Neuronal mRNA PET Imaging

描述(由申请者提供)：据估计，美国有数百万人患有毒瘾，与严重的健康问题有关，每年有数千人死亡。可卡因与多巴胺受体2(D2R)结合，是被滥用最广泛的药物。观察到更高的可卡因敏感性与D2R或MAO A表达减少相关，这似乎是违反直觉的。我们假设，体外正电子发射断层扫描(PET)成像和编码D2R或MAO A的神经元mRNAs的定量将使临床前模型和人类受试者对可卡因敏感性的实时分析成为可能。我们已经设计并展示了一种新的技术，可以从体外可视化细胞内的mRNAs。信使核糖核酸靶向剂是多肽核酸(PNA)。当放射性标记和静脉注射时，这些序列与激活基因的mRNA拷贝特异地杂交。我们添加了一种小肽类似物，以允许表达特定细胞表面受体的靶细胞摄取mRNA显像剂。最后，我们添加了一种螯合剂，将发射正电子的放射性核素结合到信使核糖核酸显像剂上，以便进行外部PET成像。尾静脉注射CCND1、IRS1、MYCC和KRAS mRNAs的mRNA显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瘤的活体小鼠的基因表达。不匹配的探针和对照细胞产生了背景信号。其他实验室已经证明，PNA偶联物可以穿越血脑屏障。为了将mRNA显像剂特异性地内吞到神经细胞中，我们需要使用一种特有的神经元受体。高水平的MU阿片受体2(MOPR)由神经细胞高表达，神经细胞也表达D2R和MAOA蛋白。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，我们提出了一项初步研究，旨在通过两个特定的目标来设计和测试新型的mRNA PET显像剂，用于检测神经细胞中表达的D2R和MAOA mRNAs。具体目标1：我们将设计、合成、提纯和鉴定D2R和MAOA mRNA显像剂，其中DAMGO专用于神经细胞内吞作用的MOPR。我们还将准备用于共聚焦显微镜研究的荧光探针。对于这两个基因靶点，我们还将合成和评估PNA和多肽错配对照。具体目标2：我们将确定D2R和MAOA mRNA显像剂在培养的Cho-Harmor细胞、错配对照细胞和不表达MOPR的对照细胞中的积聚是否大于或等于3倍。荧光探针将被用来研究内吞作用和细胞内运输。如果DAMGO不能促进细胞质定位，将选择替代脑啡肽衍生物并进行测试。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显像剂将被用于定量摄取。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mRNAs的mRNA显像剂。公共卫生相关性：我们建议开发一种基因核医学程序，以揭示和测量来自体外的脑细胞基因的活动。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动之间的联系。未来，在这项提议的范围之外，对人类脑细胞基因活动的核医学成像可能会解释为什么一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.