Christian D. McRoberts Amador

Interactions between genes or cis-regulatory elements (CREs) underlie many biological processes. High-throughput CRISPR screens have allowed researchers to assess the impact of activation or repression of gene and regulatory elements on many phenotypes. However, assessment of interactions between those genes or elements remains limited. To enable efficient highly-multiplexed control of regulatory element activity, we combine a hyper-efficient version of Lachnospiraceae bacterium dCas12a (dHyperLbCas12a) with RNA Polymerase II expression of long CRISPR RNA (crRNA) arrays. We demonstrate this system with several activation and repression domains, in cultured primary immune cells, and to differentiate induced pluripotent stem cells. We also develop approaches to use dCas12a for simultaneous activation and repression. Lastly, we demonstrate that dHyperLbCas12a effectors can be used to dissect the independent and combinatorial contributions of CREs to gene expression. These tools create possibilities for highly multiplexed control of gene expression in many biological systems. CRISPR/Cas9 screens have identified genetic contributions to many phenotypes. However, studying combinations of genes or regulatory elements remains challenging. Here, the authors use CRISPR/Cas12a to overcome those challenges and enable new approaches to study combinatorial genetic mechanisms.

Abstract Transcription factors (TFs) collaborate to regulate gene expression programs that define cell fate. In CD8 + T cells, this coordinated regulation underlies exhaustion, a dysfunctional state that constrains immunity in chronic infection and cancer. Here, we screen for cell state-specific TFs by performing pooled overexpression screens of 3,548 TF and TF isoforms in primary T cells across multiple CD8 + T cell states. We identify 82 regulators that collaborate with exhaustion-specific programs and profile their effects using perturb-SHARE-seq, connecting perturbations to changes in chromatin accessibility and gene expression across 702,314 single cells. We identify 38 reproducible regulatory programs and construct a map of 12,616 TF-program connections that shape CD8 + T cell states, nominating KLF2 as predictive of positive response to CAR-T therapy. Using seq2PRINT, a deep learning framework that predicts functional TF interactions, we identify RUNX as a “master collaborator”, a TF that broadly collaborates with other factors, and uncover a RUNX2:KLF2 interaction that specifies exhaustion-associated programs. Mutation of the RUNX2:KLF2 protein interface attenuates KLF2-mediated repression of exhaustion, while synthetic tethering of RUNX2 to KLF2 leads to an amplification of the phenotype. More broadly, we identify the collaborative action of RUNX as a driver in CD8 + T cell states, and show that tethering TFs enables the rational engineering of cell state identity for cell and gene therapies.

Abstract Tumor-infiltrating lymphocytes (TILs) are a promising autologous cell therapy to treat solid tumors. TILs are manufactured by expanding and reinfusing tumor-reactive T cells from tumor biopsies. Efficacy of TIL therapies has been limited by the heterogeneity of expanded TIL products and the high prevalence of dysfunctional exhausted CD8+ T cells (T EX ). While a subset of CD8+ TILs co-expressing CD103 and CD39 are enriched for tumor-reactive TILs across multiple cancer types, these cells are often in the T EX state with low proliferative potential. To identify regulators of human TIL proliferation, we screened an open reading frame library encoding for all human transcription factors (TFs). RELB emerged as the dominant driver of human TIL expansion with a skew towards CD8+ cells. TCR diversity was maintained after multiple days of in vitro expansion driven by RELB. Transcriptome profiling of multiple RELB-expressing TIL subtypes revealed a shift towards a memory/costimulatory-like phenotype. Using a HER2-targeting CAR and tumor co-culture model, RELB conferred improved persistence after multiple tumor challenges in vitro and improved solid tumor control in mouse xenografts in vivo . Finally, co-culture of RELB-overexpressing TILs with patient-matched tumor organoids showed an increase in TIL product polyfunctionality, tumor reactivity, and tumor killing. Collectively these results support promoting RELB expression as a strategy for broadly enabling TIL therapy for treating solid tumors.

<h3>Background</h3> T cells are part of the immune system’s adaptive defence; they specifically target and kill both virally infected and cancerous cells through antigen recognition. Unfortunately, cancer can exploit intrinsic T cell mechanisms to survive attack by the immune system. T cell exhaustion arises from chronic antigen stimulation, which shifts a portion of the T cell population to the exhausted state (T_EX), resulting in diminished T cell proliferation and tumor/viral clearance. Modern genome engineering technologies have the potential to dramatically advance T cell therapy by programming T_EX cells into desirable phenotypic states. The transcription factor (TF) TOX drives and maintains the T_EX state through epigenetic regulation of exhaustion-associated programs. <h3>Methods</h3> To recapitulate T cell exhaustion in vitro, we repeatedly stimulated primary human T cells with anti-CD3/CD28 beads to mimic chronic stimulation and confirmed induction of the exhaustion program via RNA- and ATAC-seq. To understand what other TF regulators might be driving or preventing T cell exhaustion upstream of TOX, we designed screens using an Open Reading Frame (ORF) library encoding all the TFs in the human genome with TOX protein expression as a readout. CD8+ T cells from three distinct donors were used in an acute stimulation setting (one TCR stimulation) and a chronic stimulation setting (two additional stimulations). Comparing screen results in the context of acute versus chronic stimulation facilitated identification of factors specific to the T_EX state. <h3>Results</h3> Over 100 TF ORFs were enriched in TOX-high and TOX-low bins, including both known and novel regulators of TOX. To comprehensively look at the transcriptomic, epigenomic, and functional effects top TFs had on acutely or chronically stimulated T cells, we sub pooled top TFs and screened in chronic versus acute stimulation setting with a final Phorbol myristate acetate and ionomycin (PMA/IO) stimulation using Simultaneous High-throughput ATAC and RNA expression with sequencing (SHARE-seq) as a readout. Computational analysis revealed THAP6 to be improving stimulation response and pushing away from the exhausted chromatin state. Individual validation of THAP6 confirmed downregulation of multiple T cell exhaustion markers via FACS and an improved functional response via a bead-based cytokine expression assay. <h3>Conclusions</h3> Ongoing work is focused on validating the effects of THAP6 and other top TFs on T cell function (T cell killing and cytokine expression assays), transcriptome (bulk RNA-seq), and epigenome (bulk ATAC-seq). Through epigenetic reprogramming, we hope to achieve clinically relevant T cell phenotypes to improve current immunotherapies in both viral infection and cancer contexts.