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

Julian J Albers,Angela M Krackhardt,Jürgen Ruland,Dario Gosmann,Stefan Audehm,Christof Winter,Christian Kupatt,Anja Wolf,Anja Stelzl,Tim Ammon,Katja Steiger,Silvia Thoene,Martina Anton,Florian Bassermann,Ramona Secci

doi:10.26508/lsa.201900367

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.

Highlights

In recent years, the adoptive transfer of genetically reprogrammed T cells has gained more and more momentum (Lim and June 2017; June & Sadelain, 2018)
This region is attractive as it is shared between all rearranged T cells, and a disruption in the first exon is located upstream of the functional region needed for surface expression (Eyquem et al, 2017)
We demonstrate that the specificity of T cells can be redirected by targeting an engineered TCR to the TCR alpha constant (TRAC) locus using CRISPR-Cas9 RNPs and AAV6 to functionally replace the endogenous TCR with an exogenous, tumor-reactive TCR

Summary

Introduction

The adoptive transfer of genetically reprogrammed T cells has gained more and more momentum (Lim and June 2017; June & Sadelain, 2018). With the advent of individualized cellular immunotherapies that target patient-specific antigens, it is essential to produce personalized vectors, which will further increase the effort needed for clinical translation (Krackhardt et al, 2018). Nonviral vectors such as transposons are easier to implement clinically and show a more favorable integration pattern, and only integrate in a nonspecific fashion (Tipanee et al, 2017)

Methods

Results

Conclusion