Novel compositions and methods for targeted replacement of endogenous T-cell receptor with a chimeric antigen receptor

Technology Description

This invention is a method for targeted and high-efficiency replacement of the endogenous T‐cell receptor (TCR) with a chimeric antigen receptor (CAR) by introducing a Cas9 and guide RNA (gRNA) ribonucleoprotein (RNP) that targets a genomic break to the endogenous T‐cell receptor alpha constant chain (TRAC) locus. The depletion of TCR‐positive cells by magnetic beads enriches the cells which have both the CAR knockin and TCR knockout. Also cells, which are modified successfully by homology‐directed‐repair (HDR) through minimizing non‐homologous‐end‐joining (NHEJ)‐mediated TCR knockout by the selection of gRNA targets that do not disrupt protein‐coding regions, are enriched. A set of HDR templates, which use recombinant adeno‐associated virus (rAAV) or ssDNA/dsDNA Cas9 “shuttle” hybrid templates, is adapted along with selected gRNA targets that stimulate high-efficiency HDR with reduced disruption of endogenous TCR expression in the absence of HDR.

Patent Status

Additional Patent Pending