Redirecting regulatory T cell specificity using non-viral T cell engineering
Infusion of regulatory T cells (Tregs) has emerged as an attractive approach to mitigate graft-versus-host disease (GVHD), which is the main life-threatening immune complication of allogeneic hematopoietic stem cell (HSC) transplantation. Clinical studies and preclinical models support the use of Tregs to prevent severe GVHD without eliminating the graft-versus-tumor, and indicate that antigen-specific Tregs, such as those expressing transgenic T cell receptors, can have increased potency compared to polyclonal Tregs and lower potential for off-target immunosuppression.
Generation of transgenic T cells for cellular therapy rely on transduction with viral vectors. This approach has several drawbacks: the unpredictable site of integration results in variable expression levels and mutational risk, the transgene competes with the endogenous TCR for surface expression and binding to co-receptors, and can interact to form mixed TCR dimers of unpredictable specificity. Importantly, this approach requires time-consuming production of viral supernatants which are expensive in a clinical setting. Recent advances using CRISPR-Cas9 technology offer an alternative method to insert sequences in precise genomic locations, without the need of viral vectors.
We developed a novel method to generate antigen-specific Treg cells using non-viral, CRISPR/Cas9-mediated integration of transgenic TCRs in blood-derived Tregs. This method, not previously established on human Tregs, involves the generation of a double-strand break in the constant domain of the TCR-α gene (TRAC), and the introduction via homology-directed repair using a synthetic dsDNA template of a sequence containing the transgenic TCR-β and-α chains which integrate into the TRAC locus. Simultaneous KO of the TCR-β gene eliminates endogenous TCR expression, avoiding generation of mixed TCR dimers and competition for surface expression.
Using this method, we have been able to accomplish physiological expression of transgenic TCRs in blood-derived Tregs, while preventing competition and mixed dimer formation through disruption of the endogenous TCR. Removing competition was crucial for surface expression of certain TCRs, ostensibly due to preferential pairing of the transgenic TCR-α with endogenous TCR-β chains. The transgenic Tregs generated with this method recognize their target antigen and exhibit potent suppressive capacity in vitro.
The role of Tregs as upholders of immune homeostasis makes them an exciting option to prevent or treat GVHD. Specific recognition of the recipient’s tissues by these cells would improve the clinical efficacy of such a therapy, but clinical application is limited by current methods to generate such antigen-specific Tregs. The use of non-viral methods to generate antigen-specific regulatory T cells would be advantageous but is not established in the field, so to address this gap we used CRISPR-Cas9 tools to efficiently generate functional and stable antigen-specific regulatory T cells in vitro