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

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.</p

Enabling out-of-clinic human immunity studies via single-cell profiling of capillary blood

An individual's immune system is driven by both genetic and environmental factors that vary over time. To better understand the temporal and inter-individual variability of gene expression within distinct immune cell types, we developed a platform that leverages multiplexed single-cell sequencing and out-of-clinic capillary blood extraction to enable simplified, cost-effective profiling of the human immune system across people and time at single-cell resolution. Using the platform, we detect widespread differences in cell type-specific gene expression between subjects that are stable over multiple days

Enabling out-of-clinic human immunity studies via single-cell profiling of capillary blood

An individual's immune system is driven by both genetic and environmental factors that vary over time. To better understand the temporal and inter-individual variability of gene expression within distinct immune cell types, we developed a platform that leverages multiplexed single-cell sequencing and out-of-clinic capillary blood extraction to enable simplified, cost-effective profiling of the human immune system across people and time at single-cell resolution. Using the platform, we detect widespread differences in cell type-specific gene expression between subjects that are stable over multiple days

Single cell profiling of capillary blood enables out of clinic human immunity studies

An individual’s immune system is driven by both genetic and environmental factors that vary over time. To better understand the temporal and inter-individual variability of gene expression within distinct immune cell types, we developed a platform that leverages multiplexed single-cell sequencing and out-of-clinic capillary blood extraction to enable simplified, cost-effective profiling of the human immune system across people and time at single-cell resolution. Using the platform, we detect widespread differences in cell type-specific gene expression between subjects that are stable over multiple days

Bone CLARITY: Clearing, imaging, and computational analysis of osteoprogenitors within intact bone marrow

Bone tissue harbors unique and essential physiological processes, such as hematopoiesis, bone growth, and bone remodeling. To enable visualization of these processes at the cellular level in an intact environment, we developed “Bone CLARITY,” a bone tissue clearing method. We used Bone CLARITY and a custom-built light-sheet fluorescence microscope to detect the endogenous fluorescence of Sox9-tdTomato+ osteoprogenitor cells in the tibia, femur, and vertebral column of adult transgenic mice. To obtain a complete distribution map of these osteoprogenitor cells, we developed a computational pipeline that semiautomatically detects individual Sox9-tdTomato+ cells in their native three-dimensional environment. Our computational method counted all labeled osteoprogenitor cells without relying on sampling techniques and displayed increased precision when compared with traditional stereology techniques for estimating the total number of these rare cells. We demonstrate the value of the clearing-imaging pipeline by quantifying changes in the population of Sox9-tdTomato–labeled osteoprogenitor cells after sclerostin antibody treatment. Bone tissue clearing is able to provide fast and comprehensive visualization of biological processes in intact bone tissue

Post-outburst Radio Observations of the High Magnetic Field Pulsar PSR J1119-6127

We have carried out high-frequency radio observations of the high magnetic field pulsar PSR J1119-6127 following its recent X-ray outburst. While initial observations showed no evidence of significant radio emission, subsequent observations detected pulsed emission across a large frequency band. In this Letter, we report on the initial disappearance of the pulsed emission and its prompt reactivation and dramatic evolution over several months of observation. The periodic pulse profile at S-band (2.3 GHz) after reactivation exhibits a multi-component emission structure, while the simultaneous X-band (8.4 GHz) profile shows a single emission peak. Single pulses were also detected at S-band near the main emission peaks. We present measurements of the spectral index across a wide frequency bandwidth, which captures the underlying changes in the radio emission profile of the neutron star. The high-frequency radio detection, unusual emission profile, and observed variability suggest similarities with magnetars, which may independently link the high-energy outbursts to magnetar-like behavior

Multiplexed Cre-dependent selection yields systemic AAVs for targeting distinct brain cell types

Recombinant adeno-associated viruses (rAAVs) are efficient gene delivery vectors via intravenous delivery; however, natural serotypes display a finite set of tropisms. To expand their utility, we evolved AAV capsids to efficiently transduce specific cell types in adult mouse brains. Building upon our Cre-recombination-based AAV targeted evolution (CREATE) platform, we developed Multiplexed-CREATE (M-CREATE) to identify variants of interest in a given selection landscape through multiple positive and negative selection criteria. M-CREATE incorporates next-generation sequencing, synthetic library generation and a dedicated analysis pipeline. We have identified capsid variants that can transduce the central nervous system broadly, exhibit bias toward vascular cells and astrocytes, target neurons with greater specificity or cross the blood–brain barrier across diverse murine strains. Collectively, the M-CREATE methodology accelerates the discovery of capsids for use in neuroscience and gene-therapy applications

Engineering Artificial Somatosensation Through Cortical Stimulation in Humans

Sensory feedback is a critical aspect of motor control rehabilitation following paralysis or amputation. Current human studies have demonstrated the ability to deliver some of this sensory information via brain-machine interfaces, although further testing is needed to understand the stimulation parameters effect on sensation. Here, we report a systematic evaluation of somatosensory restoration in humans, using cortical stimulation with subdural mini-electrocorticography (mini-ECoG) grids. Nine epilepsy patients undergoing implantation of cortical electrodes for seizure localization were also implanted with a subdural 64-channel mini-ECoG grid over the hand area of the primary somatosensory cortex (S1). We mapped the somatotopic location and size of receptive fields evoked by stimulation of individual channels of the mini-ECoG grid. We determined the effects on perception by varying stimulus parameters of pulse width, current amplitude, and frequency. Finally, a target localization task was used to demonstrate the use of artificial sensation in a behavioral task. We found a replicable somatotopic representation of the hand on the mini-ECoG grid across most subjects during electrical stimulation. The stimulus-evoked sensations were usually of artificial quality, but in some cases were more natural and of a cutaneous or proprioceptive nature. Increases in pulse width, current strength and frequency generally produced similar quality sensations at the same somatotopic location, but with a perception of increased intensity. The subjects produced near perfect performance when using the evoked sensory information in target acquisition tasks. These findings indicate that electrical stimulation of somatosensory cortex through mini-ECoG grids has considerable potential for restoring useful sensation to patients with paralysis and amputation