Receptor-mediated delivery of engineered nucleases for genome modification

Nucleic Acids Res. 2013 Oct;41(19):e182. doi: 10.1093/nar/gkt710. Epub 2013 Aug 16.

Abstract

Engineered nucleases, which incise the genome at predetermined sites, have a number of laboratory and clinical applications. There is, however, a need for better methods for controlled intracellular delivery of nucleases. Here, we demonstrate a method for ligand-mediated delivery of zinc finger nucleases (ZFN) proteins using transferrin receptor-mediated endocytosis. Uptake is rapid and efficient in established mammalian cell lines and in primary cells, including mouse and human hematopoietic stem-progenitor cell populations. In contrast to cDNA expression, ZFN protein levels decline rapidly following internalization, affording better temporal control of nuclease activity. We show that transferrin-mediated ZFN uptake leads to site-specific in situ cleavage of the target locus. Additionally, despite the much shorter duration of ZFN activity, the efficiency of gene correction approaches that seen with cDNA-mediated expression. The approach is flexible and general, with the potential for extension to other targeting ligands and nuclease architectures.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Cell Line
  • Cells, Cultured
  • DNA Cleavage
  • Deoxyribonucleases / genetics
  • Deoxyribonucleases / metabolism*
  • Endocytosis
  • Genomics
  • Humans
  • Ligands
  • Mice
  • Protein Engineering
  • Receptors, Transferrin / metabolism*
  • Recombinant Fusion Proteins / metabolism
  • Targeted Gene Repair / methods*
  • Transferrin / genetics
  • Transferrin / metabolism
  • Zinc Fingers

Substances

  • Ligands
  • Receptors, Transferrin
  • Recombinant Fusion Proteins
  • Transferrin
  • Deoxyribonucleases