Biology subjects

Curtis, B. C.

Publications and source records attributed to Curtis, B. C..

3 recordsLinked to original sources

bioRxiv2024

Enhancer AAVs for targeting spinal motor neurons and descending motor pathways in rodents and macaque

Experimental access to cell types within the mammalian spinal cord is severely limited by the availability of genetic tools. To enable access to lower motor neurons (LMNs) and LMN subtypes, we generated single cell multiome datasets from mouse and macaque spinal cords and discovered putative enhancers for each neuronal population. We cloned these enhancers into adeno-associated viral vectors (AAVs) driving a reporter fluorophore and functionally screened them in mouse. We extensively characterized the most promising candidate enhancers in rat and macaque and developed an optimized pan LMN enhancer virus. Additionally, we generated derivative viruses expressing iCre297T recombinase or ChR2-EYFP for labeling and functional studies, and we created a single vector with combined enhancer elements to achieve simultaneous labeling of layer 5 extratelencephalic projecting (ET) neurons and LMNs. This unprecedented LMN toolkit will enable future investigations of cell type function across species and potential therapeutic interventions for human neurodegenerative diseases.

neuroscience↗

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↗