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: 8464103
• 负责人: Philip K Liu
• 金额: $ 36.67万
• 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/8464103
• 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.

描述（由申请人提供）：具有反义序列互补的合成核酸及其在基因活性检测中的使用，使我们对生物科学所有学科中疾病的分子机制的理解提高了我们的理解。对于体内研究，可以用磷酸盐酸盐（产生SODN或SORN）来修饰寡素（ODN）或寡虫（ORN），以增加对核酸酶的抗性。我们的假设是，SORN与靶mRNA的杂种比SODN杂种更稳定。我们将比较一个模块化磁共振（MR）探针，该探针包括氧化铁纳米颗粒（SPION，T2剂），标记为SODN或SORN。目前，脑室内（ICV）通过皮质或腰椎注射是仅有少数几种临床批准的方法之一，可以将药物输送到人类的脑脊髓液（CSF）。我们已经证明，活动物的神经细胞在MR成像中以中等效率和特异性在MRNA靶向体内的SPION-SODN：（1）小鼠中的ICV递送可安全地促进小鼠大脑中SPION-SODN的全球分布而没有致死的效应，而不是由spion-sodn tarts tart（2）特定的结合（2）targe（2）tarts tarts tarts tarts tarts tarts tarts（3）显微镜（EM）表明，氧化铁位于某些末端，其中有些与MRNA所在的内质网（ER）和核具有独特的关联，（4）（4）SPION-SODN在基线（DR2*）上方的SPION-SODN保留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纤维纤维蛋白（GFAP）mRNA的效率。拟议工作的完成为新颖的基因靶向探针提供了一个平台，以及用于早期评估体内星形胶质激活的强大工具。因此，与SPION-SODN相比，Spion-SORN较少用于基因靶向，并减少了大脑中铁的积累，从而导致神经系统事件的纵向评估。我们将：AIM 1：使用超高场MRI比较小鼠中体内剂量和Sp-Sodn或Spion-SORN的摄取。我们的假设是，当使用SPION-SORN（SPION-RGFAP）靶向GFAP mRNA时，SPION保留（DR2*）将有所改善。我们将纵向比较活小鼠大脑中这两个探针的Dr2*。目标2：验证MRI与组织学评估之间的相关性。我们的假设是，可以将双标记探针的局部定位（例如，SPION-RGFAP-CY3）特异性转染到体内转基因的转基因胶质细胞，并可以在荧光，光学和电子显微镜下确认。 ICV探针输送后，我们将收集大脑样本，以进行这项相关研究。 AIM 3：使用无引物原位与cDNA进行验证靶标结合，然后使用靶特定PCR。假设是SPION-RGFAP将专门与体内GFAP mRNA靶标结合，并用作原位RT-PCR的底漆。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*与mRNA拷贝数之间的相关性。