Chimeric antigen receptor (CAR)-reprogrammed immune cells offer exciting therapeutic potential for addressing oncology, autoimmune diseases, transplant medicine, and infections. However, current approved CAR-T therapies rely on personalized manufacturing using viral gene transfer, resulting in challenges related to CAR-signaling regulation and limited accessibility due to the expensive personalized manufacturing. To overcome these limitations, we present a novel approach utilizing non-viral CRISPR-Cas gene editing to redirect T cells or natural killer (NK) cells with CARs. By employing non-viral gene editing, we transfer truncated CAR-transgenes lacking the CD3ζ activation domain into the human CD3ζ (CD247) gene, thereby creating functional CAR fusion-genes. This strategy allows effective reprogramming of various immune cell types, including conventional T cells, TCRγ/δ T cells, regulatory T cells, and NK cells. Notably, the engineered CD3ζ in-frame fusion gene in T cells eliminates TCR surface expression, substantially reducing the risk of graft-versus-host disease in allogeneic off-the-shelf applications. In a xenograft mouse model, optimized CD3ζ-CD19-CAR-T cells demonstrates remarkable leukemia control at low doses, outperforming CAR-T cells generated via TRAC gene editing. The CD3ζ-editing approach also enables reprogramming of NK cells without compromising their canonical NK cell functions in vitro. Collectively, CD3ζ gene editing represents a promising platform technology for the development of allogeneic off-the-shelf cell therapies utilizing redirected killer lymphocytes. 

Key Learning objectives:

• Non-viral gene editing with CRISPR-Cas and synthetic DNA templates allows efficient reprogramming of T cells for therapeutic applications.
• Integrating truncated CARs transgenes into the endogenous CD3ζ gene conveys potent CAR-dependent activation in various immune cells, such as T cells, regulatory T cells and natural killer cells.
• CD3ζ-edited CAR T cells are unable to express potentially allo-reactive T cell receptors and benefit from physiological CAR regulation.