Engineering Next-Generation CAR T Cells Via Genetic Screens

The successes of genetically engineered chimeric antigen receptor (CAR) T cells in B cell

leukemia and lymphomas have revolutionized the field of immunotherapy. However, durable

remission is only achieved in a subset of patients, and poor treatment efficacy has been

reported for solid tumors. These limitations often result from intrinsic dysfunctions of T cells,

such as poor proliferation, compromised effector function, or T cell exhaustion.

CRISPR-based functional genetic screens assess the impact of genetic modifications at the

genome-wide level and could greatly accelerate the design of improved T cell-based

therapies. We developed a new technology, CRISPR applied by mRNA electroporation and

lentivirus, to enable efficient genome editing and high-throughput screening in CAR T cells.

The simultaneous delivery of the CAR and a CRISPR guide RNA (gDNA) enabled single-

gene perturbation as well as genetic screens with libraries of target genes in CAR T cells

directed against various antigens. Our mRNA-based protocol for the delivery of CRISPR

modifiers extends the available tool kit for genome engineering in primary human T cells

from loss-of-function mutations to precise base editing and CRISPR activation.

We performed genome-scale fitness-based CRISPR screens in human T and CAR T cells. We

uncovered gene regulators specific to TCR-driven and CAR-driven proliferation. Moreover,

our screens robustly identified canonical immunotherapy targets such as CTLA4, along with

genes that have not been characterized in T cells. We have thus demonstrated the high

scalability and efficiency of our technology and created a comprehensive map of genetic

perturbations tuning CAR T cell performance.

Our platform is compatible with CROP-seq technology for single-cell CRISPR screens and

with pooled in vivo screening of CAR T cells in a mouse leukemia model. Thus, the

technology enables unbiased immunotherapy target discovery, and can be applied for high-

throughput characterization of functional regulators in primary human cells.

No relevant conflicts of interest to declare.