Figure 3. Multiple gene ablation by CRISPR/Cas9 to generate universal effector cells.

(a) HLA-I disruption with gRNA targeting B2M. (b) Flow chart of the protocol to generate universal effector cells as described in methods. (c) The allo-reactivity of TCR and TCR/HLA disrupted was tested with an IFNγ Elispot assay by challenging the gene-ablated T cells with irradiated allogenic PBMCs (left panel) or co-culturing allogenic PBMCs with irradiated gene-ablated T cells. Specific spots are shown on the y axis as the spots produced in the presence of stimulators minus the spots produced by the effectors alone. **P<0.01 by Mann-Whitney test. (d) Survival without severe GVHD and (e) weight loss in mice after infusion of PBS (n = 5), Cas9 Mock wild type (Cas9 Mock) T cell (n = 5), TCR ablated (TCR^neg) cells (n = 5) or TCR/HLA-I double ablated (TCR/HLA-I^neg) (n = 5). ***P<0.005 by the log-rank Mantel-Cox test. (f) Abolishment of target recognition of allogeneic T cells by disrupting MHC-I on target T cells. Allogeneic T cells were primed by dendritic cells of the same donor with gene-disrupted T cells and infused into NSG mice with TCR^neg or TCR/HLA-I^neg target T cells. Significant prolonged survival of HLA-I ablated T cells was observed by the presence of CD3^neg T cells, which is also confirmed by the failed expansion of allogeneic effector T cells (n=3). ***P<0.001**P<0.01, *P<0.05, by Mann-Whitney test.