CN117004632A - Chimeric antigen receptor modified glial cells and their uses - Google Patents

Abstract

The present invention relates to chimeric antigen receptor modified glial cells and uses thereof. In particular, the 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 them, compositions comprising said nucleic acid molecules encoding them, and Its use in the treatment and/or prevention of neurodegenerative diseases. The chimeric antigen receptor-modified glial cells of the present invention can effectively recognize and phagocytose amyloid oligomers, reduce inflammation in the brain and the release of pro-inflammatory cytokines, and provide a brand-new method for the field of neurodegenerative diseases. Treatment Strategies.

Description

Chimeric antigen receptor modified glial cells and their uses

Technical field

The present invention relates to chimeric antigen receptor modified glial cells and their uses. In particular, the 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 them, compositions comprising said nucleic acid molecules encoding them, and Its use in the treatment and/or prevention of neurodegenerative diseases.

Background technique

Neurodegenerative diseases are a general term for diseases caused by chronic progressive degeneration and loss of nerve cells, including Alzheimer's Disease (AD), Parkinson's Disease (PD), and Huntington's disease (Huntingdon's Disease, HD) and amyotrophic lateral sclerosis, etc., which are characterized by neuronal structure and dysfunction, neuron loss, cognitive and behavioral abnormalities. Current targets for treating neurodegenerative diseases include neurotransmitters, amyloid, intestinal flora, neuroinflammation, mitochondrial incompetence, oxidative stress and intracellular signaling pathways. Although these therapeutic strategies have shown some therapeutic effects in preclinical studies, most of them have no significant therapeutic effects in the clinical trial stage, so it is particularly important to develop therapies that target the pathogenesis of the disease.

AD, commonly known as Alzheimer's disease, is a neurodegenerative disease characterized by progressive memory impairment, impaired cognitive function, personality changes, and language impairment. The pathogenesis of AD is mainly due to the abnormal aggregation of Aβ and highly phosphorylated tau protein to form toxic oligomers, which lead to the formation of senile plaques and neurofibrillary tangles in the brain, causing neuronal death. Currently, AD treatment strategies under research mainly include natural compounds, immunotherapy, gene therapy, stem cell therapy and intestinal flora regulation. However, no specific therapeutic drugs targeting the cause have been developed so far.

PD is a movement disorder disease with an incidence rate of approximately 0.3%. The main pathological characteristics of PD are the death of dopamine (DA) neurons and the occurrence of Lewy bodies (Lewy bodies) formed by abnormal aggregation of α-synuclein in substantia nigra neurons. Abnormally aggregated α-synuclein can destroy the integrity of synaptic vesicles, interfere with dopamine metabolism, damage mitochondrial function, interfere with intracellular transport, and ultimately trigger the death of dopaminergic neurons.

HD is a neurodegenerative disease characterized by chorea-like symptoms, manifesting as PD-like movement disorders and ataxia, and AD-like dementia. HD is caused by the accumulation of a large amount of mHTT protein containing polyglutamine in nerve cells, leading to dysfunction and apoptosis of nerve cells in the cortex, striatum and other parts of the body.

The causes of neurodegenerative diseases are complex. Many neurodegenerative diseases are characterized by abnormal aggregation of amyloid proteins, such as amyloid beta (Aβ), microtubule-associated protein (tau), α-synuclein (α-synuclein) and Mutated huntingtin (mHTT) and other proteins, the oligomers formed by the aggregation of these protein monomers are key pathogenic factors leading to the development of AD, PD, HD, etc., and are also effective targets for the diagnosis and treatment of such diseases. Among all aggregate forms, oligomers are the most cytotoxic and are directly related to the occurrence and development of diseases. Oligomers and fibers of different amyloid proteins are deposited in different parts of the brain, damaging nerve cells in corresponding areas, leading to cognitive and behavioral disorders. Although these amyloid proteins have different primary sequences, their aggregation can form similar three-dimensional structures and have similar toxicity mechanisms. Therefore, therapeutic strategies targeting amyloid toxic oligomers are of great significance for the diagnosis and treatment of various neurodegenerative diseases.

In recent years, more and more researchers have focused on glial cells. Glial cells are another major type of cells in the nervous system besides neurons, mainly including: microglia, astrocytes, oligodendrocytes and oligodendrocyte precursor cells (OPC), etc. . In the central nervous system, glial cells play a variety of important physiological functions, such as removing foreign matter, participating in the formation of myelin around axons, regulating normal physiological functions of synapses and synaptic regeneration, and maintaining the blood-brain barrier ( BBB) integrity and the homeostasis of ions and neurotransmitters in the brain.

Microglia are widely distributed in the brain and spinal cord. They are the most important innate immune cells in the central nervous system. They play an important role in immune surveillance and endogenous immune defense in the brain and play an important role in the occurrence and development of neurodegenerative diseases. In AD, microglia play a key role in the clearance of Aβ from the brain. Aβ oligomers can interact with certain microglia surface receptors. On the one hand, they induce microglia to phagocytose and clear Aβ, protecting neurons from Aβ. On the other hand, large amounts of Aβ oligomers can overactivate microglia. Plasma cells promote the release of inflammatory factors, leading to neuroinflammation. At the same time, the phagocytosis of Aβ by overactivated microglia is inhibited, further causing the deposition of Aβ. Aβ oligomers can also activate complement-related clearance pathways, leading to abnormal phagocytosis of synapses by microglia, resulting in excessive synapse loss and cognitive decline. In addition, Aβ oligomers can cause autophagy dysfunction in microglia and reduce their ability to degrade Aβ. Therefore, microglia play a "double-edged sword" role in the pathological process of AD. Maintaining the normal activation state of microglia and accelerating the phagocytosis and clearance of Aβ toxic oligomers are the key to AD treatment. Current research believes that activated microglia can be divided into two states: M1 and M2. Microglia in the M2 state can release anti-inflammatory factors (such as IL-4, IL-10, etc.), phagocytose damaged nerve cell debris, promote tissue repair and neuron regeneration, but when microglia are overactivated Afterwards, it will turn to the M1 state and release a large number of inflammatory factors such as IL-1β, IL-6, TNF-α, ROS and NO, etc., which will have a toxic effect on nerve cells and cause nerve cell apoptosis. Therefore, maintaining the normal M2 state of microglia and reducing the release of neuroinflammatory factors is of great significance to maintaining the health of the central nervous system, and is of great value to the development of AD drugs.

Scavenger receptor SR-A is an important receptor that mediates the phagocytosis and clearance of Aβ by microglia. Studies have shown that SR-A promotes the phagocytosis and clearance of Aβ and at the same time downregulates the expression of inflammation-related genes. In the brains of AD patients, the expression level of SR-A decreases, the ability of microglia to phagocytose and clear Aβ oligomers is significantly reduced, and Aβ oligomers gradually accumulate, leading to neuronal apoptosis. Existing technology has reported a cyclic heptapeptide XD4 that can activate SR-A. This polypeptide promotes the binding of Aβ to SR-A receptors and enhances the ability of microglia to clear Aβ oligomers. Similarly, up-regulating the expression of SR-A can promote the phagocytosis and clearance of Aβ by microglia and reduce the release of neuroinflammatory factors, which is an effective gene therapy for AD. Astrocytes are the largest type of glial cells in the brain and are components of the BBB. They play an important role in controlling the transport of Aβ from the brain to the periphery and the transport of peripheral Aβ into the brain. In the brains of AD patients, astrocyte dysfunction leads to reduced Aβ transport to the periphery, causing the accumulation of Aβ in the brain and the formation of Aβ plaques. In addition, reactive astrocytes can promote the activity of β-secretase and γ-secretase, leading to the overproduction of Aβ. Aβ pathology in turn triggers astrocyte activation and dysfunction. Studies have shown that astrocyte dysfunction is also closely related to tau protein hyperphosphorylation and NFT formation. The tau protein in neurons will spread into astrocytes, causing the loss of normal physiological functions of astrocytes and further aggravating AD symptoms. Under pathological conditions, Aβ stimulates astrocytes to produce excessive reactive oxygen species (ROS). Although low levels of ROS have a certain protective effect on neurons. However, excessive ROS can induce nitrosative stress responses 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 protein in the brain. Excessive levels of Aβ and tau in the brain directly cause reactive astrocytes to damage neurons. Inhibiting astrocyte activation and reducing the release of inflammatory factors are currently commonly used methods to modify astrocyte function.

The MerTK receptor is mainly expressed on M2 astrocytes and macrophages. When activated, the receptor can mediate phagocytosis and simultaneously inhibit the release of pro-inflammatory cytokines. The MerTK structure is similar to the CAR structure in traditional CAR-T cells, including the antigen recognition region, the transmembrane region and the intracellular signal transduction region. It is an ideal receptor for CAR transformation.

Oligodendrocytes and oligodendrocyte precursor cells are the most abundant glial cells in the brain. Oligodendrocytes express MHC-I class molecules and immune receptors and have immune regulatory functions. They can regulate the functional status of microglia by expressing a variety of cytokines and chemokines. They can also express a variety of neurotrophic factors, which are important for Neurons play a supporting role. Oligodendrocyte precursor cells are widely present in the central nervous system and can proliferate and differentiate in demyelinated areas to form oligodendrocytes to repair damage caused by demyelination.

In the brains of AD patients, oligodendrocytes are easily stimulated by Aβ oligomers and undergo demyelination, leading to damage to neuronal function and neuronal death. In addition, oligodendrocytes are also highly sensitive to the inflammatory environment in the brain caused by Aβ. Oligodendrocytes are rich in microtubules, and the stability of microtubules and the formation of myelin require the participation of tau protein, and tau pathology seriously affects the myelination process and axonal transport. Therefore, in AD, oligodendrocyte status is closely related to Aβ and tau pathology.

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 that mainly contains the intracellular signaling domain that recognizes known tumor antigen-binding domains, hinge and transmembrane regions, and T cell receptors. and costimulatory domains that trigger T cell activation. After the chimeric antigen receptor binds to the corresponding antigen, it will mediate the activation of inflammatory pathways in immune cells, release a large number of cell-killing factors and pro-inflammatory factors, and kill tumor cells.

The antigen-binding domain is the extracellular part of CAR, which can mediate the killing of corresponding target cells by CAR-T cells. Cytokines, single-chain antibodies, and ligands can all be used as the antigen-binding domain of CAR, among which single-chain antibodies are the most common. common.

The hinge region and transmembrane domain of CAR are the bridge connecting the extracellular antigen-binding domain and the intracellular signaling domain. The hinge region provides sufficient flexibility to overcome the steric hindrance of antigen and scFv binding. The transmembrane domain anchors the CAR to the T cell membrane and is usually derived from type I proteins. Different transmembrane domains can affect the stability and function of CAR. Different hinge regions and transmembrane domains can be selected according to different needs.

Intracellular signaling domains typically consist of an activation domain and one or more costimulatory domains. The vast majority of CARs activate CAR-T cells through CD3ζ. However, signaling mediated by CD3ζ alone is insufficient to induce reactive proliferation of T cells, resulting in limited T cell persistence and activity in vivo. Increasing the costimulation domain can effectively solve this problem. In addition to traditional CAR, some researchers are also trying to modify other receptors, such as Megf10, TLR4, TLR2, MerTK and Fcγ, etc. to explore the therapeutic advantages of multiple receptor modifications in different diseases, which is expected to expand the application scope of CAR technology.

In view of the fact that glial cells play a "double-edged sword" role in the occurrence and development of neurodegenerative diseases, it is necessary to modify the functions of glial cells to increase their beneficial functions, reduce their harmful functions, and maintain their normal activation state. , accelerating the clearance of Aβ oligomers is a promising therapeutic strategy. However, no drugs based on such treatment strategies have yet entered the clinic. There are two main reasons. First, the existing glial cell function modification strategies mainly use non-steroidal anti-inflammatory drugs or small molecule drugs to inhibit the inflammatory state of glial cells. However, these strategies while reducing inflammation in the brain, , also reduces the ability of glial cells to clear Aβ oligomers, and therefore cannot effectively treat AD. How to resolve the contradiction between glial cells engulfing Aβ oligomers and releasing inflammatory factors, so that glial cells can phagocytose Aβ oligomers without causing the release of a large number of pro-inflammatory factors, is a strategy to transform glial cell function. the key of. Secondly, there is a lack of effective drug delivery system targeting glial cells. Some drugs used to modify microglia, such as Nec-1s, have a short half-life in the body and are easily cleared in the blood circulation. Due to the existence of the BBB, only a very small amount of drugs can enter the brain. These drugs cannot accurately target glial cells after entering the brain, resulting in high side effects. Therefore, enhancing the ability of drugs to penetrate the BBB and improving their bioavailability within the central nervous system are keys to the successful development of such drugs.

Liposome nanocarrier is an efficient gene drug delivery system and has been widely used in the research of AD and other diseases. As a non-viral vector, liposomes can overcome the shortcomings of viral vectors such as high immunogenicity, high toxicity, and limited number of genes carried. Its phospholipid bilayer membrane itself can promote the transport of various biological membranes, including the BBB, and can exist in tissues for a long time so that the target drug or gene can fully penetrate into the target. Simple "non-targeting" liposomes can enter the brain via the olfactory nerve pathway. Targeted liposomes can cross the BBB through active transport mechanisms.

Liposomes can be functionalized or modified in a variety of ways. Small molecule compounds, biological macromolecules, imaging probes, etc. can be attached to the outside of the liposomes or encapsulated in the water core or lipid bilayer, such as through polymerization. Ethylene glycol (PEG) modification can prepare a kind of "stealth" liposome, which increases the solubility of liposomes and resists interaction with plasma proteins to avoid phagocytosis by the reticuloendothelial system. Additionally, these PEG liposomes promote BBB transport. At present, a variety of new liposome gene carriers have been reported, such as magnetic nanoliposome gene carriers.

Up to now, there is still an urgent need in this field for drugs that can effectively treat and/or prevent neurodegenerative diseases. At the same time, there is no application and implementation of chimeric antigen receptor technology in the transformation of glial cells in this field. Reports on the treatment and/or prevention of neurodegenerative diseases such as AD.

Contents of the 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 trans- Membrane domain; and 3) intracellular signaling domain.

In some embodiments, the amyloid oligomer is selected from the group consisting of beta amyloid oligomers, microtubule-associated protein tau oligomers, alpha-synuclein oligomers, huntingtin oligomers, pancreatin oligomers, The group consisting of amyloid oligomers, SOD1 oligomers and TDP-43 oligomers.

In some embodiments, the amyloid oligomer is an amyloid beta oligomer, preferably the amyloid beta 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 an anti-beta amyloid oligomer. More preferably, the binding domain of the anti-β-amyloid oligomer is a scFV containing heavy chain CDR1-3 and light chain CDR1-3 of single-chain antibody W20, Aducanumab ) or Crenezumab, wherein the amino acid sequence of single-chain antibody W20 is shown in SEQ ID NO: 1, and the amino acid sequences of heavy chain CDR1-3 and light chain CDR1-3 of single-chain antibody W20 are shown in SEQ ID NO. As shown in NO: 2-7, more preferably, the binding domain of the anti-β-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 the anti-inflammatory receptor is selected from the group consisting of class A scavenger receptors. Intracellular signaling domain of the group consisting of 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 a hinge region and a costimulatory domain that triggers glial cell activation.

In some embodiments, the hinge region is derived from the group consisting of MerTK receptor FNIII domain, CD8α, CD28, IgGl and IgG4.

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, CD8α, CD3, ICOS, and CD28. membrane domain. In a further embodiment, the transmembrane domain is that derived from SR-A or MerTK.

In some embodiments, the binding domain of the anti-β-amyloid oligomer is single chain antibody W20, and the intracellular signaling domain of the chimeric antigen receptor is the intracellular class A scavenger receptor or MerTK. The signaling domain and the transmembrane domain are those derived from SRA and MerTK.

In some embodiments, the nucleic acid molecule comprises an operably linked glial cell-specific promoter. Preferably, the glial cell-specific promoter is a microglia-specific promoter, an astrocyte-specific promoter, or an astrocyte-specific promoter. Cell and/or oligodendrocyte-specific promoter, more preferably, the glial cell-specific promoter is selected from the group consisting of gfa2, GFAP, GFAP104, GfaABC1D, ALDH1L1, Cst3, CX30, CX3CR1, IBa-1 , Pdgfra, olig2 and NG2 promoter group. In further embodiments, the glial cell-specific promoter is the gfa2, CX3CR1 or olig2 promoter.

In some embodiments, the binding domain of the anti-amyloid oligomer co-expresses SR-A, MerTK receptor, glycosylation end product receptor, G protein-coupled receptor, CC class receptor, CXC receptor-like, C receptor, CX3C receptor or lamp2a receptor.

In some embodiments, the glial cells are selected from the group consisting of microglia, astrocytes, oligodendrocytes, and oligodendrocyte precursor cells. In further embodiments, the glial cells are microglia or astrocytes.

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 adenovirus vector, an adenovirus-associated vector or a lentiviral vector, preferably the vector is plasmid pGFP-N1, retroviral vector pRetroX, adenovirus Vector pDC315, pDC311, adeno-associated virus 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 microglia, astrocytes, oligodendrocytes and oligodendrocyte precursors Group of cells. In further embodiments, the glial cells are microglia or astrocytes.

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 present 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 Accepted excipients.

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. quality.

In some embodiments, the liposomes are 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 , ribose cationic lipids or their combinations, 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, sphingolipid, cerebroside, ganglioside, 16-O-monomethylPE, 16-O-dimethylPE, 18-1-trans PE, SOPE or combinations 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 combination: 1): POPC, DDAB, DSPE; (2): DSPE, DOPC, DOTAP; more preferably, the DSPE is PEG-modified DSPE, and preferably, the DSPE is DSPE-PEG2000.

In some embodiments, nanoparticles prepared using liposomes are conjugated with glial cell-specific targeting peptides. In some embodiments, the targeting peptide is selected from AS1, MG1, V9, NGR and other polypeptides.

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 present invention 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 in the preparation of a method for promoting the Drugs that remove amyloid oligomers from cells, treat and/or prevent inflammatory diseases, reduce senile plaques in the brain, treat and/or prevent neurodegenerative diseases, inhibit gliosis, and improve the level of brain synapses use.

Another aspect of the invention relates to a method 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 glial A method for cell proliferation and improving brain synaptic levels, which includes administering to a subject in need 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 Pharmaceutical compositions as described above.

The present 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, which is used to promote cells in a subject. Clear amyloid oligomers, treat and/or prevent inflammatory diseases, reduce senile plaques in the brain, treat and/or neurodegenerative diseases, inhibit gliosis, and improve brain synapse 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 play a "double-edged sword" role in the pathological changes of neurodegenerative diseases such as AD. They can not only phagocytose AβOs and have a protective effect on neurons, but also induce inflammation. The present invention adopts chimeric Antigen receptor technology functionally transforms microglia and astrocytes to improve their ability to phagocytose Aβ oligomers and prevent them from producing too many pro-inflammatory factors, thus solving the problem of functional transformation of glial cells. The main problems faced in the treatment of neurodegenerative diseases such as AD.

Specifically, in order to regulate the normal function of microglia, the present invention delivers the Aβ oligomer-specific single-chain antibody W20 and the scavenger receptor SR-A fusion gene to the brain through a liposome gene delivery system, so that W20 Together with SR-A, it is fused and expressed on the surface of microglia, transforming microglia in the brain into chimeric antigen receptor microglia (CAR-M), allowing the expressed W20 to directly target Aβ oligomers in the brain, thereby activating the linked SR-A, promoting the phagocytosis and clearance of Aβ oligomers, and reducing the release of inflammatory factors, providing a new method for the treatment of AD and other neurodegenerative diseases. Possible strategies.

The single-chain antibody W20 that specifically recognizes amyloid oligomers (its amino acid sequence is shown in SEQ ID NO: 1, the amino acid sequences of heavy chain CDR1-3 are shown in SEQ ID NO: 2-4, respectively, and the light chain CDR1- The amino acid sequences of 3 are shown in SEQ ID NO: 5-7 respectively) and were screened out using phage display technology. The antibody has strong affinity, good stability, high safety, good oligomer specificity, does not bind to monomers or fiber forms, and can also inhibit the aggregation and cytotoxicity of amyloid proteins such as Aβ. Single-chain antibody W20 is different from intact antibodies in that it can avoid the inflammatory response and excessive synaptic phagocytosis mediated by the Fc fragment of the antibody, thereby avoiding the occurrence of adverse reactions.

Class A scavenger receptor (SR-A, whose nucleotide sequence is shown in SEQ ID NO: 8) on microglia is involved in Aβ phagocytosis. Downregulation of SR-A can reduce the phagocytosis of Aβ by microglia. , and induces the accumulation of Aβ to cause neurodegenerative diseases such as AD. Up-regulating the expression of SR-A can reduce the level of Aβ in the brain and reduce the production of inflammatory factors in microglia by inhibiting the activation of the NF-κB signaling pathway. . Since neuroinflammation is closely related to the pathology of neurodegenerative diseases such as AD, upregulating SR-A and increasing the clearance of Aβ without inducing the production of inflammatory factors is an ideal strategy for the treatment of neurodegenerative diseases such as AD. . In the present invention, the antibody W20 and SR-A are co-fused and expressed on the surface of microglia, and the microglia are transformed into microglia CAR-M, a chimeric antigen receptor. At the same time, the present invention carries out PLGA modification on the liposome gene carrier, so that it has a longer blood half-life and a higher amount of brain entry, and is administered through the nose into the brain to target microglia, thereby solving the problem of microglia. The main problem faced by functional transformation. That is, the present invention designs and constructs a W20-SRA gene expression vector, prepares liposome carrier delivery particles containing the gene, characterizes the morphology, particle size and stability of the liposomes in vitro, and verifies plasmid transfection by cellular immunofluorescence experiments. ability, protein expression, and the ability of CAR-M to recognize, phagocytose, and clear Aβ oligomers. Use ELISA, QPCR, Western-blot and other methods to verify the inflammatory factor levels and polarization of CAR-M cells incubated with Aβ oligomers. state. At the same time, the present invention delivers W20-SRA liposome nanoparticles into the brains of AD transgenic mice through nasal administration, and detects the transfection ability of microglia in the mouse brain and the functional characteristics of CAR-M. The cognition of new things, Y maze and water maze were used to detect the cognitive behavioral functions of mice, and tissue immunochemistry, ELISA, western-blot and QPCR were used to detect pathological changes in their brains to investigate the therapeutic effect of CAR-M on AD mice. .

Experimental results of the present invention show that the prepared liposome gene delivery nanoparticles can transfect microglial cells such as BV2 cells, convert BV2 cells into CAR-BV2 cells, and fuse and express SRA-W20 on their surfaces. SRA-W20 liposome nanoparticles have good dispersion, uniform size, with a particle size of approximately 91.3±5.0nm, and high biological safety. SRA-W20 was successfully fused and expressed on the surface of CAR-BV2 cells. After W20 specifically binds AβOs, it can activate the fusion-expressed SR-A, activate the lysosomal pathway and autophagy system of cells, promote the phagocytosis and degradation of AβOs, reduce the inflammatory response of cells, and promote the transformation of cells from M1 type to M2 type. , and will not affect its normal phagocytic function, will not cause excessive phagocytosis of synapses, and has a neuroprotective effect. At the same time, nasal administration can bypass the blood-brain barrier, allowing the drug to enter the brain directly through the olfactory bulb, while liposome encapsulation can protect genes from degradation and promote their transmucosal transport, allowing gene carriers to enter more effectively The brain has good application prospects in the treatment of AD. That is, the CAR-M of the present invention effectively improves the cognitive function of AD mice and significantly reduces the pathological changes in the brains of AD mice. The main conclusions are as follows:

(1) CAR-M gene therapy significantly improved the cognitive function and memory of AD mice. APP/PS1 mice were administered nasally for 28 days. Behavioral experiments such as Y maze, water maze and new object recognition proved that CAR-M therapy can effectively improve the spatial memory of AD mice. Ability and cognitive level.

(2) CAR-M gene therapy significantly reduced Aβ pathology in the brains of AD mice. Compared with AD mice in the solvent control group, the number of senile plaques in the brains of AD mice treated with CAR-M was significantly reduced, and the levels of soluble and insoluble Aβ40/42 were also significantly reduced, indicating that CAR-M can effectively promote Aβ clearance.

(3) CAR-M gene therapy significantly reduced neuroinflammation in the brains of AD mice and promoted the transformation of microglia from the inflammatory M1 type to the anti-inflammatory M2 type. CAR-M therapy significantly reduced the activation of astrocytes and microglia in the brains of AD mice, and reduced the levels of inflammatory factors such as TNF-α, IL-1β, and IL-6. At the same time, CAR-M promotes the expression of microglia M2-type related genes Arg-1, IL-10, TGF-β, CD206 and other proteins, causing microglia to transform into the anti-inflammatory M2 type. Its anti-inflammatory mechanism includes inhibiting the activation of NLRP3 inflammasome and NF-κB.

(4) CAR-M gene therapy inhibits synapse loss in AD mice and improves the survival of neurons in the mouse brain. CAR-M therapy significantly increased the levels of synaptophysin and postsynaptic protein (PSD95) in the brains of AD mice and improved the survival of neurons, which is closely related to the improvement of cognitive abilities in AD mice.

(5) CAR-M gene therapy reduces the level of oxidative stress in the brains of AD mice. Compared with AD mice in the solvent control group, the GSH/GSSG ratio in the brains of AD mice treated with CAR-M was significantly increased, and the ROS levels were significantly reduced.

(6) CAR-M gene therapy is highly safe and has no toxic or side effects. CAR-M therapy does not promote excessive phagocytosis of synapses by microglia in the brains of AD mice, and long-term and large-dose administration has adverse effects on the mice's mental state, body weight, biochemical indicators, nasal mucosa, and various internal organs. No adverse effects have been seen and it is highly safe.

The present invention further verified the chimeric antigen receptor system of the present invention in astrocytes. The present invention designs and prepares W20-MerTK modified astrocytes (CAR-A). The anti-inflammatory chimeric antigen receptor W20-MerTK expression plasmid specifically expressed by astrocytes was designed and constructed, and loaded into liposome nanoparticles targeting astrocytes. Characterize the morphology, particle size, and cytotoxicity of the lipoplexes. Cell immunofluorescence, flow cytometry and other methods were used to characterize the expression of W20-MerTK in astrocytes, as well as the phagocytosis ability of Aβ oligomers and the release of inflammatory factors by modified astrocytes. The present invention also administers the lipoplex expressing W20-MerTK through the nose, and detects the expression of W20-MerTK in astrocytes in the brain of AD mice and the effect of CAR-A on Aβ oligomers in the brain. its clearance ability and its release of inflammatory factors. The improvement of memory ability of AD mice by CAR-A was tested through water maze, new object recognition and Y maze experiments, and immunohistochemistry and ELISA were used to detect pathological changes in the brains of AD mice.

Specifically, the present invention replaces the antigen-binding region (19Gly-275Asn) of MerTK (whose nucleotide sequence is shown in SEQ ID NO: 9) with a single-chain antibody fragment (W20) that specifically binds Aβ oligomers, A new anti-inflammatory chimeric antigen receptor (W20-MerTK) that recognizes Aβ oligomers was constructed. W20-MerTK expression plasmid is loaded into liposome nanoparticles targeting astrocytes. The lipoplex has uniform particle size, good dispersion, and no obvious cytotoxicity. The chimeric antigen receptor is fully expressed in astrocytes, allowing astrocytes to be programmed to CAR-A. When W20-MerTK binds to Aβ oligomers, it can activate intracellular RhoA, Rac1 and Cdc42 signals. pathway, guiding CAR-A to phagocytose Aβ oligomers. At the same time, CAR-A can activate the SOCS1/3 signaling pathway when engulfing Aβ oligomers, inhibiting cytokine receptors and reducing the positive feedback effect of cytokine receptors on cytokines. In addition, CAR inhibits the release of inflammatory factors caused by phagocytosis of Aβ oligomers by receptors such as RAGE and TLR by inhibiting the NF-κB signaling pathway, thereby improving the cellular microenvironment. Using a liposome gene delivery system, the constructed W20-MerTK plasmid was delivered into the brain through nasal administration. W20-MerTK could be successfully expressed on astrocytes in the brain and converted into CAR-A. CAR-A can effectively clear Aβ oligomers in the brain, reduce the number of Aβ plaques, and reduce the levels of Aβ40 and Aβ42 in the brain. When CAR-A clears Aβ oligomers in the brain, it can reduce the release of pro-inflammatory cytokines and In terms of inflammatory response in the brain, CAR-A gene treatment can change the phenotype of microglia from MGnD to M0, significantly increase the levels of synaptophysin and PSD95, and inhibit the loss of synapses in the brain of AD mice. Thereby improving the cognitive function of mice, astrocyte programming technology provides a new therapeutic strategy for the treatment of neurodegenerative diseases such as AD.

Experimental results show that the particle size of the obtained lipoplex is 132±19.5nm, with good dispersion and uniform size. W20-MerTK can be fully expressed in astrocytes, but not in neurons and microglia. Express. After W20-MerTK binds to Aβ oligomers, it activates the RhoA, Rac1 and Cdc42 signaling pathways in astrocytes, causing CAR-A to phagocytose Aβ oligomers. CAR-A can inhibit the NF-κB pathway and inhibit the release of inflammatory factors when engulfing Aβ oligomers. It can also significantly increase the intracellular SOCS1/3 protein level, inhibit cytokine receptors, and reduce the positive response of cells to cytokines. feedback effect. When CAR-A engulfs Aβ oligomers, it reduces the release of pro-inflammatory cytokines caused by receptors such as RAGE and TLR when phagocytosis of Aβ oligomers, thereby reducing the activation of microglia and neuronal damage, thereby improving the cell microenvironment. After intranasal administration of lipoplexes containing the W20-MerTK plasmid, the chimeric antigen receptor successfully programmed astrocytes in the brain into CAR-A. CAR-A can effectively clear Aβ oligomers in the brain, reduce the number of Aβ plaques, reduce the levels of Aβ40 and Aβ42 in the brain and the levels of pro-inflammatory cytokines in the brain, and increase the levels of anti-inflammatory cytokines. After CAR-A gene therapy, the phenotype of microglia changes from MGnD to M0, and the level of synapses in the brain significantly increases.

The CAR-M and CAR-A of the present invention are different from CAR-T. CAR-T injects a large number of CAR-T cells expanded in vitro back into the patient's body for tumor treatment at a huge cost, and may induce a cytokine storm. The present invention creatively introduces chimeric antigen receptors into glial cells such as microglia and astrocytes, and induces microglia and astrocytes to become CAR-M in situ in the brain. and CAR-A to improve the recognition and phagocytosis of amyloid oligomers by glial cells such as microglia and astrocytes without causing side effects such as significant inflammatory response and synapse loss. CAR The implementation of -M and CAR-A gene therapy is expected to overcome the shortcomings of existing treatments for neurodegenerative diseases and will provide a new strategy for the treatment of various neurological diseases.

Description of the drawings

Figure 1: Plasmid map.

Figure 2: Characterization of liposomes. (A) TEM observed the morphology of NP and SRA-W20-NP, scale bar = 200nm; (B) TEM measured the particle size distribution of NP and SRA-W20-NP; (C) DLS method was used to measure and detect NP and SRA-W20- Hydrated diameter of NP.

Figure 3: Expression of W20 and GFP on the surface of CAR-BV2 cells. (A) CAR-BV2 cells were immunostained with W20 antibody (red) and observed with a confocal microscope, scale bar = 25 μm; (B) Flow cytometry was used to analyze the proportion of CAR-BV2 cells positive for GFP expression.

Figure 4: Cytotoxicity assay of liposomes. Different concentrations of NP, SRA-NP, SRA-W20-NP and SRA-ns-scFv-NP were added to the culture medium of BV2, N2a and U87-mg cells, incubated at 37°C for 48 hours, and cell viability was determined by MTT.

Figure 5: CAR-BV2 improves the phagocytosis ability of AβOs. (A) After CAR-BV2 cells were transfected with SRA, SRA-W20 and SRA-ns-scFv plasmids, AβOs was added, and after incubation for 0, 1, 6, 12 and 24 hours, Aβ (Aβ) in CAR-BV2 cells (green) was observed. Antibody staining, red), scale bar = 25 μm; (B) Quantitative analysis of Aβ levels in CAR-BV2 cells.

Figure 6: CAR-BV2 promotes phagocytosis of AβOs through SR-A. (A) BV2 cells were transfected with SRA, SRA-W20 and SRA-ns-scFv and then added fucose (SR-A receptor antagonist) and AβOs. Cell immunofluorescence was used to detect intracellular Aβ levels. Scale bar = 25 μm; (B) Quantitative analysis of Aβ levels in CAR-BV2 cells.

Figure 7: The ability of CAR-BV2 to clear AβOs is improved. (A) In the CAR-BV2 cell culture medium transfected with SRA, SRA-W20 and SRA-ns-scFv, AβOs was added and incubated for 2 hours, and then CAR-BV2 was detected at 0, 3, 6, 12, 24 and 36 hours later. Aβ (Aβ antibody staining, red) levels in cells (green), scale bar = 25 μm. (B) Detection of Aβ levels in CAR-BV2 cells at different times by flow cytometry.

Figure 8: CAR-BV2 activates the intracellular lysosomal pathway. (A) AβOs was added to the culture medium of CAR-BV2 cells transfected with SRA, SRA-W20 and SRA-ns-scFv, and cell immunofluorescence was used to detect the level of lysosomal marker LAPM1; (B) LAMP1 signal in CAR-BV2 quantitative analysis.

Figure 9: Western-blot detects intracellular LAMP1 protein levels. (A) The transfected BV2 cell extract was subjected to SDS-PAGE electrophoresis and transferred to the membrane, and its level was detected with LAMP1 antibody; (B) Quantitative analysis of protein band density.

Figure 10: qPCR determination of the mRNA levels of pro-inflammatory and anti-inflammatory factors in CAR-BV2 cells. 4 μM AβOs was added to the CAR-BV2 cell culture medium transfected with SRA, SRA-W20 and SRA-ns-scFv and incubated for 24 h, and IL-6, TNF-α, IL-10, TGF-β, and TGF-β in the cells were measured by qPCR. YM-1 and Arg-1 mRNA levels.

Figure 11: Expression of fusion gene SRA-W20 in mouse brain. Fusion proteins GFP (green) and W20 (anti-W20 antibody stained, red) were observed by confocal microscopy, scale bar = 25 μm.

Figure 12: Y-maze test was used to test the therapeutic effect of CAR-M on AD mice. After the mice were treated with various liposome gene vectors for 28 days, the Y maze test was used to measure the number of entries (A) and residence time (B) of the mice in each group in the new arm.

Figure 13: Morris Water Maze Test (MWM) Effect of CAR-M on spatial learning and memory functions in AD mice. (A) During the training period, the latency of the mouse to find the platform; (B-D) After the platform was removed, the latency of the mouse to reach the platform area (B), the number of times the mouse crossed the platform location (C) and the time it stayed in the target quadrant (D ).

Figure 14: CAR-M treatment reduces Aβ plaques in the brains of AD mice. (A) After applying CAR-M to treat AD mice and WT mice, immunohistochemical staining was performed with 6E10 antibody, scale bar = 100 μm; (B) Quantitative analysis of Aβ plaque staining area.

Figure 15: CAR-M reduces Aβ levels in the brains of AD mice. AD and WT mice treated with CAR-M were used to measure the levels of soluble Aβ40 (A), insoluble Aβ40 (B), soluble Aβ42 (C) and insoluble Aβ42 (D) in the brains of mice using ELISA.

Figure 16: CAR-M reduces Aβ oligomer levels in the brains of AD mice. The content of Aβ oligomers in brain homogenates of AD mice was detected by ELISA.

Figure 17: CAR-M promotes Aβ phagocytosis in vivo. (A) The phagocytosis of Aβ (red) in GFP-positive (green) and Iba-1-positive (yellow) microglia in the mouse brain after treatment with various liposome gene vectors; (B) Microglia Quantitative analysis of Aβ levels phagocytosed by plasma cells, scale bar = 500 nm.

Figure 18: CAR-M gene therapy reduces oxidative stress and ROS levels in the brains of AD mice. The corresponding kits were used to measure GSH (A), GSSG (B), GSH/GSSG ratio (C), and ROS (D) levels in mouse brain homogenate.

Figure 19: CAR-M gene therapy inhibits synapse loss in the brain of AD mice. (A) Synaptophysin immunostaining (green) and PSD95 immunostaining (red) in the brains of AD and WT mice treated with different liposomes, scale bar = 200 μm; (B-D) Synaptophysin and PSD95 immunostaining areas Quantitative analysis.

Figure 20: CAR-M gene therapy reduces glial cell activation in the brains of AD mice. (A) After treatment with different liposomes and PBS, the activation of astrocytes and microglia in the mouse brain was detected by GFAP immunostaining and Iba-1 immunostaining, scale bar = 250 μm; (B) GFAP and quantitative statistics of Iba-1 positive staining area.

Figure 21: CAR-M gene therapy promotes the transformation of microglia in the brain of AD mice to M2 type.

Figure 22: Construction of CAR-A expression plasmid. gfa2: astrocyte-specific promoter; Poly(A): polyadenylation signal; ORI: origin of replication; KanR: kanamycin resistance gene.

Figure 23: ICC and Western blot analysis of complete expression of CAR in astrocytes. (A) CAR was stained using W20 and MerTK antibodies to identify intact CAR expression. Scale bar: 5 μm. (B) Western blot analysis of membrane proteins extracted from ns-CAR-A and CAR-A using W20 and MerTK antibodies.

Figure 24: CAR is specifically expressed in astrocytes. CAR lipoplexes were transfected into astrocytes (A), neuronal cells (B), and microglia (C), and laser confocal microscopy was used to detect the expression of CAR in different cells. Scale bar: 50 μm.

Figure 25: Flow cytometric analysis of the ability of CAR-A to phagocytose Aβ monomers and oligomers. Use CAR lipoplex or ns-CAR lipoplex to transfect astrocytes. After transfected astrocytes were incubated with 200 nM Aβ monomer or Aβ oligomer for 6 hours, the cells were treated with Aβ antibody. Staining and flow cytometry were used to analyze the phagocytosis ability of astrocytes towards various types of Aβ.

Figure 26: CAR reduces the transcription levels of inflammatory factors in astrocytes. Aβ oligomers were added to CAR-A, ns-CAR-A or control astrocyte cultures, and incubated at 37°C for 24 h. Proinflammatory cytokines IL1β, TNFα, iNOS, IL6(A) and anti-inflammatory cytokines were detected by qPCR. The mRNA levels of sex cytokines IL4, Arg1, CD206, and TGFβ(B).

Figure 27: Distribution of CAR lipoplexes in mice.

Figure 28: Expression of CAR in astrocytes in mouse brain. Scale bar: 50 μm.

Figure 29: CAR-A improves spatial learning and memory impairment in AD transgenic mice. After intranasal administration of liposomes, CAR lipoplexes or ns-CAR lipoplexes to WT and AD mice, the spatial learning and memory abilities of WT and AD mice were evaluated through the water maze test. During the training experiment, the movement trajectory of the mouse (A) and the change of the latency of the mouse to find the platform with time (C) were recorded. During the detection experiment, the mouse's movement trajectory (B), the latency of the mouse to reach the platform area (D), the time it stayed in the target quadrant (E) and the number of times it crossed the platform (F) were recorded.

Figure 30: CAR-A improves spatial memory impairment in AD transgenic mice. After intranasal administration of PBS, CAR lipoplexes or ns-CAR lipoplexes to WT and AD mice, the spatial memory ability of WT and AD mice was evaluated through the Y maze test. Record the mouse's movement trajectory (A), residence time in the new arm (B) and number of entries (C).

Figure 31: CAR-A in mouse brain has a strong ability to phagocytose Aβ oligomers. (A) Representative pictures of astrocytes phagocytizing Aβ oligomers in the brains of mice in different experimental groups. Scale bar: 5 μm. (B) Aβ oligomers in astrocytes were quantified using Image J software, and values were normalized relative to the AD-PBS group.

Figure 32: CAR-A reduces the levels of Aβ oligomers in the brains of AD transgenic mice. After intranasal administration of PBS, CAR lipoplexes or ns-CAR lipoplexes to WT and AD mice, a kit was used to determine the soluble Aβ oligomer content in the mouse brain homogenate.

Figure 33: CAR-A reduces the number of Aβ plaques in the brains of AD transgenic mice. After WT and AD mice were intranasally administered with PBS, CAR lipoplexes or ns-CAR lipoplexes, 6E10 antibody was used for immunostaining to detect the level of senile plaques in the brain (A), and Image J software was used to count the senile plaque areas. (B). Scale bar: 200 μm.

Figure 34: CAR-A reduces the levels of Aβ40 and Aβ42 in the brains of AD transgenic mice. After intranasal administration of liposomes, CAR lipoplexes or ns-CAR lipoplexes to WT and AD mice, the corresponding detection kits were used to measure insoluble Aβ40(A) and insoluble Aβ42(A) in the mouse brain homogenates. B) content.

Figure 35: CAR-A reduces the levels of pro-inflammatory cytokine mRNA and increases the level of anti-inflammatory cytokine mRNA in the brains of AD transgenic mice. After intranasal administration of liposomes, CAR lipoplexes or ns-CAR lipoplexes to WT and AD mice, pro-inflammatory cytokines (IL1β, TNFα, iNOS, IL6) in the brains of mice in each group were measured using qPCR. ) and the mRNA levels of the anti-inflammatory cytokines IL4, Arg1, TGFβ).

Figure 36: Western blot detection of GFAP and Iba 1 protein levels in the brains of mice in each group. After intranasal administration of liposomes, CAR lipoplexes or ns-CAR lipoplexes to WT and AD mice, Westernblot experiments were performed using GFAP and Iba 1 antibodies to detect the amounts of GFAP and Iba 1 proteins in the mouse brain (A ), and the band area was counted through Image J (B).

Figure 37: CAR-A reduces synaptic loss in the brain of AD mice. After intranasal administration of liposomes, CAR lipoplexes or ns-CAR lipoplexes to WT and AD mice, the levels of PSD95 (red) and SYN (green) in the brains of mice were detected by immunofluorescence, and colocalization indicated complete Synapse (yellow) A Scale bar: 5 μm. Use Image J software to calculate area statistics (B).

Detailed ways

definition

Amyloid oligomers refer to the form of aggregates formed by the aggregation of two or more amyloid monomer molecules. Amyloid proteins include β-amyloid, microtubule-associated protein tau, α-synuclein, huntingtin, pancreatic amyloid, superoxide dismutase 1 (SOD1), and TDP-43 proteins.

As used herein, anti-inflammatory receptors refer to glial cell surface receptors that do not promote the production of inflammatory factors, including scavenger receptor type A (SR-A), MERTK, Tyro3, Ax1, ItgB5, BAI1, ELMO, or MRC1 , Stabilins, ADGRB1, TIMs, αVβ3/αVβ5 integrin. Preferably, the anti-inflammatory receptor is SR-A or MERTK. In this article, the abbreviations SRA and SR-A for class A scavenger receptors are used interchangeably.

Chimeric antigen receptor glial cells refer to chimeric antigen receptor-modified glial cells that contain amyloid oligomer-binding domain, transmembrane domain, and intracellular signaling domain.

CAR-M refers to chimeric antigen receptor-modified microglia.

CAR-A refers to chimeric antigen receptor-modified astrocytes.

"CAR" or "chimeric antigen receptor" as used interchangeably herein refers to a molecule that contains at least three domains, i.e., a cellular antigen-binding domain (in the present invention, an anti-amyloid oligomer-binding domain). The ectodomain, the transmembrane domain, and the intracellular domain containing the intracellular signaling domain.

Therefore, when a CAR is expressed on a host cell (especially 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 outside the cell to allow the CAR to bind to the target antigen (e.g., at least 90%, 95%, 95%, 97%, 99% or 100% will exist outside the cell). In some embodiments, the antigen binding domain is an antibody, especially a single chain antibody.

In some embodiments, the antigen-binding domain is a single chain antibody that specifically targets amyloid oligomers, which can be genetically modified from an antibody known in the art that specifically targets amyloid oligomers. Operation modified. Those skilled in the art are aware of the methods of carrying out the modifications. In some embodiments, the antigen-binding domain is a W20 antibody, Aducanumab, or Crenezumab, preferably in scFV form thereof.

The transmembrane domain connects the extracellular domain containing the antigen-binding domain (i.e., the anti-amyloid oligomer binding domain in the present invention) to the intracellular signaling domain and typically spans the cell membrane of the host cell upon CAR expression and membrane targeting. . The transmembrane domain may be derived from a protein or part of a protein having both transmembrane and intracellular regions, such as CD28, and these domains or parts thereof may be included in the CAR of the invention. The transmembrane domain can be based on or derived from the transmembrane domain of any transmembrane protein. In some embodiments, it may be or may be derived from SRA, MerTK, Axl, Tyro3, Tim1, Tim4, Tim3, FcR, BAI1, DAP12, MRC1, ICOS, CD8a, CD28, CD4, CD3ζ, CD45, CD9, Transmembrane domains of CD16, CD22, CD33, CD64, CD80, CD86, CD134(OX40), CD137(4-1BB) and CD154. In further embodiments it may be or may be derived from a transmembrane domain from SRA, MerTK, Axl, Tyro3, Tim1, Tim4, Tim3, FcR, BAI1, CD4, DAP12, MRC1, CD8α, CD3, ICOS or CD28 .

As used herein, "intracellular signaling domain" refers to the portion of the CAR protein that is involved in transducing information about the efficient binding of the CAR to the target antigen (amyloid oligomers) to cells (host cells, such as glial cells). cells) to initiate cellular functions (e.g., effector cell functions). The intracellular signaling domain of the CAR is present within the host cell upon expression of the CAR (i.e., contained within the intracellular domain of the CAR), usually within the cytoplasm of the cell. This domain is capable of activating one or more normal functions of the CAR-expressing host cell. For example, if the host cell is a microglial cell, the intracellular signaling domain can improve its ability to phagocytose Aβ oligomers and modify the downstream signaling pathway of the chimeric antigen receptor so that it does not produce too much Aβ oligomers. Pro-inflammatory factors. In some embodiments, the intracellular signaling domain is selected from scavenger receptor class A (SR-A), MerTK, Tyro3, Ax1, ItgB5, BAI1, ELMO or MRC1, Stabilins, ADGRB1, TIMs, αVβ3/αVβ5 integrin intracellular signaling domain.

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 aid antigen binding to the anti-amyloid oligomer binding domain. In certain embodiments, the hinge region and/or spacer region may be an immunoglobulin hinge region, and may be a wild-type immunoglobulin hinge region or an altered wild-type immunoglobulin hinge region, such as a truncated hinge region . Other exemplary hinge regions and/or spacers that may be used include those derived from the extracellular region of a type 1 membrane protein, such as CD8a, CD4, CD28, CD7, IgGl or IgG4, which may be The wild-type hinge/spacer regions from these molecules may be altered. Preferably, the hinge/spacer region is or is derived from the hinge/spacer region of the human MerTK receptor FNIII domain, CD8a, CD4, CD28, CD7. IgD, CH3 and Fc spacers or hinges may also be used in the CARs of the invention. A 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 (eg a eukaryotic gene expression vector, especially for expression in glial cells) or a cloning vector. Possible expression vectors include, but are not limited to, plasmids, modified viruses (e.g., replication-deficient retroviruses, adenoviruses, adeno-associated viruses, and lentiviruses), transposons, or cosmids, as long as the vector is compatible with the host cell used. It can be accommodated.

Accordingly, the present invention encompasses recombinant expression vectors containing the nucleic acid molecules of the invention, as well as the regulatory sequences necessary for the transcription and translation of the protein sequences encoded by the nucleic acid molecules of the invention. Suitable regulatory sequences can be derived from a variety of sources, including bacterial, fungal, viral, mammalian or insect genes. The selection of appropriate regulatory sequences depends on the host cell chosen 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: transcription 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 chosen and the vector used, other sequences, such as origins of replication, additional DNA restriction sites, enhancers and sequences conferring transcriptional induction ability, can be introduced into the expression vector.

Examples of promoters capable of expressing CAR molecules in cells (mammalian cells) are glial cell-specific promoters. A glial cell-specific promoter refers to a promoter specific to glial cells. Preferably, the glial cell-specific promoter is selected from promoters of gfa2, GFAP, GFAP104, GfaABC1D, ALDH1L1, Cst3, CX30, CX3CR1, IBa1, Pdgfra, olig2, NG2, CNP, CD68, etc., for example, for gfa2 promoter, GFAP promoter, Cst3 promoter, GFAP104 promoter, gfaABC1D promoter or Cx30 promoter for astrocytes, olig2 promoter for oligodendrocytes or CNP promoter for microglia CD68 promoter, CX3CR1 promoter or Iba1 promoter of plasma cells.

Recombinant expression vectors can be introduced into host cells to produce transformed host cells. The terms "transformed with," "transfected with," "transformed," "transduced" and "transfection" are intended to include the transfer of a nucleic acid, such as a vector, by one of the many possible techniques known in the art. ) introduced into cells. 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 vector of the invention. Prokaryotic cells can be transformed with nucleic acids, for example, by electroporation or calcium chloride-mediated transformation. For example, nucleic acids can be introduced into mammalian cells by conventional techniques such as calcium phosphate or calcium chloride coprecipitation, 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 will also be understood that cells of the invention may comprise more than one nucleic acid or vector of the invention. In particular, 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 particularly refers to a human, a domestic animal (e.g., cat, dog, etc.), horse, mouse, rat, primate, such as monkey, cow, pig wait.

As discussed herein, degenerate variants of a defined nucleic acid or polynucleotide sequence that result from redundancy of the genetic code are also contemplated. In particular, codon-optimized nucleotide sequences that are optimized for expression within cells of a particular organism are contemplated. For example, polynucleotide sequences codon-optimized for expression in human or murine glial cells can be developed and are 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. The corresponding RNA sequences to the DNA sequences described herein are therefore encompassed. The skilled person will understand how to derive an RNA sequence encoding the same protein/polypeptide product into the DNA sequence 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 naturally occurring sequence of bases, sugars, and sugars (backbone). A sequence of linked nucleoside or nucleotide monomers. The term also includes modified or substituted sequences containing non-naturally occurring monomers or portions thereof. The nucleic acid, polynucleotide or nucleotide sequence of the invention may be a deoxyribonucleic acid sequence (DNA) or a ribonucleic acid sequence (RNA), and may include naturally occurring bases, including adenine, guanine, cytosine, thoracic acid, Glycosides and uracil. The sequence may also contain modified bases. Examples of such modified bases include aza and deazaadenine, guanine, cytosine, thymidine and uracil; and xanthine and hypoxanthine. A nucleic acid, polynucleotide or nucleotide sequence may be double-stranded or single-stranded. Nucleic acids, polynucleotides or nucleotide sequences may be wholly or partially synthetic or recombinant.

The glial cells of the present invention can be in situ glial cells, isolated primary glial cells or established glial cells.

The modified glial cells of the present invention have therapeutic effects, and can especially be used to recognize and phagocytose amyloid oligomers. During treatment, gene vectors can be delivered to the brain using methods well known in the art.

Before using the cells therapeutically, it may be desirable to subject the cells to an activation or expansion step using methods well known in the art.

The invention additionally specifically provides a population of cells, wherein at least one cell of said population comprises a nucleic acid or vector of the invention. A population of cells may comprise cells containing different nucleic acids or vectors of the invention. Thus, one cell in the population may comprise a nucleic acid of the invention encoding a first CAR, such as W20-SRA, and a second cell of the population may comprise a nucleic acid of the invention encoding a second CAR, such as W20-MerTK.

Although the nucleic acids, vectors or cells of the invention are effective against disease when used alone, additional therapeutic agents can be used in combination with the nucleic acids, vectors or cells of the invention to combat disease. Accordingly, in another embodiment of the invention, at least one additional or additional therapeutic agent (eg, other neurodegenerative disease treatment agents) may be administered to the subject. Accordingly, the nucleic acids, vectors, CARs, cells or cell populations of the present invention and other therapeutic agents (eg, other neurodegenerative disease treatments) can be administered to a subject. Accordingly, compositions or pharmaceutical compositions of the invention may comprise other active or therapeutic agents, as well as nucleic acids, vectors, CARs, cells and/or cell populations of the invention. However, it is understood that the nucleic acids, vectors, CARs, cells or cell populations of the invention and other therapeutic agents (eg, other neurodegenerative disease treatments) may be administered separately, for example, by separate routes of administration. Additionally, the nucleic acid, vector, CAR, cell or cell population of the invention and at least one other therapeutic agent (eg, other neurodegenerative diseases) may be administered sequentially or (substantially) simultaneously. They may be administered in the same pharmaceutical preparation or drug, or they may be formulated and administered separately. For sequential administration, administration can occur 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 the nucleic acid/vector/cell administration Additional therapeutic agents.

"Pharmaceutically acceptable" includes preparations that 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 compositions 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 patient's condition and the type and severity of the patient's disease, although appropriate dosages can be determined through clinical trials.

The compositions of the present invention may be administered in a single dose or in multiple doses. In particular, the composition can be administered in a single, disposable application.

The nucleic acid molecules or compositions of the present invention may be administered by any parenteral route in the form of a pharmaceutical preparation containing the active ingredient. The compositions may be administered in varying dosages depending on the condition and patient to be treated, as well as the route of administration. In any case, 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 based on the intended route of administration and standard pharmaceutical practice. For each of the described embodiments, the nucleic acid molecules or compositions of the invention can 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, granules, microgranules, dispersible granules, cachets, inhalants, aerosols for inhalation agents, patches, particle inhalants, implants, long-acting implants, injections (including subcutaneous, intramuscular, intravenous and intradermal, preferably intravenous), infusions and combinations thereof. Typically, the cells of the invention can be administered in nasal buffer, injection or infusion buffer. Exemplary formulations can be found, for example, in Remington's Pharmaceutical Sciences, 19th Edition., Grennaro, A., Editor, 1995, which is incorporated herein by reference.

The nucleic acid molecules or compositions of the invention may also be administered parenterally, such as nasal, intravenous, intraarterial, intraperitoneal, intrathecal, intracranial, topical, intramuscular, buccal, subcutaneous, transepidermal, intradural External, inhaled, intracardiac, intracerebroventricular, intraocular, intraspinal, sublingual, transdermal or transmucosal, or they may be administered by infusion techniques. They are best used in the form of sterile aqueous solutions, which may contain other substances, such as sufficient salt or glucose to make the solution isotonic with the blood. If necessary, the aqueous solution should be appropriately buffered (preferably pH 3 to 9). The preparation of suitable parenteral formulations under sterile conditions can be readily accomplished by standard pharmaceutical techniques well known to those skilled in the art.

Formulations may be presented in unit-dose or multi-dose containers, such as sealed ampoules, bags, and vials.

In some embodiments, the invention relates to the following:

Item 1. A nucleic acid molecule comprising a polynucleotide sequence encoding a chimeric antigen receptor, the chimeric antigen receptor comprising: 1) an anti-amyloid oligomer binding domain; 2) a transmembrane domain; and 3) intracellular signaling domain.

Item 2. The nucleic acid molecule according to item 1, wherein the amyloid oligomer is selected from the group consisting of β-amyloid oligomer, microtubule-associated protein tau oligomer, α-synuclein oligomer, The group consisting of huntingtin oligomers, amyloid oligomers, SOD1 oligomers and TDP-43 oligomers.

Item 3. The nucleic acid molecule according to item 1 or 2, wherein the amyloid oligomer is a β-amyloid oligomer. Preferably, the β-amyloid oligomer is AβO42 or AβO40.

Item 4. The nucleic acid molecule according to any one of items 1 to 3, wherein the binding domain of the anti-amyloid oligomer is a single-chain variable region fragment scFV. Preferably, the anti-amyloid oligomer The binding domain of the body is a binding domain of an anti-β-amyloid oligomer. More preferably, the binding domain of the anti-β-amyloid oligomer is a heavy chain CDR1-3 and a light chain CDR1 comprising the single-chain antibody W20. -3 scFV, Aducanumab or Crenezumab, in which the amino acid sequence of single-chain antibody W20 is shown in SEQ ID NO: 1, and the heavy chain CDR1-3 of single-chain antibody W20 The amino acid sequences of CDR1-3 of the light chain are shown in SEQ ID NO: 2-7 respectively. More preferably, the binding domain of the anti-β-amyloid oligomer is single-chain antibody W20.

Item 5. The nucleic acid molecule according to any one of items 1 to 4, wherein the intracellular signaling domain is an intracellular signaling domain of an anti-inflammatory receptor. Preferably, the intracellular signaling domain of the anti-inflammatory receptor is The intracellular signaling domain is an intracellular component selected from the group consisting of scavenger receptor class A (SR-A), MerTK, Tyro3, Ax1, ItgB5, BAI1, ELMO, MRC1, Stabilins, ADGRB1, TIMs, and αVβ3/αVβ5 integrins. Signaling domain.

Item 6. The nucleic acid molecule according to any one of items 1 to 5, wherein the chimeric antigen receptor further comprises one or more selected from the group consisting of a hinge region and a costimulatory structure that triggers glial cell activation. area.

Item 7. The nucleic acid molecule according to item 6, wherein the hinge region is derived from the group consisting of the MerTK receptor FNIII domain, CD8α, CD28, IgG1 and IgG4.

Item 8. The nucleic acid molecule according to any one of items 1-7, wherein the transmembrane domain is derived from SR-A, MerTK, Axl, Tyro3, Tim1, Tim4, Tim3, FcR, BAI1, CD4, DAP12, A transmembrane domain composed of MRC1, CD8α, CD3, ICOS and CD28.

Item 9. The nucleic acid molecule according to any one of items 1 to 8, wherein the binding domain of the anti-β-amyloid oligomer is a single-chain antibody W20, and the chimeric antigen receptor also has an intracellular signaling domain. It is the intracellular signaling domain of scavenger receptor type A (SR-A) or MerTK, and the transmembrane domain is derived from the transmembrane domain of SR-A or MerTK.

Item 10. The nucleic acid molecule according to any one of items 1-9, wherein the nucleic acid molecule comprises an operably linked glial cell-specific promoter. Preferably, the glial cell-specific promoter is a small Glial cell-specific promoter, astrocyte and/or oligodendrocyte-specific promoter, more preferably, the glial cell-specific promoter is selected from the group consisting of gfa2, GFAP, GFAP104, GfaABC1D , ALDH1L1, Cst3, CX30, CX3CR1, IBa-1, Pdgfra, olig2 and NG2.

Item 11. The nucleic acid molecule according to any one of items 1 to 10, wherein the binding domain of the anti-amyloid oligomer co-fuses to express SR-A, MerTK receptor, glycosylation end product receptor, G Protein-coupled receptors, CC receptors, CXC receptors, C receptors, CX3C receptors or lamp2a receptors.

Item 12. The nucleic acid molecule according to any one of items 6-11, wherein the glial cells are selected from the group consisting of microglia, astrocytes, oligodendrocytes and oligodendrocyte precursors Group of cells.

Item 13. A vector comprising the nucleic acid molecule described in any one of Items 1-12.

Item 14. The vector according to item 13, wherein the vector is a plasmid, a retroviral vector, an adenovirus vector, an adenovirus-related vector or a lentiviral vector. Preferably, the vector is plasmid pGFP-N1, reverse transcription Viral vector pRetroX, adenovirus vector pDC315, pDC311, adeno-associated virus vector pAAV or lentiviral vector pCDH.

Item 15. A cell comprising the nucleic acid molecule described in any one of Items 1 to 12 or the vector described in Item 13 or 14.

Item 16. The cell according to item 15, wherein the cell is a glial cell. Preferably, the glial cell is selected from the group consisting of microglia, astrocytes, oligodendrocytes and oligodendrocytes. A group of glial precursor cells.

Item 17. A chimeric antigen receptor encoded by the nucleic acid molecule described in any one of Claims 1 to 12, expressed by the vector described in Item 13 or 14, or expressed by the cell described in Item 15 or 16 .

Item 18. A pharmaceutical composition comprising the nucleic acid molecule described in any one of Items 1 to 12, the vector described in Item 13 or 14, the cell described in Item 15 or 16, and/or the chimeric vector described in Item 17. Combined antigen receptor, and pharmaceutically acceptable excipients.

Item 19. The pharmaceutical composition according to item 18, wherein the pharmaceutical composition is a nanoparticle prepared using liposomes. Preferably, the liposomes comprise one or more cationic lipids and/or One or more noncationic lipids.

Item 20. The pharmaceutical composition according to item 18 or 19, wherein the liposome is selected from the group consisting of 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, ribose cationic lipids or their combinations, 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, sphingolipid, cerebroside, ganglioside, 16-O-monomethylPE, 16-O-dimethylPE, 18-1 -Trans PE, SOPE or their combinations, preferably, the liposomes are selected from POPC, DDAB, DSPE, DOPC, DOTAP or their combinations; more preferably, the liposomes are selected from the following combinations : 1): POPC, DDAB, DSPE; (2): DSPE, DOPC, DOTAP; More preferably, the DSPE is PEG-modified DSPE, and preferably, the DSPE is DSPE-PEG2000.

Item 21. The pharmaceutical composition according to item 19 or 20, wherein the nanoparticles prepared using liposomes are conjugated with a glial cell-specific targeting peptide. Preferably, the targeting peptide is selected from AS1, MG1 , V9, NGR polypeptides.

Item 22. The pharmaceutical composition according to any one of items 18 to 21, wherein the dosage form of the pharmaceutical composition is nasal drops, intravenous injection, intravenous infusion, subcutaneous injection, or the pharmaceutical composition is directly Administered to the brain.

Item 23. The nucleic acid molecule described in any one of Items 1 to 12, the vector described in Item 13 or 14, the cell described in Item 15 or 16, the chimeric antigen receptor described in Item 17, or any of Items 18 to 22 The pharmaceutical composition described in one is prepared for use in 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, Use in drugs that inhibit gliosis and increase synaptic levels in the brain.

Item 24. A method for promoting the clearance of amyloid oligomers by cells in a subject, treating and/or preventing inflammatory diseases, reducing senile plaques in the brain, treating and/or preventing neurodegenerative diseases, inhibiting gliosis, and improving A method at the brain synapse level, comprising administering to a subject in need the nucleic acid molecule described in any one of Items 1-12, the vector described in Item 13 or 14, the cell described in Item 15 or 16, or Item 17 The chimeric antigen receptor or the pharmaceutical composition described in any one of items 18-22.

Item 25. The nucleic acid molecule according to any one of Items 1 to 12, the vector according to Item 13 or 14, the cell according to Item 15 or 16, the chimeric antigen receptor according to Item 17 or Items 18 to 22 The pharmaceutical composition of any one, which is used to promote cell clearance of amyloid oligomers in a subject, treat and/or prevent inflammatory diseases, reduce senile plaques in the brain, treat and/or neurodegenerative diseases, Inhibit glial cell proliferation and improve brain synapse levels.

Item 26. The use according to item 22, the method according to item 23, or the nucleic acid molecule, vector, cell, chimeric antigen receptor or pharmaceutical composition according to item 25, wherein the neurodegenerative disease is selected from the group consisting of Alzheimer's disease Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, or spinocerebellar ataxia.

The technical solutions of the present invention will be specifically described below through examples. These examples are descriptive and illustrative, and are not meant to be limiting. The reagents used in the following examples, unless otherwise noted, can be easily purchased from reagent companies such as SigmaAldrich and Merck. The test methods, unless otherwise noted, can be obtained from textbooks such as Sambrook, J., Fritsch, Found in E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Press, New York.

Example

Example 1. Construction of W20-SRA gene delivery particles and chimeric antigen receptor microglia (CAR-M).

1.1 Experimental materials and methods

1.1.1 Experimental materials

(2,3-Dioleoyl-propyl)-trimethylammonium (DOTAP) was purchased from Avanti Polar Lipids; dioleoylphosphatidylcholine (DOPC), distearoylphosphatidylethanolamine-polyethylene glycol-N- Hydroxysuccinimide (DSPE-PEG-NHS), N-hydroxysuccinimide (NHS), and N,N-diisopropylethylamine were purchased from Aladdin; Aβ42 peptide and AS1 (CLNSSQPSC) cyclic peptide were purchased from Zhongtide Biochemical Co., Ltd.; 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), BCA protein quantification kit, DMEM cell culture medium, RPMI-1640 cell culture medium , PS, and FBS were purchased from Thermo Fisher Scientific; RNA extraction kit, reverse transcription kit, and 2×SYBR Green qPCR Mix were purchased from Beijing Kangwei Century Biotechnology Co., Ltd.

The antibodies used were as follows: Iba-1 antibody (GeneTex, catalog number: GTX101495), GFAP antibody (CellSignaling Technology, catalog number: 3670S), PSD95 antibody (Abcam, catalog number: ab12093), Synaptophysin antibody (Abcam, catalog number: ab32127 ), LAMP1 antibody (Abcam, catalog number: ab24170).

1.1.2 Experimental instruments

The equipment used in the experiment of this example mainly includes: Zetasizer Nano ZS laser particle size analyzer (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 (AppliedBiosystem).

1.1.3 Main solution preparation

(1) Phosphate buffer solution (PBS): 0.62g sodium hydrogen phosphate dihydrate, 5.73g disodium hydrogen phosphate dodecahydrate, and 9g sodium chloride were dissolved in deionized water, diluted to 1L, and filtered with a 0.22 μm filter. Sterilize.

(2) 0.1% PBST: Dissolve 1mL Tween 20 in 1L PBS.

(3) 5% skim milk: Weigh 5 g of skim milk and dissolve it in 100 mL of 0.1% PBST.

(4) 10× electrophoresis buffer: Dissolve 144g glycine, 30g Tris, and 10g sodium dodecyl sulfate in deionized water and adjust the volume to 1L.

(5) 5× reducing electrophoresis loading buffer: 2.5mL 1M Tris-HCl (pH 6.8), 1g sodium dodecyl sulfate, 5mL glycerol, 0.5mL β-mercaptoethanol, 50mg bromophenol blue dissolved in deionized In water, adjust the volume to 10mL.

(6) Flow cytometry buffer: 2g bovine serum albumin was dissolved in 100mL PBS.

(7) LB medium: Dissolve 10g tryptone, 5g yeast extract, and 5g sodium chloride in deionized water, dilute to 1L, and autoclave at 121oC for 20 minutes.

Note: The relevant reagents, equipment and solutions mentioned in Examples 2, 3 and 4 but whose sources are not given are the same as those in Example 1.

1.2 Construction of plasmid

Use Nhe1 and BamH1 endonucleases to double-digest the pEGFP-N1 vector (Beijing Solebao Technology Co., Ltd., Cat. No.: P6460), and synthesize the target genes (SR-A, SRA-W20, SRA-ns-scFv) , whose nucleotide sequences are shown in SEQ ID NO: 8, 9 and 10 respectively) were double digested with Nhe1 and BamH1 endonucleases and then ligated into the vector (Figure 1).

1.3 Preparation and characterization of DNA-NP liposome complex

19.2 μmol of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC, purchased from Aladdin) and 0.2 μmol of diformyldimethylammonium bromide (DDAB, purchased from Aladdin) Latin) and 0.6 μmoL of diformyl phosphorus-phenylethanolamine (DSPE)-PEG2000 (purchased from Sigma Aldrich) were dissolved in chloroform and evaporated in a rotary evaporator. Add 1 mL of 10 mM PBS (pH=7.4) to resuspend, and perform sonication. Plasmid is added and encapsulated through a series of freeze/thaw cycles. Add exonuclease III/DNase I to remove excess plasmid DNA adsorbed on the outside of the liposome particles, and store at 4°C for use.

Transmission electron microscope (TEM, Hitachi, Japan, HT7700) samples were prepared and observed using TEM, and then dynamic light scattering instrument DLS was used to characterize the dynamic size of liposomes in aqueous solution. The results show that the two particles are well monodispersed and have a uniform size distribution (Figure 2, A), with diameters of 87.1±3.9nm and 91.3±5.0nm respectively (Figure 2, B). The hydrated diameter of NP measured by dynamic light scattering is 92.0±2.9nm, and the hydrated diameter of SRA-W20-NP is 95.1±2.5nm (Figure 2, C), and the dispersion in water is good. The NP and SRA-W20-NP solutions were placed at 4°C for 3 months. The TEM or DLS detection results showed that the particle dispersion was still good, the size distribution was uniform, and the particle size did not change significantly, indicating that the various particles prepared had good stability (data not shown).

1.4 Expression of CAR on BV2 cell surface

BV-2 cells (National Experimental Cell Resource Sharing Service Platform (NICR)) were inoculated into cells containing Opti- In the culture medium (purchased from Gibco) and the 12-well plate of the slide, the cell density was 5 × 10 ⁵ cells/well, the prepared CAR lipoplex was added, and the culture was continued for 12 hours. Cell immunofluorescence was used to observe the presence of CAR in the cells. Express the situation. The results showed that the single-chain antibody W20 was expressed on the cell membrane surface and co-localized with the GFP fluorescent protein on the cell membrane (Figure 3, Panel A), while no W20 signal was observed in BV2 cells transfected with SRA-ns-scFv. Flow cytometry was used to analyze the expression of GFP on BV-2 cells, and the rate of transfected positive cells was counted. The results showed that the rate of BV-2 cells with positive GFP expression was much greater than that of the control group (Figure 3, B).

In order to evaluate the biosafety of this liposome, different concentrations of NP, SRA-NP, SRA-W20-NP and SRA-ns-scFv-NP were added to BV2, N2a and U87-mg (National Experimental Cell Resource Sharing Service In the three cell cultures of the platform (NICR), the MTT method was used to determine cell viability after 48 hours. The results show that when the concentration of DNA-NP in each group is 1000 μg/mL, the activity of the three types of cells can still reach more than 90% of the control cells, indicating that the liposome has less cytotoxicity and better biological safety (Figure 4 ).

1.5 CAR-BV2 improves the phagocytic ability of AβOs

In order to verify the improvement of the ability of CAR-BV2 cells to phagocytose AβOs, the present invention detected the level of AβOs in BV-2 cells. BV-2 cells transfected with SRA, SRA-W20 and SRA-ns-scFv plasmids were added with AβOs and incubated for 12 h, and intracellular Aβ levels were detected by cell immunofluorescence. The results showed that compared with untransfected BV-2 cells, the phagocytosis of AβOs by cells transfected with SRA and SRA-W20 plasmids was significantly improved, and CAR(SRA)-BV2 cells and CAR(SRA-W20)-BV2 cells Internal AβOs levels increased 8-fold and 15-fold respectively (Figure 5).

1.6 CAR-BV2 promotes phagocytosis of AβOs through SR-A

In order to verify whether SR-A mediates the uptake of AβOs by CAR-BV2, the SR-A receptor antagonist fucose was added to BV2 cells transfected with SRA, SRA-W20 and SRA-ns-scFv. , using cellular immunofluorescence to detect intracellular Aβ levels. The results showed that compared with the control group, the phagocytosis of AβOs by CAR-BV2 cells was significantly reduced after adding fucose (Figure 6). It shows that AβOs are taken up through SR-A receptor-mediated endocytosis.

1.7 CAR-BV2 improves the degradation ability of AβOs

In order to verify the change in the degradation ability of AβOs after the AβOs phagocytosis ability of CAR-BV2 cells is improved, the present invention observed and measured the changes in the level of AβOs engulfed into the cells over time. The results showed that the intracellular Aβ levels gradually decreased at 0, 3, 6, 12, 24 and 36 hours after cells engulfed AβOs (Figure 7), indicating that they were continuously digested and degraded. Further cell immunofluorescence and western-blot analysis showed that in BV2 cells transfected with SRA and SRA-W20, the level of the lysosomal marker LAMP1 increased significantly (Figures 8 and 9), proving that in CAR- The ability to digest and degrade Aβ in BV2 cells is significantly improved.

1.8 CAR-BV2 reduces the production of inflammatory factors induced by AβOs

AβOs are cytotoxic and can promote BV-2 cells to produce inflammatory factors and transform in the M1 direction. The present invention uses qPCR to detect the mRNA of inflammatory factors in liposome-transfected BV-2 cells. The primers used are shown in SEQ ID NO: 11-28. (Note: The above primers are also used in Examples 2, 3 or 4)

The qPCR results showed that after AβOs stimulation, the mRNA levels of pro-inflammatory cytokines IL-6 and TNF-α significantly increased in BV2 cells, while the mRNA levels of anti-inflammatory factors such as Arg-1, TGF-β, and IL-10 decreased. In BV2 cells transfected with SR-A and SRA-W20, the levels of M1 markers TNF-α and IL-6 mRNA decreased, and the levels of M2 markers Arg1, TGF-β, and IL-10 increased, indicating that CAR-M It can inhibit the production of inflammatory cytokines induced by AβOs and promote the transformation of BV-2 cells from M1 to M2 (Figure 10).

Example 2 In vivo study of chimeric antigen receptor-modified microglia

2.1 Main experimental materials and equipment

In addition to the experimental materials and equipment used in Example 1, this example also uses the following main experimental materials and equipment: FXPRO small animal in vivo imaging system (KODAK), KD-BM paraffin embedding machine (KEDEE), RM2245 paraffin sectioning machine (Leica), water maze equipment (Chinese Academy of Medical Sciences), new object identification equipment (Chinese Academy of Medical Sciences), Y-maze equipment (Chinese Academy of Medical Sciences). 50% xylene: Equal volumes of ethanol and xylene are mixed thoroughly. Different percentages of ethanol: Different volumes of absolute ethanol and deionized water are thoroughly mixed. Citric acid antigen retrieval solution: Dissolve 3g trisodium citrate and 0.4g citric acid in deionized water and adjust the volume to 1L.

2.2 Animal experiment design

AD mice (purchased from Beijing Huafukang Biotechnology Co., Ltd.) were divided into 5 groups, with 8 mice in each group. The drug administration strategy was as follows: different liposome particles DNA-free-NP, SRA-NP , SRA-W20-NP and SRA-ns-scFv-NP were instilled into AD mice and WT mice (purchased from Beijing Huafukang Biotechnology Co., Ltd.) through the nasal cavity, respectively, 10 μL per mouse, and the dose per mouse was 10 μg, once every other day, for a total of 28 days. After the last dose, behavioral testing was performed.

2.3 Distribution of liposome gene vectors in mice

The Cy7-labeled SRA-W20-NP liposome gene vector was injected into mice by intranasal administration. After 0.5, 1, 2, 3, 6 and 12 hours, the brain, heart, liver, spleen, lung, and In organs such as the kidney, the distribution of liposomes is detected through a bioluminescence imaging system. The results show that SRA-W20-NP is distributed in various organs of mice, and the distribution status changes with time. SRA-W20-NP mostly exists in the liver, and Cy7 fluorescence signal can also be detected in the brain, and within 3 hours Peaks around. The results showed that the liposome gene vector could successfully deliver the SRA-W20-NP liposome gene vector to the brain via nasal administration (data not shown).

2.4 Expression of fusion gene W20-SRA in mouse brain

In order to examine the expression of the fusion gene W20-SRA in nerve cells in the mouse brain, the present invention used a laser confocal microscope to observe the fluorescence distribution of the fusion gene GFP and W20 in the mouse brain after nasal administration. The results showed that the fusion gene W20-SRA was successfully expressed on the cell membrane of microglia in the brain (Figure 11, A), while only the GFP fluorescence signal of the control gene SRA-ns-scFv could be observed (Figure 11, B) .

2.5 CAR-M therapy improves cognitive impairment in AD mice

CAR-M therapy was administered intranasally to AD and WT mice. After 28 days of treatment, the present invention tested the cognitive function of the mice using Y-maze and Morris water maze experiments. Y-maze and Morris water maze experiments were performed as follows.

2.5.1Y maze

The Y-maze is composed of three arms A, B, and C, and includes two test stages with an interval of 1 hour. In the first stage, the mice were allowed to explore the two arms (A, B) of the maze, and the third arm (new arm C) was blocked for 10 min. The second stage is to place the mouse on the same starting arm as in the first stage, open the new arm C, and allow it to freely enter and exit all three arms for 5 min. Use a camera to record the exploration trajectory, record the number of times the new arm is explored, and the time the new arm stays. The results showed that compared with WT mice, PBS- and DNA-free-NP-treated AD mice had no obvious preference for the new arm. After SRA-W20-NP treatment, the AD mice's residence time in the new arm (Figure 12, B) and the number of explorations (Figure 12, A) were significantly increased, indicating that the spatial memory ability was improved.

2.5.2Morris Water Maze (MWM)

The water maze consists of a pool with a diameter of 110 cm and a platform with a diameter of 10 cm. The pool contains opaque water (22±1°C). Different symbols are marked around the pool to facilitate the location and identification of mice. During the 5 consecutive days of training, the platform was placed approximately 1 cm underwater. Put the mouse into the pool from a random position and let it swim for 60 s to find the platform and stay on the platform for 10 s. The trial ended when the mouse found the platform, and the time it took to find the platform was recorded. Mice that were unable to find the platform were guided to the platform, and the latency was recorded as 60 s. After each training session, the mice were dried with a dry towel, dried with a heater, and returned to the cage. Train twice a day with an interval of 3 to 4 hours, and record the latency, swimming distance, average swimming speed and exploration mode of mice in each group. 24 hours after the last learning test, the platform was removed, a water entry point was randomly selected, the mice were placed in the water, and swam for 60 seconds. Without the platform, the memory retention ability of the mice was tested. Monitoring and recording are carried out through cameras installed above the maze, and parameters such as residence time in the target quadrant, swimming distance, average swimming speed, exploration path, and number of platform crossings are determined using software analysis.

In the MWM test to evaluate spatial memory and learning ability, during the training period, the time to reach the platform was significantly shortened in AD mice treated with SRA-NP and SRA-W20-NP compared with AD mice in the PBS control group (Fig. Figure A of 13). In the test of removing the platform, AD mice in the SRA-W20-NP or SRA-NP treatment group showed obvious spatially oriented swimming behavior, reaching The time required for the platform position was significantly shortened (Figure 13, Panel B), the platform was crossed more times (Figure 13, Panel C), and the target quadrant stayed longer (Figure 13, Panel D). In the training and test trials, there was no significant difference in the swimming speed of the mice in each group, indicating that their motor functions were not affected. These results indicate that SRA-W20-NP and SRA-NP can significantly reduce the cognitive dysfunction of AD mice, among which SRA-W20-NP improves the cognitive and memory functions of AD mice more significantly.

2.6 Determination of Aβ levels in mouse brain

In order to explore the effect of CAR-M on Aβ plaques in the brains of AD mice, the present invention used immunohistochemistry to detect the level of 6E10-positive Aβ plaques in the brains of AD mice. Specifically, mice were deeply anesthetized with sodium pentobarbital (50 mg/kg), and cardiac perfusion was performed with pre-cooled PBS (containing heparin 10 U/mL) before sacrifice. The brain was peeled off and cut into two parts along the sagittal plane. The left cerebral hemisphere was fixed in 4% paraformaldehyde at 4°C overnight and dehydrated the next day (30% ethanol for 1 hour, 50% ethanol for 1 hour, 75% Ethanol 1h, 100% ethanol 1h, 50% xylene 1h, 100% xylene 1h, 100% xylene 1h), paraffin embedding for 3h. Cut 5 μm or 10 μm thick paraffin-embedded sections on a microtome. Place the paraffin sections in a 60°C oven for 1 hour, and then place them in 100% xylene, 50% xylene, 100% alcohol, 70% alcohol, 50% alcohol and 30% alcohol for dewaxing. Soak the slices in deionized water for 10 minutes, add antigen retrieval solution and boil for 15 minutes, then cool to room temperature for later use. Wash three times with PBS and block for 1 h at room temperature in PBST containing 10% sheep serum and 0.3% Triton X-100. Subsequently, the sections were incubated with 6E10 (1:100) antibody, washed three times with PBS, 5 min each time, and then incubated with HRP-labeled secondary antibodies, washed three times with PBS, 5 min each time, and observed with a confocal microscope.

Preparation of brain homogenate: The right hemisphere obtained above was lysed and homogenized in RIPA buffer containing protease inhibitors, and then the tissue was centrifuged at 14000 g and 4°C for 30 min, and the supernatant containing soluble Aβ was collected ( RIPA soluble fraction). The insoluble pellet was resuspended in guanidine hydrochloride buffer (5.0 M guanidine hydrochloride, pH 8.0) and centrifuged at 14000g for 1 h at 4°C to obtain a supernatant containing insoluble Aβ (guanidine soluble fraction). Use IBL's Aβ40 ELISA detection kit and Aβ42 ELISA detection kit for detection.

The results showed that compared with AD mice in the PBS control group, the Aβ plaque staining area in the hippocampus of mice in the SRA-NP and SRA-W20-NP treatment groups was significantly reduced, with the SRA-W20-NP treatment group showing the most obvious reduction (Figure 14). ELISA was used to further detect the levels of soluble and insoluble Aβ40/42 in mouse brain homogenates. The results showed that both SRA-NP and SRA-W20-NP significantly reduced the levels of soluble and insoluble Aβ40 and Aβ42 in the brain (Figure 15).

2.7 Determination of Aβ oligomer levels in brain homogenates

In order to explore the effect of CAR-M on the level of Aβ oligomers in the brains of AD mice, the present invention uses W20 antibody to detect the content of Aβ oligomers in mouse brain homogenates through ELISA. The results showed that SRA-W20-NP significantly reduced the level of Aβ oligomers in the brains of AD mice (Figure 16). However, compared with PBS or empty liposomes, SRA-NP and SRA-ns-scFv-NP There was no statistical difference in the effect on Aβ oligomer levels.

2.8 Determination of the ability of CAR-M to phagocytose Aβ in the brain of AD mice

In order to examine the ability of CAR-M to phagocytose Aβ in the brains of AD mice, the present invention used immunohistochemistry to detect the levels of Aβ in GFP+ microglia in the brains of mice. The preparation and immunohistochemical processing of paraffin brain sections are as in 2.6. The primary antibodies were 6E10 (1:100) and anti-Iba-1 antibody (1:100), and the secondary antibodies were corresponding fluorescently labeled anti-IgG antibodies. The results showed that the level of phagocytosis of Aβ in microglia expressing CAR (SRA-W20) in the brains of AD mice was significantly increased (Figure 17, Panel A). Moreover, although the phagocytosis of Aβ by microglia expressing CAR (SRA) and CAR (SRA-ns-scFv) increased, both were significantly lower than those in the CAR (SRA-W20) group (Figure 17, Panel B). .

The present invention also measured the contents of GSH and GSSG in the brain homogenates of AD mice and their WT mice treated with various liposome gene carriers. The kits used were GSH and GSSG detection kits and ROS detection kits from Beyuntian Company. The experiment was performed according to the instructions of the kits. The results showed that compared to the PBS control group, SRA-W20-NP significantly reduced the GSSG levels in the brains of AD mice (Figure 18, A and B), and increased the GSH/GSSG ratio (Figure 18, C), while Neither SRA-NP nor SRA-ns-scFv-NP reached statistical differences. The present invention further detected the ROS content in the mouse brain homogenate, and the results showed that SRA-W20-NP and SRA-NP can significantly reduce the ROS level in the brain of AD mice (Figure 18, D). The above results indicate that CAR-M gene therapy can reduce oxidative stress in the brains of AD mice.

2.9 CAR-M reduces synaptic loss in the brain of AD mice

AβOs can cause synaptic dysfunction and synaptic loss in the AD brain, leading to a decline in cognitive function. Synaptophysin and PSD95 are the signature proteins of front and rear synapses respectively. Therefore, the present invention uses synaptophysin and PSD95 antibodies to evaluate the synaptic levels in the mouse brain through immunohistochemistry (Figure 19). Experimental results showed that compared with WT mice, synaptic levels in the brains of AD control mice were significantly reduced, while both SRA-W20-NP and SRA-NP treatments significantly increased synaptophysin and PSD- in the brains of AD mice. The level of 95 (Panels A, B and C of Figure 19), and the number of synapses was also significantly increased (Panel D of Figure 19). There was no statistical difference in synaptic levels between the SRA-ns-scFv-NP treatment group and the PBS control group of AD mice.

2.10 CAR-M reduces glial cell activation in the brain of AD mice

In the occurrence and development of AD, neuroinflammation caused by glial cell activation plays a key role. We detected the activation of astrocytes and microglia in the mouse brain through GFAP and Iba-1 immunostaining respectively. Compared with AD mice in the PBS and NP control groups, the GFAP and Iba-1 positive staining areas in the brains of AD mice treated with SRA-W20-NP were significantly reduced (Figure 20). These results indicate that SRA-W20-NP can significantly reduce the excessive activation of astrocytes and microglia in AD brain and reduce neuroinflammation. The therapeutic effect of SRA-W20-NP was better than that of SRA-NP.

2.11 Effect of CAR-M treatment on microglia phenotype in the brain of AD mice

In order to explore whether CAR-M can promote the transformation of microglia from the inflammatory M1 type to the anti-inflammatory M2 type, the present invention used qPCR to determine the marker gene IL- of M1 type microglia in the mouse brain after CAR-M treatment. Gene expression of 1β, TNF-α and M2 microglia marker genes Arg-1, TGF-β, IL-10 and YM-1. The results showed that SRA-W20-NP treatment could significantly increase the mRNA levels of M2-related marker genes and reduce the mRNA levels of M1-related genes in the brains of AD mice (Figure 21). It shows that CAR-M treatment changes the microglia in the brain of AD mice from the inflammatory M1 type to the anti-inflammatory M2 type.

Example 3 Construction of W20-MerTK gene delivery particles and chimeric antigen receptor astrocytes (CAR-A).

Similar to microglia, astrocytes also play a "double-edged sword" role in the occurrence and development of AD. Therefore, this embodiment also uses chimeric antigen receptor technology to functionalize astrocytes. transformation to confirm whether the technical effects of CAR-M described above are universal in glial cells.

Different from traditional chimeric antigen receptors used to program CAR-T cells, the chimeric antigen receptor constructed in this example is derived from the MerTK receptor, which is mainly expressed on M2 macrophages and astrocytes. On glial cells, activation of this receptor mediates phagocytosis and simultaneously inhibits the release of pro-inflammatory cytokines. The MerTK structure is similar to the CAR structure in traditional CAR-T cells, including the antigen recognition region, the transmembrane region and the intracellular signal transduction region. It is an ideal receptor for CAR modification, and other anti-inflammatory phagocytosis Receptor-like receptors such as Tyro3 and Axl are also expected to be used to engineer anti-inflammatory CARs through rational design. In this example, the antigen-binding region of MerTK (19Gly-275Asn) was replaced with an Aβ oligomer-specific single-chain antibody (W20) to construct a new anti-inflammatory chimeric antigen receptor that recognizes Aβ oligomers, and The functional properties of astrocytes modified by this CAR were studied in depth.

3.1 Culture of primary astrocytes and microglia

Take 5 C57BL/6J newborn mice, soak and disinfect them in 75% alcohol, take out the brains, soak the brains in HBSS to remove the blood meninges, cut the brains with the blood meninges removed using tissue scissors, and place them in a 50 mL centrifuge tube. Add 20 mL of tissue digestion solution (DMEM medium containing 0.25% trypsin and 1 mg/mL DNase I), digest at 37°C for 20 min, pass the digested cell suspension through a 70 μm cell sieve to remove undigested tissue blocks, and centrifuge at 500 g for 10 min. Collect the cells, spread them in T-75 culture flasks, and culture them at 37°C for 5-7 days. Change the culture medium every 2-3 days. After 7 days of culture, the cells were taken out and placed in a cell shaker. Shake at 220 rpm for 30 min. Put the supernatant into a centrifuge tube and centrifuge at 500 g for 10 min to collect microglia. The astrocytes are still attached to the cell culture flask, add fresh medium and continue to culture, changing the medium every 2-3 days.

3.2 Culture of primary neuronal cells

Take 5 C57BL/6J newborn mice, soak and disinfect them in 75% alcohol and then take out the brains. Soak the brains in HBSS and peel off the blood meninges and peel off the hippocampal tissue. Cut the stripped hippocampal tissue into pieces with tissue scissors and place it in 15 mL. In the centrifuge tube, add 5 mL of tissue digestion solution (DMEM medium containing 0.25% trypsin and 1 mg/mL DNase I), digest at 37°C for 20 minutes, and pass the digested cell suspension through a 70 μm cell sieve to remove incompletely digested tissue pieces. , centrifuge at 500g for 10 minutes to collect the cells, spread the cells in a 12-well plate containing cell slides (PDL coated at room temperature overnight), and culture using Neurobasal medium (containing B27, L-GlutaMAX).

3.3 Plasmid construction

Use Nhe1 and BamH1 endonucleases to double-digest the pEGFP-N1 vector, and synthesize the nucleotide sequences of the target genes (MerTK, W20-MERTK, ns-ScFv-MerTK (ns-CAR)) as shown in SEQ ID NO: 29-31) was double digested with Nhe1 and BamH1 endonucleases and then ligated into the vector (Figure 22).

3.4Synthesis and characterization of DSPE-PEG-AS1

Weigh 2 mg DSPE-PEG2000-NHS and 1.5 mg AS1 peptide (CLNSSQPSC (C-C cyclization), China Peptide Biochemical Co., Ltd.) into a round-bottomed flask, and add 5 mL dimethylformamide (containing 5 μL/mL triethylamine) , after stirring at room temperature for 72 hours, put the reaction solution into a dialysis bag (molecular weight cutoff: 1kDa), dialyze for 48 hours to remove unreacted AS1 peptide, then freeze-dry and store at -20°C, and use MALDI-TOF mass spectrometer to analyze DSPE- Molecular weight of PEG2000-AS1. Molecular weight data indicated that AS1 was successfully coupled to DSPE-PEG (data not shown).

3.5 Preparation of liposomes and lipoplexes

Dissolve 50 μmol DOTAP, 50 μmol DOPC and 5 μmol DSPE-PEG2000-AS1 in 5 mL chloroform/methanol mixed solution (v:v=2:1). After gentle stirring at room temperature for 2 h, the mixture was evaporated on a rotary evaporator to form a lipid bilayer film, 2 mL of Tris buffer (10 mM, pH 7.4) was added, and sonicated in an ice bath to generate unilamellar vesicles. Liposomes. The resulting liposomes were sequentially passed through 200 nm and 100 nm polycarbonate membranes using a liposome extruder. To prepare plasmid-liposome complexes (liposomes), the plasmid and liposome mixture (N:P=4:1) was vortexed and kept at room temperature for 15-20 min. The prepared lipoplex was then transferred to a cryogenic bottle and subjected to ten freeze-thaw cycles. The resulting lipoplex passed through 200nm and 100nm membranes in sequence.

The experimental results of dynamic light scattering show that the hydrated diameter of empty liposomes is 106±13.1nm and the PDI is 0.16. The hydrated diameter of the lipoplex is slightly larger than that of the empty liposomes, which is 132±19.5nm and the PDI is 0.19 ( data not shown). The PDI of both empty liposomes and lipoplexes was less than 0.3, indicating uniform particle size.

3.6 Expression of CAR in astrocytes

In order to characterize whether CAR can be fully expressed in astrocytes, as described in Example 1, this example transfected astrocytes with CAR lipoplexes for 24 hours, and then performed cellular immunization on astrocytes. Fluorescence analysis. The results showed that CAR expressed W20 (red fluorescence), 276-994MerTK (yellow fluorescence) and EGFP (green fluorescence) (Figure 23, Panel A). The membrane proteins of astrocytes were further extracted and Western blot analysis was performed. The results showed that the CAR receptor contained Mertk and W20 protein fragments with a molecular weight of about 200 kDa, which was consistent with the theoretical molecular weight (Figure 23, B).

In order to characterize the expression of CAR in various types of cells, this example uses CAR lipoplexes to transfect different types of cells. The results show that CAR is specifically expressed in astrocytes and in neurons and microglia. is not expressed (Fig. 24). Since the astrocyte-specific promoter (gfa2) is used in the CAR expression plasmid, CAR is only expressed in astrocytes.

3.7 Detection of Aβ phagocytosis ability of CAR-A

Flow cytometry was used to analyze the phagocytosis of Aβ oligomers by CAR-A. Specifically, after fixing the cells with 4% paraformaldehyde for 20 minutes, add 6E10 primary antibody and incubate at room temperature for 1 hour; after washing three times with flow cytometry buffer, add PE-labeled goat anti-mouse secondary antibody and incubate at room temperature for 1 hour; use flow cytometry buffer to incubate for 1 hour at room temperature. After washing three times with buffer, data were collected using a CytoFLEX LX flow cytometer and analyzed using FlowJo X10. The results showed that compared with astrocytes in the control group, the ability of CAR-A to phagocytose Aβ oligomers was significantly improved, while the ability of ns-CAR-A to phagocytose Aβ oligomers had no significant change, indicating that W20 mediated the effect of CAR on Aβ oligomers. Recognition of Aβ oligomers (Figure 25).

3.8 CAR-A does not cause inflammatory response when clearing Aβ oligomers

Fluorescent quantitative PCR was used to determine the pro-inflammatory cytokines (IL-1β, TNF-α, iNOS and IL-6) and anti-inflammatory cytokines (IL-4, Arg -1, CD206 and TGF-β) expression levels, the primers used were as described above. The results showed that when astrocytes and ns-CAR-A engulfed Aβ oligomers, pro-inflammatory cytokines were significantly increased and anti-inflammatory cytokines were significantly decreased. When CAR-A engulfed Aβ oligomers, there was no significant increase in pro-inflammatory cytokines and no significant decrease in anti-inflammatory cytokines (Figure 26). These results indicate that CAR-A does not cause an inflammatory response when phagocytosis of Aβ oligomers.

Example 4 Research on the treatment of AD mice using chimeric antigen receptor programmed astrocytes

Under AD pathological conditions, astrocytes have reduced phagocytosis of Aβ and continue to release inflammatory factors. Regulating the function of astrocytes in the brain, enhancing their ability to phagocytose Aβ, 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 fluorescence imaging and immunohistochemistry to evaluate the metabolism of liposome gene delivery particles in the body and the expression of chimeric antigen receptors in the brain; through water maze, novel identification Zhihe Y maze experiment was used to detect the effect of CAR-A on the memory ability of AD mice, and immunohistochemistry and ELISA were used to detect pathological changes in the brains of AD mice.

4.1 Experimental animals

Six-month-old APP/PS1 transgenic mice and 6-month-old male wild-type mice were purchased from Beijing Huafukang Biotechnology Co., Ltd. Mice were raised in a clean room with a room temperature of 22±2°C and a humidity of 45%±10%. The mice had free access to food and water. All animal experiments were conducted in accordance with the "Guidelines for the Care and Use of Laboratory Animals of the Chinese Public Health Service" conduct. Experiments involving mice were approved by the Animal Care and Use Committee of Tsinghua University.

4.2 Experiment

Animal experiment administration and grouping, Morris water maze experiment, Y maze experiment, new thing recognition experiment, collection and processing of mouse brain tissue, preparation of brain homogenate, immunohistochemistry and immunofluorescence were performed as described in Example 2. Analysis, determination of Aβ40 and Aβ42 in the mouse brain, real-time quantitative fluorescence PCR to determine the transcription levels of pro-inflammatory cytokines and anti-inflammatory cytokines-related genes in the mouse brain, Western blot experiment to determine GFAP in the mouse brain of different experimental groups , Iba-1 protein level.

4.3 Experimental results

4.3.1 Lipid complexes can effectively enter the brain

The results show that after nasal administration, CAR lipid complexes are mainly distributed in the brain, lung, liver and other tissues. The CY7 signal in the brain reaches the highest at 3h, and there is still CY7 signal in the brain 24h later (Figure 27), which shows that the CAR lipid complex The complex can enter the mouse brain through the nasal cavity. Immunofluorescence results showed that CAR had good co-localization with astrocytes, indicating that CAR could effectively infect and express astrocytes in the mouse brain (Figure 28).

4.3.2 CAR-A can improve the cognitive function of AD transgenic mice

After treating AD mice with CAR lipoplex for 6 weeks, Morris water maze, Y maze and novel object recognition experiments were used to test the impact of CAR-A on the cognitive function of AD transgenic mice.

First, the Morris water maze test was used to evaluate the spatial memory and learning ability of mice. During the training period, compared with wild-type mice (WT-PBS and WT-NP), AD-PBS (PBS control group AD mice), AD- The time for NP (empty vector control group AD mice) and AD-ns-CAR (non-specific scFv-CAR control group AD mice) to reach the platform was significantly increased, while the time for AD mice given CAR lipoplex to reach the platform was significantly increased. Significantly reduced (Figure 29, A and C), indicating that the CAR lipoplex can effectively improve the spatial memory and learning ability of mice. In the post-weaning test, compared with mice in the AD-PBS, AD-NP and AD-ns-CAR groups, the time for AD mice given CAR lipoplexes to first cross the target location was significantly shortened (Figure Figures B and D of Figure 29), the residence time in the target quadrant increased significantly (Figure E of Figure 29), and the number of times crossing the platform area increased significantly (Figure F of Figure 29). The above results indicate that CAR lipoplexes can significantly improve cognitive dysfunction in AD transgenic mice.

The effect of CAR-A on the short-term memory of AD mice was further evaluated through the Y maze experiment. The results showed that the number of times WT mice entered the new arm and the time they stayed in the new arm were significantly higher than those of AD-PBS, AD-NP and AD-ns-CAR, while AD mice given CAR lipoplexes had higher The residence time and the number of explorations increased significantly (Figure 30), indicating that the CAR lipoplex can significantly improve the short-term memory ability of AD transgenic mice.

4.3.3 CAR-A can reduce the level of Aβ oligomers in the brain

Immunofluorescence results showed that the levels of Aβ oligomers in astrocytes in the brains of mice in the AD-PBS, AD-NP and AD-ns-CAR groups were lower, indicating that AD-PBS, AD-NP and AD-ns- Astrocytes in the CAR group had a weak ability to phagocytose Aβ oligomers, while astrocytes modified by CAR had a significantly enhanced ability to phagocytose Aβ oligomers (Figure 31).

In this example, the ELISA method was further used to determine the content of Aβ oligomers in the mouse brain. The results showed that compared with the AD-PBS, AD-NP and AD-ns-CAR groups, the content of Aβ oligomers in the brains of mice in the AD-CAR experimental group was significantly reduced (Figure 32). The above results show that CAR-A can phagocytose a large amount of Aβ oligomers in the brains of AD mice, thereby reducing the levels of Aβ oligomers in the brains of mice.

4.3.4 CAR-A can reduce the levels of senile plaques and Aβ40/Aβ42 in the brain

The effect of CAR-A on Aβ plaque levels in the brains of AD mice was evaluated. The results of immunohistochemistry staining of Aβ plaques and Aβ plaque area statistics showed that compared with AD-PBS, AD-NP and AD-ns-CAR Compared with the AD-CAR group, the Aβ plaque area in the brain of mice in the AD-CAR group was significantly reduced (Figure 33).

The ELISA method was further used to determine the levels of insoluble Aβ40 and Aβ42 in the mouse brain. The results showed that compared with the AD-PBS, AD-NP and AD-ns-CAR groups, the levels of insoluble Aβ40 and Aβ42 in the brains of AD mice given CAR lipoplexes were significantly reduced (Figure 34). CAR-A significantly reduces the level of Aβ oligomers in the brain by engulfing Aβ oligomers, and the reduction in Aβ oligomer content further reduces the formation of Aβ plaques, thereby reducing the levels of total Aβ40 and Aβ42 in the brain.

4.3.5 CAR-A can improve the inflammatory microenvironment in the brain of AD mice

qPCR measured the levels of pro-inflammatory cytokines and anti-inflammatory cytokines in mouse brains. The results of qPCR experiments showed that compared with WT mice, mice in the AD-PBS, AD-NP and AD-ns-CAR groups had higher levels of pro-inflammatory cytokines (IL-1β, TNF-α, iNOS, IL-6) in the brains. The levels of CAR lipoplexes were significantly increased, and the levels of anti-inflammatory cytokines (IL-4, Arg-1, TGF-β) were significantly decreased, while the pro-inflammatory and anti-inflammatory cytokines in the brains of AD mice given CAR lipoplexes The levels returned to close to WT levels (Fig. 35).

4.3.6 CAR-A reduces gliosis in the brain of AD mice

Western blot method was used to measure the protein levels of GFAP and Iba-1 in the mouse brain to evaluate the proliferation of astrocytes and microglia in the mouse brain. The results showed that CAR lipoplex can effectively reduce the risk of AD mice Glial cell proliferation in the brain (Figure 36).

4.3.7 CAR-A reduces synaptic loss in the brain of AD mice

Synaptic dysfunction and synaptic loss are one of the main pathological features of AD. The presynaptic proteins synaptophysin (SYN) and postsynaptic protein 95 (PSD95) are the signature proteins of presynaptic and postsynaptic synapses respectively. This example uses immunofluorescence to immunostain presynaptic and postsynaptic proteins in the mouse brain to evaluate the synaptic level in the mouse brain. The results showed that compared with WT mice, the synaptic levels in the brains of mice in the AD-PBS and AD-NP groups were significantly reduced, and the synaptic levels in the brains of AD mice given CAR lipoplexes were significantly increased, while the AD-ns -The level of synapses in the brains of mice in the -CAR control group did not increase significantly (Figure 37).

Example 5 Research on the treatment of PD mice using chimeric antigen receptor programmed astrocytes

Under PD pathological conditions, astrocytes have reduced phagocytosis of α-synuclein and continue to release inflammatory factors. Therefore, regulating the function of astrocytes in the brain, enhancing their ability to phagocytose α-synuclein, and reducing the release of inflammatory factors is an ideal strategy for the treatment of 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 the body and the expression of chimeric antigen receptors in the brain; through the water maze , novel object cognition and Y-maze experiments were used to detect the impact of CAR-A on the memory ability of PD mice, and immunohistochemistry and ELISA were used to detect pathological changes in the brains of PD mice (data not shown).

Equivalent solution

Although various embodiments of the invention have been described and illustrated herein, one of ordinary skill in the art will readily envision one or more of the inventions being used to perform the functions described herein and/or to obtain the results described herein. Various other means and/or structures may be used to advantage, and each such change and/or modification is deemed to be within the scope of the invention. More broadly, those skilled in the art will readily appreciate that all parameters, materials, and settings described herein are intended to be exemplary and that the actual parameters, materials, and/or settings will depend on the specific circumstances in which the teachings of the present invention are utilized. application. 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 illustration only and that within the scope of the appended claims and their equivalents, the invention may be practiced otherwise than as specifically described and claimed. Any combination of two or more such features, systems, articles, materials and/or methods is included within the scope of the invention provided that such features, systems, articles, materials and/or methods are not in conflict with each other.

As used in the description and claims of the present invention, the phrase "and/or" should be understood to mean "either or both" of the elements so combined, that is, present in combination in some cases and not in other cases. element. In addition to the elements specifically identified by the "and/or" clause, other elements may optionally be present, whether related or unrelated to those specifically identified elements, unless expressly stated otherwise. Thus, as a non-limiting example, when used in conjunction with open-ended language such as "comprises," a reference to "A and/or B" in one embodiment may refer to both A and B (optionally including anything other than B). elements other than A); in another embodiment, it refers to B and A (optionally including elements other than A); in yet another embodiment, it refers to both A and B (optionally including other elements); etc.

As used herein in the specification and claims, "or" is to be understood to have the same meaning as "and/or" as defined above. For example, when separating items in a list, "or" or "and/or" should be understood to be inclusive, i.e. including a plurality of elements or at least one of the list of elements, but also more than one, and optionally, Other items not listed. Only terms that expressly indicate the contrary, such as "only one of" or "exactly one of", or when used in claims, "consisting of" will refer to exactly one element of a plurality of elements or a list of elements. Generally, when preceded by an exclusive term such as "any", "an", "only one" or "exactly one", the term "or" as used herein should only be understood to mean exclusive alternatives (i.e. , "one or the other but not both"). “Consisting essentially of” when used in a claim shall have its ordinary meaning in the field of patent law.

As used herein in the specification and claims, when referring to a list of one or more elements, the phrase "at least one" shall be understood to mean at least one element selected from any one or more elements of the list of elements, However, it 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 those specifically identified in the list of elements referred to by the phrase "at least one" may optionally be present, whether or not related to those specifically identified elements. Thus, by way of 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" "A") may refer to at least one, optionally including more than one A, without B (and optionally including elements other than B); in another embodiment, refers to at least one, optionally including more than In a B, A is absent (and optionally includes elements other than A); in yet another embodiment, refers to at least one, optionally including more than one A, and at least one, optionally including more than one A to a B (and optionally other elements); etc.

In the claims and the above description, all connectives such as "includes", "includes", "with", "has", "contains", "involves", "has", etc. are to be understood as open-ended, i.e. Means including but not limited to. Only the connectives "consisting of" and "consisting essentially of" shall be closed or semi-closed connectives respectively.

The use of sequential terms in the claims, such as "first", "second", "third", etc., to modify a claim element does not itself imply any priority of one claim element over another claim element. precedence or order, or the temporal order in which actions are performed in a method, and merely used as a label to distinguish one claim element with a certain name from another element with the same name (but in ordinal terms), to Distinguish claim elements.

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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

<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 acttcacct gaccatcagc agcctgcagc ccgaggactt cgccacctac 2760

tactgccagc agagcgacac cagccccacc accttcggcc agggcaccaa ggtggagatc 2820

aagcgctaaggatcc 2835

<210> 11

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Tnf-α forward primer

<400> 11

gattatggct cagggtccaa 20

<210> 12

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Tnf-α reverse primer

<400> 12

gctccagtga attcggaaag 20

<210> 13

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Il-1β forward primer

<400> 13

cccaagcaat acccaaagaa 20

<210> 14

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Il-1β reverse primer

<400> 14

gcttgtgctc tgcttgtgag 20

<210> 15

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> IL-6 forward primer

<400> 15

ccggagagga gacttcacag 20

<210> 16

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> IL-6 reverse primer

<400> 16

ttgccattgc acaactcttt 20

<210> 17

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> iNos forward primer

<400> 17

cacctggaac agcactctct 20

<210> 18

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> iNos reverse primer

<400> 18

ctttgtgcga agtgtcagtg 20

<210> 19

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> IL-4 forward primer

<400> 19

atccatttgc atgatgctct 20

<210> 20

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> IL-4 reverse primer

<400> 20

gagctgcaga gagctttcg 20

<210> 21

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Tgf-β forward primer

<400> 21

ttacctggat ggaagtggaa 20

<210> 22

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Tgf-β reverse primer

<400> 22

tgttatgagg aaggggacaa 20

<210> 23

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Arg1 forward primer

<400> 23

aagccaaggt taaagccact 20

<210> 24

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Arg1 reverse primer

<400> 24

cgattcacct gagctttgat 20

<210> 25

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> CD206 forward primer

<400> 25

tcagctattg gacgcgaggc a 21

<210> 26

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> CD206 reverse primer

<400> 26

tccgggttgc aagttgccgt 20

<210> 27

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Gapdh forward primer

<400> 27

tgaatacggc tacagcaaca 20

<210> 28

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Gapdh reverse primer

<400> 28

aggcccctcc tgttattatg 20

<210> 29

<211> 2985

<212> DNA

<213> 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

<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 gcggggatgac 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

agaacaccccc 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

<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 ttaaaagg 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 gagctttacct 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 tcaacggggag 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 acccccaacga 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) an anti-amyloid oligomer binding domain; 2) a transmembrane domain; and 3) Intracellular signaling domain. 2. The nucleic acid molecule according to claim 1, wherein the amyloid oligomer is a β-amyloid oligomer, preferably, the β-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 The domain is an anti-β-amyloid protein oligomer binding domain. More preferably, the anti-β-amyloid protein oligomer binding domain is a single-chain antibody W20, Aducanumab or Cretuzumab. Anti-(Crenezumab), wherein the amino acid sequence of single-chain antibody W20 is shown in SEQ ID NO: 1. 4. The 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 the anti-inflammatory receptor is The intracellular signaling domain is an intracellular component selected from the group consisting of scavenger receptor class A (SR-A), MerTK, Tyro3, Ax1, ItgB5, BAI1, ELMO, MRC1, Stabilins, ADGRB1, TIMs, and αVβ3/αVβ5 integrins. Signaling domain. 5. The nucleic acid molecule according to any one of claims 1-4, wherein the transmembrane domain is derived from SR-A, MerTK, Axl, Tyro3, Tim1, Tim4, Tim3, FcR, BAI1, CD4, DAP12, A transmembrane domain composed of MRC1, CD8α, CD3, ICOS and CD28. 6. The nucleic acid molecule according to any one of claims 1-5, wherein the binding domain of the anti-amyloid oligomer co-fuses to express SR-A, MerTK receptor, glycosylation end product receptor, G Protein-coupled receptors, CC receptors, CXC receptors, C receptors, CX3C receptors or lamp2a receptors. 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 carrier of claim 7, and a pharmaceutically acceptable excipient. Preferably, the pharmaceutical composition For nanoparticles prepared using liposomes, preferably, the liposomes contain one or more cationic lipids and/or one or more non-cationic lipids. 9. The nucleic acid molecule of any one of claims 1 to 6, the carrier of claim 7 or the pharmaceutical composition of claim 8 is prepared for use in promoting cellular clearance of amyloid oligomers in a subject. , use in medicines to treat and/or prevent inflammatory diseases, reduce senile plaques in the brain, treat and/or prevent neurodegenerative diseases, inhibit gliosis, and improve the level of brain synapses. 10. Use according to 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.