Abstract
The fusion of genome engineering and adoptive cellular therapy holds immense promise for the treatment of genetic disease and cancer. Multiplex genome engineering using targeted nucleases can be used to increase the efficacy and broaden the application of such therapies but carries safety risks associated with unintended genomic alterations and genotoxicity. Here, we apply base editor technology for multiplex gene modification in primary human T cells in support of an allogeneic CAR-T platform and demonstrate that base editor can mediate highly efficient multiplex gene disruption with minimal double-strand break induction. Importantly, multiplex base edited T cells exhibit improved expansion and lack double strand break-induced translocations observed in T cells edited with Cas9 nuclease. Our findings highlight base editor as a powerful platform for genetic modification of therapeutically relevant primary cell types.
Highlights
Base editing has been previously used to induce premature stop codons for gene knockout in mice and in mammalian cells[15,16,17,18], and to induce exon skipping by disrupting splice acceptor (SA) sites[19]
We reasoned that splice-site disruption, including splice donor (SD) sites, could be effective for gene knockout and may have several advantages over induction of premature stop (pmSTOP) codons (Supplementary Fig. 1)
Concerns surrounding DSBs are further heightened in the context of multiplex gene editing where multiple, simultaneous DSBs can compound toxicity
Summary
Base editing has been previously used to induce premature stop (pmSTOP) codons for gene knockout in mice and in mammalian cells[15,16,17,18], and to induce exon skipping by disrupting splice acceptor (SA) sites[19]. Efficient editing was observed at two pmSTOP candidate sites in exon 3, albeit at lower efficiencies than that of either splice-site disrupting sgRNA (Fig. 1f) Both the exon 1 SD and exon 3 SA sites were edited at similar frequencies, yet disruption of the exon 3 SA site resulted in the highest rate of TCR disruption as measured by loss of cellsurface CD3 expression (69.0 ± 15.3% for BE3; 83.7 ± 5.8% for BE4) (Fig. 1g). BE4 mRNA delivered with an sgRNA targeting the exon 1 SD site showed the most efficient C to T conversion of the target base (58.3 ± 2.5% for BE3; 70.3 ± 3.2% for BE4) (Fig. 1j), resulting in efficient knockout of B2M protein (79.1 ± 1.3% for BE3; 80.0 ± 3.2% for BE4) (Fig. 1k).
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