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Background Glioblastoma (GBM) poses significant challenges for CAR-T therapy due to antigenic heterogeneity and immunosuppression, making effective treatment difficult. To address these limitations, this study aims to develop a novel CAR-T therapy strategy, which knocks out intracellular immune checkpoint CISH and targets a common antigen, Fibroblast Activation Protein α (FAPα), shared by GBM cells and their stromal cells, to enhance the efficacy of CAR-T therapy for GBM.Methods FAPα expression levels and prognostic value in glioma patients were analyzed using TCGA/GEO databases. Subsequently, protein expression of FAPα in GBM cell lines was verified through the Human Protein Atlas database and flow cytometry. Further, immunohistochemistry (IHC) and immunofluorescence (IF) analyses of patient-derived histopathological sections were performed to characterize FAPα expression patterns and spatial distribution features. For CAR-T construction, a specific and high-affinity anti-FAPα nanobody was selected via yeast surface display system. CAR construction was designed and introduced into T cells at the CISH locus using cssDNA-based CRISPR-Cas9-mediated site-specific integration technology, achieving both CAR expression and CISH knockout (KO). Functional validation was conducted in vitro and in orthotopic GBM xenografts mice. Finally, their anti-GBM efficacy was validated in patient-derived GBM organoids (GBOs).Results FAPα expression was significantly elevated in GBM compared to LGG or normal brain tissue and associated with poor prognosis ( figure 1A, B). Notably, IHC and IF analysis revealed a dual localization pattern of FAPα in GBM, with enrichment in both tumor parenchyma and perivascular cellular compartments (figure 1C, D). A high-affinity FAPα-specific nanobody (clone 1A10) was subsequently isolated through yeast surface display screening (figure 1E). The non-viral CISH locus-targeted CAR-T cells were generated efficiently (figure 1F-I). CAR-T cells engineered with CISH locus-targeted 1A10-CAR integration displayed enhanced proliferative capacity and significantly improved cytotoxicity against FAPα+ targets in vitro (figure 1J,K). In GBM-bearing mice, these modified CAR-T cells achieved extended survival and superior tumor control compared to controls (figure 1L,M). In GBOs, CISH locus-targeted FAPα-CAR-T cells induced rapid structural disruption (figure 1N).Conclusions Non-viral CISH locus-specific integrated FAPα-targeting CAR-T cells generates potent anti-GBM efficacy by enhancing T cell function and targeting a common antigen shared by GBM cells and their stromal cells. Therefore, our results provides a novel strategy for improving anti-GBM efficacy of current CAR-T cells.Acknowledgements This work was supported by Hospital-Industry Collaboration Project (GRGZ2022330 and GRGZ20230520), the National Natural Science Foundation of China Grants (82373302), Science and Technology Projects of Guangzhou (2024A04J6613 and 2023A04J2362), and Science and Technology Plan Project of Guangdong (2024A0505040015).Ethics Approval This study involved the use of patient-derived pathological specimens and surgical tissues for organoid culture experiments. The protocol was approved by the Ethics Committee of Southern Hospital (Approval No. NFEC-202407-K17). All procedures complied with Chinese ethical guidelines and were conducted with written informed consent from participants.All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Southern Medical University Nanfang Hospital Experimental Animal Center (Approval No. IACUC-LAC-20240401-004).Consent Written informed consent was obtained from the patient for using surgical specimens to generate organoids and publish related research data, including microscopic images of derived organoid models. All identifiable information has been anonymized following HIPAA standards. A copy of the consent form is archived in the institutional review board office and available for editorial review.Abstract 217 Figure 1