Targeted Epigenetic Editing to Increase Adult Pancreatic Β-Cell Proliferation

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β-cell replacement therapy is potentially a curative approach in treating diabetes, as demonstrated by the success of pancreatic islet transplantation in type 1 diabetes. However, there are an insufficient number of organ donors to meet the demand of this disease, which is increasing in prevalence. One strategy to increase the supply of human β-cells for transplantation in type 1 diabetics, or to increase residual β-cell mass in type 2 diabetics, is to induce human β-cell replication. This strategy has not been implemented clinically because adult human β-cells are largely quiescent and the capacity for proliferation decreases with age. I hypothesized that changes in DNA methylation contribute to the age-related decline in proliferative capacity in human β-cells, and that altering the DNA methylome in a targeted manner could improve proliferative capacity. To investigate this hypothesis, I sought to profile the β-cell across the human lifespan, and to develop tools that permit targeted DNA methylation modifications and efficiency in measuring DNA methylation. I conducted RNA-Seq and whole-genome bisulfite sequencing (WGBS) to profile the aging human β-cell transcriptome and DNA methylome. I found that there are significant changes in gene expression with age, and in DNA methylation, particularly at islet-specific active enhancers. Further, I developed transcription activator-like effector (TALE) fusion proteins conjugated to DNA methyltransferases (DNMTs) and demonstrated that targeting TALE-DNMTs to the promoter of the CDKN2A locus, encoding the cell cycle inhibitor p16, increases proliferation in primary human fibroblasts. Finally, I developed BisPCR2, a novel technique for preparing targeted bisulfite next-generation sequencing libraries, which greatly improves the efficiency in which DNA methylation can be measured at target regions. I demonstrated the utility of this tool to validate genome-wide findings of type 2 diabetes CpG risk loci. Together, these novel datasets and epigenetic tools poise the β-cell regeneration field to investigate targeted epigenetic modifications as a strategy to improve proliferative capacity of adult human β-cells.

Klaus H. Kaestner
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