Aptamer Imaging: A Theranostic Approach to Treat Substance Abuse

项目描述（申请人提供）：本项目将开发和探索磁共振（MR）可见探针成像细胞内基因转录激活因子蛋白。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。在过去的一个世纪里，细胞和分子生物学的进步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动。在药物成瘾的研究中，细胞内激活蛋白-1 （AP-1）和核因子kappa- β (NF-k-b)这两种活性已经显示出改变。在转基因小鼠中，不能产生功能性AP-1蛋白结合在AP-1位点，这阻碍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核磁共振成像技术，并将其应用于一系列活体大脑的药物成瘾研究。为了实现对非人类灵长类动物（NHP）表观遗传过程的特异性操作，我们提出了一个假设：具有AP-1蛋白序列结构域的短双链DNA适体将作为内源性AP-1蛋白结合的诱饵。适配体将与核磁共振可见造影剂（超顺磁性氧化铁纳米颗粒- neutravidin，或SPION-NA，直径49纳米）相连接，该造影剂允许对活体动物中的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：设计一个具有高亲和力的抗核酸酶dasp -1适配体，与AP-1蛋白结合。定量罗丹明（Rhd）标记的dsAP-1适体在体外的结合。展示spon - dsap1在MRI中的传递，并比较C57black6小鼠和已知不产生或较少产生AP-1蛋白的突变株（a - fos /NSE或FosB敲除突变体）的体内MR信号。在AP-1蛋白介导的绿色荧光蛋白（GFP）表达细胞中显示Rhd-dsAP1摄取特异性[B6；DBA-Tg(Fos-tTA,Fos-EGFP*)1Mmay Tg(tetO-lacZ,tTA*)1Mmay/J]证明高剂量dasp -1阻断安非他明后小鼠AP-1蛋白诱导的活性。证明SPION-dsAP1的递送为活体非人类灵长类动物的MRI提供了一个窗口，并检测安非他明暴露后AP-1蛋白的升高。该应用程序旨在利用一种非传统的、新颖的活体系统靶向MRI技术，对基因产物的分子靶向进行高度创新和概念创造性的研究。由于转录因子结合域从啮齿动物到人类都是保守的，我们的创新技术一旦得到验证，就有可能应用于从啮齿动物到灵长类动物的治疗。

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.