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Abstract: FR-PO097

Ex Vivo Gene Editing and Pharmacologic Modification of T Cell Keap1/Nrf2 Towards Immunotherapy for Experimental AKI

Session Information

Category: Acute Kidney Injury

  • 103 AKI: Mechanisms

Authors

  • Kurzhagen, Johanna T., Johns Hopkins University , Baltimore, Maryland, United States
  • Noel, Sanjeev, 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 School of Medicine, Baltimore, Maryland, United States
Background

Adoptive transfer of Keap1 deficient T cells with enhanced Nrf2 activity has been shown to lead to significant protection from IR-induced AKI in mice (JASN 2015), and gene editing of Keap1 in human T cells enhances Nrf2 products (J Immunol 2018). Pre-clinical studies in mice with Keap1/Nrf2 gene editing and pharmacologic manipulation in AKI are needed to set the stage for human cell-based therapies.

Methods

Primary mouse CD4+ or pan T cells were isolated from B6 WT spleen by negative selection using Dynabeads. Nrf2 activity was modulated either by treating T cells with different concentrations (10nM, 20nM and 50nM) of the pharmacologic Nrf2 activator CDDO-Im or using CRISPR/Cas9 system to edit Keap1 gene. The efficiency of gene editing was assessed by using ATTO 550-labeled tracrRNA. Various T cell culture conditions were tested and viable cells were enriched using flow-sorting. Viable cells were detected by 4′6-diamidino-2-pheylindole (DAPI). Functional effects of ex vivo Nrf2 activation and Keap1 editing were assessed on Nrf2 dependent antioxidant genes NADPH dehydrogenase quinone 1 (NQO1), heme oxygenase 1 (HO-1) and Glutamate-Cysteine Ligase Catalytic Subunit (GCLC) expression using quantitative real-time PCR.

Results

Ex-vivo murine CD4+ T cell CDDO-Im treatment resulted in a dose dependent response in Nrf2 target gene expression. The higher dosage of 50nM CDDO-Im had the highest impact on NQO1 (∼16.4-fold), HO-1 (∼3.6-fold) and GCLC (∼2.8-fold) mRNA expression. T cells cultured longer before gene editing using CRISPR/Cas9 technique resulted in significantly higher (32.5%) viable CRISPR edited (ATTO 550 positive) cells compared to cells that underwent CRISPR editing soon after isolation (1%). CRISPR/Cas9 based Keap1 editing resulted in significant increase in NQO1 (∼15.5-fold), HO-1 (∼3.0-fold) and GCLC (∼2.5-fold). Changes in levels of IL-10, IFN-γ and IL-17 were measured.

Conclusion

Pharmacologic and gene editing techniques increased murine T cell Nrf2 target genes ex vivo for infusion in studies to prevent AKI and accelerate repair during AKI. These studies set the stage for immunotherapy treatment for AKI and other immune mediated kidney diseases.

Funding

  • NIDDK Support