Orthogonal CRISPR screens to identify transcriptional and epigenetic regulators of human CD8 T cell function

Abstract

The clinical response to adoptive T cell therapies is strongly associated with transcriptional and epigenetic state. Thus, technologies to discover regulators of T cell gene networks and their corresponding phenotypes have great potential to improve the efficacy of T cell therapies. We developed pooled CRISPR screening approaches with compact epigenome editors to systematically profile the effects of activation and repression of 120 transcription factors and epigenetic modifiers on human CD8+ T cell state. These screens nominated known and novel regulators of T cell phenotypes with BATF3 emerging as a high confidence gene in both screens. We found that BATF3 overexpression promoted specific features of memory T cells such as increased IL7R expression and glycolytic capacity, while attenuating gene programs associated with cytotoxicity, regulatory T cell function, and T cell exhaustion. In the context of chronic antigen stimulation, BATF3 overexpression countered phenotypic and epigenetic signatures of T cell exhaustion. CAR T cells overexpressing BATF3 significantly outperformed control CAR T cells in both in vitro and in vivo tumor models. Moreover, we found that BATF3 programmed a transcriptional profile that correlated with positive clinical response to adoptive T cell therapy. Finally, we performed CRISPR knockout screens with and without BATF3 overexpression to define co-factors and downstream factors of BATF3, as well as other therapeutic targets. These screens pointed to a model where BATF3 interacts with JUNB and IRF4 to regulate gene expression and illuminated several other novel targets for further investigation.

HyperCas12a enables highly-multiplexed epigenome editing screens

“…Recent work to experimentally test and validate thousands of CRE-gene pairs found between two and three CREs per gene, while predictive modeling of millions of CRE-promoter interactions estimates about four CREs per gene 11,12 . It is also common that coordinated expression of several genes controls aspects of cell differentiation and disease [13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28] .…”

Design and characterization of multiplex gene-edited CAR-T cells resistant to orthogonal immunosuppressive pathways within the solid tumor microenvironment

“…We believe this strategy provides proof-of-concept rationale for the design of CAR-T cell products with a synergistic combination of gene edits that confer effective anti-tumor responses. Indeed, the recent identification of other negative regulators [129][130][131][132] is likely to reveal additional target genes for interrogation, ultimately requiring a complex genome engineering strategy to generate higher-order multiplex gene-edited CAR-T cells. As more single agent therapies targeting immunosuppressive barriers in the TME fail in the clinic [133][134][135], the urgency to better understand optimal combinations of alleviating suppressive pathways rapidly increases.…”