Neuronal mRNA PET Imaging

神经元 mRNA PET 成像

• 批准号: 7773248
• 负责人: ERIC WICKSTROM
• 金额: $ 15.45万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7773248
• 关键词: Aggressive behavior; Animal Model; Antibodies; Avidin; Binding; Biotin; Blood; Blood - brain barrier anatomy; Brain; Brain imaging; CCND1 gene; Cell Surface Receptors; Cells; Cessation of life; Characteristics; Chelating Agents; Cocaine; Cocaine Dependence; Confocal Microscopy; Contralateral; Cytoplasm; DRD2 gene; Diffuse; Discipline of Nuclear Medicine; Dopamine; Dopamine Receptor; Drug Addiction; Endocytosis; Enkephalins; Figs - dietary; Fluorescent Probes; Future; Gene Activation; Gene Expression; Gene Targeting; Genes; Genetic; Health; Human; IRS1 gene; Image; Imagery; Individual; KRAS2 gene; Laboratories; Life; Ligands; Malignant neoplasm of pancreas; Malignant neoplasm of prostate; Measures; Mediating; Messenger RNA; Methaqualone; Methods; Monoamine Oxidase A; Mus; Muscle; Neurons; Nucleic Acid Probes; Oncogenes; Opioid; Peptide Nucleic Acids; Peptides; Pharmaceutical Preparations; Photons; Pilot Projects; Positron; Positron-Emission Tomography; Pre-Clinical Model; Procedures; Proteins; RNA; Radioisotopes; Radiolabeled; Relative (related person); Reporter; Scanning; Signal Transduction; Site; Structure; Surface; Tail; Testing; Tissues; Transferrin Receptor; Tyrosine; Veins; Xenograft procedure; alanylglycine; analog; anti social; base; brain cell; design; fluorophore; glycylphenylalanine; human subject; malignant breast neoplasm; mu opioid receptors; new technology; novel; peptide analog; public health relevance; radiotracer; receptor; trafficking; trait; tumor; uptake

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.

描述（由申请人提供）：据估计，美国数以百万计的人会遭受吸毒成瘾的困扰，与严重的健康问题和数千人死亡有关。与多巴胺受体2（D2R）结合的可卡因是最广泛的滥用药物。观察到较高的可卡因敏感性与降低的D2R或MAO A表达相关的观察似乎是违反直觉的。我们假设对临床前模型和人类受试者中可卡因敏感性的实时分析，对编码D2R或MAO的神经元mRNA的外部正电子发射层析成像（PET）成像和定量将实现实时分析。 我们设计并展示了一种新型技术，可以可视化体外外的细胞内mRNA。 mRNA靶向剂是肽核酸（PNA）。当放射性标记和施用静脉内时，这些序列专门杂交与活化基因的mRNA拷贝。我们添加了一个小的肽类似物，以使mRNA成像剂被表达特征性细胞表面受体的靶细胞吸收。最后，我们添加了一个螯合剂，以结合正电子发射放射性核素与mRNA成像剂允许外部PET成像。 CCND1，IRS1，MYCC和KRAS mRNA的mRNA成像剂的尾静脉给药使我们能够可视化患有乳腺癌，胰腺癌和前列腺癌异种移植的活小鼠中的基因表达。不匹配探针和对照细胞产生了背景信号。其他实验室表明，PNA结合物可以越过血脑屏障。 为了专门针对神经元细胞的mRNA成像剂的内吞作用，我们需要使用特征性的神经元受体。高水平的MU阿片受体2（MOPR）由也表达D2R和MAOA蛋白的神经元细胞高度表达。 Enkephalin衍生物N-tyr-d-ala-gly-n-me-phe-gly-ol（Damgo）与MOPR紧密结合并诱导内在化。 基于上述观察结果，我们提出了一项试点研究，以通过两个特定的目的设计和测试在神经元细胞中表达的D2R和MAOA mRNA的新型mRNA PET成像剂。 具体目标1：我们将设计，合成，纯化和表征D2R和MAOA mRNA成像剂，具有DAMGO的MOPR特异性用于神经元细胞内吞作用。我们还将准备荧光探针进行共聚焦显微镜研究。对于两个基因靶标，我们还将合成和评估PNA和肽不匹配对照。 具体目标2：我们将确定D2R和MAOA mRNA成像剂是否显示出培养物中的Cho-Harmor细胞中大于或等于3倍的积累，与不匹配的对照组以及与未表达MOPR的对照细胞相比。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，则将选择和测试替代的Enkephalin衍生物。一旦我们确定了诱导MOPR介导的内吞作用的配体，将使用放射性标记的mRNA成像剂来定量摄取。 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，以揭示和衡量体外外部脑细胞基因的活性。我们将通过我们的新方法研究可卡因敏感性与脑细胞基因活性之间的联系。将来，除了该提案的范围之外，人类脑细胞基因活性的核医学成像可能解释了为什么有些人比其他人更容易上瘾。