Molecular fluorescence lifetime sensor of pro-inflammatory signaling in diabetes

描述（由申请人提供）：预计全球T1和T2糖尿病患者数量将增加（在未来20年内约为2.3亿），因此需要持续努力开发用于早期诊断该疾病的改进方法，这可能会在早期临床干预和最终疾病预防方面取得更大成功。越来越明显的是，疾病发作时的治疗干预最有可能逆转免疫系统对胰岛素分泌β细胞的持续破坏。目前已知1型糖尿病（早期胰岛炎阶段）和2型糖尿病都有一个共同的初始病理，即从微环境开始， 由促炎信号和相关局部血管功能障碍引起的变化;在1型和2型糖尿病的起始和进展期间发生的β细胞死亡由炎性信号分子介导 IL-1 β和TNFα，后者在NF-κB（一种对许多炎症信号传导途径起关键作用的转录因子）活化后产生。鉴于上述情况，我们相信，非侵入性检测功能性NF-κB早期变化的能力最终将提供一种方法来预测可能处于内分泌胰腺潜在不可逆损伤早期阶段的患者。此外，对活化的NF-κB的功能分析将为研究新开发的旨在减缓或预防该疾病发展的疗法的纵向效应提供灵敏的手段。我们最近的工作已经验证了使用稳定的近红外（NI）荧光寡核苷酸双链体为基础的分子传感器（ODN-MS）的使用，所述分子传感器使用生物缀合物化学中的新方法作为用于检测体外活化的NF-κB的传感器。通过在基因组水平发生的NF-κB-DNA结合事件引起的NIR光子发射谱的变化进行检测。在平行的研究路线中，我们设计、合成并测试了纳米尺寸的受保护的移植共聚物（PGC）作为用于将顺磁性和光学探针递送至糖尿病小鼠模型中的胰腺的血管区室的载体（Medarova Z，et al. Diabetes 2007; 53：1318-25）。在这里，我们建议，与MGH A的研究人员合作。Martinos中心，利用这两种新型探针技术的组合强度来设计合成和测试能够检测转录因子NF-κB和DNA之间的分子相互作用的疏水核心（HC）PGC-ODN分子传感器。我们将通过在糖尿病小鼠模型中确定其在胰腺中的递送和细胞摄取来进一步测试探针用于临床翻译的适用性。最后，我们将通过对胰腺中NF-κB活性进行诊断荧光寿命体内成像来测试PGC-ODN探针检测NF-κB-DNA结合事件变化的灵敏度。为了实现这一目标，我们提出追求以下目标：目标1：使用计算机模拟和碱基间接头和末端接头方法的新组合来设计具有改善的细胞递送的优化的ODN-MS探针;目标2：在急性糖尿病小鼠模型中进行基于细胞和分离的胰岛的测定和生物分布研究的体外ODN-MS的测试递送;目标3：使用MGH A开发的最先进成像平台，研究体内荧光寿命成像作为确定正常和糖尿病小鼠中NF-κB活化程度的方式。马蒂诺斯中心将通过治疗剂（TNFα信号传导的IκB磷酸化抑制剂，BAY-11- 7082）控制疾病程度来评估成像方法检测疾病负荷的灵敏度，该治疗剂可改变NF-κB表达水平并防止NOD和NOD/Lepr小鼠中胰岛细胞的破坏。

DESCRIPTION (provided by applicant): The predicted increase in the number of people worldwide afflicted with T1 and T2 diabetes (approximately 230 million during the next 20 years) necessitates the ongoing effort to develop improved methods for the early diagnosis of the disease, which may afford greater success with early clinical intervention and ultimately, disease prevention. It is increasingly evident that therapeutic interventions at disease onset have the greatest likelihood of reversing the otherwise continuously progressing destruction of insulin-secreting ß-cells by the immune system. It is now known that both Type 1 (the early insulitis stage) and type 2 diabetes share an initial pathology that starts with microenvironmental changes brought on by pro-inflammatory signaling and associated local vascular dysfunction; ß-cells death, which occurs during the initiation and progression of both type 1 and type 2 diabetes, is mediated by the inflammatory signaling molecules IL-1ß and TNFα, which in turn are produced subsequent to the activation of NF-κB, a transcription factor central to many inflammatory signaling pathways. In view of the above we are confident that the ability to non-invasively detect early changes in functional NF-κB eventually will provide a means to predict patients who may be in the earliest stage of potentially irreversible damage to endocrine pancreas. In addition, functional analysis of activated NF-κB will provide a sensitive means for studying the longitudinal effects of newly developed therapeutics designed to slow or prevent the development of this disease. Our recent work has validated the use of stabilized near-infrared (NI) fluorescent oligonucleotide duplex- based molecular sensors (ODN-MS) created using new approaches in bioconjugate chemistry as sensors for detecting activated NF-κB in vitro. The detection is afforded by changes in NIR photon emission profiles evoked by NF-κB-DNA binding events occurring at the genomic level. In a parallel line of investigation, we have designed, synthesized, and tested nano-sized protected graft co-polymer (PGC) as a carrier for the delivery of paramagnetic and optical probes to the vascular compartment of pancreas in a mouse model of diabetes (Medarova Z, et al. Diabetes 2007; 53:1318-25). Here we propose, in collaboration with investigators from the MGH A. Martinos' Center, to harness the combined strength of both novel probe technologies to design synthesis and test a hydrophobic core (HC) PGC-ODN molecular sensor capable of detecting molecular interactions between the transcription factor NF-κB and DNA. We will further test the applicability of the probe for clinica translation by determining its delivery and cellular uptake in the pancreas in a murine model of diabetes. And finally we will test the sensitivity of the PGC-ODN probe for detecting changes in NF-κB-DNA binding events by performing diagnostic fluorescence lifetime in vivo imaging of NF-κB activity in the pancreas. Toward achieving this goal we propose to pursue the following aims: Aim 1: use in silico modeling and a novel combination of interbase linker and end-linker approaches for designing optimized ODN-MS probes with improved cell delivery; Aim 2: test delivery of ODN-MS in vitro performing cell and isolated islet-based assays and biodistribution studies in an acutely diabetic mouse model; Aim 3: investigate in vivo fluorescence lifetime imaging as a modality for determining the extent of NF-κB activation in normal and diabetic mice using a state of the art imaging platform developed at the MGH A. Martinos' Center. The sensitivity of the imaging method to detect disease burden will assessed by controlling the extent of disease via a therapeutic agent (an IκB phosphorylation inhibitor of TNFα signaling, BAY-11- 7082), which alters levels of NF-κB expression and prevents the destruction of islet cells in NOD and NOD/Lepr mice.