Orthotopic replacement of T-cell receptor α- and β-chains with preservation of near-physiological T-cell function

• DOI: 10.1038/s41551-019-0409-0
• Publication Date: 2019-06-10T16:02:43Z
• Authors: Kilian Schober; Thomas R. Müller; Füsun Gökmen; Simon Grassmann; Manuel Effenberger; Mateusz Poltorak; Christian Stemberger; Kathrin Schumann; Theodore L. Roth; Alexander Marson; Dirk H. Busch
• Keywords: Gene Editing; 0301 basic medicine; Receptors, Antigen, T-Cell, alpha-beta; T-Lymphocytes; Genetic Vectors; Gene Knockout Techniques; Genes, T-Cell Receptor; 03 medical and health sciences; Retroviridae; Antigens, Neoplasm; Transduction, Genetic; CRISPR-Associated Protein 9; Cell Line, Tumor; Humans; Transgenes; CRISPR-Cas Systems

Therapeutic T cells with desired specificity can be engineered by introducing T-cell receptors (TCRs) specific for antigens of interest, such as those from pathogens or tumour cells. However, TCR engineering is challenging, owing to the complex heterodimeric structure of the receptor and to competition and mispairing between endogenous and transgenic receptors. Additionally, conventional TCR insertion disrupts the regulation of TCR dynamics, with consequences for T-cell function. Here, we report the outcomes and validation, using five different TCRs, of the use of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) with non-virally delivered template DNA for the elimination of endogenous TCR chains and for the orthotopic placement of TCRs in human T cells. We show that, whereas the editing of a single receptor chain results in chain mispairing, simultaneous editing of α- and β-chains combined with orthotopic TCR placement leads to accurate αβ-pairing and results in TCR regulation similar to that of physiological T cells.