Development of a Fully Non-Viral 1XX-enhanced BCMA CAR-T Cell Therapy for Multiple Myeloma

Multiple myeloma (MM) is a clonal plasma cell malignancy characterized by bone marrow infiltration, monoclonal immunoglobulin production, and microenvironmental dysregulation that leads to systemic organ damage. The advent of B-cell maturation antigen (BCMA)-directed chimeric antigen receptor (CAR) T-cell therapy has induced unprecedented responses and durability for patients with relapsed/refractory MM. These outcomes are rarely observed with prior salvage strategies, although relapse remains the predominant long-term challenge for most patients. The two currently approved BCMA CAR-T cell products use viral vectors to semi-randomly insert the CAR gene, which results in heterogeneous genomic composition and variability in efficacy, safety, and product consistency. To address these challenges, we integrated targeted CRISPR genome engineering with precise CAR transgene insertion at the T-cell receptor alpha constant (TRAC) locus, 1XX CAR signaling architecture to enhance potency and durability, and non-viral manufacturing with a single-stranded DNA repair template to improve efficiency and yield. This approach confers physiological CAR expression, reduces insertional mutagenesis, and improves persistence by mitigating tonic signaling and exhaustion. Our GMP manufacturing process consistently achieved high CAR integration (37.7-72.7%) and yields across all full-scale runs and met predefined release criteria for identity, purity, safety, and quality. In NSG mouse models of MM, the UCCT-BCMA-1 product exhibited exceptionally potent tumor control, CAR-T cell expansion 100-1000-fold greater than that of lentiviral constructs, and durable clearance of myeloma cells after multiple rechallenges. These findings establish a CRISPR-edited, fully non-viral manufacturing platform for next-generation 1XX-BCMA CAR-T therapies with enhanced persistence, safety, and efficacy. One Sentence SummaryCRISPR-engineered, TRAC-targeted 1XX-BCMA CAR-T therapy with improved safety, potency, and persistence in relapsed and refractory multiple myeloma.