Measurement of Beta Cell Death in Diabetes

描述（由申请人提供）：所有形式的糖尿病的特征均为产生胰岛素的β细胞死亡。在1型糖尿病（T1 D）中，这导致对外源性胰岛素的依赖。β细胞的死亡是沉默的-它不能在体内检测到，直到它已经发展到代谢功能受损的程度。β细胞的损失仅通过可能受环境因素影响的功能研究进行评估。已经开发出可以调节T1 D病程的新疗法，但这些疗法的有效应用需要更清楚地了解病理过程，包括β细胞死亡的时间和促发因素。为了直接测量体内β细胞死亡， 在我们的I期STTR中，我们开发了一种使用新的液滴数字PCR（ddPCR）检测源自β细胞的INS DNA的测定法。具有表观遗传修饰（未甲基化CpG）的INS DNA的释放鉴定了DNA的β细胞来源。该检测试剂盒可检测约600拷贝/μl至0.7拷贝/μl范围内的未甲基化DNA，对数转换拷贝数的线性回归为0.99。在我们的研究中，我们表明，该测定可以区分T1 D个体与健康对照受试者，以及处于T1 D风险并进展为疾病的血糖正常个体与健康对照受试者。结论：ddPCR是一种检测β细胞死亡的有效方法，比巢式PCR等方法更敏感、更可行。在第二阶段，我们将解决检测开发中的关键问题，如预测代谢衰竭和缓解的能力。II期的目标是分析诊断潜力，并提高我们的检测在不同临床环境中的预后准确性。我们计划研究有发生T1 D风险的个体，进入临床缓解和加重的新发T1 D患者，囊性纤维化相关糖尿病患者，以及认为发生β细胞破坏的胰岛自体移植受体。我们将联合收割机将本试验的结果与其他数据结合，并使用逻辑回归方法进行统计分析，以确定预测β细胞死亡的变量和测量的修饰符。该II期STTR的目标是获得将该方法商业化所需的数据，作为用于监测高危患者、可能接受免疫调节剂或胰岛移植治疗的确诊T1 D患者以及其他形式糖尿病患者的诊断检测方法。我们的目标是进行研究，以支持这种方法作为糖尿病患者和糖尿病风险患者β细胞死亡的生物标志物的效用。未来的研究将涉及扩大的研究组，如2型糖尿病。

DESCRIPTION (provided by applicant): All forms of diabetes are characterized by the death of insulin producing β cells. In Type 1 diabetes (T1D) this leads to the reliance on exogenous insulin for survival. Death of β cells is silent - it cannot be detected in vivo until it has progressed to such an extent that metabolic function is impaired. The loss of β cells has only been assessed by functional studies that can be affected by environmental factors. New therapies have been developed that can modulate the course of T1D but the effective application of these therapies requires a clearer understanding of the pathologic processes including the timing and precipitants of β cell death. To directly measure β cell death in vivo, in our Phase I STTR, we developed an assay using a novel droplet digital PCR (ddPCR) that detects INS DNA derived from β cells. The release of INS DNA with epigenetic modifications (unmethylated CpGs) identifies the β cellular source of the DNA. The assay can detect unmethylated DNA between a range of approximately 600copies/μl to 0.7copies/μl, with a linear regression for the log transformed copy number of 0.99. In our studies, we showed that the assay could distinguish individuals with T1D from healthy control subjects and normoglycemic individuals who are at-risk for T1D and who progress to disease from healthy control subjects. We conclude that ddPCR is a useful method to detect β cell death and is more sensitive and feasible than other methods such as nested PCR. In Phase II we will address critical issues in assay development such as its ability to predict metabolic failure and remission. The goal of the Phase II is analyze the diagnostic potential and improve the prognostic accuracy of our assay in diverse clinical settings. We plan to study individuals at-risk for development of T1D, patients with new onset T1D who enter clinical remissions and exacerbations, patients with cystic fibrosis related diabetes, and recipients of islet autotransplants in which β cell destruction is thought to occur. We will combine the results from this assay with other data and perform statistical analyses using a logistic regression approach to identify variables that predict beta cell death and modifiers of the measurement. The goal of this Phase II STTR is to obtain the data needed to commercialize this method as a diagnostic assay for monitoring at-risk patients, and those with established T1D who may be treated with immune modulators or islet transplants, and patients with other forms of diabetes. Our goal is to perform studies that will support the utility of this approach as a biomarker of β cell death in patients with and at risk fo diabetes. Future studies will involve expanded study groups such as Type 2 diabetes.