A Viral Toolkit for Ultrasound Imaging of Cellular Activity and Gene Expression

Citation

Shivaei, Shirin (2025) A Viral Toolkit for Ultrasound Imaging of Cellular Activity and Gene Expression. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/r9gz-1482. https://resolver.caltech.edu/CaltechTHESIS:06022025-044636280

Abstract

Observing and manipulating cell dynamics in living organisms is essential for understanding biological processes and intervening when they malfunction. However, the lack of non-invasive, non-ionizing, and cost-effective imaging technologies limits our ability to study these processes in their native context. To address this gap, we developed a toolkit for ultrasound imaging of acoustic reporter gene expression in mammalian tissues using virally-delivered gas vesicle (GV) genes. We demonstrate the versatility of this toolkit across multiple applications, including tracking engineered cell-based therapies and imaging activity-dependent gene expression in the brain.

To track cell-based therapies, we developed lentiviral vectors encoding the eight genes necessary for GV expression, achieving robust ultrasound contrast in both cell lines and primary human T cells. By expressing GVs downstream of activity-dependent promoters, we monitor T cell activation in cytotoxic T cells engaged with tumor cells. In a mouse xenograft model, we then image the targeted accumulation and proliferation of GV-expressing T cells within tumors. These ultrasound measurements, which closely correlate with immunohistological analysis, provide real-time, in vivo insights into the spatial dynamics of therapeutic cells. This approach offers a powerful tool to accelerate the development and clinical translation of cell-based therapies.

We extend this technology to the brain by engineering an AAV-based system for GV expression in primary neurons. Following intracranial injection of the GV-encoding AAVs in mice, we demonstrate longitudinal imaging of in situ gene expression in the brain over several weeks. Moreover, by using immediate early gene promoters to drive GV expression, we track changes in neuronal activity in the hippocampus during seizure episodes, enabling repeated, longitudinal imaging of brain function within the same animal. Collectively, these advancements establish a robust platform for ultrasound imaging of cellular activity and gene expression in opaque tissues, with applications ranging from cancer immunotherapy to neuroscience.