Gene editing enables T-cell engineering to redirect antigen specificity for potent tumor rejection

Abstract

Adoptive transfer of TCR transgenic T cells holds great promise for treating various cancers. So far, mainly semi-randomly integrating vectors have been used to genetically modify T cells. These carry the risk of insertional mutagenesis, and the sole addition of an exogenous TCR potentially results in the mispairing of TCR chains with endogenous ones. Established approaches using nonviral vectors, such as transposons, already reduce the risk of insertional mutagenesis but have not accomplished site-specific integration. Here, we used CRISPR-Cas9 RNPs and adeno-associated virus 6 for gene targeting to deliver an engineered TCR gene specifically to the TCR alpha constant locus, thus placing it under endogenous transcriptional control. Our data demonstrate that this approach replaces the endogenous TCR, functionally redirects the edited T cells’ specificity in vitro, and facilitates potent tumor rejection in an in vivo xenograft model.

T cell receptor‐based cancer immunotherapy: Emerging efficacy and pathways of resistance

“…Recently, two groups demonstrated the feasibility of knocking in an exogenous TCR into the human TCR locus using CRISPR/Cas9 . Importantly, in work by Roth et al, both disruption of the TCR locus and replacement with an exogenous TCR template was achieved using an entirely non‐viral approach.…”

“…This was accomplished through co‐electroporation of a guide‐RNA loaded CRISPR‐Cas9 ribonucleoprotein (RNP) complex in combination with a long double stranded DNA template. In a second example, disruption of the TCR locus was also achieved through electroporation with a CRISPR‐Cas9 RNP . However, the donor DNA template was provided through infection with an adeno‐associated virus (AAV)6 viral transfer system, building upon earlier work by Eyquem et al using CARs .…”

“…Targeted integration into genomic "safe harbors", 139 including the endogenous TCR locus, can overcome many of these challenges Recently, two groups demonstrated the feasibility of knocking in an exogenous TCR into the human TCR locus using CRISPR/ Cas9. 38,146 Importantly, in work by Roth et al, 38 both disruption of the TCR locus and replacement with an exogenous TCR template was achieved using an entirely non-viral approach. This was accomplished through co-electroporation of a guide-RNA loaded CRISPR-Cas9 ribonucleoprotein (RNP) complex in combination with a long double stranded DNA template.…”

“…In a second example, disruption of the TCR locus was also achieved through electroporation with a CRISPR-Cas9 RNP. 146 However, the donor DNA template was provided through infection with an adeno-associated virus (AAV)6 viral transfer system, building upon earlier work by Eyquem et al using CARs. 36 Both the fully non-viral and AAV6 methods generated T cells with a reasonable frequency of genome integration, minimal to no genomic off-target effects, good cell viability, and evidence of in vivo anti-tumor efficacy.…”

Adoptive Cell Therapy—Harnessing Antigen-Specific T Cells to Target Solid Tumours

“…To prevent mispairing, several approaches were developed, including the introduction of an additional disulphide bond between introduced TCR α and β constant domains [142][143][144] and linking TCR α and β chains with a self-cleaving 2A peptide [145]. More recent approaches utilised CRISPR-Cas9 technology to replace endogenous TCR with exogenous one via: (1) knockout of endogenous TCR gene simultaneously with transduction with a desired TCR [146] or (2) knock-in of exogenous TCR into a TCR-α constant (TRAC) locus [147,148]. Safety and efficacy of ACT with TCR-engineered T cells is now explored intensively in clinical trials, and importantly, most of them focus on solid tumours (over 80%) [67,149].…”