Abstract: TH-PO286

Keap1 Specific CRISPR/Cas9 Gene Editing in Primary Human T Cells Increases Nrf2 Activity and Anti-Inflammatory Phenotype

Session Information

Category: Acute Kidney Injury

  • 001 AKI: Basic

Authors

  • Noel, Sanjeev, Johns Hopkins University, Baltimore, Maryland, United States
  • Lee, Sul A, Johns Hopkins University, Baltimore, Maryland, United States
  • Sadasivam, Mohanraj, Johns Hopkins University, Baltimore, Maryland, United States
  • Hamad, Abdel, Johns Hopkins University, Baltimore, Maryland, United States
  • Rabb, Hamid, Johns Hopkins University, Baltimore, Maryland, United States
Background

T lymphocytes are established mediators of acute kidney injury (AKI) and other immune mediated kidney diseases. Previous work demonstrated significant protection from AKI in mice with enhanced T lymphocyte specific nuclear factor erythroid-derived 2-like 2 (Nrf2) activity, via deletion of kelch like-ECH-associated protein 1 (Keap1). In this study we applied CRISPR technology to edit Keap1, and enhance Nrf2 activity, in human Jurkat and primary T cells to develop immune cell based therapy for humans.

Methods

We targeted Keap1 exon 2 using site specific guide RNA. Briefly, 5X105 cells were electroporated with cas9:guide RNA complex. Control cells were electroporated without cas9:guide RNA complex. Cells were harvested 72h after electroporation and assessed for Keap1 editing and qPCR based analysis of Nrf2 target genes. Edited cells were further enriched using ATTO 550 positive sorting and analyzed for Keap1 editing and immunological changes.

Results

Genomic cleavage analysis showed Keap1 editing in up to 70% Jurkat cells and 40% primary T cells. qPCR analysis in Jurkat cells showed significant (p≤0.05) increase in Nrf2 regulated antioxidant genes, Nqo1 (~11 fold), Ho-1 (~11 fold) and Gclm (~2 fold) mRNA in Keap1 edited cells compared to control cells. In primary T cells, CRISPR mediated Keap1 editing resulted in significant (p≤0.04) increase in Nqo1 (~16 fold), Ho-1 (~9 fold) and Gclm (~2 fold) compared to control cells. Enrichment of ATTO 550 positive cells improved Keap1 editing from 40% to 55% cells. qPCR analysis found significantly (p≤0.01) higher expression of Nqo1 (~7 fold) in ATTO 550 positive cells compared to control cells. Keap1 edited cells had increased (p≤0.01) frequency of CD4, CD25 and CD69 and reduced frequency of CD8 (p≤0.01) and IL-17 (p≤0.05) compared to control cells.

Conclusion

Gene editing using CRISPR/Cas9 successfully augments Nrf2 activity in primary human T lymphocytes resulting in increased expression of antioxidant genes and reduction in IL-17 expression. This sets the stage for immune cell therapy for AKI and other inflammation mediated diseases.

Funding

  • NIDDK Support