Jeffrey Cao

Background: Leukemia is a cancer in which blood-forming tissues like bone marrow generate high numbers of immature or abnormal leukocytes, suppressing the production of normal blood cells. Like most cancers, malignant cells in leukemia exhibit immune evasion and resist checkpoint inhibition, which pose challenges to therapy. To overcome this, CRISPR Cas9-gene editing is being researched as an avenue to generate modified T-cells called Chimeric Antigen Receptor T-cells (CAR T cells) that can specifically target and destroy leukemia cancer cells. Methods: Gene editing via the CRISPR-Cas9 system was used to engineer T-cells that 1) specifically target leukemia cancer cells (uniquely expressing CD19 antigen), 2) resist checkpoint (PD1, CTLA4) inhibition, and 3) have universal compatibility with little to no immune rejection. Specifically, CRISPR Cas9 was used to precisely disrupt gene regions encoding checkpoint inhibitor Programmed Death Ligand 1 Protein (PDL1), and introduce a CD19-specific CAR gene to the T-cell Receptor Alpha Constant (TRAC) gene locus.^1-3,5 These CAR T-Cells were assessed for their ability to produce effective and sustained regression of non-solid tumors in animal models. Results: CAR T Cells generated using CRIPSR Cas9 exhibited virtually no immune rejection in vivo and were able to effectively avoid checkpoint inhibition.¹ Additionally, the PD1-negative CAR T-cells not only showed enhanced efficacy compared to PD1-positive controls, but also demonstrated accelerated tumor cell clearance related to concentration.³ Furthermore, CAR T-cells generated using CRISPR Cas9 exhibited uniform CAR expression in human peripheral blood T cells and enhanced T-cell potency, outperforming CAR T cells generated using conventional gene editing methods such as Zinc finger (ZFN) or TALEN in mouse models.² Conclusions: CRISPR Cas9 is an efficient and effective technique for generating CAR T-cells with properties that enhance CAR T-cell potency, longevity, and overall efficacy in vivo, and show promise in future treatment of leukemia in human patients.^1-4 A logical next step should be to continue assessment using larger sample sizes to better quantify the risks and benefits in cancer therapy. Additional research to determine specific antigens that can be targeted by CARs would increase the robustness of CAR T-Cell function in immunotherapy for a variety of tumors. Clinical trials in human patients should then be conducted to determine effectiveness in treatment of human leukemia and lymphomas.

1. Ren J, Liu X, Fang C, Jiang S, June CH, Zhao Y. Multiplex Genome Editing to Generate Universal CAR T Cells Resistant to PD1 Inhibition. Clin Cancer Res. 2016;
2. Eyquem J, Mansilla-soto J, Giavridis T, et al. Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection. Nature. 2017;543(7643):113-117.
3. Rupp LJ, Schumann K, Roybal KT, et al. CRISPR/Cas9-mediated PD-1 disruption enhances anti-tumor efficacy of human chimeric antigen receptor T cells. Sci Rep. 2017;7(1):737.
4. Maude SL, Frey N, Shaw PA, et al. Chimeric antigen receptor T cells for sustained remissions in leukemia. N Engl J Med. 2014;371(16):1507-17.
5. Schumann K, Lin S, Boyer E, et al. Generation of knock-in primary human T cells using Cas9 ribonucleoproteins. Proc Natl Acad Sci USA. 2015;112(33):10437-42.