Objective
Type 1 diabetes (T1D) is the result of our immune system being flawed and attacking the β-cells in the pancreas that secrete insulin. This is called an autoimmune attack. When β-cells are killed, patients rely on insulin shots to keep their blood sugar under control. This disease is extremely hard to detect before the patients are already very sick, so new treatment approaches must be identified. We can now successfully generate unlimited numbers of insulin producing β-like cells from human stem cells, but once we transplant them, cells are still threatened by the same autoimmune attack that caused the disease in the first place. Genetic engineering is at the forefront of scientific advances, allowing us to alter cells at will, to ask questions and study approaches we otherwise would not be able to investigate. I propose to use techniques that we have already perfected in our lab to genetically engineer stem cell-derived insulin producing β-like cells in two different ways so that they are protected. My studies will provide critical insights on how engineering approaches can prevent immune rejection of transplanted β-cells from immune cells that cause the autoimmune disease. This information is essential to understanding how this key autoimmune response can detect and destroy insulin producing cells in a human context and will potentially open the door for improved transplantation survival without the need for systemic immune suppression.
Background Rationale
To ensure that our immune system responds properly to different threats, it has developed many internal checks and balances. Type 1 diabetes (T1D) occurs when the immune system is flawed and attacks the cells in the pancreas that produce insulin in what is called an autoimmune attack. When insulin-producing β¬-cells are killed, patients must rely on insulin shots to keep their blood sugar under control. Insulin injections successfully extend patients’ lives, but the swings in blood sugar levels over multiple years or decades can cause serious life-threatening complications like hypoglycemia, blindness, stroke, heart attack, and kidney failure. Transplanting β-cells that secrete insulin back into patients is a promising treatment option for T1D, but a lack of donor material, side effects of anti-rejection medications, and poor cell survival after transplantation are major challenges for wide-spread use. We can now successfully generate unlimited numbers of insulin producing β-like cells from human stem cells, effectively addressing donor organ shortage, but these cells are still threatened by the human immune system once transplanted. Genetic engineering is at the forefront of scientific advances, allowing us to ask and study unique questions that would otherwise be inaccessible. I propose to genetically engineer stem cell-derived insulin producing cells to have high amounts of programmed death-ligand 1 (PD-L1) and a unique modified version of epidermal growth factor receptor (EGFR) so that they are protected from autoimmune attack. PD-L1 can provide localized immune suppression, however, only manages to inhibit autoimmune attacks partially. Engineered EGFR will serve as an anchor to attract immune cells that have powerful immune tolerance abilities, T regulatory cells (Tregs). I will test the ability of both strategies alone and in combination using innovative human model systems both in the cell culture and preclinical animal models. I predict that the combined strategy provides complete protection from recurring autoimmunity and enables long term graft function without the need for systemic immune suppression. My studies are essential to understanding how key autoimmune responses attack insulin-producing β-cells in a human context and will potentially open the door for much improved treatment options for patients suffering from T1D.
Description of Project
The immune system has a natural way of keeping itself in balance to make sure it responds appropriately to different threats but does not attack its own tissues. Type 1 diabetes (T1D) is a disease where the immune system is flawed and attacks the β-cells in the pancreas that produce insulin in what is called an autoimmune attack. When insulin-producing β-cells are killed, patients must rely on insulin shots to keep their blood sugar under control. Insulin injections successfully extend patients’ lives, but the swings in blood sugar levels over multiple years or decades can cause serious life-threatening complications like hypoglycemia, blindness, stroke, heart attack, and kidney failure. Transplanting β-cells that make insulin back into patients is a promising treatment option for T1D, but a lack of donors, side effects of systemic anti-rejection medications, and poor cell survival after transplantation are major challenges for wide-spread use. We can now successfully generate unlimited numbers of insulin producing β-like cells from human stem cells (sBCs), which can address the need for more donor organs, but sBCs are still threatened by the human immune system once transplanted. Genetic engineering is at the forefront of scientific advances, allowing us to alter cells at will. I propose to genetically engineer sBCs so that they are protected from immune attack in a localized manner. Specifically, I will do this by manipulating an important molecule called programmed death-ligand 1 (PD-L1), which is found on certain cells within the body. PD-L1 helps the immune system tolerate its own tissues and prevent harmful autoimmune reactions. Scientists have been exploring ways to use the PD-L1 molecule to improve the immune system's ability to accept transplanted tissues and prevent rejection. By increasing the amount of PD-L1 on sBCs, we may be able to transplant these cells without the patient needing to take systemic anti-rejection medicine. Specifically, my studies will focus on recurring autoimmune attack which has been shown to persist even in the face of systemic immune suppression. Recurring autoimmunity against cell replacement therapy is currently not studied effectively, largely due to the absence of appropriate model systems. In addition to protecting sBCs by PD-L1, I will also manipulating a different immune population known as regulatory T cells (Tregs). Tregs are a type of immune cell that play an important role in keeping the rest of the immune system under control and preventing autoimmune diseases like T1D in healthy individuals. Unfortunately, in patients with T1D, Tregs do not function properly, which is part of the reason they get sick. We will use Tregs that we modified to recognize specifically a genetic modification on sBCs that I introduce. This strategy ensures that the Tregs only protect the desired cells, sBCs in a localized manner. I will study individual and combined approach using innovative and novel humanized model systems, including preclinical animal models. I anticipate that the combined approach will provide localized immune protection for sBC grafts and enable long term graft function without the need for systemic immune suppression. My studies are essential to understanding how this key autoimmune response can see insulin producing cells in a human context and will potentially open the door for improved transplantation survival without the secondary risks facing patients with T1D.
Anticipated Outcome
I predict that my studies will show that an increase in programmed death-ligand 1 (PD-L1) on the surface of stem cell-derived β-like cells (sBCs) will be able to reduce the ability for autoimmune T cells to kill insulin producing sBCs. I anticipate that transplanting PD-L1 engineered insulin-producing sBCs will display reduced autoreactive T cell infiltration, lessened cell death within the graft, and continued survival and function of the transplanted insulin-producing cells compared to controls. My work in this proposal will lay the foundation for further research by providing important technical and biological knowledge into the protective abilities of PD-L1. In addition to PD-L1, this proposal seeks to harness the protective abilities of regulatory T cells (Tregs). I predict that the stable increased overexpression of truncated epidermal growth factor receptor (EGFRt) on sBCs will demonstrate the ability to recruit and activate designer CAR-Tregs to reduce the killing of autoreactive T cells. I also anticipate that combining both protective approaches will significantly suppress transplant destruction by autoreactive T cells and allow long term function of sBC grafts in humanized preclinical animal models. My studies are essential to further our understanding how key autoimmune responses can recognize and destroy insulin producing sBCs in a human context and will provide critical insights that will improved insulin producing cell transplantation survival and long term function without the need for systemic immune suppression. Thus, my proposed studies have the potential to directly impact the lives of individuals affected by T1D positively.
Relevance to T1D
I have been studying different aspects of Type 1 diabetes (T1D) for my entire scientific training and am determined to contribute to providing a cure for patients suffering from T1D. Cell replacement therapy using cadaveric islets has been shown to serve as a practical cure but for only a limited number of patients, and they also require systemic immune suppression. Recent advances by several labs, including my postdoctoral lab, and subsequent startup companies, have shown that stem cell derived β-like cells (sBCs) can serve as an unlimited source for cell therapy. Indeed, preliminary results from clinical trials transplanting sBCs into patients achieves insulin independence, further highlighting the stellar prospect of this approach. However, patients still rely on systemic immune suppression that carries considerable complications and likely excludes this approach form the majority of T1D patients, many of which are children. My proposal seeks to test if a combinatorial genome engineering approach harnessing known surface molecules that provide localized immune modulation in conjugation with localized enrichment of tolerance inducing immune cells to provide complete protection of sBC grafts and long-term function without the need for systemic immune suppression. In addition to these innovative genome engineering approaches, my proposal will take advantage of novel human model systems that directly test T1D autoimmunity in a human context, a critical progression from current models that test only allogeneic (human to human) rejection. I anticipate that my study will advance our understanding of key autoimmune attack processes in a human setting, provide critically needed human model system to test novel protection ideas to the field, and describe a dual approach that enables improved cell therapy without the need for systemic immune suppression. Thus, I believe my study will positively impact the lives of many individuals affected by T1D.