Using CRISPR/Cas9 to Knock Out GM-CSF in CAR-T Cells

Journal of Visualized Experiments : JoVE
Rosalie M SternerSaad S Kenderian

Abstract

Chimeric antigen receptor T (CAR-T) cell therapy is a cutting edge and potentially revolutionary new treatment option for cancer. However, there are significant limitations to its widespread use in the treatment of cancer. These limitations include the development of unique toxicities such as cytokine release syndrome (CRS) and neurotoxicity (NT) and limited expansion, effector functions, and anti-tumor activity in solid tumors. One strategy to enhance CAR-T efficacy and/or control toxicities of CAR-T cells is to edit the genome of the CAR-T cells themselves during CAR-T cell manufacturing. Here, we describe the use of CRISPR/Cas9 gene editing in CAR-T cells via transduction with a lentiviral construct containing a guide RNA to granulocyte macrophage colony-stimulating factor (GM-CSF) and Cas9. As an example, we describe CRISPR/Cas9 mediated knockout of GM-CSF. We have shown that these GM-CSFk/o CAR-T cells effectively produce less GM-CSF while maintaining critical T cell function and result in enhanced anti-tumor activity in vivo compared to wild type CAR-T cells.

Citations

Jan 18, 2020·Briefings in Functional Genomics·Chenggong LiYu Hu
Feb 11, 2020·The Journal of Clinical Investigation·Stephen M Ansell, Yi Lin
Sep 13, 2020·Cells·Simone HagerMatthias Bros
May 22, 2021·Inflammation Research : Official Journal of the European Histamine Research Society ... [et Al.]·Yeison Santamaria-Alza, Gloria Vasquez
Jul 30, 2021·Stem Cell Research & Therapy·Ehsan RazeghianFarhad Motavalli Khiavi
Sep 30, 2021·Journal of Oncology Pharmacy Practice : Official Publication of the International Society of Oncology Pharmacy Practitioners·Yunjung H ShinMarc G Sturgill

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