Objective
The past decade has seen enormous efforts by the T1D research community leading to the successful and unlimited generation of human β cells from pluripotent stem cells. Having set this foundation for islet replacement therapy in T1D, new challenges need to be addressed to enhance function and survival of β cells upon transplantation. Poor survival of grafts is due to the adverse environment β cells encounter upon transplantation leading to their cell death. Immunosuppression can partially prevent this process but also results in multiple adverse side-effects, including impaired β cell function and reduced quality of life. Recent reports suggest that β cells from T1D patients play an active role in their own destruction. Identifying β cell-specific mechanisms involved in this process, and whose depletion could protect against cell death upon transplantation would improve outcomes of islet replacement therapy.
The overarching objective of this project is to remove candidate β cell proteins that contribute and/or lead to β cell death when exposed to inflammatory conditions and to identify conditions for enhanced stem cell-derived β cell function. Using CRISPR/Cas9 gene editing, we will investigate if perturbation of the candidate proteins leads to protection and enhanced survival of both donor and stem cell-derived β cells. Similarly, we will induce expression of a candidate protein to enhance β cell function. In both cases, we will characterize the effects in cell culture, then validate our results through transplantation of the modified β cells into mice.
For these experiments, we chose a T1D mouse model in which native β cells are destroyed prior to transplantation, making them dependent on transplanted β cells. At the end of the experiment, we will recover the donor and stem cell-derived β cell grafts and evaluate the conditions preventing β cell destruction and/or enhancing β cell function. We will initially focus on the protective effect induced by removal of the cytokine protein IL32. IL32 loss in stem cell-derived β cells protects from cell death upon transplantation. Our preliminary data using CRISPR/Cas9 targeting IL32 in human islets confirm that its loss protects from inflammation in conditions mimicking T1D-like inflammation. In parallel, we will investigate a new approach for enhancing the function of SC-β cells. We found that the transcription factor RXRG is required for β cell function but is not expressed in SC-β cells. Here we propose to accelerate SC-β cell maturation by RXRG induction and activation. We identified a compound capable of inducing RXRG expression and insulin levels. This drug is bioavailable after in vivo injection in mice and here we will test its application for in vivo maturation of β cells.
Specific objectives include:
1: Genetic depletion of IL32 in donor and SC-β cells and subsequent evaluation of protective roles against inflammation in vitro and in vivo. This will include identification of gene functions affected by IL32.
2: Genetic and small-molecule based activation of RXRG to induce SC-β cell function. This aim will also explore combinatorial interventions for protection against apoptosis and enhancement of SC-β cell function.
3: Pharmacological maturation of SC-β cell function after transplantation.
Our unique genetic tools for donor and SC-islets introduce possibilities to test whether removal of specific proteins can increase survival following transplantation. This aim introduces novel strategies to prevent loss of β cell function, which we anticipate will decrease the number and iteration of required transplantations.
Background Rationale
Efforts in the last decade have led to the generation of replacement β cells from renewable sources, such as human pluripotent stem cells. Despite this groundbreaking progress, major challenges in islet transplantation therapy for T1D remain. Long-term survival of transplanted β cells is poor, likely due to inflammatory environments. Immunosuppressive intervention can reduce this destruction but also entails undesired complications, such as impaired β cell function. Thus, identification of strategies to improve post transplantation survival and function of transplanted β cells is a necessity for islet replacement therapy.
Accumulating evidence points to a central role of β cells in their own vulnerability to cell death as a mechanism driving T1D pathogenicity. Identification and modulation of β cell-specific pathways involved in cell death would allow for intervention enhancing β cell survival upon transplantation. We will characterize the candidate cytokine IL32, for which our preliminary data and published studies suggest that its loss protects β cells from T1D-like inflammation.
IL32 loss in stem cell-derived β cells was recently shown to confer protection against immune destruction following transplantation into mice. In human islets, we and others could demonstrate that IL32 expression is significantly upregulated upon exposure to T1D-like inflammation. We hypothesize that increased IL32 expression during inflammation leads to β cell death. Using CRISPR/Cas9, we depleted IL32 from human islet cells and showed that loss of IL32 leads to decreased expression of proinflammatory effectors after culture in T1D-like inflammation. We propose to characterize the protective effects mediated by loss of IL32 and identify the molecular pathways affected by IL32 and responsible for increased cell death in inflammatory conditions.
In addition, we propose to enhance the function of stem cell-derived β cells before and after transplantation. Specifically, we discovered that the transcription factor RXRG is required for β cell function and is not expressed in stem cell-derived β cells. This transcription factor is induced 3-month post transplantation in stem cell-derived β cells, coinciding with their maturation. In pilot studies, we identified a compound capable of inducing RXRG expression and insulin levels. Importantly, this drug is bioavailable after injection into mice. Within the scope of this research plan, we will: a) genetically induce RXRG expression in stem cell-derived β cells; b-c) pharmacologically activate RXRG expression in stem cell-derived β cells to induce their maturation b) before and c) after transplantation.
Altogether, our approach will ensure identification of novel, β cell-specific pathways with high potential for translation into novel therapeutic targets to protect transplanted β cells from death while enhancing their function.
Description of Project
Diabetes mellitus is a disease of pandemic proportions affecting hundreds of millions of patients worldwide. It is the result of impaired insulin secretion, the only blood glucose lowering hormone in the human body. Autoimmune destruction of the insulin-secreting pancreatic β cells leads to type 1 diabetes (T1D), which typically manifests in childhood and leads to lifelong dependence on exogenous insulin. However, supplementing insulin through injections can result in hypoglycemic episodes, and usually fails to fully normalize blood glucose levels. Vascular damage and other long-term complications cannot be fully prevented by this standard treatment. Replacement of lost β cells can be achieved through transplantation of donor islets, the micro-organs β cells are situated in. Advances in stem cell biology now also allow for the generation of β cells in the laboratory in unlimited quantity, providing an alternative for a tighter control of blood glucose.
Despite this groundbreaking progress, major challenges in islet transplantation therapy for T1D persists. Only a small proportion of the transplanted insulin-producing β cells survive the transplantation procedure, likely the result of multiple stressors, including an inflammatory environment. Immunosuppression can partially prevent this process, but undesired side-effects reduce overall life quality and β cell function. Thus, novel methods to improve β cell survival and function would provide a major step forward towards therapeutic application of stem cell-derived β cells. Recent evidence suggests that β cells of T1D patients are involved in their own autoimmune destruction, exemplified by the discovery of several β cell genes that drive β cell death following transplantation.
This research plan will investigate if removal of genes involved in β cell death leads to improved survival of transplanted β cells from both donor- and stem cell-derived β cells. We will validate these effects prior to and after transplantation into mice in conditions resembling the cytokine-induced proinflammatory environment of patients with T1D. Specifically, our studies will decode the roles of IL32, a cytokine upregulated in the blood and β cells of autoantibody positive and T1D patients. Loss of IL32 in stem cell- derived β cells transplanted into mice protected against β cell death. Further, human islets exposed to proinflammatory cytokines that mimic T1D conditions have excessively elevated levels of IL32. Our pilot studies perturbing IL32 in human islets using CRISPR/Cas9 and exposing them to T1D-like proinflammatory conditions decreased proinflammatory responses. We propose to remove IL32 in donor- or stem cell-derived β cells before transplantation into mice exposed to human immune cells. Our studies will clarify if loss of IL32 can reduce β cell death upon transplantation and the duration of this protective effect.
In addition, this proposal will investigate a new approach for enhancing the function of (SC-)β cells before, but also after transplantation. Specifically, we discovered the transcription factor RXRG, required for β cell function but not expressed in stem cell β cells. We propose to mature stem cell-derived β cells by activating RXRG, taking advantage of a compound that we found to induce RXRG expression and insulin levels. This drug is bioavailable after in vivo injection in mice and here we will test its application for in vivo maturation of stem cell-derived β cells.
This project will lead to significant, innovative contributions to prevent β cell death and enhancement of their function upon transplantation into T1D patients.
Anticipated Outcome
Within this project, we anticipate to discover novel, protective mechanisms for β cell survival and enhanced function following transplantation. Specifically, we will uncover β cell genes whose depletion leads to protection of donor and stem cell-derived (SC) β cells from the consequences of T1D-like inflammation.
In our first aim, we will confirm the protective effects of IL32 perturbation against inflammation-induced β cell death from our findings and reports of others. We will study this process and, most importantly, anticipate replication of these findings upon transplantation of IL32-depleted (SC-)β cells into mice. Systematic recovery of grafts following transplantation, T1D-like inflammation and/or exposure to human immune cells, will allow us to discern how IL32 depletion protects from cell death. This aim will also characterize the molecular changes associated with IL32 loss. Understanding the molecular pathways leading to increased β cell survival will be important for the development of a specific, pharmacological agents with high translational potential. We are experienced in studying loss-protective interventions in β cells, which we have successfully adapted to IL32. In the unlikely scenario that our predicted outcomes are not achieved, we will take advantage of the flexibility of our genetic system to target other highly promising protective candidates. Furthermore, we recently showed feasibility of targeting multiple genes simultaneously, which we could explore if needed.
In our second aim, we will enhance the function of SC-β cells by inducing the expression of a transcription factor, RXRG. It is required for mature function of β cells but not expressed in SC-β cells. This transcription factor is induced 3-month post-transplantation of SC-β cells coinciding with their functional maturation. In pilot studies, we identified a compound capable of inducing RXRG expression and insulin levels. We anticipate to enhance the function of SC-β cells by genetic and pharmacologic activation of RXRG. This aim will include identification of molecular pathways induced by RXRG activation. While our pilot studies suggest that RXRG can enhance function of SC-β cells, we acknowledge that additional factors may be required to achieve this goal. In this case, we will test additional candidates or combinations of them. Finally, in our third aim we anticipate observing enhanced maturation and function of SC-β cells after transplantation and administration of the RXRG-inducing compound. If the effect is not sufficient to induce functional SC- β maturation, we will include further candidate compounds.
In sum, our strong, innovative and highly efficient pipeline will ensure the completion of our overarching goal, which is the study and intervention of β cell effectors as a strategy to prevent β cell death and loss of function upon transplantation.
Relevance to T1D
Efforts over the past >10 years have led to significant improvement in the generation of stem cell-derived (SC) β cells with promising clinical trials ongoing. Still, a major challenge persists: transplanted β cells are exposed to T1D-like inflammatory environments of T1D patients. Recent evidence suggests that proteins present in β cells play a role in in immune-mediated β cell death leading to loss of β cell function. Identifying these proteins and their mode of action is key to enhance β cell survival upon transplantation. The work proposed here focuses on β cell-specific regulators and cytokines that are upregulated during inflammation and might therefore mediate immune-related β cell death in T1D or after transplantation. Conversely, we explore molecular pathways impaired in T1D(-like) environments and which are required for mature β cell function. Modulating cytokines and transcription factors susceptible to dysregulation in β cells upon inflammation will allow for a novel approach to efficiently reduce inflammation-induced β cell death, as well as loss of function following transplantation. Our unique genetic studies in donor and SC-derived β cells are the ideal platform to answer these important questions.