Objective
We performed a large-scale genetic screen in which we used genetics to delete cell surface proteins called G protein coupled receptors (or GPCRs) from human islet cells to identify which GPCRs controlled beta cell replication.
This 3-year project has four objectives.
Objective 1 is to confirm that the GPCR genes we identified control β cell proliferation in human islets and an animal model. Here we will remove or ‘silence’ each of the GPCR target candidates and monitor beta cell proliferation in human islets. We will confirm the specificity of the GPCRs by reintroducing them to restore cell cycle arrest – the critical experiment to demonstrate that you have a “real” target. We will also study the impact on beta cell function in this context – insulin secretion, cell survival – so predict any potential effects of inhibiting the GPCRs on beta cell function.
Objective 2 is to establish whether deletion of the GPCR candidates can promote β cell proliferation in the context of a whole animal. This is a pre-clinical step prior to moving t human studies. Specifically, we will generate β cell-specific knockout mice for each candidate individually, and measure beta cell proliferation. We expect that more beta cells will proliferate when the candidate GPCRs are removed. We will also evaluate β cell function by tests of oral and intraperitoneal glucose tolerance, insulin tolerance, refeeding and meal tolerance, and arginine tolerance Our lead candidate will be GPR3, for which we are generating a β cell-specific GPR3 KO mouse. Should we observe increased proliferation and increased beta cell mass/function, we propose to perform a complete metabolic analysis of this mouse to be able to predict effects in the beta cell on the whole organism. As an alternative approach, we would use RNAi to silence the GPCRs in human islets and transplant these under the kidney capsule of mice. This Objective will proceed in parallel with Objective 1.
Objective 3 is to understand how the GPCR candidates affects proliferation and function of human stem cell (SC)-derived (SCβ) cells. We will test the idea that the GPCR candidates may promote not only cell cycle arrest, but also affect cell function of the cells that are being proposed as potential alternative to islet transplantation. Specifically, we will introduce the GPCR candidates into SCβ cells and monitoring of proliferation rates and β cell functional outputs in vitro and after transplantation into STZ-treated immunodeficient mice. This will allow us to assess whether the GPCRs can reverse diabetes more effectively than control SC cells. We predict that increased levels of GPCRs will improve beta cell function and recovery from hyperglycemia.
Objective 4 is to bring a lead candidate molecule through preclinical testing in rodents. Here we will perform dose response, toxicity and efficacy, in rodents including a long-term safety study, with parallel dosing in human islets as a precursor to clinical studies in humans. The requirements for testing of the selected candidate in pharmacologic (ADME) and short-term and long-term toxicology tests in animals will depend on the depth in which the drug has already been tested prior to this proposed use. The proposed preclinical studies will be discussed with regulatory authorities, bearing in mind the age(s) of the specific patient population for which the drug is intended. Specifically, we will monitor impact on blood sugars, kidney, CV effects already in the animal studies, as well as reproductive toxicity. The mouse models developed in Aim 2 will be to guide initial dosing and formulation studies.
Background Rationale
Islet replacement has brought us closer to a cure for T1D, but is still limited by donor tissue availability, the need for lifelong immune suppression, and the failure of the transplanted islets to function and survive. An exciting alternative to transplanting islets is to transplant stem cell-derived beta (SCβ) cells. SCβ cells offer the promise of an unlimited supply of β cells for transplant, these too are subject to loss of function and cell death, even when surrounded by devices designed to protect them from the immune system.
Beta cell regeneration is an exciting alternative to replacement that centers around reactivating the replication program in a patient’ own β cells to restore lost cells and re-establish control of blood sugar levels. To date, the field has focused on screening large libraries of small molecules for drugs that can make human β cells divide. While this approach is perhaps the fastest road to identifying a potential therapeutic, a downside to this approach is unwanted targets, resulting in undesirable effects including replication of non-β cells. Despite repeated efforts, small molecules that specifically promote regeneration of human β cells have yet to be discovered. As β cell replication is controlled by genes, we believe that achieving careful, controlled proliferation will require a careful dissection of what genes determine when a beta cell starts and stops growing. To date our understanding of the genes that govern β cell proliferation in humans is poor; determining which specific genes stop β cell from dividing is a critical unmet need. Once identified, we could design strategies to target these key genes to help restart β cell replication to repopulate islets. In conjunction with an approach that blocks immune attack of β cells, our findings could also be useful in disease prevention.
Using our robotic genetic screening system, we have identified 9 cell surface proteins called GPCRs that govern the replication of human β cells. This is exciting for several reasons. First, GPCRs are critical regulators of islet biology. Human islets have numerous GPCRs on their cell surface, and they control many aspects of β cell function, thus it stands to reason that GPCRs would be control replication. Second, GPCRs are also the target of 1/3 of all drugs that are approved for use in humans by the Food and Drug Administration. Thus, GPCRs have a rich history of success as druggable targets with high therapeutic potential - the path to translating new results is clear. Third, despite the centrality of GPCRs to islet function, a comprehensive exploration of GPCR function in β cell replication has not been performed. Fourth, small molecules may face a lower regulatory burden than stem cell-based therapies, which means a faster road from discovery to clinic. Recent work in the field has shown us that β cells make a choice: to be functional and secrete insulin after a meal, or to replicate, but not both. And work from the stem cell field has shown us that SCβ cells make insulin but are not fully mature – they don’t secrete insulin effectively. We hypothesize that the same GPCRs that stop β cell from dividing may also play a role in β cell maturation. This project will determine if inhibiting the 9 GPCRs we identified will restart β cell replication in the human islet and in the mouse, and we will determine whether introducing the 9 GPCRs into SCβ cells can improve the ability of SCβ cells to restore normal blood sugar levels in diabetic mice. Thus, knowledge gained herein will identify strategies to regenerate human β cells but will also make SCβ cells work better.
Description of Project
Our vision is to establish a complete understanding of the biology of human β cells, the cells that make and secrete insulin after a meal. The inability to produce enough insulin due to loss of pancreatic β cells results in type 1 diabetes mellitus (T1D). The eventual goal is to get these cells to replicate again. While it is widely understood that treating T1D will require increasing the number and health of β cells, our current knowledge of the genes that control when a human β cell divides - why it stops dividing - are lacking.
To address this, we will use cutting-edge genetic technology to provide a complete understanding of which genes stop mature adult human β cells from dividing. Indeed, using this world-class technology, we have identified 9 candidate genes that shut down cell division in adult human β cells. We have gone on to show how removing one of these genes, called GPR3, makes adult β cells once again replicate, or ‘regenerate’. Each of these genes are members of the gene family called G protein coupled receptors, which are the target of 1/3 of all FDA -approved drugs. The path forward to translation with these targets is well-paved.
This project will explore how inhibiting GPR3 - and the other remaining candidates - stops β cells from dividing and identify a strategy to target this gene to promote the regeneration of a patient’s own β cells. Knowledge gained in this work will not only further our understanding of how we might regenerate β cells but will also be applied to with cell replacement strategies where we attempt to use these genes to make stem cell-derived β cells work even better. In
conjunction with an approach that blocks immune attack of beta cells, our findings could also be useful in disease prevention.
Anticipated Outcome
General: We identified 9 candidate genes in our screen that when deleted resulted in human beta cell proliferation. By identifying genes that guide β cell proliferation, we anticipate developing new therapeutic strategies that can restore functional β cell mass in patients with long-standing T1D. Our proposed experiments will use genetic approaches to unequivocally establish novel gene targets to re-establish β cell replication. This will allow us to fine tune the regenerative response of beta cells to patient-specific requirements and ultimately restore β cell mass and insulin production for improved regulation of glycemia in people living with T1D.
Specific Outcomes include:
The outcome for Project 1 is that we will confirm that a subset of these is in fact bona fide targets whose presence or absence dictates replication behavior in human β cells. We will then confirm how these confirmed genes also affect the function of β cells in human islets. These outcomes will go together with those from Project 2, where we will confirm in a whole animal (the mouse) whether deletion of the GPCR candidates results in proliferation of β cells.
In Project 3, we anticipate that introduction of the candidate GPCRs will help to stop replication and support the maturation of immature SCβ cells into mature SCβ cells that can secret insulin more effectively. This knowledge would help to further the main barrier facing the use of SCβ as surrogate islets – lack of insulin secretion. Any candidate that shows improved insulin secretion in the lab will then be tested for its ability to restore euglycemia in a diabetic mouse model.
Project 4 will establish preclinical groundwork for use of an existing inhibitor of any candidate GPCR that passes the validation stages in Projects 1 and 2, including short- and long-term toxicity.
Relevance to T1D
The inability to produce enough insulin due to loss of pancreatic beta cells, the cells that make and secrete insulin after a meal, results in type 1 diabetes mellitus (T1D). A central challenge in treatments for T1D has been inappropriate loss of function and survival of transplanted islets. Two alternative approaches include using stem-cell derived β cells, which to date don’t secret insulin efficiently, or attempting to get a patient’s own β cells to replicate to replace lost cells.
It is widely appreciated that treating T1D will require addressing autoimmunity and increasing the number and health of β cells. However, our current knowledge of the cell surface proteins, or “receptors”, that control human β cell replication are lacking. Our vision is to establish a complete understanding of what cell surface receptors on human β cells result in them losing the ability to replicate early in life, and to develop a strategy to prevent this. From the regeneration side, the eventual goal is to use drugs that inhibit the cell surface receptors that tell adult β cell to stop dividing. This would be a major step forward in our understanding of how to replace lost β cells in people living with T1D.
If we are successful in finding a receptor that we can target with a drug and can demonstrate increased survival of islets cells after transplantation in mice, from the replacement side we will also evaluate the ability of the lead drug to promote the function of β-like cells derived from stem cells. Such a drug would also potentially be an exciting tool that could be developed to prevent T1D.