Imaging endogenous gene expression in brain cancer in vivo with 111In-peptide nucleic acid antisense radiopharmaceuticals and brain drug-targeting technology.
Imaging endogenous gene expression in brain cancer in vivo with 111In-peptide nucleic acid antisense radiopharmaceuticals and brain drug-targeting technology.
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发表时间：
2004-10
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通讯作者：
Toyofumi Suzuki;Dafang Wu;F. Schlachetzki;Jian Yi Li;R. Boado;W. Pardridge
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作者：
Toyofumi Suzuki;Dafang Wu;F. Schlachetzki;Jian Yi Li;R. Boado;W. Pardridge
如果反义药物能够穿过生物膜屏障，将靶细胞细胞质中的血室与信使RNA（mRNA）分子分开，则可以使用序列特异性反义放射性药物对内源性基因表达进行成像。本研究旨在使用肽核酸（PNA）反义试剂对脑癌中的内源性基因表达进行成像，所述肽核酸（PNA）反义试剂经修饰以允许（a）螯合111 In放射性核素和（B）附着于脑靶向系统，所述脑靶向系统递送PNA穿过血脑屏障（BB B）和肿瘤细胞膜。方法设计与大鼠胶质纤维酸性蛋白（GFAP）mRNA或大鼠小窝蛋白-1 α（CAV）mRNA反义的PNA。PNA含有氨基末端二亚乙基三胺五乙酸部分以螯合111 In和羧基末端ε-生物素基赖氨酸残基，这使得能够附接至递送系统。后者包括链霉亲和素（SA）和鼠OX 26单克隆抗体的大鼠转铁蛋白受体（TfR），这是由硫醇-醚接头连接。对照PNA不与SA-0X 26缀合。在成年Fischer CD 344大鼠脑内注射大鼠RG 2胶质细胞后发生脑肿瘤。用共聚焦显微镜和北方印迹法检测GFAP和CAV基因在肿瘤中的表达。结果：如果PNA不靶向TfR，则由于没有PNA穿过BBB的转运，任何脑结构的成像都是不可能的。111 In-GFAP-PNA与SA-OX 26递送系统的缀合物不能成像脑癌，这是由于脑胶质瘤中GFAP mRNA的下调。相比之下，脑癌选择性成像111 In-CAV-PNA共轭SA-OX 26由于上调CAV基因表达的脑癌。结论利用药物靶向技术，PNA反义放射性药物在体内显像内源性基因表达是可能的。PNA反义试剂与靶向配体的连接使得反义放射性药物能够穿过生物膜屏障并接近细胞内靶mRNA分子。
UNLABELLED 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-1alpha (CAV) mRNA. The PNA contained an amino-terminal diethylenetriaminepentaacetic acid moiety to chelate 111In and a carboxyl-terminal epsilon-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.