Exosome mediated in vivo FAP targeting CAR-T cell therapy in atherosclerosis
M Yi, J LiAbstract
Background
Atherosclerosis is the primary pathological basis of coronary heart disease, in which the phenotypic transformation of vascular smooth muscle cells constitutes a key pathological step in plaque progression. Fibroblast activation protein (FAP) is scarcely expressed in normal tissues but is significantly upregulated in modulated smooth muscle cells within plaques, and is associated with collagen degradation and plaque instability. Studies have confirmed that FAP gene deletion reduces plaque area and increases fibrous cap thickness in mice. Single-cell multi-omics studies have identified FAP-positive cells within plaques as an important cell subpopulation arising from abnormal differentiation of smooth muscle cells. In recent years, CAR-T cell therapy has been widely applied beyond oncological diseases, and the delivery of mRNA via vectors to generate FAP-CAR-T cells in vivo has demonstrated fibrosis suppressive effects in various tissue diseases.
Purpose
This study aims to construct a T cell-targeted exosome delivery system to generate FAP-CAR-T cells in vivo and explore its feasibility for treating atherosclerosis by eliminating modulated smooth muscle cells.
Methods
293T cells were transfected with FAP-CAR mRNA-MS2bs and anti-CD5 scFv-LAMP-2B-MS2 plasmids. The exosome secreted by 293T cells were isolated and the FAP-CAR mRNA loading and anti-CD5 expression were verified by rt-PCR and flow cytometry. Purified exosomes were co-cultured with T cells, and the proportion of T cells expressing FAP-CAR was detected by flow cytometry. The cytotoxic effect of FAP-CAR-T cells on induced fibromyocytes was evaluated through cytotoxicity assays. In vivo, exosomes were injected into atherosclerosis,mouse model and plaque burden was evaluated using Oil Red O staining.
Results
Exosomes loaded with FAP-CAR mRNA and displaying anti-CD5 single-chain antibodies on their membrane surface (CD5/Exo-FAP-CAR) were constructed. FAP-CAR expression rate of T cells was 61.2±5.8% after T cells co-cultured with CD5/Exo-FAP-CAR. The killing rate of fibromyocytes by FAP-CAR-T cells was 7-fold higher than that of control T cells at an effector-to-target ratio of 5:1 (T cells: fibromyocytes). FAP-CAR positive rate of T cells in mouse splenic was 29.2%±3.1% after tail vein injection of CD5/Exo-FAP-CAR at a dose of 10⁸ exosomes/100g body weight. The administration of CD5/Exo-FAP-CAR significantly reduced the aortic plaque area by 28% in a mouse model of atherosclerosis.
Conclusions
Our data provide causal evidence that exosomes can serve as in vivo delivery vehicles to transport FAP-CAR mRNA to T cells, enabling CAR-T cell immunotherapy in vivo to specifically target and eliminate FAP-positive cells within atherosclerotic plaques, thereby reducing plaque area. This study highlights the potential application of cell-targeted interventions in the treatment of atherosclerosis.