Objective Chimeric antigen receptors(CARs)are synthetic receptors that redirect and reprogram T cells to mediate tumor rejection.CAR-T cells have shown remarkable efficacy in cancer immunotherapy,particularly in the treatment of blood cancers.However,the therapeutic application of CAR-T cells against solid cancers has lagged behind.This issue is due,at least in part,to the restricted accumulation and survival of transferred CAR-T cells and endogenous immune cells in solid tumors.Previous study has reported the lncRNA NKILA promotes the apoptosis of tumorresponsive CTLs via enhancing the sensitivity to tumor-derived FasL,providing a novel strategy that targeting NKILA can enhance the therapeutic efficacy of immune cell transferring for breast cancer.In breast cancer,the human epidermal growth factor receptor 2(HER2)is one of the most studied tumor-associated antigens(TAAs)for targeted therapy.Whether NKILA could be the target of HER2 CAR-T cell therapy remains unclear.Methods HER2-specific T cells were generated by the transfer of genes that encode chimeric antigen receptor(CAR)and CRISPR/Cas9 genome editing was used to knockout NKILA.We constructed a novel,humanized HER2 CAR,containing HER2 single-chain variable fragment(scFv)region of antigen-specific mAb and T-cell intracellular signaling chains made up of 4-1BB and CD3δ,and transduced with CRISPR/Cas9 system targeted to NKILA promoter.The knockout efficiency of NKILA was determined by the expression of NKILA assayed by qRT-PCR and Northern blotting.Moreover,the DNA of T cells tranduced with NKILAKO CRISPR/Cas9 lentivirus was collected to analyze the editing effect.An interferon γ ELISPOT assay and cytotoxicity experiments were used to evaluate the antitumor immune response of CAR T cells in coculture with HER2+tumor cells in vitro.Furthermore,NOD/SCID mice bearing SKBR3 tumor or HER2+PDX were treated with HER2 CAR T cells to evaluate antitumor activity.The accumulation and survival of CTLs in tumor xenograft were detected by immunohistochemistry.Results HER2 CAR and NKILAKO plasmid were successfully constructed,and transduced in CD8+T cells.The expanded HER2 CAR T cells expressed a central memory phenotype and specifically reacted against HER2+tumor cell lines,rather than HER2-tumor cell lines.Furthermore,HER2 CAR-T cells with NKILAKO showed more cytotoxicity towards to HER2+cells in vitro,compared with HER2 CAR-T cells.Mechanistically,HER2 CAR-T cells with NKILAKO suffered less apoptosis to tumor-derived FasL by dissociation NKILA with p65/Iκ B complex and reactivation of NFκ B pathway,leading to anti-apoptotic genes transcription.Furthermore,NKILA knockout in HER2 CAR-T cells upon transferring to the NOD/SCID mice inoculated with HER2+PDX or SKBR3 tumor xenograft enhances CTL survival and infiltration in the tumor xenografts,and thus effectively inhibits tumor growth.Conclusions Our findings show that novel HER2 scFv-based,HER2-specific CAR T cells not only recognized and killed HER2+breast cancer cells ex vivo but also induced regression of experimental breast cancer in vivo.Furthermore,targeting the lncRNA NKILA by CRISPR/Cas9 genome editing may provide a novel strategy to enhance the therapeutic efficacy of immune cell transferring for breast cancer.These findings uncover facets of CAR immunobiology and underscore the potential of CRISPR/Cas9 genome editing to advance immunotherapies.