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G - Cell Therapies -> Cell Therapies (Includes development of somatic, embryonic and induced pluripotent stem cells or other therapeutic cell populations, and issues related to cell expansion or processing)

810: Generation of Islet-specific Engineered Regulatory T Cells (EngTregs) for Immune Tolerance Induction in Type 1 Diabetes Using a Novel Dual-editing Strategy

Type: Presidential Symposium

Presentation Details
Session Title: Presidential Symposium and Presentation of Top Abstracts
Location: Hall E
Start Time: 5/18/2022 14:45
End Time: 5/18/2022 15:00

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-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.

Peter J. Cook1, Martina S. Hunt1, Soo Jung Yang2, Emma Mortensen2, Jane Buckner2, David Rawlings1

1Seattle Childrens Research Institute, Seattle, WA,2Benaroya Research Institute, Seattle, WA
 M.S. Hunt: None.

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