Abstract

Regulatory T cells (T_regs) are a subset of CD4+ T lymphocytes, characterised by high expression of FOXP3 transcription factor. T_regs are responsible for maintaining immune homeostasis and self-tolerance by suppressing the effector function of conventional CD4+ and CD8+ T cells, as well as inhibiting other immune cell subsets. Therefore, dysfunction or loss of T_regs can lead to a break in tolerance and drive autoimmunity. Gene engineering of patient T_regs for improved stability and function aims to provide a potentially curative treatment for severe autoimmunity in inflammatory bowel disease, rheumatoid arthritis, and more, while differentiating from current T_regTherapies. To develop an engineered-T_reg cell therapy, we must first identify novel genes for T_reg stabilisation through a whole genome screening approach. To this aim, we have built a new capability for arrayed CRISPR screening in primary human T_reg cells that will validate hits arising from a whole genome pooled screen. We have developed an automation workflow that allows primary T_regs to be CRISPR-edited by Cas9-sgRNA ribonucleoprotein electroporation at scale in 384-well plates. To test the editing workflow, we targeted both neutral and control genes important to T_reg function, achieving ≥87% KO efficiency at all loci. We then combined this editing workflow with a newly-developed 384-well flow-staining protocol that uses the iQue Flow Cytometer. We optimised a 10-colour flow panel that allows us to assess markers of T_reg stability and function, and achieved robust staining with no signal spill-over between channels. Having achieved high editing efficiencies and generated reliable flow data, this capability has enabled arrayed CRISPR screening in primary T_regsfor the first time in AstraZeneca (AZ). This new capability will be utilised as part of our cross-functional collaboration in AZ, with the aim to identify novel targets for an autologous ex-vivo CRISPR edited T_reg celltherapy in our future portfolio.