Aptamer Imaging: A Theranostic Approach to Treat Substance Abuse

适体成像：治疗药物滥用的治疗诊断方法

基本信息

批准号：
8473196
负责人：
Philip K Liu
金额：
$ 32.25万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-07-01 至 2014-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8473196
关键词：
AIDS neuropathy Abstinence Acute Affect Affinity Age Amphetamines Anesthesia procedures Anesthetics Animals Antibodies Antisense DNA Astrocytes Award Base Pairing Behavior Binding Biological Assay Biological Markers Biopsy Bipolar Disorder Blood Blood Glucose Brain Brain region Buffers Bypass Caliber Cardiovascular system Catheters Cell Surface Proteins Cells Cerebral Ischemia Cerebrospinal Fluid Cerebrum Chronic Clinical Complex Consensus Sequence Contrast Media Corpus striatum structure Custom Cytokine Receptors DNA Detection Development Diabetes Mellitus Diagnostic Disease Dopamine Antagonists Dose Drug Addiction Electrophoretic Mobility Shift Assay Environment Enzyme-Linked Immunosorbent Assay Enzymes Epigenetic Process Epilepsy Evaluation Exposure to Eye FOS gene Family Fluorescein Fluorescence Frequencies Functional Magnetic Resonance Imaging Future Gender General Anesthesia General Hospitals General Population Generations Genes Genetic Genetic Transcription Genomics Goals HIV-1 Head Head Movements Health Heart Arrest Hour Human Human Activities Image Imaging Techniques Immediate-Early Genes Immunoprecipitation Immunosorbents Implant In Vitro Incubated Individual Indwelling Catheter Infection Infusion procedures Injection of therapeutic agent Intelligence Interleukin-1 Interleukins Intervention Iron Isothiocyanates JUN gene Juice Knock-out Knockout Mice Knowledge Label Laboratories LacZ Genes Learning Length Letters Life Linear Regressions Link Locomotion Lung diseases Macaca mulatta Magnetic Resonance Magnetic Resonance Imaging Maps Massachusetts Measures Mediating Medical Memory Mental Depression Mental Health Mental disorders Messenger RNA Methods Modeling Modification Molecular Molecular Biology Molecular Target Molecular and Cellular Biology Monitor Monkeys Morphologic artifacts Motion Motor Mouse Strains Mus Myocardial Infarction NF-kappa B Needle Sharing Neuroglia Neurologic Neurons Nuclear Extract Nucleic Acids Operative Surgical Procedures Peripheral Pharmaceutical Preparations Plasma Play Population Positron-Emission Tomography Posture Preparation Primates Procedures Process Productivity Promega Promoter Regions Protein Binding Proteins Psyche structure Puncture procedure Quarantine RNA Race Reaction Reagent Recombinants Recovery Reflex action Research Research Personnel Resistance Rest Rewards Rhodamine Risk Rodent Role Running Sampling Scanning Schizophrenia Series Shapes Signal Pathway Signal Transduction Signaling Pathway Gene Site Small Interfering RNA Societies Source Specificity Spinal Cord Spinal Puncture Spottings Substance abuse problem Surface Antigens Symptoms System TNF gene Techniques Technology Testing Therapeutic Time TimeLine Tissues Toxic effect Training Transcription Coactivator Transcription Factor AP-1 Transcription factor genes Transfection Transgenic Mice Transgenic Organisms Translating Translational Research Treatment Efficacy Tumor Necrosis Factor-alpha United States United States National Institutes of Health Urine Variant Vertebral column Visual seizure Weight addiction anticancer treatment aptamer awake base biological adaptation to stress brain cell brain repair clinical application crosslink cytokine design disability dosage driving force drug of abuse ds-DNA experience in vivo indexing innovation inorganic phosphate iron oxide jun D Proteins member molecular imaging mutant nanometer nanoparticle nervous system disorder neuromuscular activity neuropsychiatry neurotoxicity neutravidin nonhuman primate novel nuclease pathogen protein metabolite public health relevance relating to nervous system research study sample fixation severe mental illness theranostics tool transcription factor uptake

项目摘要

DESCRIPTION (provided by applicant): This project will develop and explore Magnetic Resonance (MR) visible probes to image intracellular gene transcription activator proteins. Drug addiction is a major health problem that severely hampers the productivity of many members of our society. While studies indicate that drug addiction results from combined influences of genes and environment, recent studies suggest that epigenetic modifications of gene transcription factors may play an important role in the development of addictions in humans. Advances in cellular and molecular biology in the past century have led to identification of novel gene markers, the signaling pathways they influence and gene activities they regulate. Two such activities that they have shown modification in in studies of drug addiction are intracellular activator protein-1 (AP-1) and nuclear factor kappa- beta (NF-k-b). The inability to produce a functional AP-1 protein to bind at the AP-1 site in a transgenic mouse blocks sensitization of drugs of abuse. With biopsy the only source of tissue for conventional assays, such studies are not permitted in humans. We aim to develop and investigate a targeted MR imaging technique and to apply it for studies of drug addiction in a series of live brains. With a long-term goal of enabling specific manipulation of epigenetic process to non-human primates (NHP), we formulate a hypothesis: short double- stranded (ds) DNA aptamers with the sequence domain for AP-1 protein will function as decoys for endogenous AP-1 protein binding. The aptamers will be linked to an MR-visible contrast agent (superparamagnetic iron oxide nanoparticles-NeutrAvidin, or SPION-NA, 49 nm in diameter), which permits imaging AP-1 proteins in live animal subjects. We have outlined the following steps to validate our hypothesis: Step 1. Design a nuclease-resistant dsAP-1 aptamer with high affinity for AP-1 protein binding Step 2. Quantify rhodamine (Rhd)-labeled dsAP-1 aptamer binding in vitro Step 3. Demonstrate SPION-dsAP1 delivery for MRI and compare in vivo MR signal in C57black6 mice and a mutant strain known to produce no or less AP-1 protein (A-FOS/NSE or FosB knockout mutants) Step 4. Demonstrate Rhd-dsAP1 uptake specificity in cells that express green fluorescence protein (GFP) directed by AP-1 protein [B6;DBA-Tg(Fos-tTA,Fos-EGFP*)1Mmay Tg(tetO-lacZ,tTA*)1Mmay/J] Step 5. Demonstrate dsAP-1 at high dose blocks AP-1 protein-induced activity in mice after amphetamine Step 6. Demonstrate that SPION-dsAP1 delivery allows a window for MRI in live non-human primates and detects elevation of AP-1 protein after amphetamine exposure This application is designed to enable highly innovative and conceptually creative research of molecular targeting of gene products using an unconventional, novel targeted MRI technique for in vivo systems. Because transcription factor binding domains are conserved from rodents to humans, our innovative technique, once validated, has potential for theranostic application, from rodents to primates.

描述（由申请人提供）：该项目将开发和探索磁共振（MR）可见探针来对细胞内基因转录激活蛋白进行成像。吸毒成瘾是一个重大的健康问题，严重影响了我们社会许多成员的生产力。虽然研究表明药物成瘾是基因和环境共同影响的结果，但最近的研究表明基因转录因子的表观遗传修饰可能在人类成瘾的发展中发挥重要作用。上个世纪细胞和分子生物学的进步导致了新基因标记、它们影响的信号传导途径和它们调节的基因活性的鉴定。他们在药物成瘾研究中显示出两种此类活性的改变，即细胞内激活蛋白 1 (AP-1) 和核因子 kappa-β (NF-k-b)。转基因小鼠无法产生与 AP-1 位点结合的功能性 AP-1 蛋白，从而阻断了滥用药物的致敏作用。由于活检是常规检测的唯一组织来源，因此不允许在人类中进行此类研究。我们的目标是开发和研究一种靶向磁共振成像技术，并将其应用于一系列活体大脑中的药物成瘾研究。为了实现对非人类灵长类动物 (NHP) 的表观遗传过程进行特异性操作的长期目标，我们提出了一个假设：具有 AP-1 蛋白序列结构域的短双链 (ds) DNA 适体将充当内源性 AP-1 蛋白结合。适配体将与 MR 可见造影剂（超顺磁性氧化铁纳米颗粒 - NeutrAvidin 或 SPION-NA，直径 49 nm）连接，从而允许在活体动物受试者中对 AP-1 蛋白进行成像。我们概述了以下步骤来验证我们的假设：步骤 1. 设计对 AP-1 蛋白结合具有高亲和力的核酸酶抗性 dsAP-1 适体步骤 2. 定量罗丹明 (Rhd) 标记的 dsAP-1 适体体外结合步骤3. 展示用于 MRI 的 SPION-dsAP1 传递，并比较 C57black6 小鼠和已知不产生或产生较少产生的突变株的体内 MR 信号AP-1 蛋白（A-FOS/NSE 或 FosB 敲除突变体）步骤 4. 在 AP-1 蛋白 [B6;DBA-Tg(Fos-tTA) 指导下表达绿色荧光蛋白 (GFP) 的细胞中展示 Rhd-dsAP1 摄取特异性,Fos-EGFP*)1Mmay Tg(tetO-lacZ,tTA*)1Mmay/J] 步骤 5. 以高剂量块展示 dsAP-1安非他明后小鼠体内 AP-1 蛋白诱导的活性步骤 6. 证明 SPION-dsAP1 递送可为活体非人类灵长类动物提供 MRI 窗口，并检测安非他明暴露后 AP-1 蛋白的升高该应用旨在实现高度创新以及使用非常规、新颖的体内系统靶向 MRI 技术对基因产物进行分子靶向的概念性创造性研究。由于转录因子结合域从啮齿动物到人类都是保守的，因此我们的创新技术一旦经过验证，就有可能在从啮齿动物到灵长类动物的治疗诊断中得到应用。