Engineering Tools to Probe and Manipulate the Immune System at Single-Cell Resolution

Citation

Dobreva, Tatyana (2022) Engineering Tools to Probe and Manipulate the Immune System at Single-Cell Resolution. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/n3rs-ft69. https://resolver.caltech.edu/CaltechTHESIS:10172021-215439860

Abstract

My thesis focuses on developing experimental and computational tools to probe and manipulate cellular transcriptomes in the context of human health and disease. Chapter 1 and 2 focus on published work where we leverage single-cell RNA sequencing (scRNA-seq) to understand human immune variability, characterize cell-type specific biases of multiple viral variants within an animal, and assess temporal immune response in the brain to delivery of genetic cargo via an adeno-associated virus (AAV). Chapter 3 and 4 present progress I have made on tools for exporting RNA extracellularly and engineering of a transcription factor for modulating macrophage state.

For probing cellular transcriptome states, we have developed a platform using multiplexed single-cell sequencing and out-of-clinic capillary blood extraction to understand temporal and inter-individual variability of gene expression within immune cell types. Our platform enables simplified, cost-effective profiling of the human immune system across subjects and time at single-cell resolution. To demonstrate the power of our platform, we performed a three day time-of-day study of four healthy individuals, generating gene expression data for 24,087 cells across 22 samples. We detected genes with cell type-specific time-of-day expression and identified robust genes and pathways particular to each individual, all of which could have been missed if analyzed with bulk RNA-sequencing. Also, using scRNA-seq, we have developed a method to screen and characterize cellular tropism of multiple AAV variants. Additionally, I have looked at AAV-mediated transcriptomic changes in animals injected with AAV-PHP.eB three days and twenty-five days post-injection. I have found that there is an upregulation of genes involved in p53 signaling in endothelial cells three days post-injection.

In the context of manipulating cellular transcriptomic states, I demonstrate that a fusion between RNA targeting enzyme, dCas13, and capsid-forming neuronal protein, Arc, is able to form a capsid-like structure capable of encapsulating RNA. I also present methods and preliminary data for tuning macrophage states through mutations in transcription factor EB (TFEB) using scRNA-seq as a readout.

Thesis Files