Use of fap protein as a target for intervention in skeletal muscle aging

Abstract

This invention belongs to the field of skeletal muscle aging intervention, specifically involving the use of FAP protein as a target for intervention in skeletal muscle aging. After analyzing human skeletal muscle single-cell / monocyte transcriptome data, this invention found that FAP protein is highly expressed in senescent skeletal muscle cells. Based on this target, CAR-M cells were constructed, verifying that FAP protein can clear senescent FAP. + The invention relates to the role of fibroblasts and the promotion of skeletal muscle cell differentiation. Furthermore, the CAR-M therapy developed in this invention for intervening in skeletal muscle aging is a therapy that specifically eliminates senescent cells highly expressing FAP. Simultaneously, the "elimination-repair" dual-function CAR-M cell therapy developed in this invention possesses multiple functions, including eliminating senescent cells, repairing MuSCs niches, promoting muscle regeneration, and enhancing the function of senescent skeletal muscle organoids. In summary, this invention provides novel targets and corresponding cell therapies for treating skeletal muscle aging, offering new options for its treatment.

Description

Technical Field

[0001] This invention belongs to the field of skeletal muscle aging intervention, specifically involving the use of FAP protein as a target for intervention in skeletal muscle aging. Background Technology

[0002] Skeletal muscle aging is a progressive degenerative process involving multiple cells and factors, essentially stemming from the disruption of the dynamic balance among muscle fibers and their microenvironment components. However, due to the complexity of skeletal muscle tissue structure and the heterogeneity of muscle fiber composition, our understanding of the mechanisms of skeletal muscle aging remains limited. Current research suggests that the decline in skeletal muscle function during aging is the result of a coordinated disruption of muscle fibers, muscle stem cells (MuSCs), fibroblasts / adipogenic progenitors (FAPs), fibroblasts, the extracellular matrix, the vascular system, the immune system, and the nervous system. MuSCs are the core executors of muscle regeneration, and their function is primarily regulated by the dynamics of FAPs and macrophages within the microenvironment. FAPs are a population of mesenchymal cells in the muscle stroma, each with different functions in muscle regeneration. Following muscle injury, FAPs are activated, proliferate, and expand, providing a transiently favorable microenvironment to promote MuSC-mediated muscle regeneration. However, with increasing age, FAPs become dysregulated, shifting from a regenerative phenotype to a fibrotic phenotype and accumulating abnormally, leading to collagen deposition and muscle fibrosis. At the same time, changes in macrophage immune defense and the secretion of TGFβ1 cause abnormal FAP clearance and further drive the fibrotic storm, prompting the remodeling of the muscle microenvironment.

[0003] The persistent accumulation of senescent interstitial cells (especially dysfunctional free radical adducts) in muscle tissue is a core contributing factor to skeletal muscle fibrosis and functional decline in the elderly. Therefore, targeted clearance of senescent cells has become an important research direction for the intervention of sarcopenia. Recent studies have found that cell therapy has shown excellent performance in targeted clearance of senescent cells. Chimeric antigen receptor T-cell (CAR-T) therapy can effectively clear senescent cells. For example, CAR-T cell therapy specifically targeting uPAR can clear senescent cells and delay the aging phenotype; in vivo CAR-T therapy targeting uPAR with LNP-oRNA can specifically clear senescent immune cells, thereby alleviating uPAR-related liver fibrosis and rheumatoid arthritis; CAR-T cells targeting NKG2D can clear senescent cells in aged mice and non-primates and increase muscle fiber size, grip strength, range of motion, and maximum speed of movement, thereby improving muscle function and delaying aging. However, CAR-T cells still have significant limitations, such as life-threatening CAR-T cell-related toxicity and poor tissue infiltration ability, which severely limits their application in the treatment of sarcopenia.

[0004] On the other hand, chimeric antigen receptor macrophages (CAR-M) make it possible to specifically clear senescent cells in skeletal muscle due to their ability to easily infiltrate solid tissues and precisely target and engulf target cells. However, strategies for using CAR-M cell therapy to intervene in skeletal muscle aging have not yet been reported. More importantly, conventional CAR-M has a single function, only capable of clearing target cells and lacking tissue repair capabilities, while also potentially posing a risk of tissue damage.

[0005] Therefore, there is an urgent need to develop new targets and strategies for skeletal muscle aging intervention to address sarcopenia. Summary of the Invention

[0006] Based on the above, this invention aims to provide a novel target for intervening in skeletal muscle aging, and to develop a CAR-M cell therapy for skeletal muscle aging intervention based on this target. Furthermore, the cell therapy is modified to obtain a dual-function CAR-M cell therapy that simultaneously clears senescent FAPs, releases the regeneration-promoting factor IGF-1 to repair the aging microenvironment of skeletal muscle, activates the proliferation and differentiation of muscle satellite cells, and ultimately synergistically promotes skeletal muscle regeneration, improves muscle function and motor ability in the elderly, and provides a novel treatment strategy for skeletal muscle aging and sarcopenia that combines targeting, safety, and regenerative repair potential.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: On the one hand, the present invention provides the use of FAP-targeting formulations in the preparation of agents for intervening in skeletal muscle aging.

[0008] Further, the FAP includes the FAP protein and / or the FAP gene, wherein the FAP protein includes the amino acid residue sequence shown in SEQ ID NO: 1; and the FAP gene includes the nucleotide sequence shown in SEQ ID NO: 2.

[0009] The amino acid residue sequence (SEQ ID NO: 1) of the FAP protein is as follows: MKTWLKTVFGVTTLAALALVVICIVLRPSRVYKPEGNTKRALTLKDILNGTFSYKTYFPNWISEQEYLHQSEDDNIVFYNIETRESYIILSNSTMKSVNATDYGLSPDRQFVYLESDYSKLWRYSYTATYYIYDLQNGEFVRGYELPRPIQYLCWSPVGSKLAYVYQNNIYLKQRPGDPPFQITYTGRENRIFNGIPDWVYEEEMLATKYALWWSPDGKFLAYVEFNDSDIPIIAYSYYGDGQYPRTINIPYPKAGAKNPVVRVFIVDTTYPHHVGPMEVPVPEMIASSDYYFSWLTWVSSERVCLQWLKRVQNVSVLSICDFREDWHAWECPKNQEHVEESRTGWAGGFFVSTPAFSQDATSYYKIFSDKDGYKHIHYIKDTVENAIQITSGKWEAIYIFRVTQDSLFYSSNEFEGYPGRRNIYRISIGNSPPSKKCVTCHLRKERCQYYTASFSYKAKYYALVCYGPGLPISTLHDGRTDQEIQVLEENKELENSLRNIQLPKVEIKKLKDGGLTFWYKMILPPQFDRSKKYPLLIQVYGGPCSQSVKSVFAVNWITYLASKEGIVIALVDGRGTAFQGDKFLHAVYRKLGVYEVEDQLTAVRKFIEMGFIDEERIAIWGWSYGGYVSSLALASGTGLFKCGIAVAPVSSWEYYASIYSERFMGLPTKDDNLEHYKNSTVMARAEYFRNVDYLLIHGTADDNVHFQNSAQIAKALVNAQVDFQAMWYSDQNHGISSGRSQNHLYTHMTHFLKQCFSLSD The nucleotide sequence of the FAP gene (SEQ ID NO: 2) is as follows: After analyzing human skeletal muscle single-cell / monocyte transcriptome data, this invention found that FAP protein is highly expressed in senescent skeletal muscle cells, and constructed CAR-M cells based on this target to verify the function of FAP protein in clearing senescent fibroblasts and promoting skeletal muscle cell differentiation.

[0010] Furthermore, the reagents used to intervene in skeletal muscle aging can promote myoblastic differentiation of aging skeletal muscle organoids and / or enhance the function of aging skeletal muscle organoids and / or improve motor function and / or muscle function in elderly individuals.

[0011] The core mechanism by which chimeric antigen receptor macrophages targeting FAP protein (FAP CAR-M) improve age-related functional decline in skeletal muscle lies in the dual synergistic effect of "clearance + repair". In the microenvironment of aging skeletal muscle, abnormally accumulated FAP-positive senescent cells (including fibro-adipogenic progenitor cells and fibroblasts) occupy the ecological niche of muscle satellite cells (MuSCs) by secreting inhibitory factors such as TGFβ1, inducing chronic inflammation and fibrosis, forming a vicious cycle that inhibits myogenic differentiation. FAPCAR-M specifically recognizes and engulfs FAP-positive senescent cells through its chimeric antigen receptor, on the one hand, relieving TGFβ1-mediated transcriptional repression, releasing ecological niche space, and alleviating inflammation and fibrosis; on the other hand, the bifunctional CAR-M actively activates the myogenic differentiation program of MuSCs through the PI3K / Akt / mTOR signaling pathway via the intracellular segment of IGF-1, upregulating myosin heavy chain (MyHC) expression and promoting myotube formation. In live aged mice, this series of cellular and tissue repairs ultimately translates into a decrease in the proportion of central nuclear fibers in muscle tissue, reduced fibrosis, and a comprehensive improvement in motor abilities such as running capacity, limb grip strength, and voluntary movement distance and speed. Therefore, the effects of promoting myogenic differentiation, enhancing organoid contractile function, and improving motor ability in aged individuals are natural manifestations of the same therapeutic mechanism across three progressive dimensions: molecular and cellular, isolated tissue function, and in vivo overall behavior. Together, they constitute a complete chain of evidence from mechanistic repair to functional reconstruction.

[0012] Furthermore, the senescent cells include any one or more of MuSCs, FAPs, and fibroblasts that highly express FAP protein.

[0013] Furthermore, the senescent cells include senescent cells induced by any one or more of cell replication, drugs, physical factors, or oncogenes.

[0014] It should be understood that, theoretically, the reagent can eliminate senescent cells caused by any factor. Factors that induce senescent cells include, but are not limited to, physical and chemical factors (such as doxorubicin, radiation) and biological factors (such as oncogenes).

[0015] In some specific embodiments, MuSCs / FAPs / fibroblasts are treated with drugs to construct a cell senescence model; more specifically, the drugs include any one or more combinations of H2O2, palbociclib, and etoposide.

[0016] Furthermore, the FAP-targeting formulation includes any one or more of FAP antibodies, siRNAs, or FAP signaling pathway inhibitors. Furthermore, the therapeutic agent for skeletal muscle aging includes a chimeric antigen receptor that targets the FAP protein.

[0017] Furthermore, the chimeric antigen receptor comprises an extracellular segment, a transmembrane domain, and an intracellular segment; the intracellular segment comprises an intracellular signal transduction domain and a co-stimulatory domain, wherein the co-stimulatory domain comprises any one of CD28, 4-1BB, FcRr, TLR2, IL4R, and IL10R.

[0018] This invention constructs macrophages (FAP CAR-M) that target FAP protein and contain multiple chimeric antigen receptors with different co-stimulatory domains. It was found that macrophages that express chimeric antigen receptors with the IL10R co-stimulatory domain have the strongest phagocytic effect and anti-inflammatory effect, and are most suitable for clearing senescent cells that highly express FAP protein. Therefore, the IL10R co-stimulatory domain is preferred.

[0019] In theory, any factor can be attached to the back of a chimeric antigen receptor (CAR) structure to enable the CAR to achieve a certain function.

[0020] In some specific embodiments, the intracellular segment also includes IGF-1 (IGF-1 inserted into the intracellular segment), and the IGF-1 factor has the function of promoting muscle repair and growth.

[0021] Preferably, the intracellular segment contains both a CD28 co-stimulatory domain and an IGF-1 factor.

[0022] In some configurations, the IGF-1 factor is located after the CD28 co-stimulatory domain. The complete CAR structure is CD8leader-scFv-CD8Hinge-CD8TM-CD28-CD3ζ, with IGF-1 preferably located after the intracellular activation region CD3ζ to minimize disruption of the preceding intact CAR structure.

[0023] In some configurations, the IGF-1 factor is located before or after EGFP, which is a marker gene for selection. In other configurations, the IGF-1 factor is located between T2A and P2A, with IGF-1 linked to the preceding CAR structure via T2A and EGFP linked to the preceding CAR structure via P2A. This design ensures that the combination of IGF-1 and CD28 achieves optimal results without affecting the marker gene.

[0024] To further enhance the function of CAR-M in promoting myoblast differentiation and muscle regeneration, this invention, based on the conventional CAR structure, introduces IGF-1 (IGF-1) via T2A after CD3ζ in the intracellular activation region. Figure 14 (B) This yields a CAR structure with a dual "cleanup-repair" function. IGF-1 (Insulin-like Growth Factor 1) is a "core regulator" of muscle growth, repair, and metabolic balance. It maintains muscle mass and function through multiple mechanisms, including activating muscle satellite cells, promoting protein synthesis, and inhibiting catabolism. Introducing IGF-1 into the CAR aims to promote muscle repair and growth. It's important to understand that, theoretically, without disrupting the intact CAR structure, the location of IGF-1 attachment can be determined based on experimental conditions; it can be placed either before or after the CAR structure.

[0025] Furthermore, the therapeutic agent for skeletal muscle aging also includes cells modified with a chimeric antigen receptor targeting the FAP protein, wherein the cells include any one or more of monocytes, macrophages, or dendritic cells. In some specific embodiments, the cells are macrophages.

[0026] In another aspect, the present invention provides the use of a formulation targeting FAP in the preparation of a medicament for improving skeletal muscle histological lesions in elderly individuals, characterized in that the improvement of skeletal muscle histological lesions in elderly individuals includes reducing the proportion of central nucleus muscle fibers in muscle tissue.

[0027] In aging skeletal muscle, the presence of central nucleus muscle fibers is a marker of chronic muscle degeneration and abnormal regeneration. Due to the continuous damage, inflammation, and fibrosis in the aging microenvironment, the regeneration process of muscle fibers is disordered, and newly generated muscle fibers cannot mature normally and migrate their nuclei to the periphery, resulting in an increased proportion of central nuclei.

[0028] FAP CAR-M fundamentally improves the inhibitory senescent microenvironment that hinders normal muscle fiber maturation by clearing senescent FAP-positive cells and (in bifunctional CAR-M) secreting IGF-1, enabling newly formed muscle fibers to complete the full maturation process and properly locate the cell nucleus to the periphery, thereby reducing the proportion of central nucleus muscle fibers at the overall histological level.

[0029] In another aspect, the present invention provides a chimeric antigen receptor that targets the FAP protein, wherein the FAP protein comprises the amino acid residue sequence shown in SEQ ID NO: 1 or a sequence that is 60%, 70%, 80%, 85%, 90%, or more than 95% homologous to the amino acid residue sequence shown in SEQ ID: 1; the chimeric antigen receptor comprises an extracellular segment, a transmembrane domain, and an intracellular segment; the intracellular segment comprises an intracellular signal transduction domain and a co-stimulatory domain, wherein the co-stimulatory domain comprises any one of CD28, 4-1BB, FcRr, TLR2, IL4R, and IL10R.

[0030] Further, CD28 comprises the amino acid residue sequence shown in SEQ ID NO: 16, or a sequence that is 60%, 70%, 80%, 85%, 90%, or more than 95% homologous to the amino acid residue sequence shown in SEQ ID: 16; 4-1BB comprises the amino acid residue sequence shown in SEQ ID NO: 17, or a sequence that is 60%, 70%, 80%, 85%, 90%, or more than 95% homologous to the amino acid residue sequence shown in SEQ ID: 17; FcRr comprises the amino acid residue sequence shown in SEQ ID NO: 18, or a sequence that is 60%, 70%, 80%, 85%, 90%, or more than 95% homologous to the amino acid residue sequence shown in SEQ ID: 18; IL4R comprises the amino acid residue sequence shown in SEQ ID NO: 19, or a sequence that is 60%, 70%, 80%, 85%, 90%, or more than 95% homologous to the amino acid residue sequence shown in SEQ ID: 19; IL10R comprises the amino acid residue sequence shown in SEQ ID NO: 19; The amino acid residue sequence shown in SEQ ID: 20, or a sequence that is 60%, 70%, 80%, 85%, 90%, or more than 95% homologous to the amino acid residue sequence shown in SEQ ID: 20; the TLR2 includes the amino acid residue sequence shown in SEQ ID NO: 21, or a sequence that is 60%, 70%, 80%, 85%, 90%, or more than 95% homologous to the amino acid residue sequence shown in SEQ ID: 21.

[0031] Preferably, the co-stimulatory domain is IL10R.

[0032] Furthermore, the intracellular segment also includes IGF-1, which comprises an amino acid residue sequence as shown in SEQ ID NO: 47, or a sequence that is 60%, 70%, 80%, 85%, 90%, or more than 95% homologous to the amino acid residue sequence shown in SEQ ID: 47.

[0033] In some embodiments, the co-stimulatory domain includes CD28, and the intracellular segment also includes IGF-1.

[0034] Further, the extracellular segment includes an scFv that recognizes the FAP protein, the scFv comprising a heavy chain and a light chain; the amino acid sequence of the heavy chain is shown in SEQ ID NO: 3, and the amino acid sequence of the light chain is shown in SEQ ID NO: 7, or a sequence that is 60%, 70%, 80%, 85%, 90%, or more than 95% homologous to the amino acid sequences shown in SEQ ID NO: 3 and / or SEQ ID NO: 7.

[0035] In some specific implementations, the scFv comprises a heavy chain and a light chain, which are connected by a Linker (SEQ ID NO: 11).

[0036] In another aspect, the present invention provides a cell expressing a chimeric antigen receptor, wherein the chimeric antigen receptor is as described above, and the cell is one or more of monocytes, macrophages, dendritic cells, and T cells.

[0037] In some specific embodiments, the cells are macrophages.

[0038] In another aspect, the present invention provides a method for preparing chimeric antigen receptor-expressing cells, which uses lentivirus to infect cells to obtain cells expressing chimeric antigen receptors.

[0039] In some embodiments, the cells are immune cells, and more specifically, the cells are one or more of monocytes, macrophages, dendritic cells, and T cells.

[0040] In another aspect, the present invention provides the use of the chimeric antigen receptor or the cells expressing the chimeric antigen receptor in the preparation of agents for treating skeletal muscle aging and / or clearing senescent fibroblasts and / or repairing the aging microenvironment of skeletal muscle and / or promoting myoblastic differentiation.

[0041] In another aspect, the present invention provides the use of a co-stimulatory domain in enhancing the phagocytic capacity and / or anti-inflammatory function of macrophages modified with chimeric antigen receptors targeting FAP proteins, wherein the co-stimulatory domain is IL10R.

[0042] In another aspect, the present invention provides the use of IGF-1 factor in enhancing the myogenic differentiation function of macrophages modified with chimeric antigen receptors targeting FAP protein, wherein the chimeric antigen receptors include a co-stimulatory domain, and the co-stimulatory domain is CD28.

[0043] The beneficial effects of this invention include: 1. This invention provides a novel target for the treatment of skeletal muscle aging: FAP protein / gene; 2. This invention develops a novel CAR-M cell therapy for skeletal muscle aging intervention based on FAP protein. The CAR-M cells can specifically target FAP protein and then clear senescent cells that highly express FAP. Furthermore, the co-stimulatory domain in the CAR structure on the CAR-M cells is optimized to enhance the phagocytic capacity and anti-inflammatory effect of the CAR-M cells, with IL10R being the preferred component. 3. Based on the above-mentioned CAR-M cells, this invention introduces IGF-1 factor into the intracellular segment to further develop a "clearance-repair" dual-function CAR-M cell therapy, which has multiple functions such as clearing senescent cells, repairing MuSCs niches, and promoting muscle regeneration. The combination of IGF-1 factor and CD28 co-stimulatory domain is preferred. Attached Figure Description

[0044] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0045] Figure 1 Analysis of single-cell / monuclear transcriptome data from human skeletal muscle; among which, Figure 1 FAP in the formula: fibroblast activation protein; Figure 2 FAP in young and old skeletal muscle cells + Cell flow cytometry analysis; Figure 3 FAP in young and old skeletal muscle cells + Quantitative analysis of cell flow cytometry results; among which, Figure 3 A in the text is Figure 2 FAP in flow cytometry + Statistical results of cell proportions; Figure 3 B in the text is Figure 2 Statistical results of average fluorescence intensity of FAP in flow cytometry analysis; Figure 4 Analysis of FAP expression in skeletal muscle cell subsets of young mice Figure 5 Analysis of FAP expression in skeletal muscle cell subsets of aged mice Figure 6 The aging models of MuSCs were induced by hydrogen peroxide (H2O2), palbociclib + trametinib, and etoposide, respectively. Figure 7 Statistical results of SA-β-Gal positive cells in a senescent MuSCs cell model; Figure 8 Flow cytometry detection of FAP-positive cells in an etoposide-induced MuSCs aging model; Figure 9 : FAP expression level in an etoposide-induced aging model of MuSCs; Figure 10 : Etoposide-induced Fibroblast aging model; Figure 11 Standardized FAP in an etoposide-induced Fibroblast aging model + Cell distribution map; Figure 12 FAP expression level in an etoposide-induced Fibroblast aging model; Figure 13 Statistical results of the average fluorescence intensity of FAP protein in the etoposide-induced Fibroblast aging model; Figure 14 : CAR structure diagram; where, Figure 14 In the diagram, A represents a conventional CAR structure. Figure 14 B in the diagram represents a dual-function CAR structure for "clearing and repairing". Figure 15 Functional analysis results of FAP CAR-M cells; among which, Figure 15 In the figure, A represents the phagocytic efficiency of FAP CAR-M containing different costimulatory domains on target cells, and VT in 15 represents the empty vector, namely pLVX-EF1α-IRES-ZsGreen1. Figure 15 In this context, B represents the IL-4 level in the cell supernatant; Figure 15 C in the text represents the IL-6 level in the cell supernatant; Figure 15 In this context, D represents the INF-α level in the cell supernatant. Figure 15 E in the text represents the IL-10 level in the cell supernatant; Figure 16 Phagocytosis of senescent MuSCs (snMuSCs) by FAP CAR-M; Figure 17Phagocytosis of senescent fibroblasts (snFibroblasts) by FAP CAR-M cells; Figure 18 Immunofluorescence staining of MyHC, a marker gene for myoblastic differentiation, after CAR-M cells cleared FAP-positive cells; among which, Figure 18 VT in the text refers to the empty carrier. Figure 18 CD28 in: FAP CAR-M expressing the CD28 co-stimulatory domain, Figure 18 CD28-IGF-1 in FAP CAR-M: FAP CAR-M that expresses CD28 co-stimulatory domain and secretes IGF-1. Figure 18 IL10R in: FAP CAR-M expressing the IL10R co-stimulatory domain, Figure 18 IL10R-IGF-1 in FAP CAR-M: expressing the IL10R co-stimulatory domain and secreting IGF-1; Figure 19 Statistical analysis of MyHC immunofluorescence staining results; among which Figure 19 In this context, A represents the myotubular fusion index; Figure 19 In this context, B represents the number of myotubes; Figure 20 IGF-1 level in cell supernatant.

[0046] Figure 21 CAR-M enhances the function of aging muscle organoids; among which, Figure 21 In the image, A represents a 3D image of an aging muscle organoid. Figure 21 B in the figure represents calcium flow signal in aging muscle organoids; Figure 22 Quantification results of calcium flow signals in aging muscle organoids; Figure 23 : Schematic diagram of CAR-M local intramuscular injection strategy; Figure 24 The number of electric shocks administered during a 10-minute running test in mice; Figure 25 Mouse muscle strength test; among which, Figure 25 In this context, A represents the suspension time of the mice in the suspension experiment; Figure 25 In this context, B represents the forelimb strength of the mouse. Figure 25 In this context, C represents the limb strength of the mouse. Figure 26 : Movement trajectory diagram of mice in the mine experiment; Figure 27 : Figure 27 In this context, A represents the distance the mice in the mine experiment moved. Figure 27 In this context, B represents the average speed of the mice in the mine experiment. Figure 27 In this context, C represents the maximum speed of the mice in the mine experiment; Figure 28 Mouse muscle histology; among which, Figure 28 In the diagram, A represents the HE staining result of muscle tissue. Figure 28 B in the figure represents the results of Laminin1 immunofluorescence staining in muscle tissue; Figure 29 Statistical analysis results of mouse muscle histology; among which, Figure 29 In this context, A represents the average area of ​​muscle fibers. Figure 29 In this context, B represents the number of muscle fibers per unit area. Figure 29 In this context, C represents the proportion of central nucleus muscle fibers. Detailed Implementation

[0047] The following description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

[0048] To better understand this invention, definitions and explanations of relevant terms are provided below.

[0049] (1) Chimeric antigen receptor (CAR): refers to a molecule that comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain capable of activating or enhancing an immune response. In some implementations, the extracellular antigen-binding domain of the CAR includes an antibody single-chain variable region fragment scFv.

[0050] (2) Single-chain fragment variable (scFv): The scFv includes the variable heavy chain region and light chain region of the fusion antibody. The scFv may be derived from Fab (instead of from the antibody, for example, obtained from a Fab library). In some embodiments, the scFv is fused to a transmembrane domain and then to an intracellular signaling domain.

[0051] (3) CDR: The complementarity-determining region amino acid sequence of the antibody, which is a hypervariable region of the heavy and light chains of immunoglobulins.

[0052] (4) "Substantially identical" or "substantially homologous": This means that the polypeptide or nucleic acid molecule exhibits at least about 50% homology or identical nucleic acid sequence (e.g., any amino acid sequence described in this invention) to a reference amino acid sequence (e.g., any nucleic acid sequence described in this invention). In some embodiments, such a sequence is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, or at least about 85%. It is at least about 90%, at least about 95%, at least about 99%, or at least about 100% homologous to the sequence of the amino acid or nucleic acid used for comparison.

[0053] (5) Macrophages: These are myeloid immune cells that develop from monocytes after they emerge from blood vessels. They are widely distributed in various organs of the body. Their main physiological functions in normal tissues are: to mediate specific immune responses by processing and presenting antigens; to phagocytose and degrade necrotic cells, debris, and foreign bodies as fixed or free cells, thereby participating in non-specific reactions in the body; and to activate lymphocytes or other immune cells by secreting inflammatory factors, thereby coordinating the inflammatory process.

[0054] (6) Chimeric Antigen Receptor Macrophages (CAR-M): refers to an engineered macrophage that is genetically modified to enable it to target and eliminate specific pathogens, abnormal cells or target cells.

[0055] (7) Myogenic differentiation: refers to the highly programmed and coordinated biological process by which myogenic progenitor cells, mainly myoblasts, emerge from their resting state after being stimulated by specific signals (such as injury or load), undergo proliferation, migration, and fusion, and finally form new multinucleated myotubes with contractile function, and further mature into muscle fibers. It is the cellular basis for skeletal muscle development, growth, and repair and regeneration after injury.

[0056] (8) Skeletal muscle aging: Skeletal muscle aging is not a single event, but a comprehensive decline state that is age-related, progressive, and driven by multiple factors. It is characterized by a significant decline in skeletal muscle mass, strength, and function (the three together are called muscle fitness), accompanied by metabolic homeostasis imbalance, tissue degeneration, and impaired regenerative and repair capabilities. The core clinical phenotype of this pathophysiological state is sarcopenia.

[0057] (9) Skeletal muscle aging microenvironment: The skeletal muscle aging microenvironment refers to the dynamically changing local homeostatic system composed of muscle fibers and all supporting cells, extracellular matrix components, and signaling molecules (nutrient factors, inflammatory factors, hormones, etc.) flowing within them during the aging process. This microenvironment changes from supporting regeneration to inhibiting regeneration and promoting degeneration, which is an important factor driving and aggravating skeletal muscle aging.

[0058] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the antibodies used are listed in Table 1; and the materials and reagents used are commercially available unless otherwise specified.

[0059] Table 1. Antibodies used in this invention

[0060] Example 1: Materials and Methods 1.1 Immunofluorescence staining Each experiment should have at least 3 biological replicates. Taking a 48-well plate as an example, the specific operating steps are as follows: (1) Discard the culture medium, wash the cells with PBS 1-2 times, add 100 µL of 4% paraformaldehyde to each well, and fix the cells at room temperature for 30 min; wash the cells with PBS 3 times, 5 min each time; (2) Add 100 µL of PBS containing 0.1% Triton X-100 to each well and permeate the cells at room temperature for 20 min; wash the cells with PBS 3 times, 5 min each time; (3) Add 100 µL of immunofluorescence rapid blocking buffer to each well, block at room temperature for 15 min, discard the blocking buffer, and do not wash; dilute the primary antibody according to the antibody instructions, add 100 µL of the diluted primary antibody to each well, and incubate overnight at 4°C; (4) Wash cells with PBS 3 times, 5 min each time; add 100 µL of the corresponding fluorescent secondary antibody to each well and incubate at 37°C in the dark for 1 h; wash cells with PBS 3 times, 5 min each time. (5) Add 100 µL of ready-to-use DAPI solution to each well, incubate at room temperature in the dark for 5 min, and wash the cells with PBS 1-2 times; (6) Add 100 µL of PBS to each well, and record the images using a fluorescence microscope. Take at least 5 different fields of view for each well.

[0061] 1.2 Flow cytometry analysis (1) Trypsin digestion of adherent cells, followed by washing with PBS 1-2 times. Resuspend cells in 100 μL PBS; (2) Add the corresponding direct-label primary antibody according to the antibody dilution ratio, and incubate on ice in the dark for 30 min; (3) Centrifuge at 400×g for 5 min, wash with PBS 2-3 times, and then perform flow cytometry analysis.

[0062] 1.3 Construction of a Cellular Senescence Model (1) H2O2 induction model: stimulate cells (preferably MuSCs and fibroblasts) with H2O2 at a final concentration of 200 μM for 4 h, and then replace with fresh culture medium for 3-5 days.

[0063] (2) Palbociclib + Trametinib induced model: cells (preferably MuSCs and fibroblasts) were treated with a final concentration of 1 μM Palbociclib and 50 nM Trametinib for 48 h, and then cultured in fresh medium for 3-5 days.

[0064] (3) Etoposides induction model: cells (preferably MuSCs and fibroblasts) were induced with a final concentration of 10 μM Etoposides for 24 h, and then cultured in fresh medium for 3-5 days.

[0065] 1.4 Cellular senescence β-galactosidase staining (1) For cells cultured in 6-well plates, aspirate the cell culture medium, wash once with PBS, add 1 mL of β-galactosidase staining and fixing solution, and fix at room temperature for 15 min; (2) Remove the cell fixative and wash the cells three times with PBS for 3 min each time; (3) Prepare the staining working solution according to the instructions, add 1 mL of staining working solution to each well, and incubate at 37ºC overnight; (4) Optical microscope imaging and storage, at least 5 different fields of view for each well.

[0066] 1.5 CAR vector construction and plasmid extraction (1) The conventional CAR structure consists of extracellular fragments, a transmembrane domain (TM domain), and intracellular fragments in sequence. See the diagram below for details. Figure 14The extracellular segment A includes the signal peptide CD8 leader, a single-chain variable region fragment of the FAP-specific antibody (anti-FAP scFv), and the CD8 hinge region. The structure of the anti-FAP scFv is the anti-FAP heavy chain variable region (VH) -- Linker -- anti-FAP light chain variable region (VL). Both the VH and VL regions contain three CDR regions, corresponding to anti-FAP VH CDR1~3 and anti-FAP VLCDR 1~3, respectively. The transmembrane domain is CD8-TM. The intracellular segment includes a co-stimulatory domain, a CD3ζ activation region (intracellular signal transduction domain), and a P2A-linked EGFP tag. The co-stimulatory domain includes any one of CD28, 4-1BB, FcRr, TLR2, IL4R, and IL10R. Any amino acid and nucleotide sequences provided in this embodiment are preferred sequences. Specific sequences are shown in Table 2. Table 2 Sequences involved in CAR

[0067]

[0068]

[0069]

[0070] (2) The "clearance-repair" bifunctional CAR structure, based on the conventional CAR structure, introduces IGF-1 via T2A after CD3ζ in the intracellular activation region (see Table 3 for specific sequences). Figure 14 The B in the example. The co-stimulatory domain includes any one of CD28, 4-1BB, FcRr, TLR2, IL4R, and IL10R, with specific sequences shown in Table 2. Any amino acid and nucleotide sequences provided in this example are preferred sequences.

[0071] Table 3. Sequences involved in CAR

[0072] Apart from the sequences provided above, the sequences for the remaining "clean-repair" dual-function CAR structures are exactly the same as those for conventional CAR structures.

[0073] (3) The complete sequences of the above conventional CAR structure and the "clearance-repair" bifunctional CAR structure were sent to Genewise Biotech for gene synthesis, and then... Eco RI and MluThe I restriction site was ligated to the lentiviral vector pLVX-EF1α-IRES-ZsGreen1, where a Kozak sequence was added to the beginning of the CAR structure sequence to enhance translation initiation efficiency; the plasmid was extracted using an endotoxin-free plasmid extraction kit.

[0074] 1.6 Lentiviral Packaging (1) Transfection was initiated when HEK 293T cells were cultured at 37℃ in a 5% CO2 incubator until the confluence reached 80-90%; (2) Prepare the cell transfection system as shown in Table 4, wherein DNA is added in a mass ratio of 1:2:4 for the envelope plasmid pMD2.G, the packaging plasmid psAX2, and the target plasmid. Table 4. Lentiviral Packaging HEK 293T Cell Transfection System

[0075] (3) Carefully add solution B to solution A, mix slowly by blowing and aspirating, and incubate at room temperature for 30 min; (4) Carefully add the mixture of A and B dropwise to the culture dish, and replace the culture medium with fresh culture medium after 6 h; (5) Collect cell culture supernatant at 48 h and 72 h after transfection, filter to remove cell debris using a 0.45 μm cell filter, and concentrate the virus by ultracentrifugation at 30,000 rpm. (6) Resuspend the virus in DMEM and store it in a refrigerator at -80°C.

[0076] 1.7 CAR-M Preparation (1) The bilateral tibias and femurs of 4-week-old C57BL / 6 male mice were separated, and the muscles and connective tissues were removed; (2) Cut open both ends of the tibia and femur, and use a syringe to repeatedly flush out all bone marrow cells with culture medium; centrifuge and wash with PBS 1-2 times; (3) Culture the cells in 10 cm culture dishes with DMEM + 10% FBS + 1% PS + 50 ng / mL M-CSF medium for 3 consecutive days, changing the medium until the confluence reaches 90%-100%, and then pass the cells. (4) Digest the cells, passage them into 6-well plates, and infect them with viruses when the confluence reaches 60%-70%; (5) Add concentrated virus solution at 100 μL / well, add polybrene at a ratio of 1:1,000, and replace with fresh culture medium 24 hours after infection.

[0077] 1.8 Enzyme-linked immunosorbent assay (ELISA) (1) Prepare Cytokine Standard according to the ELISA kit instructions, and dilute Cytokine Standard according to the concentration gradient; (2) Add 100 μL of Cytokine Standard or sample to each well, and add 100 μL of dilution buffer as a blank control well; (3) Add 50 μL of Biotinylated Antibody working solution to each well; mix well, cover with sealing film, and incubate at 37°C for 90 min; (4) Remove the liquid from the well and add 300 μL of 1×Washing Buffer working solution to each well; let it stand for 1 min and then discard the liquid from the well; repeat 4 times, and wipe dry on filter paper each time; (5) Add 100 μL of Streptavidin-HRP working solution to each well; cover with a sealing film and incubate at 37°C for 30 min; (6) Repeat step (4); (7) Add 100 μL TMB to each well and incubate at 37°C in the dark for 10-20 min; (8) Add 100 μL of Stop Solution to each well to terminate the reaction; (9) Terminate the reaction and measure the absorbance at 450 nm wavelength using an enzyme-linked immunosorbent assay (ELISA) reader within 10 min.

[0078] 1.9 CAR-M phagocytosis effect detection (1) Senescent MuSCs cells were labeled with CellTracker CM-Dil for 30 min and then co-cultured with CAR-M for 48 h. The images were stored using fluorescence microscopy.

[0079] (2) Senescent fibroblasts were labeled with CellTracker CM-Dil for 30 min, and young fibroblasts were labeled with CellTracker Deep Red for 30 min. They were then co-cultured with CAR-M for 48 h and the images were stored using fluorescence microscopy.

[0080] 1.10 Construction of co-culture system and induction of myogenic differentiation (1) Construct an etoposides-induced senescent fibroblast model. For specific implementation methods, refer to “Method 1.3” in Example 1. (2) Preparation of CAR-M, the specific implementation plan is described in “Method 1.5” in Example 1.

[0081] (3) Senescent fibroblasts, MuSCs and CAR-M were co-cultured in a ratio of 2:3:3. After 48 h, the culture medium was replaced with myogenic differentiation induction medium (H-DMEM+2% HS+1% double antibiotic) to induce myogenic differentiation of MuSCs.

[0082] (4) Perform MyHC immunofluorescence staining on the myotubes, count the number of cell nuclei in the myotubes and the number of myotubes, and calculate the myotube fusion index, where the myotube fusion index = number of cell nuclei in the myotubes / total number of cell nuclei × 100%.

[0083] 1.11 Construction of aging muscle organoids and evaluation of organoid function (1) Construct an etoposide-induced senescent fibroblast model. For specific implementation methods, refer to “Method 1.3” in Example 1.

[0084] (2) Construct FAP CAR-M and "Clear-Repair" dual-function FAP CAR-M. For specific implementation plan, refer to "Method 1.7" in Example 1.

[0085] (3) Construct a co-culture system of senescent muscle organoids and CAR-M, with a total cell count of 9×10⁶ cells for each organoid. 4 All cells were resuspended in complete medium containing 50% Matrigel (Corning, 354230) at a ratio of 1:4:10:4 for young fibroblasts, senescent fibroblasts, MuSCs and CAR-M. The cells were carefully dropped into a low-adsorption culture dish at a ratio of 10 μL, inverted at 37°C for 45 min to form spheroids, and the spheroids were carefully scraped off and cultured in fresh complete medium for 24 h.

[0086] (4) Myogenic differentiation induction medium (H-DMEM + 2% HS + 1% antibiotics) to induce myogenic differentiation of MuSCs. Change the differentiation medium with fresh medium once a day for 5 consecutive days.

[0087] (5) Electrically stimulate organoids, capture calcium flow imaging videos, and calculate the maximum fluorescence brightness ratio.

[0088] (6) Organoids were subjected to MyHC immunofluorescence staining and microscopic imaging.

[0089] 1.12 Local intramuscular injection of CAR-M and evaluation of animal behavior and motor ability (1) Construct FAP CAR-M and "Clear-Repair" dual-function FAP CAR-M. For specific implementation plan, refer to "Method 1.7" in Example 1.

[0090] (2) CAR-M was injected into the gastrocnemius muscle of 23-month-old aged mice. 50 μL of cell suspension was injected into each leg of each mouse, for a total of 1.0 × 10⁻⁶ cells. 6 10 μL of cells were injected twice, 15 days apart. Five injection sites were selected in the gastrocnemius muscle, and 10 μL was injected at each site. Behavioral and motor function of the mice were analyzed on day 35 after the first injection.

[0091] (3) Running experiment: Before the formal running test, the mice were trained twice to form a memory, once every other day. The training conditions were 5 m / min for 5 min and 10 m / min for 5 min, for a total of 10 min. The conditions for the formal running test were 15 m / min for 10 min, and all other conditions were kept the same. The number of times the mice were shocked within 10 min was recorded.

[0092] (4) Suspension test: Before the formal experiment, the mice were trained to grasp the clothes hanger with their front paws, and the training was repeated twice. After the training, the mice rested for 10 minutes before the formal experiment was conducted. The mice were measured three times, and rested for 2 minutes after each test. The suspension time of the mice was recorded.

[0093] (5) Muscle strength test: Use a small animal gripping force tester to test the gripping force of the forelimbs and limbs, and record the gripping force.

[0094] (6) Mine experiment: ANYmaze behavioral analysis software was used to record the trajectory, distance, average speed and maximum speed of the mice in the mine.

[0095] 1.13 Muscle Histological Analysis (1) Take the gastrocnemius muscle of a mouse, embed the muscle tissue using liquid nitrogen-isopentane freezing method, and perform frozen sectioning.

[0096] (2) The muscle tissue was stained with HE according to the conventional HE staining method and scanned using a digital pathological slide scanning system.

[0097] (3) Perform Laminin immunofluorescence staining on muscle tissue. The immunofluorescence staining method is the same as "Method 1.1" in Example 1, and the digital pathological slide scanning system is used for scanning and imaging.

[0098] (4) Use Image J to calculate the area of ​​muscle fibers, the number of muscle fibers per unit area, and count the number of central nucleus muscle fiber cells, and calculate the proportion of central nucleus fibers. Central nucleus fiber proportion = number of central nucleus fibers / total number of muscle fibers × 100%.

[0099] Example 2: High expression of FAP protein in aging skeletal muscle 2.1 FAP protein expression is upregulated in senescent skeletal muscle cells To explore new therapeutic targets for skeletal muscle aging, this study analyzed human skeletal muscle single-cell / monocyte transcriptome data (CNP0004394, CNP0004495), and analyzed differentially expressed proteins in young and aged skeletal muscle. It was found that fibroblast activation protein (FAP) was highly expressed in aged FAP cells and aged tendon cells compared to the younger group. Figure 1 This indicates that FAP protein is highly expressed in aging skeletal muscle and has the potential to become a target for intervening in skeletal muscle aging.

[0100] To further verify the expression level of FAP protein in skeletal muscle cells at different stages of aging, this study collected muscle tissue from young (2-month-old) and aged (20-month-old) mice. Flow cytometry analysis showed that FAP expression was high in aged skeletal muscle cells. + The proportion of cells increased significantly ( Figure 2 , Figure 3 (A and B in the text), FAP in the aging skeletal muscle FAPs subset + The proportion of cells also increased significantly, approximately three times that of the younger group, with FAP in the MuSCs subpopulation. + The proportion of cells was not different between young and old skeletal muscle cells. Figures 4-5 These results indicate the accumulation of FAPs in aging skeletal muscle, suggesting that FAPs can serve as a marker of FAP subsets in aging skeletal muscle.

[0101] 2.2 FAP expression is upregulated in senescent cell models In this embodiment, three MuSCs-induced aging models were constructed using hydrogen peroxide (H2O2), palbociclib + trametinib, and etoposide, respectively, to further explore the expression pattern of FAP protein. The specific experimental steps were the same as those described in Example 1.

[0102] The results showed that all three induction methods could induce senescence in MuSCs to varying degrees. Figure 6 The proportion of SA-β-Gal positive cells after H2O2 and P+T induction was about 15%, while the proportion of SA-β-Gal positive cells after Etop induction was as high as 70%. Figures 6-7 Flow cytometry analysis revealed increased FAP expression levels in all three induced MuSCs aging models. + The cell ratio also increased ( Figures 8-9 ).

[0103] Furthermore, in the Etopside-induced fibroblast senescence model, FAP +The proportion of cells also increased significantly, and the expression level of FAP increased significantly. Figures 10-13 ).

[0104] Based on all the above experimental results, this embodiment fully demonstrates that FAP protein is highly expressed in senescent skeletal muscle cells, but lowly expressed in normal skeletal muscle cells. It is preliminarily believed that FAP protein can serve as a target for clearing senescent skeletal muscle cells and intervening / treating skeletal muscle aging.

[0105] Example 3: Preparation of FAP-targeting chimeric antigen receptor macrophages (FAP CAR-M) according to Figure 14 The diagram illustrates the construction of a chimeric antigen receptor (FAP CAR) targeting FAP. A conventional FAP CAR structure consists of an intracellular domain, a transmembrane domain, and an extracellular domain. The intracellular domain includes the signal peptide CD8 leader, a single-chain variable region fragment of the FAP-specific antibody (anti-FAP scFv), and the CD8 hinge region; the transmembrane domain is CD8-TM; the extracellular domain includes a co-stimulatory domain, a CD3ζ activation region, and a p2A-linked EGFP tag (…). Figure 14 In the A), the co-stimulatory domains include any one of CD28, 4-1BB, FcRr, TLR2, IL4R, and IL10R. Figure 14 ).

[0106] It is important to understand that the reason why this invention constructs CARs containing different co-stimulatory domains is twofold: first, to select co-stimulatory domains with high phagocytic efficiency for target cells; and second, to consider CAR-M subtypes. Since the elements in the CAR structure may initially be used more for tumor-related research or treatment, CAR-Ms for tumor research are usually M1 type that produce pro-inflammatory effects. However, based on the fact that the aging microenvironment is prone to inflammation, this invention tends to screen for M2 type CAR-Ms with anti-inflammatory effects.

[0107] To further enhance the function of CAR-M in promoting myoblast differentiation and muscle regeneration, this invention, based on the conventional CAR structure, introduces IGF-1 (IGF-1) via T2A after CD3ζ in the intracellular activation region. Figure 14(B) This yields a CAR structure with a dual "cleanup-repair" function. IGF-1 (Insulin-like Growth Factor 1) is a "core regulator" of muscle growth, repair, and metabolic balance. It maintains muscle mass and function through multiple mechanisms, including activating muscle satellite cells, promoting protein synthesis, and inhibiting catabolism. Introducing IGF-1 into the CAR aims to promote muscle repair and growth. It's important to understand that, theoretically, without disrupting the intact CAR structure, the location of IGF-1 attachment can be determined based on experimental conditions; it can be placed either before or after the CAR structure.

[0108] The complete sequences of the above-mentioned conventional CAR structure and the "clear-repair" dual-function CAR structure are obtained through... Eco RI and Mlu The I restriction site was ligated to the lentiviral vector pLVX-EF1α-IRES-ZsGreen1 Vector, in which a Kozak sequence was added to the beginning of the CAR structure sequence to enhance translation initiation efficiency; then, the CAR virus was packaged in HEK 293T cells and then infected with mouse bone marrow-derived macrophages (BMDM) to prepare CAR-M cells, with the specific steps being the same as described in Example 1.

[0109] This invention employs a co-expression approach, linking IGF-1 to the CAR structure via T2A to achieve CAR-M expression and IGF-1 secretion. It should be noted that any scheme capable of achieving CAR-M IGF-1 secretion can be used as an alternative. For example, a conditional release IGF-1 secretion strategy, which couples CAR signaling activation with the CRISPRa system, triggers endogenous IGF-1 expression after CAR-M recognizes and engulfs target cells.

[0110] This invention uses lentiviruses to prepare CAR-M. It should be noted that any transfection method can be used as an alternative, including but not limited to adenovirus infection, LNP delivery of CAR mRNA, etc.

[0111] Example 4: Functional Investigation of FAP CAR-M Cells To further verify the function of the FAP protein and develop a novel cell therapy for intervening in skeletal muscle aging, this embodiment constructed macrophages (FAP CAR-M) expressing the FAP protein and containing conventional chimeric antigen receptors with different co-stimulatory domains, and investigated the function and characteristics of the macrophages. The specific steps were the same as those described in Examples 1 and 3.

[0112] CAR-M cells expressing conventional CAR structures were co-cultured with senescent fibroblasts for 48 h, and flow cytometry analysis was performed. The results showed that CAR-M cells containing different co-stimulatory domains all exhibited high phagocytic activity against senescent target cells. Among them, FAP CAR-M containing the IL10R co-stimulatory domain (IL10R-FAP CAR-M) showed the highest phagocytic efficiency. Figure 15 (A in the middle).

[0113] Furthermore, FAP CAR-M containing different co-stimulatory domains also exhibit phagocytic activity against senescent MuSCs and fibroblasts. Figures 16-17 Among them, CD28-FAP CAR-M and IL10R-FAP CAR-M both exhibited excellent phagocytic function; at the same time, FAP CAR-M targeted phagocytosis of senescent fibroblasts, but did not target young fibroblasts. Figure 17 This demonstrates the targeting specificity of the macrophages and minimal impact on normal cells. In summary, FAP CAR-Ms containing different co-stimulatory domains can simultaneously and specifically target multiple senescent cells expressing or overexpressing FAP proteins, with minimal impact on young cells. Among them, IL10R-FAP CAR-M exhibits the highest targeting phagocytic efficiency.

[0114] In addition to phagocytic function, FAP CAR-Ms should also possess anti-inflammatory effects (see Example 3 for details). Therefore, to screen for FAP CAR-Ms with anti-inflammatory effects, this example also tested the levels of pro-inflammatory and anti-inflammatory factors secreted by different FAP CAR-Ms. The results showed that, compared with CAR-Ms containing other co-stimulatory domains, only CAR-Ms containing the IL10R co-stimulatory domain produced lower levels of pro-inflammatory factors IL-6 and TNF-α and higher levels of the anti-inflammatory factor IL-10. Figure 15 The B~E in the text means that CAR-M containing the IL10R co-stimulatory domain has a lower probability of causing an inflammatory response in the body and belongs to the M2 type CAR-M.

[0115] In summary, this embodiment verifies the effectiveness of FAP as a target for treating skeletal muscle aging. It also explores the targeting specificity, phagocytic function, and anti-inflammatory effects of FAP CAR-Ms containing different co-stimulatory domains. Among them, IL10R-FAP CAR-M has the best overall performance.

[0116] Example 5: FAP CAR-M can eliminate senescent FAP-positive cells and promote myoblastic differentiation. To investigate the effect of FAP CAR-M on myogenic differentiation, this example co-cultured CAR-M with senescent fibroblasts and MuSCs, and induced myogenic differentiation of MuSCs, as detailed in Example 1. The results showed that, compared with the control group VT, the FAP CAR-M group (i.e., the corresponding...)... Figure 18 The myotubular fusion index was significantly increased in the CD28 group and the IL10R group. Figure 18 , Figure 19 In A), there was no significant difference in the number of myotubes ( Figure 18 , Figure 19 (B in the text). The above results indicate that CAR-M scavenges senescent FAP. + In addition to promoting the myogenic differentiation of MuSCs, FAP CAR-M can also significantly enhance the application of FAP CAR-M.

[0117] To further enhance the repair function of FAP CAR-M, this embodiment introduces IGF-1 factor on the basis of conventional CAR structure to prepare FAP CAR-M with dual "clearance-repair" function. The specific construction principle and process are described in Examples 1 and 3.

[0118] Compared to conventional FAP CAR-M, FAP CAR-M that secretes IGF-1 (corresponding to...) Figures 18-20 The CD28-IGF-1 group and the IL10R-IGF-1 group can secrete high levels of IGF-1 regeneration factor (IGF-1). Figure 20 In summary, the above results indicate that the bifunctional CAR-M was successfully constructed. Co-culturing it with MuSCs and FAPs revealed that when the co-stimulatory domain of the CAR was CD28, the introduction of IGF-1 significantly improved the myoblastic differentiation capacity of the FAP CAR-M, i.e., the myotube fusion index was significantly increased. Figure 18 , Figure 19 (A in the text); while when the co-stimulatory domain is IL10R, the synergistic effect of IGF-1 is not obvious.

[0119] In summary, on the one hand, this embodiment demonstrates that FAP CAR-M can not only clear senescent FAP-positive cells, but also promote myoblast differentiation; on the other hand, it demonstrates that the introduction of IGF-1 can enhance the function of FAP CAR-M to a certain extent, with FAP CAR-M containing the CD28 co-stimulatory domain being preferred.

[0120] Example 6: FAP CAR-M can enhance the function of aging muscle organoids To further confirm the function of FAP CAR-M, this embodiment uses the CD28 co-stimulatory domain as an example to construct FAP CAR-M and a "clearance-repair" bifunctional FAP CAR-M. These were co-cultured with senescent Fibroblasts and MuSCs, and then induced to differentiate into muscle organoids for 5 days. MyHC immunofluorescence staining showed that, compared with the control group VT, the muscle organoids in the FAP CAR-M group under 3D culture conditions formed significantly more myotubes. Figure 21 In the A group, calcium flow signaling was also significantly enhanced, and the dual-function CAR-M with "clearance-repair" properties had a stronger ability to enhance calcium flow signaling. Figure 21 B in Figure 22 These results further demonstrate that FAP CAR-M can promote myogenic differentiation and improve the function of muscle organoids.

[0121] Example 7: FAP CAR-M can improve motor ability and muscle function in aged mice. To confirm the function of FAP CAR-M in vivo, this embodiment uses the CD28 co-stimulatory domain as an example to construct FAP CAR-M and a "clearance-repair" dual-function FAP CAR-M. The CAR-M was then injected into the gastrocnemius muscle of 23-month-old aged mice twice, 15 days apart. Behavioral and motor function tests were performed on day 35 after the first injection. Figure 23 The results showed that, compared with the control groups PBS and VT, FAP CAR-M treatment effectively enhanced the running ability of aged mice. Figure 24 ), limb strength ( Figure 25 (C in the text), while also increasing the movement distance of older mice ( Figure 26 and Figure 27 A) Average speed of motion ( Figure 26 and Figure 27 (B) and maximum speed ( Figure 26 and Figure 27 (C) Muscle histological analysis revealed that, compared with the control groups PBS and VT, FAP CAR-M did not affect muscle fiber size (C). Figure 28 A~B in Figure 29 (A~B in the original text), but it can effectively reduce the proportion of central nucleus muscle fibers in aged mice ( Figure 28 A and B in Figure 29 (C in the text). These results collectively demonstrate that FAP CAR-M can effectively improve motor function and muscle function in aged mice.

[0122] The foregoing has shown and described the basic principles and main features of the present invention and its advantages. It will be apparent to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments, and that the present invention can be implemented in other specific forms without departing from the spirit or basic characteristics of the present invention. Therefore, the embodiments should be regarded as exemplary and non-limiting in all respects. The scope of the present invention is defined by the appended claims rather than the foregoing description. Therefore, it is intended that all changes falling within the meaning and scope of the equivalents of the claims be included within the present invention, and no reference numerals in the claims should be regarded as limiting the scope of the claims.

Claims

1. The use of a formulation targeting FAP in the preparation of agents for intervening in skeletal muscle aging, characterized in that, The FAP is the FAP protein and / or the FAP gene, wherein the FAP protein comprises an amino acid residue sequence as shown in SEQ ID NO: 1, and the FAP gene comprises a nucleotide sequence as shown in SEQ ID NO:

2.

2. The use as described in claim 1, characterized in that, The reagents for intervening in skeletal muscle aging can promote myoblastic differentiation of aging skeletal muscle organoids and / or enhance the function of aging skeletal muscle organoids and / or improve motor function and / or muscle function in elderly individuals; the agents targeting FAP include any one or more of FAP antibodies, siRNAs, or FAP signaling pathway inhibitors.

3. The use as described in claim 2, characterized in that, The senescent cells include senescent cells induced by any one or more of cell replication, drugs, physical factors, or oncogenes.

4. The use as described in claim 3, characterized in that, The senescent cells include any one or more of the following: skeletal muscle satellite cells that highly express FAP, fibro-adipogenic progenitor cells, and fibroblasts.

5. The use as described in claim 1, characterized in that, The intervention agent for skeletal muscle aging includes a chimeric antigen receptor that targets the FAP protein.

6. The use as described in claim 5, characterized in that, The chimeric antigen receptor includes an extracellular segment, a transmembrane domain, and an intracellular segment; the intracellular segment includes a co-stimulatory domain, which includes any one of CD28, 4-1BB, FcRr, TLR2, IL4R, and IL10R.

7. The use as described in claim 6, characterized in that, The intracellular segment further includes IGF-1; and / or the co-stimulatory domain includes CD28, and the intracellular segment further includes IGF-1.

8. The use as described in claim 1, characterized in that, The skeletal muscle aging intervention agent also includes cells modified with a chimeric antigen receptor targeting the FAP protein.

9. The use as described in claim 8, characterized in that, The cells include any one or more of monocytes, macrophages, dendritic cells, and T cells.

10. Use of formulations targeting FAP in the preparation of drugs for improving skeletal muscle function decline in elderly individuals via local intramuscular injection.

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