Engineering Sonogenetic EchoBack-CAR T cells

Introduction: : Chimeric Antigen Receptor (CAR) T-cell therapy has shown remarkable success against blood cancers but challenges remain for solid tumors due to on-target off-tumor toxicities (OTOT), T-cell exhaustion, and limited persistence post-infusion. Notably, OTOT has caused severe adverse effects in vital organs, leading to fatalities.
Advancements in synthetic biology, including sonogenetics, offer remote-controlled gene activation with high spatiotemporal precision, potentially mitigating OTOT in CAR T-cell therapies. These technologies enable efficient targeting of antigens with suboptimal specificity, easing antigen selection constraints for solid tumor treatment. However, challenges remain due to limited inducibility and transient CAR expression, necessitating strategies to enhance CAR expression sustainability for therapeutic efficacy.
To address this, we engineered ultrasound-controllable sonogenetic CAR T cells using an ultrasensitive heat-shock promoter screened from a library, integrated with a positive feedback loop from CAR signaling to drive CAR expression. This system enables long-lasting CAR expression upon focused-ultrasound (FUS) stimulation, increasing both inducibility and sustainability. We term this innovation Sonogenetic EchoBack CAR-T, providing a novel strategy for precise and durable CAR expression to enhance therapeutic potential.

This work is currently published on CELL.

Reference:
Liu L*, He P*, Wang Y*, Ma F, Li D, Bai Z, Qu Y, Zhu L, Yoon CW, Yu X, Huang Y, Liang Z, Zhang Y, Liu K, Guo T, Zeng Y, Zhou Q, Chung HK, Fan R, Wang Y. Engineering sonogenetic EchoBack-CAR T cells. Cell. 2025 Mar 23:S0092-8674(25)00271-5. doi: 10.1016/j.cell.2025.02.035. Epub ahead of print. PMID: 40179881.

Materials and

Methods: : We engineered EchoBack-CAR T cells, a sonogenetically controlled CAR-T therapy, utilizing a highly heat-sensitive promoter evolved via sort-seq and integrated with a positive feedback loop. The heat shock elements (HSE) of the promoter was optimized using site-saturation mutagenesis, generating a library of 16,384 variants, which were screened using high-throughput FACS sorting to identify promoters with high inducibility and low basal leakage. The top-performing promoter was selected, showing enhanced CAR expression in response to short-pulsed heat shock.
To sustain CAR expression, we designed a positive feedback loop incorporating NFκB, NFAT and CRE-SRE responsive elements in to the heat shock promoter,ensuring prolonged CAR expression since intrinsic CAR activation signaling will promote further CAR production. This design mitigates CAR downregulation, a major limitation of previous inducible systems.
We evaluated the cytotoxicity of EchoBack-CAR T cells against GD2+ and PSMA+ tumors using in vitro killing assays, 3D tumor spheroids, and in vivo mouse models. The EchoBack system was further assessed for induction kinetics, durability, tumor suppression, safety, and toxicity. We also compared it with conventional CAR-T approaches. Single-cell RNA sequencing (scRNA-seq) was performed to analyze the transcriptomic profiles of EchoBack-CAR vs. standard CAR-T cells to uncover potential mechanisms of reduced exhaustion and enhanced persistence.

Results, Conclusions, and Discussions:: We developed EchoBack-CAR T cells, an ultrasound controllable CAR-T therapy that integrates an optimized heat-shock promoter with a positive feedback loop, enabling sustained CAR expression upon focused ultrasound (FUS) stimulation. EchoBack-CAR T cells demonstrated robust anti-tumor effects in glioblastoma and prostate cancer models while reducing off-tumor toxicity, a major concern in conventional CAR-T therapies.
Compared to standard CAR-T cells, EchoBack-CAR showed better tumor suppression, even after transient stimulation, and showed higher cytotoxicity and reduced exhaustion in scRNA-seq analysis. FUS-controlled activation ensures that CAR expression decays upon leaving the tumor site, reducing systemic toxicity while maintaining strong anti-tumor responses. The modular heat shock element and feedback loop design allow for sustained functionality and adaptability across different tumor targets. Our high-throughput sort-seq platform provides a powerful scalable approach for promoter optimization, expanding applications beyond CAR-T therapies.
EchoBack-CAR integrates synthetic biology and immunotherapy, demonstrating a promising clinically viable strategy for controllable and durable CAR-T therapy against solid tumors, bridging fundamental research with clinical translation.

Acknowledgements and/or References (Optional):: We thank all co-authors of this work for their contribution.
We thank Professor Michel Sadelain at the Memorial Sloan Kettering Cancer Center (MSK) for insightful discussion and suggestions. The authors would like to thank the Molecular Genomics Core at USC for the processing of the single-cell RNA-seq experiment. This work was supported in part by grants from the National Institutes of Health (NIH) (EB029122, GM140929, HL121365, HD107206, and CA262815 to Yingxiao Wang; NIH K01EB035649 to L.L.; and K01EB034321 to H.K.C.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.