title: Intracellular delivery of an antisense oligonucleotide via endocytosis of a G protein-coupled receptor.
abstract: 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.
authors.affiliation: Division of Molecular Pharmaceutics, UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599, USA.
authors.forename: Xin
authors.initials: X
authors.lastname: Ming
authors.forename: Md Rowshon
authors.initials: MR
authors.lastname: Alam
authors.forename: Michael
authors.initials: M
authors.lastname: Fisher
authors.forename: Yongjun
authors.initials: Y
authors.lastname: Yan
authors.forename: Xiaoyuan
authors.initials: X
authors.lastname: Chen
authors.forename: Rudolph L
authors.initials: RL
authors.lastname: Juliano
mesh_terms: D045744:Cell Line, Tumor; D004705:Endocytosis; D006801:Humans; D007700:Kinetics; D049409:Luciferases, Firefly / Q000032:analysis; D016376:Oligonucleotides, Antisense / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D018004:Receptors, Bombesin / Q000378:metabolism*
chemical_list: D016376:Oligonucleotides, Antisense; D018004:Receptors, Bombesin; D049409:Luciferases, Firefly