Producing CAR T cells carrying large lentiviral transgenes has long posed a manufacturing challenge for next-generation cell therapies. Now, researchers at the University of Pennsylvania have introduced an optimized workflow that significantly improves the efficiency of generating CAR T cells with lentiviral transgenes exceeding 10 kilobases.
Chimeric antigen receptor (CAR) T cell therapies have transformed treatment for several blood cancers, but next-generation designs increasingly incorporate multiple engineered receptors to address antigen escape and tumor resistance. These larger constructs, however, strain the packaging limits of lentiviral vectors used to deliver CAR genes into T cells. As transgene size approaches or exceeds ~9 kb, viral titers and transduction efficiency fall sharply, complicating manufacturing and often requiring cell sorting to enrich engineered cells without increasing their total numbers.
The Penn team developed a step-by-step workflow designed to address this bottleneck. The protocol combines optimized lentivirus production conditions with adjustments to T cell culture and transduction parameters, including the use of a transduction enhancer and controlled culture surface-to-volume ratios.
Across a series of test constructs ranging from 5.7 kb to 10.1 kb, the optimized workflow markedly improved transduction performance compared with standard methods. For the largest construct, measuring 10.1 kb, transduction efficiency increased by roughly 10- to 12-fold and functional viral titers improved by about an order of magnitude. When the full production workflow was applied, T-cell transduction rates increased by up to 14.8-fold compared with conventional approaches.
Importantly, the modified protocol supported robust T-cell expansion without signs of toxicity and removed the need for cell sorting to enrich engineered cells. The authors report routinely achieving transduction rates of around 60 to 70 percent for ~9 kb constructs and 15 to 20 percent for 10 kb vectors.
By improving the efficiency of lentiviral delivery for larger genetic constructs, the workflow could help address a key manufacturing bottleneck in the development of more complex CAR T cell designs. As the authors write, “clinical translation of this workflow may also help reduce the manufacturing costs of lentivirus-modified cell therapy products.”
The authors also suggest the approach could be adapted for additional immune cell types, such as natural killer cells or macrophages, potentially broadening its therapeutic applications.
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