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 inflammatory 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. Thus, alternatives to immunosuppression are being intensively sought. Recent reports suggest that β cells from T1D patients play an active role in their own destruction. Identifying β cell-specific mechanisms involved in their cell death, and whose repression could protect against it upon transplantation can improve outcomes of islet replacement therapy.
The overarching objective of this project is to block the activity of candidate β cell proteins that contribute and/or lead to β cell death when exposed to inflammatory conditions. Using CRISPR/Cas9 gene editing systems, we will investigate if perturbation of the candidate proteins leads to protection and enhanced survival of both donor and stem cell-derived β cells. First, we will characterize the effects in cell culture, then validate our results through transplantation of the modified β cells into mice.
At the end of the transplantation experiments, we will recover the donor and stem cell-derived β cells and evaluate the conditions preventing β cell destruction. This will include evaluation of pro-apoptosis (or cell death) markers, inflammation markers, and insulin levels. We will initially focus on the protective effect of blockage of the candidate protein HIVEP2. HIVEP2 is a transcription factor for which our preliminary data suggests that its inactivation improves β cell survival during inflammation. Importantly, HIVEP2 levels are increased in β cells of T1D patients and following exposure to T1D-like inflammation.
Our specific objectives include:
1: Genetic elimination of HIVEP2 in donor and stem cell-derived β cells and subsequent evaluation of protective roles against inflammation in vitro and in vivo.
2: Identification of HIVEP2 downstream targets, both in non-cytotoxic conditions and upon exposure to proinflammatory cytokines. This will help identify additional presumptive protective candidates against cell death in T1D environments.
3: Identification of the mechanisms dysregulating HIVEP2 in T1D. This will include genetic perturbation of cytokine-induced regulatory regions of HIVEP2, selectively activated in T1D-like inflammation.
Our unique genetic tools for donor and stem cell-derived islets introduce possibilities to test whether elimination of specific protein functions can reduce immune destruction following transplantation.
Background Rationale
Efforts in the last decade have led to the generation of replacement β cells from renewable sources, such as stem cells. Despite this groundbreaking progress, a major challenge in islet transplantation therapy for T1D remains. The residual auto- and alloimmune host environment will lead to destruction of the transplanted β cells. Immunosuppressive intervention can reduce this destruction but also entails undesired complications, including impaired β cell function. Thus, identification of alternatives to immunosuppression 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 central mechanism driving T1D pathogenicity. Identification and modulation of β cell-specific mechanisms involved in cell death would constitute a viable approach to avoid immune destruction of β cells upon transplantation. We will investigate a candidate genes, HIVEP2, for which our preliminary data suggests that its loss will protect β cells from T1D cytokine-proinflammation and immune-based destruction.
HIVEP2 loss in a murine β cell line was recently shown to confer protection against immune destruction following transplantation into mice. In human islets, we and others could demonstrate that HIVEP2 expression is significantly upregulated in T1D and also upon exposure to a cytokine cocktail that mimics the proinflammatory environment generated by infiltrated immune cells in the islets of T1D patients. We hypothesize that increased HIVEP2 expression during inflammation leads to β cells death. Using CRISPR/Cas9, we introduced mutations into HIVEP2 in human islet cells corroborating this hypothesis: loss of HIVEP2 leads to decreased expression of proinflammatory effectors and reduced cell death following culture with T1D-relevant proinflammatory cytokines. Despite this beneficial effect of HIVEP2 loss in T1D-like inflammation, it also negatively affected important β cell functions, such as insulin secretion. This is likely due to several pathways regulated by HIVEP2. In support of this possibility, subcellular localization of HIVEP2 changes upon exposure for 24 to cytokines, leading to HIVEP2 translocation from the cytoplasm to the nucleus. We propose to characterize the downstream targets of HIVEP2 and identify the specific pathways responsible for increased cell death in inflammatory conditions. Identification of targets induced upon inflammation will identify inflammation-specific druggable targets and avoid undesired effects on β cell function. Further, investigating the mechanisms that lead to increased expression of HIVEP2 in β cells during inflammation could provide a better understanding of the processes involved in T1D progression. We hypothesize that inflammation-induced signals upregulate HIVEP2 and are therefore responsible for its detrimental actions during inflammation. This project aims to identify the mechanisms responsible for HIVEP2 upregulation in inflammation.
Altogether, our rationale will ensure identification of novel, β cell-specific mechanism with high potential for translation into novel therapeutic targets to protect transplanted β cells from death. Furthermore, it could be easily adapted to additional relevant candidates.
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, a major challenge in islet transplantation therapy for T1D persists. Transplanted insulin-producing β cells are exposed to the host immune system which recognizes both non-host tissues, but also specifically β cells through the persisting autoimmune disease. Immunosuppression can partially prevent this process, but undesired side-effects reduce overall life quality. In addition, this treatment impairs β cell function, reducing the output of the transplanted cells. Thus, alternatives to immunosuppression therapy 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 or blocking of genes or proteins 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 transplantation, but also in vivo, following transplantation into mice in conditions resembling the immune environment of patients with T1D. Specifically, our studies will decode the roles of HIVEP2, a gene that regulates other genes, and for which evidence suggests a pro-death role in conditions that resemble T1D. First, loss of HIVEP2 in a murine β cell line transplanted into mice protected against β cell death. Second, human β cells of T1D patients have elevated levels of HIVEP2. Third, human islets exposed to a cocktail of proinflammatory cytokines that mimic T1D conditions also have excessively increased levels of HIVEP2. Forth, we perturbed HIVEP2 in human islets using CRISPR/Cas9 and exposed them to T1D-like proinflammatory conditions, which reduced cell death and decreased proinflammatory responses. We propose to remove HIVEP2 in donor- or stem cell-derived β cells for transplantation into mice. Our studies will clarify if loss of HIVEP2 can reduce β cell death upon transplantation and the duration of this protective effect. They will also identify downstream potentially druggable targets of HIVEP2, and mechanisms leading to the excessive activation of HIVEP2 in T1D-like proinflammatory conditions.
Anticipated Outcome
Within this project, we anticipate to discover novel, protective mechanisms for β cell survival following transplantation. Specifically, we will uncover β cell genes whose depletion leads to protection of both donor and stem cell-derived β cells from the consequences of inflammation and/or immune attack.
In our first aim, we will confirm the protective effects of HIVEP2 perturbation against inflammation-induced β cell death. Our preliminary findings show that HIVEP2 loss is protective against β cell death following short-term culture. We will further study this process and, most importantly, anticipate replication of these findings upon transplantation of HIVEP2-depleted islet cells into mouse models. Systematic recovery of grafts at different time points following transplantation and inflammation will allow us to discern how and at which stage, HIVEP2 depletion protects from cell death. Our second aim will focus on investigating downstream targets of HIVEP2. We expect to identify several pathways involved in various aspects of HIVEP2 function, such as susceptibility to β cell death and function. Understanding the signaling cascade leading to increased β cell death will be important for the development of a specific intervention with high translational potential. In our third aim, we expect to identify signals leading to dysregulated HIVEP2 expression, which will inform about T1D onset and progression. In the unlikely scenario that our predicted outcomes are not achieved, we will adapt our flexible genetic system to target additional highly promising protective candidates. Furthermore, we recently showed the feasibility of performing targeting of multiple genes simultaneously, which we could also explore if needed.
In sum, our strong, innovative and highly efficient and adaptable pipeline will ensure the completion of our overarching goal, which is the study and intervention of β cell effectors as strategy to prevent β cell destruction upon transplantation.
Relevance to T1D
Efforts over the past >10 years have led to significant improvement in the generation of stem cell-derived β cells with promising clinical trials ongoing. Yet, a major challenge persists: transplanted β cells are exposed to the immune system of T1D patients, including the autoimmune component specifically targeting β cells. Recent evidence suggests that proteins present in β cells play a role in in immune-mediated β cell death. Identifying these proteins and their mechanisms is key to enhance β cell survival upon transplantation. The work proposed here focuses on β cell-specific regulators that are upregulated during inflammation and might therefore mediate the effects of proinflammatory cytokines released by the immune system on β cells in T1D or an inflammatory transplantation setting in general. Modulating transcription factors and cytokine-induced chromatin regulatory elements, susceptible to dysregulation in β cells upon inflammation, will allow for an efficient approach to prevent autoimmune attack and destruction of β cells following transplantation. Our unique genetic studies in donor and SC-derived β cells will provide insights into these novel mechanisms and offer novel approaches to prevent or reduce immune destruction following transplantation for both donor and stem cell-derived β cells.