Synecta™ T1细胞衍生纳米颗粒与Flowfect®连续流转染提升CD19靶向CAR-T细胞的非病毒工程化效率和制造可扩展性用于血液系统恶性肿瘤

Background: Autologous CAR-T cell therapies have transformed outcomes in B-cell lymphomas, leukemias, andother indications, yet manufacturing duration, cost, process variability, and dependence on viralvectors continue to limit broader adoption. Improved processes are essential to deliver moreeffective cell therapies and enable broader adoption. Efficient non-viral gene editing requiresactivation that preserves viability and stem-like phenotypes, unlike CD3/CD28 activators, and adelivery system that achieves high editing efficiency without compromising cell health.BlueWhale Bio's Synecta™ T1 cell-derived nanoparticles (CDNPs) mimic physiologic APC signalingvia membrane-bound OKT-3, CD86, 4-1BBL, IL-7, and IL-15/IL-15RA. Kytopen's Flowfect Tx®continuous flow cell engineering technology maximizes yields of engineered cells, whileprocessing hundreds of billions of cells in minutes. Here we present the synergies of thesetechnologies to enhance CRISPR-mediated knock-in (KI) of a CD19-CAR at the TRAC locus. Methods: Primary healthy donor (HD) human T cells were activated for 2-3 days with Synecta T1 orcomparator activation technologies and then gene-edited using Flowfect Tx® platform to deliverCas9/sgRNA RNP and a CD19-CAR HDR template. KI and knock-out (KO) efficiency, viability,activation, CD4/CD8 ratio, and expansion were quantified through Day 8. Parallel experimentsevaluated Synecta T1 activation immediately after Flowfect Tx transfection in naïve T cells. Results: Synecta T1 induced >90% CD69⁺CD25⁺ activation while preserving CD3 expression. By Day 8, Tcells activated with Synecta and transfected Flowfect Tx® platform achieved >40% CD19-CAR KIand >90% TRAC KO, exceeding comparator activation technologies (<25% KI) and improving bothKI and KO efficiencies. Synecta T1 supported >80% viability and >5-fold expansion, yielding thehighest CAR-positive cell numbers, while maintaining a favorable CD4/CD8 profile. In the naïveworkflow, delivering RNP with a nanoplasmid HDR donor first and then activating with SynectaT1 increased KI-positive cells and expansion compared with unstimulated control, producingmarkedly higher CAR⁺ output. Conclusions: Synecta T1 and Flowfect Tx® platform enable efficient non-viral genome editing and rapidexpansion, supporting a streamlined, non-viral manufacturing approach for CD19-CAR-T cells. Byimproving KI and KO efficiency, Synecta T1 enhances CRISPR-based editing and supports targetedCAR insertion under endogenous promoters. Together, these technologies deliver a scalable workflow that achieves effective therapeutic doses quickly while preserving cell fitness. Futurestudies will evaluate activation strategies aligned with Day-2 or Day-0/1 Flowfect Tx® transfection.