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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

¹ Department of Medicine, UCLA School of Medicine, Los Angeles, California
² Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas

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.

Key Words: blood-brain barrier • peptide nucleic acid • transferrin receptor • streptavidin • monoclonal antibody

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