Lentiviral Surface Engineering via Tris(2-Carboxyethyl)Phosphine-mediated HEK-293T Cell Modification

초록

This study presents an effective surface modification strategy for lentiviral vectors (LVs) by engineering HEK-293 T producer cells with tris(2-carboxyethyl)phosphine (TCEP) and thiol-reactive biomolecules. Treatment of HEK-293 T cells with 0.5 mM TCEP selectively reduced extracellular disulfide bonds, exposing free thiol groups for covalent conjugation while maintaining > 90% cell viability. Fluorescein isothiocyanate (FITC) labeling confirmed efficient surface modification, with 96.5 +/- 2.7% of TCEP-treated cells exhibiting uniform fluorescence, compared to 12.3 +/- 3.1% in FITC-only controls. Flow cytometry revealed dose-dependent modification, achieving > 95% efficiency at 1 mM FITC. Maleimide-functionalized chondroitin sulfate (CS-M) was conjugated to TCEP-treated cells, producing LVs with a - 18 mV shift in zeta potential, indicative of stable surface coating. Functional assays in HeLa cells demonstrated that CS-M-modified LVs retained infectivity comparable to unmodified controls, with equivalent GFP expression levels (p > 0.05). This method leverages natural viral budding mechanisms to integrate cell-surface modifications into LV envelopes, bypassing complex post-production engineering. The approach enables rapid (< 1 h) and scalable viral vector customization, offering a viable platform for attaching therapeutic cargos. These advances address critical limitations in conventional LV surface engineering, positioning cell-mediated modification as a robust alternative for next-generation gene therapy applications.

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