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(未来20年约2.3亿人)，这需要不断努力开发更好的疾病早期诊断方法，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，发病时的治疗干预最有可能逆转免疫系统对胰岛素分泌细胞的持续破坏。现在已经知道，1型糖尿病(早期胰岛炎期)和2型糖尿病都有始于微环境的初始病理。 由促炎信号和相关的局部血管功能障碍引起的变化；在1型和2型糖尿病的发生和发展过程中发生的？细胞死亡是由炎症信号分子介导的。 IL-1？和肿瘤坏死因子α，在激活核因子-κB后产生，后者是许多炎症信号通路的核心转录因子。有鉴于此，我们相信，非侵入性检测早期功能性NF-κB变化的能力最终将提供一种手段来预测可能处于潜在不可逆胰腺内分泌损害的早期阶段的患者。此外，对激活的核因子-κB的功能分析将为研究新开发的治疗方法的纵向效应提供敏感的手段，这些药物旨在减缓或预防这种疾病的发展。我们最近的工作验证了使用稳定的近红外荧光寡核苷酸双链分子传感器(ODN-MS)作为体外检测激活的NF-κB的传感器。该检测是通过在基因组水平上发生的NF-κB-DNA结合事件引起的近红外光子发射谱的变化来实现的。在平行的研究中，我们设计、合成和测试了纳米级受保护的接枝共聚物(PGC)作为载体，将顺磁性和光学探针输送到糖尿病小鼠模型的胰腺血管间(Medarova Z，等人)。糖尿病2007；53：1318-25)。在这里，我们建议与MGH A.Martinos中心的研究人员合作，利用这两种新探针技术的组合强度来设计、合成和测试一种疏水核心(HC)pGC-ODN分子传感器，该传感器能够检测转录因子NF-κB和DNADNA之间的分子相互作用。我们将通过在糖尿病小鼠模型中测定其在胰腺的递送和细胞摄取来进一步测试该探针在Clinica翻译中的适用性。最后，我们将通过对胰腺中的NF-κB活性进行体内诊断荧光寿命成像来测试pGC-ODN探针在检测NF-κB-DNA结合事件变化方面的敏感性。为实现这一目标，我们建议追求以下目标：目标1：用于电子计算机建模，以及碱基间连接物和末端连接物方法的新组合用于设计优化的寡核苷酸-MS探针，从而提高细胞的递送性；目标2：测试寡核苷酸-MS在体外的递送情况，以执行基于细胞和隔离胰岛的分析以及在急性糖尿病小鼠模型中的生物分布研究；目标3：利用MGH A.Martinos‘Center开发的最先进的成像平台，研究体内荧光寿命成像作为确定正常和糖尿病小鼠NF-κB激活程度的一种手段。成像方法检测疾病负担的敏感性将通过一种治疗剂(一种IκB肿瘤坏死因子α信号的磷酸化抑制剂BAY-11-7082)控制疾病的程度来评估，该治疗剂可以改变NOD和NOD/LEPR小鼠的NF-κB表达水平，防止胰岛细胞的破坏。

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.