All forms of diabetes are characterized by the death of insulin producing ? cells. In Type 1 diabetes (T1D) this leads to the reliance on exogenous insulin for survival. Death of ? 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 ? 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 ? cell death. To directly measure ? 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 ? cells. The release of INS DNA with epigenetic modifications (unmethylated CpGs) identifies the ? cellular source of the DNA. The assay can detect unmethylated DNA between a range of approximately 600copies/?l to 0.7copies/?l, 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 ? 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 ? 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 ? cell death in patients with and at risk fo diabetes. Future studies will involve expanded study groups such as Type 2 diabetes.
All forms of diabetes involve death of cells. We have developed a droplet digital PCR assay to measure cell death in vivo. We propose to apply this new assay to various clinical setting in order to determine the diagnostic value of our assay.
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