A versatile CRISPR-Cas13d platform for multiplexed transcriptomic regulation and metabolic engineering in primary human T cells.

CRISPR technologies have begun to revolutionize T cell therapies; however, conventional CRISPR-Cas9 genome-editing tools are limited in their safety, efficacy, and scope. To address these challenges, we developed multiplexed effector guide arrays (MEGA), a platform for programmable and scalable regulation of the T cell transcriptome using the RNA-guided, RNA-targeting activity of CRISPR-Cas13d. MEGA enables quantitative, reversible, and massively multiplexed gene knockdown in primary human T cells without targeting or cutting genomic DNA.

Enhanced T cell effector activity by targeting the Mediator kinase module.

T cells are the major arm of the immune system responsible for controlling and regressing cancers. To identify genes limiting T cell function, we conducted genome-wide CRISPR knockout screens in human chimeric antigen receptor (CAR) T cells. Top hits were and , components of the Mediator kinase module.

c-Jun overexpression in CAR T cells induces exhaustion resistance.

Chimeric antigen receptor (CAR) T cells mediate anti-tumour effects in a small subset of patients with cancer, but dysfunction due to T cell exhaustion is an important barrier to progress. To investigate the biology of exhaustion in human T cells expressing CAR receptors, we used a model system with a tonically signaling CAR, which induces hallmark features of exhaustion. Exhaustion was associated with a profound defect in the production of IL-2, along with increased chromatin accessibility of AP-1 transcription factor motifs and overexpression of the bZIP and IRF transcription factors that have been implicated in mediating dysfunction in exhausted T cells.

Development of Cationic Quaternary Ammonium Sulfonamide Amino Lipids for Nucleic Acid Delivery.

Lipid nanoparticles (LNPs) currently comprise the most effective carrier class for the delivery of small RNAs. Among lipid carriers, charge-unbalanced lipids are relatively unexplored synthetically. Herein, we developed and evaluated a novel collection of compounds for small interfering RNA (siRNA) delivery, termed cationic quaternary ammonium sulfonamide amino lipids (CSALs).