Molecular Therapy: Methods & Clinical Development (Jun 2020)

Rapid Delivery of Nanobodies/VHHs into Living Cells via Expressing In Vitro-Transcribed mRNA

  • Xuechen Zhou,
  • Rui Hao,
  • Chen Chen,
  • Zhipeng Su,
  • Linhong Zhao,
  • Zhuojuan Luo,
  • Wei Xie

Journal volume & issue
Vol. 17
pp. 401 – 408

Abstract

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Intracellular antigen labeling and manipulation by antibodies have been long-thought goals in the field of cell research and therapy. However, a central limitation for this application is that antibodies are not able to penetrate into the cytosol of living cells. Taking advantages of small sizes and unique structures of the single-domain antibodies, here, we presented a novel approach to rapidly deliver the nanobody/variable domain of heavy chain of heavy-chain antibody (VHH) into living cells via introducing its coding mRNA, which was generated by in vitro transcription. We demonstrated that actin-green fluorescent proteins (GFP) and Golgi-GFP can be recognized by the anti-GFP nanobody/VHH, vimentin can be recognized by the anti-vimentin nanobody/VHH, and histone deacetylase 6 (HDAC6) can be recognized by the anti-HDAC6 nanobody/VHH, respectively. We found that the anti-GFP nanobody expressed via in vitro-transcribed (IVT) mRNA can be detected in 3 h and degraded in 48 h after transfection, whereas the nanobody expressed via plasmid DNA, was not detected until 24 h after transfection. As a result, it is effective in delivering the nanobody through expressing the nanobody/VHH in living cells from its coding mRNA.