Tu156 - Versatile and Efficient Integration of Large Therapeutic Transgenes in Human T Cells via Nuclease-mediated Editing and Serine Integrases

Abstract Text: Adoptive T-cell therapy using gene-modified cells expressing target-specific receptors has emerged as a powerful treatment modality for malignancies and autoimmune diseases. However, current gene transfer methods, such as lentiviral transduction and homology-directed DNA repair (HDR), are limited in their ability to integrate large DNA sequences, restricting our potential to engineer T cells.
To address these challenges, we developed PASTA (programmable and site-specific transgene addition), a method enabling efficient, site-specific integration of large transgene cassettes. PASTA functions in two phases: Phase 1 integrates a small DNA landing pad into the genome using CRISPR-Cas-mediated HDR. Phase 2 employs a large serine integrase (e.g., Bxb1) to insert a circular DNA vector at the landing pad. This “one-pot” process is initiated by a single transfection, simplifying the procedure and making it suitable for clinical application.
We demonstrate that 'one-pot' PASTA allows efficient transgene integration at multiple sites relevant for T cell engineering (TRAC, B2M, CD3E). The integrase-mediated strategy outperforms conventional HDR by 9-fold (range: 2.5-14-fold) when introducing large transgenes exceeding 6.5 kilobases, and achieving integration rates of >20% of transfected T cells. Consequently, 'one-pot' PASTA enables the precise incorporation of multicistronic constructs and co-expression of multiple antigen receptors and a safety switch. Resulting multi-antigen targeting CAR T cell are currently under evaluation in B cell lymphoma models.
Overall, 'one-pot' PASTA is a versatile and efficient non-viral gene transfer platform for enhanced T cell engineering, offering a simpler and more flexible alternative to prime editing-based approaches like PASSIGE and PASTE.