Innovative Genetic Technologies For Identifying Novel Regulatory Mechanisms Controlling Human b-Cell Maturation and Function

Diabetes mellitus affects 563 million people worldwide. All forms of diabetes are consequence of impaired function or survival of pancreatic islet b cells. Importantly, hallmark functions of human pancreatic β cells improve in the years after birth. Recent studies have revealed numerous potential regulators of adult islet function, many of which are not expressed in mouse or immature human β-like cells. Understanding the genetic and molecular mechanisms regulated by these factors in adult human islets could uncover novel druggable targets for diabetes treatment and improve outcomes with β-cells from renewable sources. However, experimental approaches for genetic modeling and modulation of adult human islets are still limited.

Multiple studies have shown that transcription factors play a critical role in regulating β-cell maturation. During my postdoctoral studies, I identified novel transcriptional regulators of mature islet function, SIX2 and SIX3. I also combined primary human islet organoid systems (“pseudoislets”) with a variety of genetic approaches, including lenti-shRNAs and lenti-CRISPR, to study their roles in the adult β-cell and discover how they are regulated by non-coding mechanisms. Following a more extensive analysis, I have now identified multiple additional human transcription factors (including RXRG, NR0B1, MXI1, ONECUT2 and ESR1) that, like SIX2 and SIX3, increase their expression with age in humans. Importantly, these are unique to human beta cell/islet maturation: they are not expressed in adult mouse islet cells or β-like cells from renewable sources.

In this proposal, I will use my novel pseudoislet-genetics approach combined with lenti-shRNAs to target a subset of these recently identified candidates and explore how their depletion affects glucose-stimulated insulin secretion. Overall, the experiments proposed here should prioritize novel regulators of adult human β-cell function.

While these are important advances, further refinements in genetic manipulation of primary human islets will simplify the ‘scaling’ of these gene-targeting approaches, and will introduce novel possibilities for discovery, such as multiplexed targeting of distinct regulatory elements. Thus, in this proposal, I will adapt CRISPR/Cas9 ribonucleoprotein (RNP) electroporation methods for use in adult primary islets. I have designed proof-of-concept experiments to validate this approach, including targeting of the transcription factor PDX1, followed by experiments designed to simultaneously target two regulatory elements of the key beta cell enzyme, PCSK1. These studies will constitute the basis for future studies of epistatic interactions in primary islets.

These considerations lead to the following Specific Aims:
Specific Aim 1: Elucidate The Roles Of Novel Age-Dependent TFs in Primary Human β-Cells.
Specific Aim 2: Establish CRISPR-RNP-Mediated Gene Editing in Primary Human β-Cells.
Collectively, these studies bear the potential to significantly increase our understanding of the genetic mechanisms governing human adult β-cell function, and to expand our toolkit for genetic interrogation of human islets.