Intracellular delivery of an antisense oligonucleotide via endocytosis of a G protein-coupled receptor

Nucleic Acids Res. 2010 Oct;38(19):6567-76. doi: 10.1093/nar/gkq534. Epub 2010 Jun 15.


Gastrin-releasing peptide receptor (GRPR), a member of the G protein-coupled receptor superfamily, has been utilized for receptor-mediated targeting of imaging and therapeutic agents; here we extend its use to oligonucleotide delivery. A splice-shifting antisense oligonucleotide was conjugated to a bombesin (BBN) peptide, and its intracellular delivery was tested in GRPR expressing PC3 cells stably transfected with a luciferase gene interrupted by an abnormally spliced intron. The BBN-conjugate produced significantly higher luciferase expression compared to unmodified oligonucleotide, and this increase was reversed by excess BBN peptide. Kinetic studies revealed a combination of saturable, receptor-mediated endocytosis and non-saturable pinocytosis for uptake of the conjugate. The K(m) value for saturable uptake was similar to the EC(50) value for the pharmacological response, indicating that receptor-mediated endocytosis was a primary contributor to the response. Use of pharmacological and molecular inhibitors of endocytosis showed that the conjugate utilized a clathrin-, actin- and dynamin-dependent pathway to enter PC3 cells. The BBN-conjugate partially localized in endomembrane vesicles that were associated with Rab7 or Rab9, demonstrating that it was transported to late endosomes and the trans-golgi network. These observations suggest that the BBN-oligonucleotide conjugate enters cells via a process of GRPR mediated endocytosis followed by trafficking to deep endomembrane compartments.

Publication types

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

MeSH terms

  • Cell Line, Tumor
  • Endocytosis*
  • Humans
  • Kinetics
  • Luciferases, Firefly / analysis
  • Oligonucleotides, Antisense / chemical synthesis
  • Oligonucleotides, Antisense / chemistry
  • Oligonucleotides, Antisense / metabolism*
  • Receptors, Bombesin / metabolism*


  • Oligonucleotides, Antisense
  • Receptors, Bombesin
  • Luciferases, Firefly