Neuronal mRNA PET Imaging

描述（由申请人提供）：据估计，美国有数百万人患有药物成瘾，与严重的健康问题和每年数千人死亡有关。与多巴胺受体2（D2 R）结合的可卡因是最广泛滥用的药物。更大的可卡因敏感性与降低的D2 R或MAO A表达相关的观察似乎违反直觉。我们假设，外部正电子发射断层扫描（PET）成像和定量的神经元mRNA编码D2 R或MAO A将使实时分析可卡因的敏感性在临床前模型和人类受试者。 我们已经设计并展示了一种新的技术来可视化来自体外的细胞内mRNA。mRNA靶向剂是肽核酸（PNA）。当放射性标记并静脉注射时，这些序列与活化基因的mRNA拷贝特异性杂交。我们添加了一种小肽类似物，以使mRNA成像剂被表达特征性细胞表面受体的靶细胞摄取。最后，我们添加了一种螯合剂，将正电子发射放射性核素与mRNA成像剂结合，以允许外部PET成像。 尾静脉给药CCND 1、IRS 1、MYCC和KRAS mRNA的mRNA成像剂使我们能够可视化携带乳腺癌、胰腺癌和前列腺癌异种移植物的活小鼠中的基因表达。错配探针和对照细胞产生背景信号。其他实验室已经证明PNA缀合物可以穿过血脑屏障。 对于特异性地将mRNA显像剂内吞到神经元细胞中，我们需要使用特征性神经元受体。高水平的μ阿片受体2（MOPR）由也表达D2 R和MAOA蛋白的神经元细胞高度表达。脑啡肽衍生物N-Tyr-D-Ala-Gly-N-Me-Phe-Gly-ol（DAMGO）与MOPR紧密结合并诱导内化。 基于上述观察结果，我们提出了一个试点研究，设计和测试新的mRNA PET成像剂的D2 R和MAOA的mRNA在神经元细胞中表达，通过两个特定的目标。 具体目标1：我们将设计，合成，纯化和表征D2 R和MAOA mRNA显像剂与DAMGO特异性MOPR神经元细胞内吞。我们也将准备荧光探针共聚焦显微镜研究。对于这两种基因靶点，我们还将合成和评估PNA和肽错配对照。 具体目标二：我们将确定D2 R和MAOA mRNA成像剂在培养物中的CHO-HArMOR细胞中是否显示出比错配对照和不表达MOPR的对照细胞大3倍或等于3倍的累积。荧光探针将用于研究内吞作用和细胞内运输。如果DAMGO未能促进细胞质定位，则将选择并检测替代脑啡肽衍生物。一旦我们确定了诱导MOPR介导的内吞作用的配体，放射性标记的mRNA成像剂将用于定量摄取。 与我们的假设一致的结果将允许在动物模型中测试神经元mRNA的mRNA成像剂。公共卫生相关性：我们建议开发一种遗传核医学程序，从体外揭示和测量脑细胞基因的活性。我们将用我们的新方法研究可卡因敏感性和脑细胞基因活性之间的联系。在未来，超出这个建议的范围，人类脑细胞基因活动的核医学成像可能会解释为什么有些人比其他人更容易上瘾。

DESCRIPTION (provided by applicant): Millions of individuals in the US are estimated to suffer from drug addiction, associated with aggravated health problems and thousands of deaths annually. Cocaine, which binds to the dopamine receptor 2 (D2R), is the most widely abused drug. The observation that greater cocaine sensitivity is associated with reduced D2R or MAO A expression seems counterintuitive. We hypothesize that external positron emission tomographic (PET) imaging and quantitation of neuronal mRNAs that encode D2R or MAO A will enable realtime analysis of cocaine sensitivity in preclinical models and human subjects. We have designed and demonstrated a novel technology to visualize intracellular mRNAs from outside the body. The mRNA targeting agents are peptide nucleic acids (PNAs). When radiolabeled and administered i.v., these sequences hybridize specifically to mRNA copies of activated genes. We added a small peptide analog to allow the mRNA imaging agents to be taken up by target cells that express a characteristic cell surface receptor. Finally, we added a chelator to bind a positron-emitting radionuclide to the mRNA imaging agents to permit external PET imaging. Tail vein administration of mRNA imaging agents for CCND1, IRS1, MYCC, and KRAS mRNAs have enabled us to visualize gene expression in live mice bearing breast cancer, pancreas cancer, and prostate cancer xenografts. Mismatch probes and control cells yielded background signals. Other laboratories have demonstrated that PNA conjugates can cross the blood-brain barrier. For endocytosis of mRNA imaging agents specifically into neuronal cells, we need to use a characteristic neuronal receptor. High levels of mu opioid receptor 2 (MOPR) are highly expressed by neuronal cells that also express D2R and MAOA proteins. An enkephalin derivative, N-Tyr-D-Ala-Gly-N-Me-Phe-Gly-ol (DAMGO), binds tightly to MOPR and induces internalization. Based on the above observations, we propose a pilot study to design and test novel mRNA PET imaging agents for D2R and MAOA mRNAs expressed in neuronal cells, through two specific aims. Specific Aim 1: We will design, synthesize, purify, and characterize D2R and MAOA mRNA imaging agents with DAMGO specific for MOPR for neuronal cell endocytosis. We will also prepare fluorescent probes for confocal microscopy studies. For both gene targets, we will also synthesize and evaluate PNA and peptide mismatch controls. Specific Aim 2: We will determine whether or not the D2R and MAOA mRNA imaging agents show greater than or equal to 3-fold greater accumulation in CHO-HArMOR cells in culture, vs. mismatch controls, and vs. control cells that do not express MOPR. Fluorescent probes will be used to study endocytosis and intracellular trafficking. If DAMGO fails to promote cytoplasmic localization, alternate enkephalin derivatives will be selected and tested. Once we have identified a ligand that induces MOPR-mediated endocytosis, Radiolabeled mRNA imaging agents will be used to quantitate uptake. Results consistent with our hypothesis would permit testing of mRNA imaging agents for neuronal mRNAs in animal models. PUBLIC HEALTH RELEVANCE: We propose to develop a genetic nuclear medicine procedure to reveal and measure the activity of brain cell genes from outside the body. We will study connections between cocaine sensitivity and brain cell gene activity with our new method. In the future, beyond the scope of this proposal, nuclear medicine imaging of brain cell gene activity in humans might explain why some individuals become addicted more easily than others.