Haizhou Liu

Traumatic brain injury (TBI) disrupts the blood–brain barrier (BBB) and compromises endothelial function, leading to persistent neurological dysfunction. Although miR‐15a/16‐1 deletion has recently been shown to be neuroprotective in brain injury, the endothelial pathways mediating long‐term structural and functional recovery after TBI remain unknown. Here, we demonstrate endothelial cell‐specific deletion of miR‐15a/16‐1 is sufficient to confer neurorestoration after TBI by reducing acute BBB leakage, preserving white and gray matter integrity, and accelerating sensorimotor and cognitive recovery. Mechanistically, transcriptomic profiling identified SYNE1 as a direct downstream target of miR‐15a/16‐1 , validated via in‐silico binding prediction and 3’‐UTR luciferase assays. Crucially, endothelial cell‐targeted AAV‐mediated knockdown of SYNE1 abrogated the neurorestorative effects of miR‐15a/16‐1 deletion, indicating SYNE1 is required for its function. Furthermore, single‐cell RNA‐seq analysis of human TBI tissue demonstrates significant downregulation of SYNE1 in endothelial cells within the contusion core relative to pericontusional endothelium. Together, these findings define a previously unrecognized miR‐15a/16‐1– SYNE1 axis that promotes endothelial repair after TBI, with significant translational potential.

The chimeric antigen receptor (CAR) T cell therapy targeting cluster of differentiation 19 (CD19)‐positive malignancies has shown considerable efficacy in clinical settings. However, the potential genotoxicity of viral‐based CAR‐T therapy, their considerable manufacturing cycle and high costs, prompts questions about its safety and leads to limitations in clinical application. Aiming to explore a safe, efficient, and low‐cost mRNA‐based CAR‐T therapy, we characterize a nonviral, mRNA‐based approach utilizing a current good manufacturing practice (cGMP)‐compatible electroporation (EP) platform to generate anti‐CD19, anti‐CD22, and tandem anti‐CD19/CD22 CAR‐T products with a turn‐around time of less than 48 h. The protocol yields a more than 90% cellular viability with a CAR expression of exceeding 60% within the 24 h of transfection. The mRNA‐based CAR‐T products have a significantly enhanced T cell activation profile with CD69 upregulation, production of tumor necrosis factor‐alpha (TNF‐α), interleukin‐2 (IL‐2), and Granzyme B, and higher Ki67 expression level. Robust in vitro tumor neutralization by the mRNA‐based CAR‐T was observed compared to control. Interestingly, we have observed that the short‐term cell cryopreservation after EP resulted in a 50% reduction of cellular expansion. However, the cryopreservation and thaw did not have an observed negative impact on the in vitro tumor neutralization. Lastly, we have evaluated the metabolic activity of the mRNA‐based CAR‐T products, which has a clearly inducible and significant increase in basal respiration (by 60%) and a 2‐fold spare respiratory capacity (SRC) upon exposure with the antigen positive target cells. We believe this approach holds promise as a viable alternative that addresses the limitations of current CAR‐T therapies, offering a potential solution for future nonviral CAR‐T clinical application.

In recent years, chimeric antigen receptor T‐cell (CAR‐T) therapy has made groundbreaking progress in the treatment of various cancer types, particularly hematological malignancies. In the meantime, various preclinical and clinical studies have extensively explored dual‐target CAR‐T therapies which can be designed to recognize two antigens simultaneously based on the immunophenotype of tumor cells. Compared with single‐target CAR‐T approach, dual‐target CAR‐T therapies demonstrate varying degrees of superior antitumor CAR effects, prevent antigen escape and relapse, reduce on‐target off‐tumor effects, and ensure durable responses in different types of cancer. These advantages highlight the potential future prospects in this field, showing varying degrees of advancement in preclinical and clinical studies. Herein, we aimed to review different dual‐target CAR‐T studies conducted on a wide range of tumor models, summarizing the selection of target combinations, the efficacy and safety demonstrated in preclinical and clinical settings, the existing limitations, and the potential future directions of this promising therapeutic strategy.