Discovery and Functional Interrogation of Diabetes Risk Factors Through CRISPR-Mediated Perturbation Screening
Abstract
Diabetes is a serious global health problem characterized by high levels of blood glucose due to insulin deficiency. Malfunction of insulin-secreting beta cells is a hallmark of diabetes. Yet, regulators that contribute to impaired beta-cell function in diabetes have not been fully uncovered, nor have their impacts on beta-cell physiology and pathology. To improve current diabetes treatment and to develop effective targeted therapies, it is critical to systematically identify diabetes-risk factors and to understand their roles in regulating beta-cell function under both normal and diabetic conditions. However, there is a major challenge in achieving these goals. In human genome, there are ~20,000 protein-coding genes, making the genome-wide identification of potential diabetes-risk genes a daunting task. To overcome this challenge, we propose to incorporate large-scale, unbiased screening approaches. Here, we plan to use two complementary systems to systematically identify and interrogate genetic regulators of human beta-cell function: (1) human pluripotent stem cell (hPSC)-derived beta cells and (2) primary human islets. hPSC-derived beta cells provide easy access to large quantities of beta cells for high-throughput screening. While primary human islets enable the examination of fully differentiated beta cells from both healthy and type 2 diabetes (T2D) individuals. Moreover, primary human islets can best represent the physiological and pathological conditions of the human tissue. Therefore, we expect to uncover regulatory mechanisms in primary islets that cannot be easily detected using other systems (e.g., mouse model). Importantly, the use of hPSCs and primary human islets would allow us to readily translate our findings into therapeutic purposes. Specifically, we plan to conduct a genome-wide CRISPR screen in hPSC-derived insulin-secreting beta cells to identify previously unknown diabetes-risk genes that can impair beta-cell function. To facilitate the genome-scale screen, we have built a directed pancreatic differentiation platform using hPSCs to generate beta-like cells that closely resemble human beta cells. In unpublished work, we have identified developmental regulators of beta cells using the hPSC-differentiation platform. This supports the feasibility of the proposed screen. By uncovering previously unrecognized regulators and molecular mechanisms, the proposed screen will bring novel perspectives in designing targeted treatment for regulating beta-cell function and also provide guidance for improving current hPSC-based pancreatic differentiation. We also plan to perform large-scale CRISPR-based screens in primary human islets targeting potential diabetes-risk (~60-70) genes for functional interrogation. Target genes are selected from the unpublished screen (~50 genes) mentioned above that are also likely to control beta-cell function. We will also include top hits (~10-20 genes) from the proposed genome-wide screen in hPSC-derived beta cells as our targets. Screens will be done in both healthy and T2D islets. By comparing the gene perturbation results from the two conditions, we will be able to identify genes with the ability to ameliorate phenotypes caused by dysfunctional beta cells in diabetes. The candidate genes will be further validated through a smaller-scale screen using T2D islets. Validated hits can be further investigated for the purpose of developing targeted treatment for diabetes. It is highly innovative to use large-scale CRISPR-based screens to identify and functionally interrogate diabetes risk factors at the beta-cell stage. So far, no such screens have been conducted. This gap needs to be filled for developing effective targeted therapeutics and improving current diagnostic tools for diabetes. Our findings can also be used to improve current hPSC-based beta cell differentiation protocols for the generation of large quantify of high-quality beta cells for cell replacement treatment. Th
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Dec 28, 2022
- Source ID
- W81XWH2210202
Entities
People
- Nan Zhang
Organizations
- Sloan-Kettering Institute
- United States Army