Sunday, May 11, 2025
5/11/2025
Engineering RNA export for measurement and control of cells in vivo - Abstract RNA offers access to cell behaviors: sequencing RNA deciphers cell states and delivering RNA to cells enables control of their states. However, current methods for interfacing with cells via RNA have critical limitations. Detecting cellular RNA generally requires destruction of the analyzed cells, which makes it difficult to track the dynamic behaviors of individual living cells across time. Delivering therapeutic RNA to specific target cells within tissue also remains a challenge, which impedes biomedical deployment of RNA circuits to repair or eliminate harmful cells in vivo. Here, I propose to address these challenges by developing a new method to export RNA from living cells, which can be used to measure cell dynamics non-destructively and deliver RNA circuits from cell to cell. In preliminary work, I established genetically encoded RNA exporters, inspired by viruses, that efficiently package and secrete RNA from mammalian cells within protective nanoparticles. I showed that sequencing exported RNA enables information to be obtained non-destructively from living cells, and delivering exported RNA from cell to cell enables control of cell states, which paves the way to RNA export-based reporter and delivery platforms. To realize this potential, I will develop fully synthetic RNA exporters, based on de novo designed proteins, which are amenable to engineering and will enable targeted export of specific RNA species (Aim 1). I will create reporter systems that leverage RNA export and sequencing to measure dynamic single-cell states in living animals, including clonal ancestry, signaling pathway activity, and transcriptome profiles (Aim 2). I will establish a platform to deliver RNA from engineered cells to non-engineered target cells and utilize it to reprogram cell fates in situ (Aim 3). Together, this work will provide a versatile toolkit for measuring and manipulating cells in living animals. I also propose a rigorous training program to help me realize my career goal of leading an innovative independent research group. The research environment at Caltech is extraordinary and offers opportunities to interact with world-leading scientists and engineers. I will collaborate with and receive essential technical training from Dr. Pamela Bjorkman (expert in protein nanoparticle engineering), Dr. Long Cai (expert in multiplexed in situ readout), and Dr. Min Yu (expert in cancer metastasis). I will also mentor students and present my work at meetings to hone my communication skills and broaden my scientific network. The K99/R00 award will provide me with time and resources needed to initiate an ambitious research program at the interface of synthetic biology and genomics to understand and improve human health.
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