Coupled Long Terminal-CRISPR Gene Editing for the Redirection of T cells

Genome editing of T cells has broadened the clinical scope of chimeric antigen receptor (CAR) treatments to include allogeneic T cells from mismatched donors, previously constrained in their use due to HLA barriers and allo-recognition. Initial proof-of-concept applications are underway using transcription activator-like effector nuclease (TALEN) edited ‘universal’ T cells devoid of TCRαβ expression and depleted of CD52 – the target antigen for the lymphodepleting antibody Alemtuzumab. While efficient, editing effects were unlinked, resulting in variable yields of heterogenous T-cell populations, complicating cell dosing strategies. Here, a novel ‘terminal’ vector platform was developed, that couples transgene expression with clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 scission effects through precise incorporation of sgRNA element(s) into the ∆U3 3’ lentiviral long terminal repeat (LTR). Transient delivery of Cas9 mRNA by electroporation to ‘terminal’ transduced primary T cells resulted in highly efficient TRAC locus modification yielding homogenous CAR+ (greater than 96%) TCR- (greater than 99%) cells capable of robust in vivo anti-leukaemic effects. The strategy has now been extended to the simultaneous disruption of TCR and HLA class I genomic targets resulting in efficient multiplexed effects. While these approaches rely on nuclease mediated cleavage and cell mediated repair of double standed DNA (dsDNA) breaks, the associated risk of chromosomal translocations is yet to be fully elucidated. Base editing through targeted chemical deamination circumvents dsDNA breaks, and instead offers the possibility of seamless nucleotide conversion for the disruption of coding sequence. sgRNA sequences targeting regions of TCRα and β genes, B2M and CD52 genes were delivered to primary T cells. Transient delivery of codon optimized base editor 3 (coBE3) resulted in high levels of ‘on-target’ base conversion, with protein knockout comparable to that achieved by spCas9 mRNA, while chromosomal translocation events remained undetected using PCR based screening. While additional characterization of CAR T cells with multiple deamination mediated edits is underway, these processes are scalable and amenable to adaption for GMP-compliant manufacturing, and provide a route to translational application.