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

• 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
• 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

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，促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，寡聚DNA（ODN）或寡聚RNA（ORN）可以用硫代磷酸酯修饰（产生sODN或sORN）以增加对核酸酶的抗性。我们的假设是，sORN与靶mRNA的杂交体比sODN杂交体更稳定。我们将比较一个模块化的磁共振（MR）探针，包括超顺磁性氧化铁纳米粒子（SPION，T2剂）标记的sODN或sORN。目前，通过皮质或腰椎穿刺进行脑室内（ICV）注射是临床上批准的将药物递送至人类脑脊液（CSF）的少数方法之一。我们已经通过MR成像证明，活体动物的神经细胞在体内mRNA靶向中以中等效率和特异性摄取SPION-sODN：（1）小鼠中的ICV递送安全地促进SPION-sODN在小鼠脑中的整体分布而没有致死作用，（2）通过逆转录（RT）SPION-sODN与靶mRNA的体内引发已经显示出特异性结合，（3）电子显微镜（EM）的结果显示，氧化铁位于末端，一些与内质网（ER）和mRNA所在的核具有独特的关联。（4）SPION-sODN保留高于基线（DR 2 *）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）靶向星形胶质细胞特异性胶质细胞酸性蛋白（GFAP）mRNA的效率。该工作的完成为新型基因靶向探针提供了一个平台，同时也为星形胶质细胞体内活化的早期评估提供了一个有力的工具。因此，SPION-sORN比SPION-sODN更少用于基因靶向并减少铁在脑中的积累，从而导致神经系统事件的纵向评估。我们将：目的1：使用超高场MRI比较SPION-sODN或SPION-sORN在小鼠中的体内剂量和摄取。我们的假设是，SPION保留（DR 2 *）将改善SPION-sORN（SPION-Rgfap）用于靶向GFAP mRNA。我们将纵向比较这两种探针在活小鼠脑中的DR 2 *。目的2：探讨MRI与组织学检查的相关性。我们的假设是双标记探针的共定位（例如，SPION-Rgfap-Cy 3）可以在体内特异性地转染到转基因小鼠的GFP表达胶质细胞中，并可以在荧光、光学和电子显微镜下证实。我们将在ICV探针递送后收集脑样本用于该相关性研究。目的3：利用无引物原位RT和靶特异性PCR技术研究靶结合。假设SPION-Rgfap将在体内特异性结合GFAP mRNA靶点，并作为原位RT-PCR的引物。我们将收集大脑样本，量化PCR结果，并使用疾病模型系统建立MRI DR 2 * 和mRNA拷贝数之间的相关性。 公共卫生相关性：胶质细胞活化在疾病过程中起着重要作用。然而，胶质细胞活化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胶质细胞激活的特异性信使RNA的基因靶向机制。我们将应用一种新的基于核酸的探针，利用磁共振成像（MRI）来研究活体动物中的神经胶质激活。在此提出的工作，初步应用于活体动物模型，具有重要意义的MRI为基础的分析在遗传水平上的神经生理事件。细胞内RNA的高分辨率MRI有望转化为活体受试者的分子生物学;允许实时纵向MRI在未来的神经科学和医学研究中的临床前评价应用。