Imaging gene expression in the brain in vivo in a transgenic mouse model of Huntington's disease with an antisense radiopharmaceutical and drug-targeting technology

Disease-specific genes of unknown function can be imaged in vivo with antisense radiopharmaceuticals, providing the transcellular transport of these molecules is enabled with drug-targeting technology. The current studies describe the production of 16-mer peptide nucleic acid (PNA) that is antisense around the methionine initiation codon of the huntingtin gene of Huntington's disease (HD). Methods: The PNA is biotinylated, which allows for rapid capture by a conjugate of streptavidin and the rat 8D3 monoclonal antibody (mAb) to the mouse transferrin receptor (TfR), and contains a tyrosine residue, which enables radiolabeling with ¹²⁵I. The reformulated PNA antisense radiopharmaceutical that is conjugated to the 8D3 mAb is designated ¹²⁵I-PNA/8D3. This form of the PNA is able to access endogenous transferrin transport pathways at both the blood- brain barrier and the brain cell membrane and undergoes both import from the blood to the brain and export from the brain to the blood through the TfR. Results: The ability of the PNA to hybridize to the target huntingtin RNA, despite conjugation to the mAb, was shown both with cell-free translation assays and with ribonuclease protection assays. The ¹²⁵I-PNA/8D3 conjugate was administered intravenously to either littermate control mice or to R6/2 transgenic mice, which express the exon 1 of the human HD gene. The mice were sacrificed 6 h later for frozen sectioning of the brain and quantitative autoradiography. The studies showed a 3-fold increase in sequestration of the ¹²⁵I-PNA/8D3 antisense radiopharmaceutical in the brains of the HD transgenic mice in vivo, consistent with the selective expression of the HD exon-1 messenger RNA in these animals. Conclusion: These results support the hypothesis that gene expression in vivo can be quantitated with antisense radiopharmaceuticals, providing these molecules are reformulated with drug-targeting technology. Drug targeting enables access of the antisense agent to endogenous transport pathways, which permits passage across the cellular barriers that separate blood and intracellular compartments of target tissues.