68Gallium labeling of antisense oligonucleotide for in vivo PET imaging of 5HT1A receptor gene expression

Objectives A marked reduction in the expression of 5HT1A receptors could be seen in the patients of neurodegenerative disorders. To detect the 5HT1A receptor expression, the antisense oligonucleotide was modified at 5’ terminal azide and carboxyl-dT as the last base of the oligo, with a C3-spacer at the 3’ terminus of this oligonucleotide. Production of 68 Ga radiopharmaceuticals demands highly efficient labeling chemistry and, therefore, highly efficient bifunctional triazacyclononane-propargyl diacetate ligand was used for click conjugation to load gallium.

Methods 5’Azide(N3)-hexyl derivative of phosphorothioate 5HT1A specific antisense oligodinucleotide (Azide-C6)-5’-GGTGGTGATGTGGTGTTGTT-3’-COOH(N3-ATSO-5HT1A) was conjugated to a macrocyclic chelating agent 2,2'-(7-(prop-2-ynyl)-1,4,7-triazonane-1,4-diyl)diacetic acid (NO2A-propargyl) through terminal Azide using click chemistry approach. The oligonucleotide conjugate was radiolabeled and evaluated in animal models. Cellular uptake studies were done in hippocampal neurons.

Results [68Ga]-NO2A-Triazole-5HT1A-ATSO was obtained in a 45-55% radiochemical yield. Purification is performed by reversed phase chromatography on a C18 nucleosil column (Rt= 5.2 min for product). The whole procedure took 35 min and the specific activity was found to be 400 mCi/µmol at the end of the synthesis. The study demonstrated gene expression in the brain in vivo could be imaged with antisense radiopharmaceuticals that are conjugated to a brain mRNA-targeting system.

Conclusions We conclude that 68Ga-labeling of oligonucleotides using click conjugation is a feasible and convenient approach for in vivo imaging and quantification of oligonucleotide biokinetics in living animals with PET.

Research Support This work is supported by the DRDO Project INM-311(3.1).