KEAP1 Editing Using CRISPR/Cas9 for Therapeutic NRF2 Activation in Primary Human T Lymphocytes.

Oxidant stress modifies T lymphocyte activation and function. Previous work demonstrated that murine T cell-specific kelch like-ECH-associated protein 1 ( Keap1 ) deletion enhances antioxidant capacity and protects from experimental acute kidney injury. In this study, we used CRISPR technology to develop clinically translatable human T cell-specific KEAP1 deletion. Delivery of KEAP1 exon 2 specific Cas9:guide RNA in Jurkat T cells led to significant (∼70%) editing and upregulation of NRF2-regulated antioxidant genes NADPH dehydrogenase quinone 1 ( NQO1 ) (up to 11-fold), heme oxygenase 1 ( HO1 ) (up to 11-fold), and GCLM (up to 2-fold). In primary human T cells, delivery of KEAP1 exon 2 target site 2-specific ATTO 550-labeled Cas9:guide RNA edited KEAP1 in ∼40% cells and significantly ( p ≤ 0.04) increased NQO1 (16-fold), HO1 (9-fold), and GCLM (2-fold) expression. To further enrich KEAP1 -edited cells, ATTO 550-positive cells were sorted 24 h after electroporation. Assessment of ATTO 550-positive cells showed KEAP1 editing in ∼55% cells. There was no detectable off-target cleavage in the top three predicted genes in the ATTO 550-positive cells. Gene expression analysis found significantly ( p ≤ 0.01) higher expression of NQO1 mRNA in ATTO 550-positive cells compared with control cells. Flow cytometric assessment showed increased ( p ≤ 0.01) frequency of CD4-, CD25-, and CD69-expressing KEAP1 edited cells whereas frequency of CD8- ( p ≤ 0.01) and IL-17- ( p ≤ 0.05) expressing cells was reduced compared with control cells. Similar experimental conditions resulted in significant KEAP1 editing, increased antioxidant gene expression, and frequency of CD69 and IL-10 positive cells in highly enriched KEAP1 -edited regulatory T cells. KEAP1 -edited T cells could potentially be used for treating multiple human diseases.