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Abstract: SU-OR01

Keap1 Gene Edited T Cells Using CRISPR/Cas9 Improve Outcomes from Experimental AKI

Session Information

Category: Acute Kidney Injury

  • 103 AKI: Mechanisms

Authors

  • Kurzhagen, Johanna T., Johns Hopkins Medical Institutions Campus, Johns Hopkins Medicine, Baltimore, Maryland, United States
  • Noel, Sanjeev, Johns Hopkins Medical Institutions Campus, Johns Hopkins Medicine, Baltimore, Maryland, United States
  • Gharaie, Sepideh, Johns Hopkins Medical Institutions Campus, Johns Hopkins Medicine, Baltimore, Maryland, United States
  • Sadasivam, Mohanraj, Johns Hopkins Medical Institutions Campus, Johns Hopkins Medicine, Baltimore, Maryland, United States
  • Lee, Sul A, Johns Hopkins Medical Institutions Campus, Johns Hopkins Medicine, Baltimore, Maryland, United States
  • Gong, Jing, Johns Hopkins Medical Institutions Campus, Johns Hopkins Medicine, Baltimore, Maryland, United States
  • Arend, Lois J., Johns Hopkins Medical Institutions Campus, Johns Hopkins Medicine, Baltimore, Maryland, United States
  • Hamad, Abdel, Johns Hopkins Medical Institutions Campus, Johns Hopkins Medicine, Baltimore, Maryland, United States
  • Rabb, Hamid, Johns Hopkins Medical Institutions Campus, Johns Hopkins Medicine, Baltimore, Maryland, United States
Background

T cells have been demonstrated to modulate responses in ischemic AKI, and Keap1/Nrf2 pathway regulates T cell functions. We hypothesized that using the gene editing technique CRISPR/Cas9 could set the stage for T cell therapy for human AKI. We therefore edited Keap1 gene in murine CD4 T cells using CRISPR technology and tested its effects in an ischemic AKI model.

Methods

Primary CD4 T cells isolated from spleen of WT mice were used for ex vivo CRISPR/Cas9 mediated Keap1 editing. Control CD4 T cells were electroporated without sgRNA and Cas9. Keap1 editing was confirmed by sequencing, qRT PCR of Nrf2 target genes and immunophenotyping using flow cytometry. Keap1 edited CD4 T cells were studied under hypoxic or normoxic conditions in vitro. For in vivo studies, Keap1 edited or control CD4 T cells were adoptively transferred into T cell deficient nu/nu mice (n=6-8/group). Mice were subjected to bilateral IR surgery, serum creatinine, histology, kidney immune cell trafficking and cytokine production were measured.

Results

Keap1 editing resulted in significant increase in Nrf2 targets Nqo1 (8.5-fold), Hmox1 (4.4-fold) and Gclc (2-fold) mRNA. Keap1 edited CD4 T cells displayed higher expression of Hif1a mRNA than control cells in hypoxic conditions (1.5-fold, p=0.02). Ifng mRNA expression was significantly decreased in Keap1 edited cells compared to control cells under normoxic (0.4-fold, p=0.04) or hypoxic (0.6-fold, p=0.01) conditions. nu/nu mice that received Keap1 edited CD4 T cells showed significantly reduced SCr (0.55±0.05mg/dl vs 1.14±0.19mg/dl, p=0.05) at 24h compared to mice that received control cells. They also had significantly reduced percentage of necrotic tubules (40±16% vs 63±15%, p=0.055). Kidney Keap1 edited CD4 T cells had significant reduction in TNFα protein expression (43±9% vs 54±7%, p=0.03) compared to control cells post IRI. Kidney CD4 IFN-γ (p=0.16), CD25 (p=0.16) and CD69 (p=0.46) expression was similar in Keap1 edited and control cells.

Conclusion

CRISPR/Cas9 mediated Keap1 editing increases murine CD4 T cell Nrf2 regulated antioxidant gene expression and modifies responses to in vitro hypoxia. Adoptive transfer of ex-vivo Keap1 edited CD4 T cells ameliorates ischemic AKI in mice. These results are important to set the stage for T cell-based therapy for AKI in humans.

Funding

  • NIDDK Support