Abstract Details

G2 - Immune Targeting and Approaches with Genetically-Modified Cells and Cell Therapies (Including CAR-T, CAR-NK, TCR editing)

1814: Non-Viral Senolytic CRISPR CAR T Cell Products Autonomously Refine into Pure Cell Therapies

Type: Poster Session

Poster Board Number: 1814

Presentation Details

Session Title: Friday Posters: Immune Targeting and Approaches with Genetically-Modified Cells and Cell Therapies






Cellular senescence is a biological process that reduces proliferation and helps to prevent the propagation of damaged cells. Controlling senescence within the body can be critical, as recent discoveries have identified its role in the pathophysiology of various disorders. Lysing senescent cells within the body using engineered cells can provide a new therapeutic modality to treat disease or prevent tissue dysfunction. Here, we exploit nonviral CRISPR genome editing reagents to efficiently generate T cells that express chimeric antigen receptors (CARs) against a senescence-associated cell surface protein, urokinase plasminogen activator receptor (uPAR). CAR T cells targeting uPAR were efficiently produced by knocking out the endogenous T cell receptor, alpha chain, gene (TRAC), and inserting a CAR transgene driven by the TRAC promoter. CAR T cells targeting murine and human uPAR proteins exhibit specific and significant cytotoxicity in vitro against induced and patient-derived senescent cells with limited off-target cytotoxicity against healthy dividing cells. During manufacturing, human TRAC-CAR cell products are autonomously refined into highly enriched CAR+ memory T cells, with purities reaching >98% CAR+/TRAC-/CD45RO+ phenotypes within weeks (Figure). Facile manufacturing of gene-edited CAR T cell products could enable new senolytic therapy for aging and various senescence-associated pathologies.

huPAR-TRAC-CAR T Cells target senescent uPAR+ T cells in culture and display a central memory phenotype after manufacturing. (A) Schematic of huPAR-TRAC-CAR T cells eliminating uPAR positive senescent cells. (B) Summary of flow cytometry for CAR transgene integration of huPAR-TRAC-CAR T cells on days 7 and 17 post-nucleofection. huPAR-TRAC-CAR T cells showed significantly increased mCherry expression from day 7 to 17 relative to Untransfected T cells. (C) Summary of flow cytometry of CAR-2A-mCherry/CD45RO double positivity from day 7 to 17. huPAR-TRAC-CAR (blue) n=3, untransfected control (grey) n=2. Ordinary one-way ANOVA; *p≤0.05; **p≤0.01; ***p≤0.001; ****p≤0.0001. ANOVA, analysis of variance.

Plain Language Summary

Cellular senescence is a natural process that limits the growth of cells in the body to prevent them from causing damage. Recent discoveries show that controlling this process is crucial for the treatment of various diseases. Here we suggest a new way to help control senescence by using engineered cells to eliminate senescent cells that are contributing to disease. We utilize a gene-editing technique called CRISPR to create T cells that can target and destroy these problematic senescent cells. The generated engineered T cells are very effective in killing senescent cells without harming healthy cells. Additionally, the process of making these cells also led to a high percentage of memory T cells, which could be beneficial for future treatments related to aging. This research opens up the possibility of developing new therapies for aging and conditions linked to cellular senescence to help people live a longer and healthier life.

Lauren E. Sarko^1,2, Jeremiah Riendeau^3,4, Claire Shepley¹, David Givand¹, Roshini Traynor¹, Lei Zhao⁵, Mackenzie Mnuk¹, Tyler Ulland², Mahau Dey⁵, Melissa Skala^3,4, Krishanu Saha<sup>1,3

1</sup>Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, WI,²Department of Pathology and Laboratory Medicine, University of Wisconsin-Madison, Madison, WI,³Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI,⁴Morgridge Institute for Research, Madison, WI,⁵Department of Neurological Surgery, University of Wisconsin-Madison, Madison, WI"