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Background Chimeric antigen receptor (CAR) T cell therapies targeting BCMA including two FDA-approved products have shown great efficacy in relapsed/refractory multiple myeloma (RRMM). However, safety and manufacturing challenges associated with viral vectors limit scalability and potentially product consistency. UCCT-BCMA-1 is the first, fully non-viral CRISPR-edited autologous CAR T cell product born out of academic GMP programs in the US, generated using CRISPR/Cas9-mediated targeted insertion of a second-generation BCMA-directed CAR into the T Cell Receptor Alpha Chain (TRAC) locus. The construct incorporates the modified 1XX CD3z intracellular signaling domain designed to enhance persistence and potency 1 and is delivered in using hybrid single-stranded DNA with enhanced efficiency.2 Methods We have validated a cGMP-compliant, fully non-viral CRISPR/Cas9 engineering platform ( figure 1) using autologous CD3+ T cells electroporated with SpyFi Cas9-sgRNA RNPs, enabling precise and efficient CAR integration at the TRAC locus through hybrid ssDNA-mediated homology-directed-repair (HDR). Pharmacology and toxicology studies include antigen-specific cytokine secretion, cytotoxicity assessed in vitro and in vivo using OPM2 xenograft models in NSG mice, genome integrity assessed by off-target and cytogenetics analyses. CMC studies evaluated product identify and purity using flow cytometry and digital PCR-based assay, and characterized residual process impurities.Results cGMP-compliant process qualification runs (n=4) confirmed robust and reproducible manufacturing, yielding TCR-negative, CD5 + UCCT-BCMA-1 cells with high CAR expression (40-70%), balanced CD4+/CD8+ distribution, and favorable immunophenotype. In vitro assays showed potent, antigen-specific cytotoxicity and polyfunctional cytokine responses, supporting a balanced effector profile. In vivo, UCCT-BCMA-1 induced complete tumor clearance in two independent xenograft models using OPM2 multiple myeloma tumors, at doses ≥66k CAR+ cells with no treatment-related toxicity or weight loss (n=7/group and n=10/group, respectively). Cas9 protein was undetectable by Day 7. Off-target analysis via GUIDE-seq and rhAmpSeq revealed two low-frequency (<0.1%) events in non-coding regions with no pathogenic relevance. Cytogenetics and cytokine-independent growth assays confirmed genomic stability and lack of oncogenic potential.Conclusions UCCT-BCMA-1 has successfully received IND-clearance to initiate Phase-I clinical trial, as demonstrated by its potent preclinical efficacy, and a favorable safety margin with minimal off-target editing. Its non-viral manufacturing platform enhances safety, scalability, and patient accessibility. Importantly, this program is part of an open-source sharing network, enabling broader access to protocols, data, and technology to accelerate the development of safe and effective gene-edited cell therapies across the field.References Feucht J, Sun J, Eyquem J, Ho YJ, Zhao Z, Leibold J, Dobrin A, Cabriolu A, Hamieh M, Sadelain M. Calibration of CAR activation potential directs alternative T cell fates and therapeutic potency. Nat Med. 2019 Jan;25(1):82-88.Shy BR, Vykunta VS, Ha A, Talbot A, Roth TL, Nguyen DN, Pfeifer WG, Chen YY, Blaeschke F, Shifrut E, Vedova S, Mamedov MR, Chung JJ, Li H, Yu R, Wu D, Wolf J, Martin TG, Castro CE, Ye L, Esensten JH, Eyquem J, Marson A. High-yield genome engineering in primary cells using a hybrid ssDNA repair template and small-molecule cocktails. Nat Biotechnol. 2023 Apr;41(4):521-531.Ethics Approval All animal studies conducted followed a protocol approved by the UCSF Institutional Animal Care and Use Committee.Abstract 247 Figure 1Overview of non-viral engineering platform