Journal Article

Unleashing the potential of a low CpG Passer transposon for superior CAR-T cell therapy

Di, Meng, Huang, Qian, Shao, Meiqi, Zeng, Jianyao, Wang, Xiaodie, Cheng, Jingbo, Fang, Yuan, Liu, Tao, Sun, Yan, Zhang, Pingjing, Wang, Pei, Qian, Qijun

2025

Overview

To date, the non-viral vector Chimeric Antigen Receptor (CAR) T cell preparation platform, exemplified by transposons, has demonstrated significant potential in tumor immunotherapy and yielded positive results in multiple clinical trials. Nonetheless, non-methylated CpG sequences within plasmid DNA can elicit an inflammatory response via Toll-like receptor 9 (TLR9) during CAR-T cell preparation, adversely affecting transgene expression. Additionally, de novo DNA methylation programs promote T cell exhaustion, which poses a significant limitation for CAR-T cell therapy applications. High-throughput liquid protein chip and CBA analyses were utilized to determine the expression levels of inflammatory factors. Flow cytometry and luciferase reporter assays were employed for mutation screening. BALB/c mice and M-NSG mice were used to evaluate the inflammatory response and efficacy of LCG CAR-T , with TIL grouping detected via immunohistochemistry. In this study, we modified the newly discovered Passer (JL) transposon to construct a low-CpG content transposon for CAR-T cell (LCG CAR-T cell) preparation. experiments demonstrated that LCG CAR-T cells prepared using this new transposon exhibited stronger cytotoxicity. In animal models, LCG CAR-T cells significantly inhibited tumor growth and increased the populations of CD4+CAR-T cells and tumor-infiltrating lymphocytes. Furthermore, LCG CAR-T cells modulated pro-inflammatory cytokine release, thereby reducing inflammatory responses and surpassing the effects observed with unmodified CAR-T cells. Collectively, our results demonstrate the high safety and efficacy of non-viral, low CpG Passer transposon CAR-T cells, offering new avenues for improving CAR-T cell efficacy while minimizing inflammation.

• Publisher: Frontiers Media SA,Frontiers Media S.A
• Subject: Animal models / Animals / Antigens / CD4 antigen / Cell culture / Cell Line, Tumor / Cell therapy / Cells / chimeric antigen receptor T cell / Chimeric antigen receptors / Clinical trials / CpG islands / CpG Islands - genetics / CpG motif / Cytotoxicity / DNA Methylation / DNA Transposable Elements - genetics / Female / Flow cytometry / Humans / Immunohistochemistry / Immunology / Immunotherapy / Immunotherapy, Adoptive - methods / Inflammation / Lymphocytes / Lymphocytes T / Lymphocytes, Tumor-Infiltrating - immunology / Lymphocytes, Tumor-Infiltrating - metabolism / methylation / Mice / Mice, Inbred BALB C / Nucleotide sequence / Passer / Proteins / Receptors, Chimeric Antigen - genetics / Receptors, Chimeric Antigen - immunology / TLR9 protein / Toll-like receptors / Transgenes / transposon / Transposons / Tumor-infiltrating lymphocytes / Vectors (Biology)