Generation of Islet-Specific Engineered Regulatory T Cells (EngTregs) for Immune Tolerance Induction in Type 1 Diabetes Using a Novel Dual-Editing Strategy
Peter J. Cook1, Martina S. Hunt1, Soo Jung Yang2, Emma Mortensen2, Jane Buckner2, David Rawlings1
1 Seattle Childrens Research Institute, Seattle, WA, 2 Benaroya Research Institute, Seattle, WA
Adoptive regulatory T-Cell (Treg) therapies represent a potentially transformative cell-based therapy to promote immune tolerance following stem cell or solid organ transplantation, and in autoimmune diseases including type 1 diabetes (T1D). Key technical hurdles, however, may limit broad clinical application including the rarity of natural (Treg), the requirement for antigen-specificity to modulate tissue-specific disorders, and requirement to compete in vivo for engraftment and survival. To address all three challenges, we have developed a dual-HDR-based gene editing approach to simultaneously induce constitutive expression of the (Treg) master transcriptional regulator, FOXP3, and replace the endogenous T-cell receptor (TCR) with a tissue-specific TCR. As previously described, this approach introduces a ubiquitous promoter (MND) into the FOXP3 locus immediately downstream of the (Treg)-specific demethylated region (TSDR), resulting in high level FOXP3 expression leading to a stable (Treg) phenotype and function. Concurrently, editing within the first coding exon of the TRAC locus deletes the endogenous TCR and replaces it with a defined TCR. Finally, to simultaneously select for cells with successful integration of both HDR templates (in vitro and in vivo), we designed donor cassettes also containing a heterodimeric, chemically-induced signaling complex (CISC) that mimics IL-2 signaling in response to an exogenous dimerizer.
In this study, we focused on generation of engineered (Treg) (EngTregs) products for prevention and/or treatment of T1D. We utilized a TCR specific for the islet antigen, IGRP, previously identified from clonally expanded CD4+ Teff cells in a T1D patient. Using CRISPR-based tools, we observed initial FOXP3/TRAC dual-editing rates ranging from 2% to 15% in healthy donor derived CD4+ T cells. Following expansion in dimerizer (rapamycin), dual-edited cells were enriched to >85% purity based on expression of both FOXP3 and the islet TCR. Droplet digital PCR analysis before and after rapamycin enrichment confirmed positive selection for each on-target editing event, and negative selection for low-frequency chromosomal translocations (resulting from double[1]strand breaks at both chromosomes X and 14). Enriched cells were cryopreserved, and subsequent analysis demonstrated a robust (Treg) immunophenotype and secretome switch from pro-inflammatory to immunosuppressive cytokines. Functionally, dual-edited Ag-specific EngTregs strongly suppressed the activation and proliferation of Teff cells expressing a matched islet TCR demonstrating potent direct suppression. Importantly, Ag-specific EngTregs also suppressed Teff cells expressing a different islet TCR in a mixed activation co-culture, demonstrating significant bystander suppression. Thus, dual-edited, Ag-specific, CISC expressing EngTregs represent a promising cell-based therapy for T1D simultaneously predicted to mediate tissue-specific bystander suppression and CISC-mediated enhanced in vivo survival and engraftment. Finally, this dual-editing platform is readily adaptable for use with alternative TCR (or CAR) cassettes designed to target a range of tissue-specific autoimmune or inflammatory diseases.