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<p>This study presents an effective surface modification strategy for lentiviral vectors (LVs) by engineering HEK-293&#xa0;T producer cells with tris(2-carboxyethyl)phosphine (TCEP) and thiol-reactive biomolecules. Treatment of HEK-293&#xa0;T cells with 0.5&#xa0;mM TCEP selectively reduced extracellular disulfide bonds, exposing free thiol groups for covalent conjugation while maintaining &gt; 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 &gt; 95% efficiency at 1&#xa0;mM FITC. Maleimide-functionalized chondroitin sulfate (CS-M) was conjugated to TCEP-treated cells, producing LVs with a − 18&#xa0;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 (<i>p</i> &gt; 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 (&lt; 1&#xa0;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.</p>

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

  • 期刊论文

  • 发表日期：

    2025年7月29日

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  • Su-Hwan Kim

摘要

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.