Biology subjects

Spurrell, C. H.

Publications and source records attributed to Spurrell, C. H..

6 recordsLinked to original sources

bioRxiv2024

Genome-wide CRISPRa screens nominate modulators of CAR T cell survival within distinct tumor cytokine milieus

Chimeric Antigen Receptor (CAR) T cell therapy has revolutionized the treatment of B cell malignancies and translating this success to other cancers remains an ongoing clinical objective. Next-generation T cell products in development aim to genetically modulate many facets of cell behavior, for which gene-nominating platforms provide a useful framework for prioritization. Among competing screening approaches, CRISPR activation (CRISPRa) technology permits gain-of-function (GoF) gene surveys at genome-wide scale, but routine implementation in primary T cells has been stymied by high cell requirements ([~]107 - 108) and abbreviated activity. Here, we describe a novel cell manufacturing schema using an all-in-one transposon-based gene delivery system coupled with CAR-restricted cell expansion to generate yields (109) of primary T cells bearing CAR and CRISPRa transgenes that are well above the threshold needed for genome-scale screening. CRISPRa activity is sustained via the inclusion of divergent, duplicate Elongation Factor 1 core/human T-cell leukemia virus (EF1-HTLV) hybrid promoters; while guide RNA representation is preserved through late lentiviral transduction, thus preventing bottlenecking and premature candidate pruning. CRISPRa-CAR T cells manufactured via this pipeline retain potent on-target gene-overexpression (>85% target+) across varied cell subsets (e.g. Tim-3+Lag3+ or serial-challenge) and timescales (>14 days). When deployed to survival-based genome-wide selection landscapes, CRISPRa-CAR pools nominate known and novel endogenous genes capable of enhancing CD8+ CAR T survival in cytokine-rich (e.g. MYC, FUT6, IRF4, GSE1) and cytokine-depleted (e.g. CSF2RB, STAT6, IRF4, GSE1) settings of tumor challenge. This system will have broad utility for therapy-enhancing gene discovery.

immunology↗

bioRxiv2023

Rationally designed modular STAT-activating scaffolds enforce cell-intrinsic transcriptional programs augmenting the anti-tumor potency of CAR T cells

Chimeric antigen receptor (CAR)-expressing T cells can mediate anti-tumor responses in a variety of preclinical models and clinical settings, however, strategies to enhance anti-tumor potency is the subject of intense investigation. Signals emanating from gamma-c cytokine receptors modulate the transcriptional state of activated T cells impacting proliferation, survival, differentiation, and effector functioning through the STAT family of transcription factors. Design of ligand-independent cell-intrinsic cytokine STAT activation scaffolds is a conceptually attractive strategy to provide CAR T cells with a surrogate for exogenous cytokine support. Here, we designed a series of ligand-autonomous STAT inducer (LASI) scaffolds comprised of an extracellular identification tag, a homodimerizing transmembrane domain, and a membrane proximal IL7R Box1 domain followed by STAT5 and/or STAT3 docking sequences derived from IL7R and IL21R, respectively. We constructed LASI scaffolds having STAT5 (LASI-5), STAT3 (LASI-3), and combined STAT5 and STAT3 (LASI-5+3) docking domains and then interrogated their impact in primary human CD8+ anti-CD19 (4-1BB:zeta) CAR T cells. While LASI-5 expression had limited effects on CAR T cells, LASI-3 transcriptional programming was found to be indispensable to achieving anti-tumor functional enhancement associated with limited terminal differentiation, heightened T cell proliferation in response to antigen, and dampened expression of exhaustion-associated genes. Moreover, CAR T cells supplemented with LASI-3 or 5+3 displayed superior potency against human leukemia tumors in NSG mice. LASI-5+3 mediated the highest magnitude of CAR T cell engraftment in vivo that evolved into a fatal lymphoproliferative syndrome. However, the same efficacy enhancement was achieved with LASI-3 without the lymphoproliferative complication. Our findings provide a rationale for utilization of constitutively expressed LASI-3 to enhance the anti-tumor potency of CAR T cells, the need to regulate the activity of LASI-5+3, and a generalizable scaffold design for studying additional combinations of STAT family transcription factors.

bioengineering↗

bioRxiv2022

A single-cell multi-omic atlas spanning the adult rhesus macaque brain

Cataloging the diverse cellular architecture of the primate brain is crucial for understanding cognition, behavior and disease in humans. Here, we generated a brain-wide single-cell multimodal molecular atlas of the rhesus macaque brain. Altogether, we profiled 2.58M transcriptomes and 1.59M epigenomes from single nuclei sampled from 30 regions across the adult brain. Cell composition differed extensively across the brain, revealing cellular signatures of region-specific functions. We also identified 1.19M candidate regulatory elements, many novel, allowing us to explore the landscape of cis-regulatory grammar and neurological disease risk in a cell-type-specific manner. Together, this multi-omic atlas provides an open resource for investigating the evolution of the human brain and identifying novel targets for disease interventions.

neuroscience↗

bioRxiv2022

Human iPSC Derived Enamel Organoid Guided by Single-Cell Atlas of Human Tooth Development

Tooth enamel secreted by ameloblasts is the hardest material in the human body, acting as a shield protecting the teeth. However, the enamel is gradually damaged or partially lost in over 90% of adults and cannot be regenerated due to a lack of ameloblasts in erupted teeth. Here we use sci-RNA-seq to establish a spatiotemporal single-cell atlas for the developing human tooth and identify regulatory mechanisms controlling the differentiation process of human ameloblasts. We reveal key signaling pathways involved between the support cells and ameloblasts during fetal development and recapitulate those findings in a novel human ameloblast in vitro differentiation from iPSCs. We furthermore develop a mineralizing enamel organ-like 3D organoid system. These studies pave the way for future regenerative dentistry and therapies toward genetic diseases affecting enamel formation.

developmental biology↗

bioRxiv2022

Single-cell analysis of chromatin and expression reveals age- and sex-associated alterations in the human heart

Sex differences and age-related changes in the human heart at the tissue, cell, and molecular level have been well-documented and many may be relevant for cardiovascular disease. However, how molecular programs within individual cell types vary across individuals by age and sex remains poorly characterized. To better understand this variation, we performed single-nucleus combinatorial indexing (sci) ATAC- and RNA-Seq in human heart samples from nine donors. We identify hundreds of differentially expressed genes by age and sex. Sex dependent alterations include pathways such as TGF{beta} signaling and metabolic shifts by sex, evident in both transcriptional alterations and differing presence of transcription factor (TF) motifs in accessible chromatin. Age was associated with changes such as immune activation-related transcriptional and chromatin accessibility differences, as well as changes in the relative proportion of cardiomyocytes, neurons, and perivascular cells. In addition, we compare our adult-derived ATAC-Seq profiles to analogous fetal cell types to identify putative developmental-stage-specific regulatory factors. Finally, we train predictive models of cell-type-specific RNA expression levels utilizing ATAC-Seq profiles to link distal regulatory sequences to promoters, quantifying the predictive value of a simple TF-to-expression regulatory grammar and identifying cell-type-specific TFs.

genomics↗

bioRxiv2019

Genome-Wide Fetalization of Enhancer Architecture in Heart Disease

Heart disease is associated with re-expression of key transcription factors normally active only during prenatal development of the heart. However, the impact of this reactivation on the genome-wide regulatory landscape in heart disease has remained obscure. Here we show that pervasive epigenomic changes occur in heart disease, with thousands of regulatory sequences reacquiring fetal-like chromatin signatures. We used RNA-seq and ChIP-seq targeting a histone modification associated with active transcriptional enhancers to generate genome-wide enhancer maps from left ventricle tissue from 18 healthy controls and 18 individuals with idiopathic dilated cardiomyopathy (DCM). Healthy individuals had a highly reproducible epigenomic landscape, consisting of more than 31,000 predicted heart enhancers. In contrast, we observed reproducible disease-associated gains or losses of activity at more than 7,500 predicted heart enhancers. Next, we profiled human fetal heart tissue by ChIP-seq and RNA-seq. Comparison with adult tissues revealed that the heart disease epigenome and transcrip-tome both shift toward a fetal-like state, with 3,400 individual enhancers sharing fetal regulatory properties. Our results demonstrate widespread epigenomic changes in DCM, and we provide a comprehensive data resource (http://heart.lbl.gov) for the mechanistic exploration of heart disease etiology.

genomics↗