Abstract
Type 1 Diabetes mellitus (T1D) is an autoimmune disease targeting pancreatic ß-cells that affects over 8 million
people worldwide. Standard treatment for T1D is limited to administration of exogenous insulin, which can result
in hypoglycemic episodes, and fails to fully prevent micro- and macro- vascular complications. Thus, deriving
glucose-responsive insulin-producing ß-cells from renewable sources is an index goal of regenerative medicine.
Yet, current human stem cell-derived ß-cells produce insulin at lower levels than native ß-cells and fail to
recapitulate regulated insulin secretion typical of mature adult islet ß-cells. This limits their therapeutic potential
and reflects a significant gap in our knowledge regarding mechanisms regulating human islet maturation. Exciting
recent studies identified human-specific age-dependent gene expression changes, including novel transcription
factors, such as RXRG, not expressed in mouse or immature human ß-like cells, that might control functional
maturation of human islet cells. My Preliminary Data shows that RXRG loss in human adult ß-cells results in
impaired glucose-stimulated insulin secretion. Recent studies also showed that gradual turnover of enhancers
drives lineage progression and maturation. I have found a presumptive enhancer regulatory element of RXRG
that becomes differentially accessible with age in adult ß-cells compared to immature ß-cells and a-cells, as
measured by H3K27Ac occupancy. My objective here is to discover mechanisms that induce maturation of
human ß-cells, using two approaches. In Aim 1, I will investigate the regulatory roles of the novel
transcriptional regulator of ß-cell maturation RXRG. This will involve knock down of RXRG, and then ß-cell
specific RNA-seq and CUT&RUN to identify RXRG downstream targets. In addition, I will activate RXRG
endogenous promoter using the CRISPRa system and evaluate chromatin accessibility changes induced by
activation of this maturation-required transcription factor. In Aim 2, I will study the non-coding regulatory
mechanisms activating RXRG expression to induce ß-cell maturation. Since these mechanisms do not occur
in mouse or stem cell-derived human ß-cells, and are specific to adult ß-cells, the studies I proposed will be
performed in adult human islets using the pseudoislet-genetics platform I developed during my postdoctoral
studies. Here, I will use novel technological advancements, including CRISPR-Cas9 ribonucleoprotein (RNP)
editing of human islet cells, to delete the presumptive enhancer element of RXRG and evaluate its role in
regulating RXRG expression. Finally, I will activate RXRG expression in juvenile islets using CRISPRa to activate
RXRG promoter, enhancer or both, and evaluate if RXRG expression enhances coordinated glucose-dependent
insulin secretion in adulthood. Altogether, findings from this proposal will uncover novel pathways leading to ß-
cell maturation, that could be exploited towards efforts with ß-cells from renewable sources. Equally importantly,
these studies provide a novel toolkit for genetically manipulating previously “unstudiable” or inaccessible non-
replicating mature and immature ß-cells.