DNA-based MR Probes for Imaging mRNA Transcripts in vivo

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，促进了我们对生物科学所有学科中疾病的分子机制的理解。在体内研究中，oligoDNA （ODN）或oligoRNA （ORN）可以用硫代酸修饰（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的杂交比sODN杂交更稳定。我们将比较由超顺磁性氧化铁纳米颗粒（SPION，一种T2试剂）标记的sODN或sORN组成的模块化磁共振（MR）探针。目前，经皮质或腰椎穿刺的脑室内（ICV）注射是临床批准的为数不多的将药物输送到人类脑脊液（CSF）的方法之一。我们已经通过磁共振成像证明活体动物的神经细胞以中等的效率和特异性在mRNA靶向中吸收SPION-sODN：(1) ICV在小鼠体内的传递安全促进了SPION- sODN在小鼠脑内的全局分布，无致死效应；(2)体内SPION- sODN通过逆转录（RT）与靶mRNA的特异性结合已被证明；(3)电镜（EM）结果显示氧化铁位于末端，与内质网（ER）和mRNA所在的细胞核具有独特的关联。(4) SPION-sODN保留率高于基线的变化（DR2*）与基因活性成正比（线性回归= 1.0）。我们的目的是评估SPION-sORN（和SPION-sODN）靶向星形胶质细胞特异性胶质纤维酸性蛋白（GFAP） mRNA的效率。这项工作的完成为新的基因靶向探针提供了一个平台，也为早期评估星形胶质细胞在体内的激活提供了一个有力的工具。因此，与SPION-sODN相比，较少的SPION-sORN用于基因靶向，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。目的1：利用超高场MRI比较小鼠体内spon - sodn或spon - sorn的剂量和摄取。我们的假设是，当SPION- sorn （SPION- rgfap）靶向GFAP mRNA时，SPION保留（DR2*）会得到改善。我们将纵向比较这两种探针在活体小鼠大脑中的DR2*。目的2：验证MRI与组织学评估之间的相关性。我们的假设是双标记探针（如spon - rgfp - cy3）的共定位可以特异性转染到体内表达gfp的转基因小鼠的胶质细胞中，并可以在荧光显微镜、光学显微镜和电子显微镜下得到证实。我们将在ICV探针投放后采集脑样本进行相关研究。目的3：利用无引物原位RT到cDNA，然后进行目标特异性PCR验证目标结合。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。我们将收集脑样本，定量PCR结果，并利用疾病模型系统建立MRI DR2*与mRNA拷贝数之间的相关性。

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.