CRISPR-Cas9-Mediated TIM3 Knockout in Human Natural Killer Cells Enhances Growth Inhibitory Effects on Human Glioma Cells

• Published in: International journal of molecular sciences Vol. 22; no. 7; p. 3489
• Main Authors: Morimoto, Takayuki, Nakazawa, Tsutomu, Matsuda, Ryosuke, Nishimura, Fumihiko, Nakamura, Mitsutoshi, Yamada, Shuichi, Nakagawa, Ichiro, Park, Young-Soo, Tsujimura, Takahiro, Nakase, Hiroyuki
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 28.03.2021; MDPI
• Subjects: Apoptosis; Brain cancer; Brain Neoplasms - genetics; Brain Neoplasms - metabolism; Brain Neoplasms - therapy; Brain tumors; Cell activation; Cell death; Cell growth; Cell Line, Tumor; CRISPR; CRISPR-Cas Systems; CRISPR-Cas9; Cytokines; Cytotoxicity; Effector cells; Endonuclease; Gene expression; Gene Expression Regulation, Neoplastic; Gene Knockout Techniques; Genome, Human; Genomes; Glioblastoma; Glioma; Glioma - genetics; Glioma - metabolism; Glioma - therapy; Glioma cells; Growth inhibition; Hepatitis A Virus Cellular Receptor 2 - genetics; Humans; Immunoglobulins; Immunoreceptor tyrosine-based inhibition motif; Immunotherapy; Immunotherapy - methods; Kaplan-Meier Estimate; Killer Cells, Natural - metabolism; Ligands; Lymphocytes; Lymphocytes T; Medical prognosis; Mucin; Mutation; Natural killer cells; NK cell; Oligonucleotide Array Sequence Analysis; PD-1 protein; Proteins; RNA, Guide, CRISPR-Cas Systems - metabolism; TIM3; Transgenes; Tumor cells; Tumors; glioblastoma; TIM3; CRISPR-Cas9; NK cell
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms22073489

Glioblastoma (GBM) is the most common and aggressive primary malignant brain tumor in adults. Natural Killer (NK) cells are potent cytotoxic effector cells against tumor cells inducing GBM cells; therefore, NK cell based- immunotherapy might be a promising target in GBM. T cell immunoglobulin mucin family member 3 (TIM3), a receptor expressed on NK cells, has been suggested as a marker of dysfunctional NK cells. We established TIM3 knockout in NK cells, using the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9). Electroporating of TIM3 exon 2- or exon 5-targeting guide RNA- Cas9 protein complexes (RNPs) inhibited TIM3 expression on NK cells with varying efficacy. T7 endonuclease I mutation detection assays showed that both RNPs disrupted the intended genome sites. The expression of other checkpoint receptors, i.e., programmed cell death 1 (PD1), Lymphocyte-activation gene 3 (LAG3), T cell immunoreceptor with Ig and ITIM domains (TIGIT), and TACTILE (CD96) were unchanged on the TIM3 knockout NK cells. Real time cell growth assays revealed that TIM3 knockout enhanced NK cell-mediated growth inhibition of GBM cells. These results demonstrated that TIM3 knockout enhanced human NK cell mediated cytotoxicity on GBM cells. Future, CRISPR-Cas9 mediated TIM3 knockout in NK cells may prove to be a promising immunotherapeutic alternative in patient with GBM.