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Imaging Endogenous Gene Expression in Brain Cancer In Vivo with ¹¹¹In-Peptide Nucleic Acid Antisense Radiopharmaceuticals and Brain Drug-Targeting Technology

¹ College of Pharmacy, Nihon University, Chiba, Japan
² Department of Radiology, Wayne State University, Detroit, Michigan
³ Department of Neurology, Regensburg University, Regensburg, Germany
⁴ Department of Medicine, UCLA, Los Angeles, California

Imaging endogenous gene expression with sequence-specific antisense radiopharmaceuticals is possible if the antisense agent is enabled to traverse the biologic membrane barriers that separate the blood compartment from messenger RNA (mRNA) molecules in the cytoplasm of the target cell. The present studies were designed to image endogenous gene expression in brain cancer using peptide nucleic acid (PNA) antisense agents that were modified to allow for (a) chelation of the ¹¹¹In radionuclide and (b) attachment to a brain targeting system, which delivers the PNA across both the blood–brain barrier (BBB) and the tumor cell membrane. Methods: PNAs were designed that were antisense to either the rat glial fibrillary acidic protein (GFAP) mRNA or the rat caveolin-1 (CAV) mRNA. The PNA contained an amino-terminal diethylenetriaminepentaacetic acid moiety to chelate ¹¹¹In and a carboxyl-terminal -biotinyl lysine residue, which enabled attachment to the delivery system. The latter comprised streptavidin (SA) and the murine OX26 monoclonal antibody to the rat transferrin receptor (TfR), which were joined by a thiol-ether linker. Control PNAs were not conjugated to SA-OX26. Brain tumors developed after the intracerebral injection of rat RG2 glial cells in adult Fischer CD344 rats. GFAP and CAV gene expression in the tumor in vivo was monitored by confocal microscopy and Northern blotting with GFAP and CAV complementary DNAs. Results: If the PNA was not targeted to the TfR, then no imaging of any brain structures was possible, owing to the absence of PNA transport across the BBB. Conjugation of the ¹¹¹In-GFAP-PNA to the SA-OX26 delivery system did not image brain cancer, owing to the downregulation of the GFAP mRNA in brain glial tumors. In contrast, brain cancer was selectively imaged with the ¹¹¹In-CAV-PNA conjugated to SA-OX26 owing to upregulation of CAV gene expression in brain cancer. Conclusion: Imaging endogenous gene expression in vivo with PNA antisense radiopharmaceuticals is possible if drug-targeting technology is used. Attachment of the PNA antisense agent to the targeting ligand enables the antisense radiopharmaceutical to traverse biologic membrane barriers and access intracellular target mRNA molecules.

Key Words: glial fibrillary acid protein • caveolin-1 • blood–brain barrier • transferrin receptor • biotin

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