Non-Hybridization Saturable Mechanisms Play a Role in the Uptake of⁶⁸Ga-Labeled LNA–DNA Mixmer Antisense Oligonucleotides in Rats

Abstract

Oligonucleotides (ODN) are key molecules for the aim of preventing translation of a gene product or monitoring gene expression in tissues. However, multiple methodological and biological hurdles need to be solved before in vivo application in humans will be possible. For positron emission tomography (PET) investigations, a 20-mer DNA-locked nucleic acid (LNA) mixmer ODN specific for rat chromogranin-A mRNA was labeled with (68)Ga and its uptake was examined in vivo in rats with and without blocking of scavenger receptors by polyribonucleotides. In addition, uptake studies of (68)Ga-LNA were performed with respect to time and concentration in human and rat cell lines. The human cell lines did not express the target mRNA. Both polyinosinic acid (poly-I) and polyadenylic acid (poly-A) reduced the uptake in rat tissues and in human cell lines. Poly-I was found to be more effective in the liver whereas poly-A was more effective in the kidney. In addition, the blockade by poly-I was statistically significant in the pancreas, adrenal gland, bone marrow, intestine, testis, urinary bladder, muscle, parotid gland, and heart, whereas poly-A also caused significant reduction in pancreas, adrenal gland, and bone marrow but not as much as in kidney. Cell culture study showed a 2-phase dose-dependent uptake characteristic with a saturable and a passive diffusion-like phase; however, these 2 phases were not so well expressed in the rat cell line. The results suggest that scavenger receptors or other saturable processes unrelated to hybridization may be involved in the tissue uptake of (68)Ga-LNA and in the clearance of antisense ODN through the liver, kidney, spleen, and bone marrow. The fact that these processes may be sequence-dependent suggests that proof of in vivo hybridization through imaging may not be obtained by only comparing sense and antisense sequences and proving dose-dependency.

Prospective of 68Ga-Radiopharmaceutical Development

“…The oligonucleotides varied in size (15-30 mer), sequence and basic structure: phosphodiester, phosphorothioate, 2'- O -methyl phosphodiester, locked nucleic acid (LNA), LNA-DNA mixmer (LNA and DNA nucleotides in alternation along the sequence), and peptide nucleic acid 316-320. The uptake of the 68 Ga-labelled analogues in non-hybridization specific tissue was most likely mediated by scavenger receptors.…”

Continued rapid growth in 68Ga applications: update 2013 to June 2014

“…Feasibility of in vivo targeting of mRNA for the development of imaging and therapeutic agents specific to oncogenes with point mutations was investigated using various analogues of antisense oligonucleotides. The oligonucleotides varied in size (15–30 mer), sequence and basic structure: phosphodiester, phosphorothioate, 2'‐ O ‐methyl phosphodiester, locked nucleic acid (LNA), LNA‐DNA mixmer (LNA and DNA nucleotides in alternation along the sequence) and peptide nucleic acid . The organ distribution of 68 Ga‐labelled analogues in healthy rats demonstrated variation dependent on the sequence and length of the oligonucleotides.…”

A practical guide to the construction of radiometallated bioconjugates for positron emission tomography

“…136–138 2′-Deoxyphosphodiester (PO), 2′-deoxyphosphorthioate (PS), 2′- O -methyl phosphodiester (OMe), and locked nucleic acid (LNA) oligonucleotides have been synthesized and radiolabeled for gene expression imaging. In all cases, DOTA has been used as the chelator for 68 Ga and is incorporated into the oligonucleotide using a DOTA-SNHS bifunctional chelate.…”