg acaaggtact 1980
ggtccaatat ggctgaatga agtgatgtgt tttggaagag aatcatctat tgagaactgt 2040
aaaatcaacc agtggggagt actaagctgt tcacattcag aagatgctgg ggtcacttgt 2100
acttcaatgg cagaagttca gttgcttgaa agcggtggag ggttggtcca acctggtgga 2160
agcctgcgct tgtcatgtgc cgcctcaggg tttaccttta gttcttacgc tatgtcatgg 2220
gtacgccaag cacctgggaa aggattggaa tgggtttccg gaatctctaa cttgggtctc 2280
accaccgggt acgcagattc cgttaaggga cgcttcacca tttctcggga caattccaag 2340
aatactctct atcttcagat gaactccctt agggcagagg ataccgcagt gtattactgc 2400
gctaagacca catccaggtt cgattattgg ggacagggaa ctttggtcac tgtttcctct 2460
ggtggcggag gctcaggtgg cgggggcagt ggagggggag gctcaacaga catccaaatg 2520
acccaatccc ccagttcatt gtctgccagt gtgggagacc gcgtcaccat aacctgccgg 2580
gccagtcaat ccattagttc ataccttaac tggtatcagc aaaagcctgg gaaagctcca 2640
aagttgttga tttataaagc tagttatttg cagtccgggg tcccctccag gttcagtggc 2700
agcggaagtg gtactgattt cacactcacc attagcagtt tgcagcccga ggactttgca 2760
acctactatt gccaacaaac tcatagacct ccagttacct tcggtcaagg cactaaagta 2820
gagataaagc gggggcccta aggatcc 2847
<210> 10
<211> 2835
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> GFP-SRA-SCFV
<400> 10
gctagcatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag 60
ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc 120
acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg 180
cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac 240
atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc 300
atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac 360
accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg 420
gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag 480
aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag 540
ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac 600
aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac 660
atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac 720
aagtccggac tcagatctcg agctatgaca gagaatcaga ggctctgccc tcatgaacga 780
gaggatgctg actgcagttc agaatccgtg aaatttgacg cacgttcaat gacagcatcc 840
cttcctcaca gcactaaaaa tggcccctcc gttcaggaga agttgaagtc cttcaaggct 900
gccctcattg ctctctacct ccttgtgttt gcagtactaa tacctgttgt tggaatagtg 960
acagctcagc ttttgaattg ggaaatgaag aactgcttag tttgttcacg taacacaagt 1020
gatacatctc aaggtcctat ggaaaaagaa aataccagta acgtggaaat gagatttaca 1080
attatcatgg cacacatgaa ggacatggag gagagaatcc aaagcatttc aaactcaaaa 1140
gccgacctta tagacacggg acgcttccag aatttcagca tggcaactga ccaaagactt 1200
aatgatattc ttctgcagtt aaattccttg attttgtcag tccaggaaca tgggaattca 1260
ctggatgcaa tctccaagtc cttgcagagt ctgaatatga cactgcttga tgttcaactc 1320
catacagaaa cactgcatgt cagagtccgt gaatctacag caaagcaaca ggaggacatc 1380
agtaaattgg aggaacgtgt gtacaaagta tcagcagaag tccagtctgt gaaagaagaa 1440
caagcgcacg tggaacagga agtaaaacag gaagtgagag tattgaacaa catcaccaac 1500
gacctcagac tgaaggactg ggaacactca cagacactga aaaacatcac cttcattcaa 1560
gggcctcctg gaccccaagg tgaaaaggga gacagagggc ttactggaca aactggtcca 1620
cctggtgctc caggaataag aggtattcca ggtgttaaag gtgatcgggg acaaattggc 1680
ttccctggag gtcgaggaaa cccaggagca ccaggaaagc cagggaggtc gggatctcct 1740
ggacctaaag gacaaaaggg agagaagggg agtgtaggcg gatcaacccc ccttaagaca 1800
gttcgactgg ttggtggtag tggagcccat gagggccgag tggagatctt ccaccaaggc 1860
cagtggggca caatctgtga tgatcgctgg gatatacggg ctggacaagt tgtctgccgg 1920
agtctaggat accaagaagt tctagctgtg cacaagagag ctcactttgg acaaggtact 1980
ggtccaatat ggctgaatga agtgatgtgt tttggaagag aatcatctat tgagaactgt 2040
aaaatcaacc agtggggagt actaagctgt tcacattcag aagatgctgg ggtcacttgt 2100
acttcagagg tgcagctgct ggagagcggc ggcggcctgg tgcagcccgg cggcagcctg 2160
cgcctgagct gcgccgccag cggcttcacc ttcagcagct acgccatgag ctgggtgcgc 2220
caggcccccg gcaagggcct ggagtgggtg agcaccatct actacgccgg cagcaacacc 2280
tactacgccg acagcgtgaa gggccgcttc accatcagcc gcgacaacag caagaacacc 2340
ctgtacctgc agatgaacag cctgcgcgcc gaggacaccg ccgtgtacta ctgcgccaag 2400
ggctactaca ccttcgacta ctggggccag ggcaccctgg tgaccgtgag cagcggcggc 2460
ggcggcagcg gcggcggcgg cagcggcggc ggcggcagca ccgacatcca gatgacccag 2520
agccccagca gcctgagcgc cagcgtgggc gaccgcgtga ccatcacctg ccgcgccagc 2580
cagagcatca gcagctacct gaactggtac cagcagaagc ccggcaaggc ccccaagctg 2640
ctgatctact acgccagcaa cctgcagagc ggcgtgccca gccgcttcag cggcagcggc 2700
agcggcaccg acttcaccct gaccatcagc agcctgcagc ccgaggactt cgccacctac 2760
tactgccagc agagcgacac cagccccacc accttcggcc agggcaccaa ggtggagatc 2820
aagcgctaag gatcc 2835
<210> 11
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> Tnf-alpha Forward primer
<400> 11
gattatggct cagggtccaa 20
<210> 12
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> Tnf-alpha reverse primer
<400> 12
gctccagtga attcggaaag 20
<210> 13
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> Il-1 beta Forward primer
<400> 13
cccaagcaat acccaaagaa 20
<210> 14
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> Il-1 beta reverse primer
<400> 14
gcttgtgctc tgcttgtgag 20
<210> 15
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> IL-6 Forward primer
<400> 15
ccggagagga gacttcacag 20
<210> 16
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> IL-6 reverse primer
<400> 16
ttgccattgc acaactcttt 20
<210> 17
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> iNos Forward primer
<400> 17
cacctggaac agcactctct 20
<210> 18
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> iNos reverse primer
<400> 18
ctttgtgcga agtgtcagtg 20
<210> 19
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> IL-4 Forward primer
<400> 19
atccatttgc atgatgctct 20
<210> 20
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> IL-4 reverse primer
<400> 20
gagctgcaga gactctttcg 20
<210> 21
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> Tgf-beta Forward primer
<400> 21
ttacctggat ggaagtggaa 20
<210> 22
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> Tgf-beta reverse primer
<400> 22
tgttatgagg aaggggacaa 20
<210> 23
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> Arg1 Forward primer
<400> 23
aagccaaggt taaagccact 20
<210> 24
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> Arg1 reverse primer
<400> 24
cgattcacct gagctttgat 20
<210> 25
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> CD206 Forward primer
<400> 25
tcagctattg gacgcgaggc a 21
<210> 26
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> CD206 reverse primer
<400> 26
tccgggttgc aagttgccgt 20
<210> 27
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> Gapdh Forward primer
<400> 27
tgaatacggc tacagcaaca 20
<210> 28
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> Gapdh reverse primer
<400> 28
aggcccctcc tgttattatg 20
<210> 29
<211> 2985
<212> DNA
<213> Homo sapiens (Homo sapiens)
<400> 29
atggttctgg ccccactgct actggggctg ctgctgctac ccgcgctctg gagtggaggc 60
actgccgaga agtgggaaga gaccgagcta gatcagctat tttcagggcc tttaccaggg 120
agactcccag tcaaccacag gccattctct gctcctcact ccagccggga ccagctgcca 180
ccaccccaga ctggaagatc acatccagca cacacagccg ctccccaggt gacctccaca 240
gcatcaaagc tcctacctcc tgttgcgttt aatcacacca ttggacacat agtactgtcg 300
gaacataaaa atgtcaaatt taattgctcc atcaatattc ctaacacata ccaagaaaca 360
gctggcattt catggtggaa agatggaaag gaattgctcg gggcacatca ttcaatcaca 420
cagttttatc ctgatgagga aggggtatca ataattgcat tgttcagcat agccagtgtg 480
cagcgctcag acaatgggtc gtacttctgt aagatgaagg tgaacaatag agagattgta 540
tctgatccca tatacgtgga agttcaagga ctcccttact ttattaagca gcctgagagt 600
gtgaatgtca ccagaaacac agccttcaac ctcacctgcc aggccgtggg ccctcctgag 660
cccgtcaata tcttctgggt tcaaaatagc agccgtgtta atgaaaaacc ggaaaggtcc 720
ccgtctgtcc taaccgtacc tggtctgaca gagacagcag tcttcagctg tgaggcccac 780
aatgacaaag gactgacggt gtccaagggt gtacatatca acatcaaagt aatcccctcc 840
ccgcccactg aagtccatat cctcaacagt acagcacaca gcatcctggt ctcctgggtc 900
cctggttttg atggctactc cccacttcag aactgcagca ttcaggtcaa ggaagctgac 960
cggctgagta atggctcagt catggttttt aatacctctg cttcgccaca tctgtatgag 1020
atccagcagc tgcaagccct ggctaattac agcatcgctg tgtcctgtcg gaatgagatt 1080
ggctggtctg cagtaagccc ttggattctg gccagcacaa cagaaggagc tccatctgta 1140
gcacctttaa acatcactgt gtttctgaac gaatctaaca atatcctgga tattagatgg 1200
acgaagcctc caattaagcg gcaggatggg gaactggtgg gctaccggat atctcacgtg 1260
tgggaaagcg cagggactta caaagagctt tctgaagaag tcagccagaa tggcagctgg 1320
gctcagattc ctgtccaaat ccacaatgcc acctgcacag tgagaatcgc ggccattact 1380
aaagggggca tcgggccctt cagtgagcca gtgaatatca tcattcctga acacagtaag 1440
gtagattacg caccctcgtc aaccccagcc cctggcaaca ccgactctat gttcatcatc 1500
ctcggctgct tctgtggatt cattttaatc gggttaattt tgtgtatttc tctggccctc 1560
agaaggagag tccaggaaac aaagtttggg ggagcattct ctgaggagga ttcccaactg 1620
gtcgtaaatt atagagcgaa gaagtccttc tgccggcgag ccatcgagct taccttgcag 1680
agcctgggag tgagcgagga gctgcagaat aagctggaag atgttgtgat tgacagaaac 1740
cttctggttc tcggcaaagt tctgggtgaa ggagagtttg ggtctgtaat ggaaggaaat 1800
ttgaagcaag aagatgggac ttctcagaag gtggcagtga agaccatgaa gttggacaac 1860
ttttctcaac gggagatcga ggagtttctc agcgaagcag catgcatgaa agacttcaac 1920
cacccaaatg tcatccgact tctaggcgtg tgtatagaac tgagctctca aggcatcccg 1980
aagcccatgg tgattttacc cttcatgaaa tacggagacc tccacacctt cctgttatat 2040
tcccgattaa acacaggacc caagtacatt cacctgcaga cactactgaa gttcatgatg 2100
gacattgccc agggaatgga gtatctgagc aacaggaatt ttcttcatag ggatttggca 2160
gctcgaaact gcatgttgcg ggatgacatg actgtctgcg tggcagactt tggcctctca 2220
aagaagattt acagtggtga ttattaccgc caaggccgca ttgccaaaat gcctgtgaag 2280
tggatcgcca tcgagagcct ggcggaccga gtctacacaa gcaaaagtga cgtgtgggct 2340
tttggcgtga ccatgtggga aataacaaca cggggaatga ctccctatcc cggagttcag 2400
aaccatgaga tgtacgacta ccttctccac ggccacaggc tgaagcagcc tgaggactgc 2460
ttggatgaac tgtatgacat catgtactct tgctggagtg ctgatccctt ggatcgaccc 2520
accttctctg tgttgaggct gcagctggaa aagctctccg agagtttgcc tgatgcgcag 2580
gacaaagaat ccatcatcta catcaatacc cagttgctag agagctgcga gggcatagcc 2640
aatgggccct cactcacggg gctagacatg aacattgacc ctgactccat cattgcctct 2700
tgcacaccag gcgctgccgt cagcgtggtc acggcagaag ttcacgagaa caaccttcgt 2760
gaggaaagat acatcttgaa tgggggcaat gaggaatggg aagatgtgtc ctccactcct 2820
tttgctgcag tcacacctga aaaggatggt gtcttaccgg aggacagact caccaaaaat 2880
ggcgtctcct ggtctcacca tagtacacta cccttgggga gcccatcacc agatgaactt 2940
ttatttgtag atgactcctt ggaagactct gaagttctga tgtga 2985
<210> 30
<211> 3653
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> W20-MERTK-GFP
<400> 30
gctagcatgg cagaagttca gttgcttgaa agcggtggag ggttggtcca acctggtgga 60
agcctgcgct tgtcatgtgc cgcctcaggg tttaccttta gttcttacgc tatgtcatgg 120
gtacgccaag cacctgggaa aggattggaa tgggtttccg gaatctctaa cttgggtctc 180
accaccgggt acgcagattc cgttaaggga cgcttcacca tttctcggga caattccaag 240
aatactctct atcttcagat gaactccctt agggcagagg ataccgcagt gtattactgc 300
gctaagacca catccaggtt cgattattgg ggacagggaa ctttggtcac tgtttcctct 360
ggtggcggag gctcaggtgg cgggggcagt ggagggggag gctcaacaga catccaaatg 420
acccaatccc ccagttcatt gtctgccagt gtgggagacc gcgtcaccat aacctgccgg 480
gccagtcaat ccattagttc ataccttaac tggtatcagc aaaagcctgg gaaagctcca 540
aagttgttga tttataaagc tagttatttg cagtccgggg tcccctccag gttcagtggc 600
agcggaagtg gtactgattt cacactcacc attagcagtt tgcagcccga ggactttgca 660
acctactatt gccaacaaac tcatagacct ccagttacct tcggtcaagg cactaaagta 720
gagataaagc gggggcccaa agtaatcccc tccccgccca ctgaagtcca tatcctcaac 780
agtacagcac acagcatcct ggtctcctgg gtccctggtt ttgatggcta ctccccactt 840
cagaactgca gcattcaggt taaggaagct gaccggctga gtaatggctc agtcatggtt 900
tttaatacct ctgcttcgcc acatctgtat gagatccagc agctgcaagc cctggctaat 960
tacagcatcg ctgtgtcctg tcggaatgag attggctggt ctgcagtaag cccttggatt 1020
ctggccagca caacagaagg agctccatct gtagcacctt taaacatcac tgtgtttctg 1080
aacgaatcta acaatatcct ggatattaga tggacgaagc ctccaattaa gcggcaggat 1140
ggggaactgg tgggctaccg gatatctcac gtgtgggaaa gcgcagggac ttacaaagag 1200
ctttctgaag aagtcagcca gaatggcagc tgggctcaga ttcctgtcca aatccacaat 1260
gccacctgca cagtgagaat cgcggccatt actaaagggg gcatcgggcc cttcagtgag 1320
ccagtgaata tcatcattcc cgaacacagt aaggtagatt acgcaccctc gtcaacccca 1380
gcccctggca acaccgactc tatgttcatc atcctcggct gcttctgtgg attcatttta 1440
atcgggttaa ttttgtgtat ttctctggcc ctcagaagga gagtccagga aacaaagttt 1500
gggggagcat tctctgagga ggattcccaa ctggtcgtaa attatagagc gaagaagtcc 1560
ttctgccggc gagccatcga gcttaccttg cagagcctgg gagtgagcga ggagctgcag 1620
aataagctgg aaggtgagca agggcgagga gctgttttgt gattgacaga aaccttctgg 1680
ttctcggcaa agttctgggt gaaggagagt ttgggtctgt aatggaagga aatttgaagc 1740
aagaagatgg gacttctcag aaggtggcag tgaagaccat gaagttggac aacttttctc 1800
aacgggagat cgaggagttt ctcagcgaag cagcatgcat gaaagacttc aaccacccaa 1860
atgtcatccg acttctaggc gtgtgtatag aactgagctc tcaaggcatc ccgaagccca 1920
tggtgatttt acccttcatg aaatatggag acctccacac cttcctgtta tattcccgat 1980
taaacacagg acccaagtac attcacctgc agacactact gaagttcatg atggacattg 2040
cccagggaat ggagtatctg agcaacagga attttcttca tagggatttg gcagctcgaa 2100
actgcatgtt gcgggatgac atgactgtct gcgtggcaga ctttggcctc tcaaagaaga 2160
tttacagtgg tgattattac cgccaaggcc gcattgccaa aatgcctgtg aagtggatcg 2220
ccatcgagag cctggcggac cgagtctaca caagcaaaag tgacgtgtgg gcttttggcg 2280
tgaccatgtg ggaaataaca acacggggaa tgactcccta tcccggagtt cagaaccatg 2340
agatgtacga ctaccttctc cacggccaca ggctgaagca gcctgaggac tgcctggatg 2400
aactgtatga catcatgtac tcttgctgga gtgctgatcc cttggatcga cccaccttct 2460
ctgtgttgag gctgcagctg gaaaagctct cagagagttt gcctgatgcg caggacaaag 2520
aatccatcat ctacatcaac acccagttgc tagagagctg cgagggcata gccaatgggc 2580
cctcactcac ggggctagac atgaacattg accctgactc catcattgcc tcttgcacac 2640
caggcgctgc cgtcagcgtg gtcacggcag aagttcacga gaacaacctt cgtgaggaaa 2700
gatacatctt gaatgggggc aatgaggaat gggaagatgt gtcctccact ccttttgctg 2760
cagtcacacc tgaaaaggat ggtgtcttac cggaggacag actcaccaaa aatggcgtct 2820
cctggtctca ccatagtaca ctacccttgg ggagcccatc accagatgaa cttttatttg 2880
tagatgactc cttggaagac tctgaagttc tgatgggggg tggaggctct gtgagcaagg 2940
gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg 3000
gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc 3060
tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc 3120
tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagaag cacgacttct 3180
tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg 3240
gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg 3300
agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca 3360
actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggcta 3420
acttcaaggt tcgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc 3480
agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc 3540
agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg 3600
tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtaagga tcc 3653
<210> 31
<211> 3725
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> SCFV-MERTK-GFP
<400> 31
gctagcatgg ttctggcccc actgctactg gggctgctgc tgctacccgc gctctggagt 60
gaacaaaaac tcatctcaga agaggatctg gaggtgcagc tgctggagag cggcggcggc 120
ctggtgcagc ccggcggcag cctgcgcctg agctgcgccg ccagcggctt caccttcagc 180
agctacgcca tgagctgggt gcgccaggcc cccggcaagg gcctggagtg ggtgagcacc 240
atctactacg ccggcagcaa cacctactac gccgacagcg tgaagggccg cttcaccatc 300
agccgcgaca acagcaagaa caccctgtac ctgcagatga acagcctgcg cgccgaggac 360
accgccgtgt actactgcgc caagggctac tacaccttcg actactgggg ccagggcacc 420
ctggtgaccg tgagcagcgg cggcggcggc agcggcggcg gcggcagcgg cggcggcggc 480
agcaccgaca tccagatgac ccagagcccc agcagcctga gcgccagcgt gggcgaccgc 540
gtgaccatca cctgccgcgc cagccagagc atcagcagct acctgaactg gtaccagcag 600
aagcccggca aggcccccaa gctgctgatc tactacgcca gcaacctgca gagcggcgtg 660
cccagccgct tcagcggcag cggcagcggc accgacttca ccctgaccat cagcagcctg 720
cagcccgagg acttcgccac ctactactgc cagcagagcg acaccagccc caccaccttc 780
ggccagggca ccaaggtgga gatcaagcgc aaagtaatcc cctccccgcc cactgaagtc 840
catatcctca acagtacagc acacagcatc ctggtctcct gggtccctgg ttttgatggc 900
tactccccac ttcagaactg cagcattcag gttaaggaag ctgaccggct gagtaatggc 960
tcagtcatgg tttttaatac ctctgcttcg ccacatctgt atgagatcca gcagctgcaa 1020
gccctggcta attacagcat cgctgtgtcc tgtcggaatg agattggctg gtctgcagta 1080
agcccttgga ttctggccag cacaacagaa ggagctccat ctgtagcacc tttaaacatc 1140
actgtgtttc tgaacgaatc taacaatatc ctggatatta gatggacgaa gcctccaatt 1200
aagcggcagg atggggaact ggtgggctac cggatatctc acgtgtggga aagcgcaggg 1260
acttacaaag agctttctga agaagtcagc cagaatggca gctgggctca gattcctgtc 1320
caaatccaca atgccacctg cacagtgaga atcgcggcca ttactaaagg gggcatcggg 1380
cccttcagtg agccagtgaa tatcatcatt cccgaacaca gtaaggtaga ttacgcaccc 1440
tcgtcaaccc cagcccctgg caacaccgac tctatgttca tcatcctcgg ctgcttctgt 1500
ggattcattt taatcgggtt aattttgtgt atttctctgg ccctcagaag gagagtccag 1560
gaaacaaagt ttgggggagc attctctgag gaggattccc aactggtcgt aaattataga 1620
gcgaagaagt ccttctgccg gcgagccatc gagcttacct tgcagagcct gggagtgagc 1680
gaggagctgc agaataagct ggaaggtgag caagggcgag gagctgtttt gtgattgaca 1740
gaaaccttct ggttctcggc aaagttctgg gtgaaggaga gtttgggtct gtaatggaag 1800
gaaatttgaa gcaagaagat gggacttctc agaaggtggc agtgaagacc atgaagttgg 1860
acaacttttc tcaacgggag atcgaggagt ttctcagcga agcagcatgc atgaaagact 1920
tcaaccaccc aaatgtcatc cgacttctag gcgtgtgtat agaactgagc tctcaaggca 1980
tcccgaagcc catggtgatt ttacccttca tgaaatatgg agacctccac accttcctgt 2040
tatattcccg attaaacaca ggacccaagt acattcacct gcagacacta ctgaagttca 2100
tgatggacat tgcccaggga atggagtatc tgagcaacag gaattttctt catagggatt 2160
tggcagctcg aaactgcatg ttgcgggatg acatgactgt ctgcgtggca gactttggcc 2220
tctcaaagaa gatttacagt ggtgattatt accgccaagg ccgcattgcc aaaatgcctg 2280
tgaagtggat cgccatcgag agcctggcgg accgagtcta cacaagcaaa agtgacgtgt 2340
gggcttttgg cgtgaccatg tgggaaataa caacacgggg aatgactccc tatcccggag 2400
ttcagaacca tgagatgtac gactaccttc tccacggcca caggctgaag cagcctgagg 2460
actgcctgga tgaactgtat gacatcatgt actcttgctg gagtgctgat cccttggatc 2520
gacccacctt ctctgtgttg aggctgcagc tggaaaagct ctcagagagt ttgcctgatg 2580
cgcaggacaa agaatccatc atctacatca acacccagtt gctagagagc tgcgagggca 2640
tagccaatgg gccctcactc acggggctag acatgaacat tgaccctgac tccatcattg 2700
cctcttgcac accaggcgct gccgtcagcg tggtcacggc agaagttcac gagaacaacc 2760
ttcgtgagga aagatacatc ttgaatgggg gcaatgagga atgggaagat gtgtcctcca 2820
ctccttttgc tgcagtcaca cctgaaaagg atggtgtctt accggaggac agactcacca 2880
aaaatggcgt ctcctggtct caccatagta cactaccctt ggggagccca tcaccagatg 2940
aacttttatt tgtagatgac tccttggaag actctgaagt tctgatgggg ggtggaggct 3000
ctgtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg 3060
gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg 3120
gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc 3180
tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaaga 3240
agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct 3300
tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg 3360
tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca 3420
agctggagta caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg 3480
gcatcaaggc taacttcaag gttcgccaca acatcgagga cggcagcgtg cagctcgccg 3540
accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact 3600
acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc 3660
tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtaag 3720
gatcc 3725
Claims (10)
1. A nucleic acid molecule comprising a polynucleotide sequence encoding a chimeric antigen receptor comprising: 1) A binding domain for an anti-amyloid oligomer; 2) A transmembrane domain; and 3) an intracellular signaling domain.
2. The nucleic acid molecule of claim 1, wherein the amyloid oligomer is a beta amyloid oligomer, preferably the beta amyloid oligomer is aβo42 or aβo40.
3. The nucleic acid molecule according to claim 1 or 2, wherein the binding domain of the anti-amyloid oligomer is a single chain variable region fragment scFV, preferably the binding domain of the anti-amyloid oligomer is a binding domain of an anti-beta-amyloid oligomer, more preferably the binding domain of the anti-beta-amyloid oligomer is a single chain antibody W20, a Du Nashan antibody (aducanaumab) or a kerrimab (Crenezumab), wherein the single chain antibody W20 has the amino acid sequence as set forth in SEQ ID NO: 1.
4. A nucleic acid molecule according to any one of claims 1-3, wherein the intracellular signaling domain is an intracellular signaling domain of an anti-inflammatory receptor, preferably the intracellular signaling domain of an anti-inflammatory receptor is an intracellular signaling domain selected from the group consisting of a class a scavenger receptor (SR-a), merTK, tyro3, ax1, itgB5, BAI1, ELMO, MRC1, stabilins, ADGRB1, TIMs and αvβ3/αvβ5 integrins.
5. The nucleic acid molecule of any one of claims 1-4, wherein the transmembrane domain is derived from a transmembrane domain consisting of SR-A, merTK, axl, tyro3, tim1, tim4, tim3, fcR, BAI1, CD4, DAP12, MRC1, CD8 a, CD3, ICOS and CD 28.
6. The nucleic acid molecule of any one of claims 1-5, wherein the binding domain of the anti-amyloid oligomer co-fusion expresses an SR-A, merTK receptor, a glycosylated end product receptor, a G protein-coupled receptor, a CC-type receptor, a CXC-type receptor, a C-receptor, a CX3C receptor, or a lamp2a receptor.
7. A vector comprising the nucleic acid molecule of any one of claims 1-6.
8. A pharmaceutical composition comprising the nucleic acid molecule of any one of claims 1-6 or the vector of claim 7, and a pharmaceutically acceptable excipient, preferably the pharmaceutical composition is a nanoparticle prepared using a liposome, preferably the liposome comprises one or more cationic lipids and/or one or more non-cationic lipids.
9. Use of the nucleic acid molecule of any one of claims 1-6, the vector of claim 7 or the pharmaceutical composition of claim 8 in the manufacture of a medicament for promoting cellular clearance of amyloid oligomers, treating and/or preventing inflammatory diseases, reducing senile plaques in the brain, treating and/or preventing neurodegenerative diseases, inhibiting glioblastoma, increasing brain synapse levels in a subject.
10. The use of claim 9, wherein the neurodegenerative disease is selected from the group consisting of alzheimer's disease, parkinson's disease, huntington's disease, amyotrophic lateral sclerosis, or spinocerebellar ataxia.
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