DNA-based MR Probes for Imaging mRNA Transcripts in vivo

用于体内 mRNA 转录物成像的基于 DNA 的 MR 探针

基本信息

批准号：
8182704
负责人：
Philip K Liu
金额：
$ 39.22万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-07-05 至 2015-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8182704
关键词：
Actins Animal Model Animals Astrocytes Autopsy Binding Biological Assay Biological Models Biological Process Biological Sciences Brain Caliber Cell Culture Techniques Cell Nucleus Cells Cerebrospinal Fluid Cerebrum Complementary DNA Contrast Media Correlation Studies DNA Data Detection Development Discipline Disease Disease model Dose Electron Microscope Electron Microscopy Electrons Electrophoresis Endoplasmic Reticulum Endosomes Ensure Evaluation Event Fluorescence Future Gel Gene Dosage Gene Targeting Genes Genetic Glial Fibrillary Acidic Protein Goals Histology Human Hybrids Image In Situ In Vitro Injection of therapeutic agent Investigation Iron Label Life Linear Regressions Link Magnetic Resonance Magnetic Resonance Imaging Measurement Medical Research Messenger RNA Methods Microscopy Modification Molecular Molecular Biology Mus Neuraxis Neuroglia Neurologic Neurons Neurosciences Nucleic Acids Optics Peptides Pharmaceutical Preparations Play Polymerase Chain Reaction Predisposition Process Proteins RNA Rattus Resistance Resolution Reverse Transcription Rhodamine Role Route Running Sampling Signal Transduction Specificity Spinal Puncture Sprague-Dawley Rats Techniques Time Tissues Transcript Transfection Transgenic Mice Translations Work base brain tissue imaging probe improved in vivo iron oxide molecular imaging nanoparticle novel nuclease phosphorothioate preclinical evaluation promoter protein expression synthetic nucleic acid tool trafficking uptake

项目摘要

DESCRIPTION (provided by applicant): Synthetic nucleic acids with antisense sequence complementary to mRNA, and their use for gene activity detection, have advanced our understanding of the molecular mechanisms of diseases in all disciplines of the biological sciences. For in vivo investigations, oligoDNA (ODN) or oligoRNA (ORN) can be modified with phosphorothioate (yielding sODN or sORN) to increase resistance to nucleases. Our hypothesis is that hybrids of sORN with target mRNA is more stable than sODN hybrids. We will compare a modular magnetic resonance (MR) probe comprising supraparamagnetic iron oxide nanoparticles (SPION, a T2 agent) labeled with sODN or sORN. At present, intracerebroventricular (ICV) injection via cortical or lumbar puncture is one of only a few clinically approved methods to deliver drugs to the cerebral spinal fluid (CSF) in humans. We have demonstrated that neural cells of live animals take up SPION-sODN with moderate efficiency and specificity in mRNA targeting in vivo by MR imaging: (1) ICV delivery in mice safely facilitates global distribution of SPION- sODN in mouse brains without lethal effect, (2) specific binding has been shown by in vivo priming of SPION- sODN to target mRNA by reverse transcription (RT), (3) results from electron microscopy (EM) show that iron oxide is located in the end some with a unique association to the endoplasmic reticulum (ER) and nuclei where mRNA is located, (4) changes in SPION-sODN retention above baseline (DR2*) are positively proportional to gene activities (linear regression = 1.0). Our goal is to evaluate the efficiency of SPION-sORN (and SPION-sODN) for targeting astroglia-specific glial fibrillary acidic protein (GFAP) mRNA. Completion of the proposed work provides a platform for novel gene targeting probes as well as a powerful tool for early evaluation of astroglia activation in vivo. Therefore, less SPION-sORN than SPION-sODN is used for gene targeting and reduces accumulation of iron in the brain, leading to longitudinal assessment of neurologic events. We will: Aim 1: Compare in vivo dose and uptake of SPION-sODN or SPION-sORN in mice using ultra-high field MRI. Our hypothesis is that SPION retention (DR2*) will improve when SPION-sORN (SPION-Rgfap) is used to target GFAP mRNA. We will longitudinally compare DR2* of these two probes in the brains of live mice. Aim 2: Validate the correlation between MRI and histological assessments. Our hypothesis is that co- localization of dual-labeled probe (e.g., SPION-Rgfap-Cy3) can be specifically transfected to GFP-expressing glia of transgenic mice in vivo, and can be confirmed under fluorescent, optical and electron microscopes. We will collect brain samples after ICV probe delivery for this correlation study. Aim 3: Validate target binding using primer-free in situ RT to cDNA followed by target specific PCR. The hypothesis is that SPION-Rgfap will bind specifically to GFAP mRNA target in vivo and serve as a primer for in situ RT-PCR. We will collect brain samples, quantify the PCR results, and establish the correlation between MRI DR2* and mRNA copy numbers, using disease model systems. PUBLIC HEALTH RELEVANCE: Glial activation plays an important role in the disease process. However, glial activation can only be detected in postmortem brain samples. The work outlined in this application investigates the mechanism of gene targeting for specific messenger RNA of glia activation in the central nervous system. We will apply a novel nucleic acid- based probe to investigate glial activation in live animals, using magnetic resonance imaging (MRI). The work proposed here, with initial application in live animal models, has important implications for MRI-based analyses of neurophysiologic events at the genetic level. High-resolution MRI of intracellular RNA holds promise for translation to molecular biology in live subjects; to permit real-time longitudinal MRI in future applications for neuroscience and preclinical evaluation in medical research.

描述（由申请人提供）：具有与 mRNA 互补的反义序列的合成核酸及其用于基因活性检测的用途，增进了我们对生物科学所有学科中疾病分子机制的理解。对于体内研究，可以用硫代磷酸酯修饰寡DNA (ODN) 或寡RNA (ORN)（产生sODN 或sORN）以增加对核酸酶的抗性。我们的假设是 sORN 与目标 mRNA 的杂交体比 sODN 杂交体更稳定。我们将比较包含用 sODN 或 sORN 标记的超顺磁性氧化铁纳米颗粒（SPION，一种 T2 剂）的模块化磁共振 (MR) 探针。目前，通过皮质或腰椎穿刺进行脑室内（ICV）注射是少数临床批准的将药物输送到人体脑脊液（CSF）的方法之一。我们通过 MR 成像证明，活体动物的神经细胞以中等效率和特异性在体内 mRNA 靶向中摄取 SPION-sODN：（1）小鼠体内 ICV 递送安全地促进 SPION-sODN 在小鼠大脑中的整体分布，而没有致命作用， (2) SPION-sODN 通过逆转录 (RT) 体内引发与靶标 mRNA 的特异性结合已显示出来，(3) 电子显微镜 (EM) 结果显示氧化铁位于末端一些与内质网 (ER) 和 mRNA 所在的细胞核具有独特的关联，(4) SPION-sODN 保留高于基线 (DR2*) 的变化与基因活性成正比（线性回归 = 1.0）。我们的目标是评估 SPION-sORN（和 SPION-sODN）针对星形胶质细胞特异性胶质纤维酸性蛋白（GFAP） mRNA 的效率。拟议工作的完成为新型基因靶向探针提供了一个平台，也为体内星形胶质细胞激活的早期评估提供了强大的工具。因此，与 SPION-sODN 相比，用于基因靶向的 SPION-sORN 更少，并减少了大脑中铁的积累，从而实现了神经系统事件的纵向评估。我们将：目标 1：使用超高场 MRI 比较小鼠体内 SPION-sODN 或 SPION-sORN 的剂量和摄取。我们的假设是，当使用 SPION-sORN (SPION-Rgfap) 靶向 GFAP mRNA 时，SPION 保留 (DR2*) 将得到改善。我们将纵向比较这两种探针在活体小鼠大脑中的 DR2*。目标 2：验证 MRI 和组织学评估之间的相关性。我们的假设是，双标记探针（例如，SPION-Rgfap-Cy3）的共定位可以特异性转染到转基因小鼠体内表达GFP的神经胶质细胞中，并且可以在荧光、光学和电子显微镜下证实。我们将在 ICV 探针交付后收集大脑样本以进行相关研究。目标 3：使用无引物原位 RT 与 cDNA 验证靶标结合，然后进行靶标特异性 PCR。假设 SPION-Rgfap 将在体内特异性结合 GFAP mRNA 靶标，并作为原位 RT-PCR 的引物。我们将使用疾病模型系统收集大脑样本，量化 PCR 结果，并建立 MRI DR2* 和 mRNA 拷贝数之间的相关性。公共卫生相关性：神经胶质激活在疾病过程中发挥着重要作用。然而，神经胶质细胞的激活只能在死后的大脑样本中检测到。本申请中概述的工作研究了中枢神经系统中神经胶质细胞激活的特定信使 RNA 的基因靶向机制。我们将应用一种新型的基于核酸的探针，利用磁共振成像（MRI）来研究活体动物中的神经胶质激活。这里提出的工作最初应用于活体动物模型，对基于 MRI 的遗传水平神经生理事件分析具有重要意义。细胞内 RNA 的高分辨率 MRI 有望在活体受试者中转化为分子生物学；允许实时纵向 MRI 未来应用于神经科学和医学研究的临床前评估。