Optimized Workflow for Self-Delivering siRNA-Mediated Gene Knockdown in Unstimulated Human CD4+ T Cells
Anangi, B.
Show abstract
T-cell-based therapies have transformed cancer treatment, yet dissecting the molecular networks that govern T-cell function remains technically challenging. Gene manipulation in primary human T cells typically relies on electroporation or viral vectors, approaches that often compromise viability, alter cell physiology, or introduce genotoxic risks. Accell small interfering RNAs (siRNAs) are chemically modified, self-delivering siRNAs that bypass the need for transfection reagents or viral systems. Despite their potential, Accell siRNAs have been underutilized in human T cells. Here, using freshly isolated, unstimulated primary CD4+ T cells, we optimized conditions for Accell siRNA uptake and gene expression knockdown. We demonstrate efficient and reproducible delivery of Accell siRNAs across multiple serum-free media, with Accell delivery medium providing the highest uptake. Using a GAPDH-targeting Accell siRNA pool, we achieved robust transcript knockdown without loss of viability, and knockdown efficiency was modestly enhanced by CD3 stimulation. This streamlined workflow enables rapid assessment of gene function in resting human T cells without genetic modification or pre-activation, expanding the toolkit available for immunology and therapeutic research.
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