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 the potential of engineered 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. 

‘One-pot’ PASTA facilitates the precise incorporation of multicistronic constructs, offering a simpler and more flexible alternative to approaches like PASSIGE and PASTE. Leveraging serine integrases, PASTA enhances T-cell engineering capabilities, enabling co-expression of therapeutic transgenes such as antigen receptors, safety markers, and cytokines, thereby boosting therapeutic efficacy and safety.

Beyond T-cells, ‘one-pot’ PASTA holds promise for treating genetic disorders involving large deletions, providing new treatment strategies for unmet clinical needs.