Precise spatiotemporal control of cellular activities in deep tissues is a key challenge in modern biomedicine. Current cell therapies aim to develop safe and effective methods for remote and localized activation of therapeutic genes and cells. With its high spatiotemporal resolution and ability to penetrate soft tissues, ultrasound has emerged as a promising tool for this purpose.
This research focused on developing and evaluating an ultrasound-based method for cell stimulation that enables robust and reproducible Ca²⁺ influx and activation of Ca²⁺-responsive transcription factors. We analyzed the effects of ultrasound parameters on Ca²⁺ signaling and the subsequent activation of engineered transcription factors. Using NFAT-based, Ca²⁺-responsive transcription factors engineered with custom DNA-binding domains, we redirected these factors to specific target genes while preventing the activation of endogenous NFAT-regulated genes. The system's functionality was validated both in vitro and in vivo in mouse xenograft models. Cells were transplanted either subcutaneously or into the peritoneal cavity of mice, ultrasound stimulation triggered the expression of firefly luciferase or the anti-inflammatory cytokine interleukin-10 (IL-10).
To demonstrate the therapeutic potential of this approach, cells engineered to express IL-10 upon ultrasound stimulation were encapsulated in biocompatible tubes and surgically implanted into the peritoneal cavity of mice with acute colitis. This strategy significantly alleviated symptoms of DSS-induced colitis, as shown by improved colon morphology and histopathology.
To enhance the modularity of the transcription factors and expand sonogenetic toolset, we also developed split Ca²⁺-responsive transcription regulators (SCaTR). These included bi- and tripartite variants, which, upon ultrasound stimulation, induced the expression of luciferase or IL-10. We utilized natural and engineered DNA-binding domains, truncated NFAT proteins as Ca²⁺ signal transducers, activation domains, and coiled-coil motifs for dimerization.
To facilitate the clinical translation of this ultrasound-based method, we optimized ultrasound parameters to reduce the stimulation time from two hours to 30 minutes by increasing Ca²⁺ signal amplitude and duration, which elevated cumulative intracellular Ca²⁺ concentrations.
This work underscores the potential of ultrasound as a precise tool for spatiotemporal modulation of genetically engineered cells, paving the way for safe and targeted therapies.
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