Gene editing of the human T cell receptor locus enables optimized in vitro testing of autoreactive T cell function in type 1 diabetes.
The generation of antigen specific T cells and subsequent implementation as “living drugs” has garnered great interest in the immunotherapy space. One of the main caveats to this approach is the potential for heterologous chain pairing between the α- and β-chains of the endogenous and de-novo T cell receptors (TCRs). The consequences of chain mispairing can range from reduced antigen specific functionality to off-target autoimmune reactivity. The latter is an important consideration when designing therapies for autoimmune and inflammatory diseases, specifically with regards to the introduction of regulatory T cells (Treg) adoptive cell therapy. To date, infusions of polyclonal Tregs have demonstrated safety but have failed to reverse established type 1 diabetes (T1D). This demonstrates the need for the generation of safe and effective antigen-specific Tregs capable of recognizing β-cells in T1D. We have previously shown the ability to generate GAD-specific Tregs which maintain lineage stability and suppressive phenotype. To extend this work, we deleted the endogenous TCR α-chain gene (TRAC) via CRISPR/cas9 in CD8+T cells. We achieved >90% editing efficiency through ribonucleoprotein (RNP) complex mediated single guide RNA delivery. Moreover, we found TRAC-KO CD8+ T cells displayed increased peptide MHC-dextramer staining, CD69 and CD25 upregulation after stimulation with cognate antigen, and increased antigen-specific killing of β-cell lines when compared to mock guide treated T cells. We believe this workflow can be applied to generate antigen specific Tregs, which we expect to provide increased suppressive capacity. Hence, we present a TCR-gene editing protocol in primary human T cells that results in efficient deletion of the endogenous TCR α-chain and robust enhancement of antigen-specific T cell function.
L.D. Peters: None. A.L. Posgai: None. T.M. Brusko: Board Member; Self; OneVax, LLC. Consultant; Self; Caladrius Biosciences, Inc., Sanofi-Aventis. Speaker’s Bureau; Self; CSL Behring.
National Institutes of Health (P01AI42288, R01DK106191, T325T32DK108736-03HIRN, UC4 P0063595)