Aptamer Imaging: A Theranostic Approach to Treat Substance Abuse

描述(申请人提供)：该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阻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识别，它们影响的信号通路和它们调节的基因活动。他们在药物成瘾研究中显示出两种修饰的活性是细胞内激活蛋白-1(AP-1)和核因子-kappa-β(NF-k-b)。在转基因小鼠中，无法产生功能强大的AP-1蛋白与AP-1结合，这会阻止药物滥用的敏感化。由于活组织检查是常规检测的唯一组织来源，因此此类研究不允许在人类身上进行。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的药物成瘾研究。为了能够对非人类灵长类动物(NHP)的表观遗传过程进行特定的操作，我们提出了一个假设：带有AP-1蛋白序列结构域的短双链(DS)DNA适配子将作为内源性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-中性亲和素，或Spion-NA，直径49 nm)，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.设计与AP-1蛋白结合具有高亲和力的核酸酶抗性DSAP-1适配子步骤2.定量罗丹明(RHD)标记的DSAP-1适配子体外结合步骤3.展示Spion-dsAP1在磁共振中的传递，并在体内比较C57Black6小鼠和已知不产生或少产生AP-1蛋白的突变株(A-FOS/NSE或FosB基因敲除突变体)的MR信号；步骤4.证明RHD-dsAP1在表达AP-1蛋白[B6；DBA-TG(Fos-TTA，Fos-EGFP*)1MMay TG(Teto-Lacz，TTA*)1MMay/J]步骤5.展示高剂量的DSAP-1在苯丙胺后阻断AP-1蛋白诱导的小鼠活动步骤6.证明Spion-dsAP1递送允许在活体非人类灵长类动物中进行核磁共振成像，并检测到苯丙胺暴露后AP-1蛋白的升高这项应用旨在使用非传统的、针对体内系统的新型靶向核磁共振成像技术来实现高度创新和概念上的创造性研究基因产品的分子靶向。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，就有可能在从啮齿动物到灵长类动物的治疗中应用。

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.