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

• 批准号: 8076922
• 负责人: Philip K Liu
• 金额: $ 33.59万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 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/8076922
• 关键词: Affect; Affinity; Age; Amphetamines; Animals; Binding; Biological Assay; Biopsy; Brain; Caliber; Cells; Cerebrum; Contrast Media; Development; Dose; Drug Addiction; Environment; Epigenetic Process; Exposure to; Fluorescence; Future; Gender; General Population; Genes; Genetic Transcription; Goals; Health; Human; Human Activities; Image; Imaging Techniques; In Vitro; Knock-out; Label; LacZ Genes; Life; Link; Macaca mulatta; Magnetic Resonance; Magnetic Resonance Imaging; Modification; Molecular Target; Molecular and Cellular Biology; Mus; NF-kappa B; Play; Primates; Productivity; Protein Binding; Proteins; Race; Research; Resistance; Rhodamine; Rodent; Role; Sampling; Series; Signal Pathway; Signal Transduction; Site; Societies; Source; Specificity; Substance abuse problem; System; Techniques; Tissues; Transcription Coactivator; Transcription Factor AP-1; Transcription factor genes; Transgenic Mice; addiction; aptamer; awake; clinical application; design; drug of abuse; ds-DNA; in vivo; innovation; iron oxide; member; mutant; nanoparticle; neutravidin; nonhuman primate; novel; nuclease; public health relevance; relating to nervous system; theranostics; transcription factor; uptake

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. PUBLIC HEALTH RELEVANCE: Narrative (Aptamer Imaging in Non-human Primates: A Theranostic Strategy for Substance Abuse) This project will develop and explore magnetic resonance (MR) visible probes to image intracellular gene transcription activator proteins. The underlying mechanism that is associated with gene activities regulated by activator proteins after exposure to drugs of abuse is not totally understood, but it occurs across the general population, affecting all ages, genders and races. There are few assays to assess intracellular proteins in the brain except in biopsy or post mortem samples. We will examine if this novel technique offers the promise of imaging important, but previously unmeasurable, aspects of live cerebral gene activities in the awake rhesus monkey (Macaca mulatta) before a crucial leap to future clinical applications in neural activity of humans.

描述（由申请人提供）：本项目将开发和探索磁共振（MR）可见探针，以成像细胞内基因转录激活蛋白。吸毒成瘾是一个严重妨碍我们社会许多成员生产力的重大健康问题。虽然研究表明药物成瘾是基因和环境综合影响的结果，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起重要作用。在过去的世纪中，细胞和分子生物学的进展导致了新的基因标记的鉴定，它们影响的信号通路和它们调节的基因活性。在药物成瘾的研究中，他们已经显示出两种这样的活性是细胞内激活蛋白-1（AP-1）和核因子κ-β（NF-k-B）。在转基因小鼠中不能产生功能性AP-1蛋白以结合在AP-1位点阻断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，因此不允许在人体中进行此类研究。我们的目标是开发和研究一种有针对性的磁共振成像技术，并将其应用于一系列活体大脑中药物成瘾的研究。为了实现对非人灵长类（NHP）的表观遗传过程的特异性操纵的长期目标，我们提出了一个假设：具有AP-1蛋白的序列结构域的短双链（ds）DNA适体将充当内源性AP-1蛋白结合的诱饵。适体将连接到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），其允许在活动物受试者中成像AP-1蛋白。我们已经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适体体外定量罗丹明（Rhd）标记的dsAP-1适体结合在C57 black 6小鼠和已知不产生或产生较少AP-1蛋白的突变株（A-FOS/NSE或FosB敲除突变体）中证明SPION-dsAP 1的MRI递送，并比较体内MR信号步骤4。在表达AP-1蛋白指导的绿色荧光蛋白（GFP）的细胞中证明Rhd-dsAP 1摄取特异性[B6;DBA-Tg（Fos-tTA，Fos-EGFP*）1 Mmay Tg（tetO-lacZ，tTA*）1 Mmay/J]步骤5.证明高剂量dsAP-1阻断苯丙胺后小鼠中AP-1蛋白诱导的活性步骤6。证明SPION-dsAP 1递送允许在活体非人类灵长类动物中进行MRI的窗口并检测苯丙胺暴露后AP-1蛋白的升高该应用旨在使用非常规的新型靶向MRI技术对体内系统进行基因产物分子靶向的高度创新和概念创造性研究。由于转录因子结合结构域是保守的从啮齿动物到人类，我们的创新技术，一旦验证，有潜力的治疗诊断应用，从啮齿动物到灵长类动物。 公共卫生相关性：叙述（非人类灵长类动物的适体成像：药物滥用的治疗诊断策略）本项目将开发和探索磁共振（MR）可见探针，以成像细胞内基因转录激活蛋白。与暴露于滥用药物后由激活蛋白调节的基因活性相关的潜在机制尚未完全了解，但它发生在一般人群中，影响所有年龄，性别和种族。除了活组织检查或死后样本外，很少有测定法来评估脑中的细胞内蛋白质。我们将研究这种新的技术是否提供了成像的承诺重要的，但以前无法测量的，在清醒的恒河猴（猕猴）的大脑基因活动的活的方面，在人类神经活动的未来临床应用的一个关键飞跃。