Objective
With more than 500 million people living with diabetes, this disease continues to burden health care systems around the world. In particular, the costs associated with the therapeutic management of type 1 diabetes (T1D) in the US alone have recently been estimated to be over $800 billion dollars over a lifetime. Hence it is critical to seek new and more efficient therapies for T1D, and most importantly, find a cure. The development of scalable protocols to manufacture pancreatic cells from human pluripotent stem cells, including human Embryonic Stem Cells (hESCs) or induced-Pluripotent Stem Cells (iPSCs) has enabled efforts to produce an unlimited supply of insulin-producing cells that can be implanted into patients with T1D to take over automatic production of insulin within the body. With over twenty years of experience in basic islet and stem cell biology, the Nagy and Kieffer Labs are dedicated to advancing this approach. Firstly, we aim to provide several sources of stem cells which can be used to produce functional beta cells. ESCs and iPSCs have already been engineered with the FailSafe™ and immune cloaking (iACT) technologies and will now be used to generate beta cells. The Kieffer Lab will lead the research work on the conversion of the various cell lines harboring the FailSafe™ and the iACT technologies. Following differentiation, cells will be examined using various functional assays with comparison to isolated human islets. Next, we will assess the therapeutic effect of cell implants in various rodent models of diabetes. We will evaluate the long-term acceptance and the function of human FailSafe™ and cloaked beta cells in treating diabetes. In addition to rodent models of disease, we will also develop a FailSafe™ and cloaked marmoset iPSC line and test the functionality of these cells in the non-human primate, marmoset model of diabetes. We believe these preclinical studies in marmosets will support the path to simplifying cell replacement therapies for T1D to human medicine. This project envisions a conduit to provide safe and universal sources of off-the-shelf, therapeutic beta cells to treat or even cure T1D.
Background Rationale
The endocrine system is comprised of a network of glands essential for development, body homeostasis and metabolism. Disorders of these endocrine glands can lead to long-term severe consequences to the health of an individual. For example, disorders of the pancreas can lead to type 1 diabetes mellitus (T1D). T1D is caused by autoimmune-mediated destruction of beta cells in the pancreatic islets. This in turn results in the loss of endogenous insulin production and the inability to properly regulate blood glucose, leading to high blood glucose levels (hyperglycemia). Patients living with T1D require lifelong administration of insulin in attempt to maintain normal blood glucose levels to minimize risk of secondary complications including cardiovascular disease, stroke, retinopathy, kidney failure and nephropathy. However, manual regulation of blood glucose levels is difficult and can lead to low glucose-induced coma and even to death. Although several efforts have been made to develop better treatments, such as transplantation of donor pancreatic tissues and immunotherapies, a cure for T1D has remained elusive. In collaboration with ViaCyte, Kieffer and colleagues recently showed the first clinical evidence that stem cell-derived beta cells can trigger meal-regulated insulin production in patients with T1D. Stem cells have the ability to self-renew, to differentiate into different cell types and to be genetically modified. Therefore, they offer a great potential source for therapeutic cells to treat T1D. Two remaining challenges to address to allow for successful application are cell safety and requirement for the use of chronic immunosuppression to prevent rejection of transplanted non-self cells. The Nagy Lab has devised solutions to overcome these two hurdles. For safety, we developed a genome-editing solution that employs an inducible kill-switch to genes essential for cell division. The kill-switch is activated with an FDA-approved prodrug (ganciclovir) that eliminates rapidly dividing/potentially tumourigenic cells. This solution for cell safety (FailSafe™) and proof of its efficacy was recently published. This system also mathematically quantifies the safety level of cell therapies, allowing patients and clinicians to make informed decisions. To counteract immune rejection, we developed an induced allogeneic cell tolerance (iACT) system that modulates the function of various immune cell types without genetic alteration. This solution offers the long-term acceptance of stem cell derivatives. Incorporating the FailSafe™ and iACT systems into stem cells could generate long-term, safe and immune-privileged therapeutic cells for treating T1D and other diseases.
Description of Project
Type 1 diabetes mellitus (T1D) is an autoimmune disorder of the pancreas, an endocrine gland critical for body homeostasis and metabolism. Attack from one’s own immune system (autoimmunity) against pancreatic beta cells results in the loss of beta cell mass, leading to a decrease in the production of insulin, which plays a key role in regulating blood glucose. This decrease causes an imbalance of glucose levels, which, when untreated, leads to elevated blood glucose levels (hyperglycemia), the classic symptom of T1D. While T1D is generally diagnosed in childhood and adolescence, it is not uncommon to manifest in adults. T1D patients require lifelong exogenous administration of insulin in an attempt to maintain normal blood glucose levels along with a healthy lifestyle to minimize the risk of complications of diabetes (i.e. cardiovascular disease, stroke, retinopathy, kidney failure, neuropathy). Unfortunately, manual delivery of insulin and monitoring one’s blood glucose levels is imperfect and can lead to hypoglycemic-induced coma or even death. Transplantation of donor pancreatic islet tissue has become available in medical practice, and this treatment can dramatically improve patient’s quality of life by preventing diabetic complications associated with episodes of hypo- or hyper-glycemia. However, this transplant therapy is limited due to shortage of cadaveric islet donors. In addition, the durability of transplanted cells is limited and there is a need for long-term immunosuppression therapy to prevent immune-based rejection. It is thus imperative to develop novel and effective therapies for T1D, and the Nagy and Kieffer Labs are dedicated to achieving this. We are pursuing an alternative strategy involving the use of lab cultivated insulin-producing islet cells obtained from an unlimited supply of human stem cells. Moreover, our stem cells are genetically modified to escape immune detection, and also to harbor an inducible kill-switch for safety, which could enable transplants in patients without the need for immunosuppression. The Nagy Lab developed a genome-editing solution called FailSafe™ that employs an inducible kill-switch to rapidly eliminate dividing, potentially tumorigenic cells. This reduces potential tumor risks posed by engrafted therapeutic cells. The solution also enables mathematical quantification of cell replacement, allowing patients and clinicians to make informed risk/benefit decisions about cell therapies. To address the issue of immune rejection, the Nagy Lab identified eight immune-modulatory genes which, when overexpressed, could do this without disrupting systemic immune function, which we termed induced-Allogeneic Cell Tolerance or iACT. The overall objective is to combine these platform technologies developed in the Nagy Lab to generate safe and universal sources of human pluripotent stem cells which can avoid immune rejection without need for immune suppression. The Kieffer Lab optimized protocols by which human stem cells can be coaxed into functional islet cells capable of reversing diabetes in animal models. Moreover, in collaboration with industry, they have recently shown the first clinical evidence that stem cell-derived beta-cells can trigger meal-regulated insulin production in patients with T1D. In collaboration with the Kieffer Lab, the Nagy Lab will devise solutions to treat or even cure T1D by generating FailSafe™ and iACT-engineered, insulin-producing beta cells. Importantly, the Nagy Lab has derived stem cells from the marmoset and will implement these technologies in these unique cells. Following conversion of these to beta cells, they will be tested in marmosets with diabetes by the Martinez-Trujillo Lab, which specializes in non-human primate models. Collectively, these studies will pave the way toward future clinical testing of this novel cell-based insulin replacement strategy for T1D.
Anticipated Outcome
T1D is an ideal candidate for cell replacement therapy. The two platform technologies originating in Nagy Lab (FailSafe™ and cloaking (iACT)) offer solutions to cell safety concerns in cell therapy and with the autoimmune challenge found in T1D. The FailSafe™ system, which can be induced via administration of an FDA-approved prodrug, ganciclovir, to eliminate rapidly dividing cells, does not interfere with cell function and physiology. Mice with all their cells containing the FailSafe™ genome edit showed normal glucose homeostasis with a functional pancreas. The cloaking system (iACT), firstly developed and characterized in the mouse, has been fully adapted and introduced into FailSafe™ human pluripotent stem cells. We have demonstrated that FailSafe™ and iACT-engineered pluripotent stem cells can form differentiated tissue that survives in immune-competent recipients without administration of immune suppressive drugs. Furthermore, FailSafe™ and cloaked human pluripotent stem cells can suppress the inflammatory response generated from non-self (allogeneic) immune cells, as measured in various in vitro assays. Both the FailSafe™ and the iACT technologies are protected intellectual property (IP) and are exclusively licensed to a start-up company, panCELLa, Inc. The company’s mandate is to manufacture clinical-grade human pluripotent stem cell lines with the FailSafe™ and cloaking technologies built in. panCELLa can provide these cells to partners who are targeting different diseases. The Kieffer Lab has optimized differentiation protocols with human pluripotent stem cells to generate functional islet beta cells capable of reversing diabetes in rodents. Both our novel ESC and iPSC lines will be used to generate beta cells by the Kieffer lab, and the resulting cell products will be evaluated with comparison to primary human islets. We will next assess the therapeutic effect of transplanting the cell products in various diabetic rodent models. In addition, the Nagy Lab situated in the Australian Regenerative Medicine Institute (ARMI) at Monash university, in Melbourne, has established a pluripotent stem cell line from a common marmoset. This non-human primate marmoset stem cell line, soon to be engineered with our platform technologies, will lead to preclinical studies, driving acceleration of this novel cell therapy for T1D. We envision that our T1D-focused research coupled with the combination of the two platform technologies will promote the translation of this novel approach into a potential cure for T1D.
Relevance to T1D
T1D involves loss of pancreatic beta cells, resulting in a decreased production of insulin and leading to an imbalance of whole-body glucose levels. Thus, individuals with T1D would be ideal candidates for cell replacement therapy to restore insulin production and glucose homeostasis. Islet transplantation has become available in medical practice, whereby insulin-producing islets can be purified from cadaveric pancreas and transplanted via the portal vein. This procedure improves the quality of patient’s life by preventing complications of diabetes associated with episodes of hypo- or hyperglycemia. However, the loss of pancreatic beta cells in patients with T1D is caused by attack from their own immune system (the so called ‘autoimmunity’). Patients who undertake islet transplantations are thus required to take immunosuppressive drugs in order to protect the transplanted islet cells from immune rejection. Although immunosuppression provides a level of cell safety as a malfunctional graft can presumably be eliminated by cessation of immunosuppression, it leaves patients at risk for opportunistic infections. In addition, islet transplantation cannot be generalized in medical practice due to the shortage of cadaveric islet donors and limited durability of the transplanted cells. Clinical evidence of islet transplantations has demonstrated the importance of finding an alternative strategy for transplanting cells. Genetically modified stem cells capable of escaping immune detection could ultimately enable transplants in patients without the need for immunosuppression. Moreover, generating pancreatic beta cells from stem cells will provide an unlimited source of insulin-producing cells ready for transplantation. We harnessed genome engineering and unbiased screens to develop a novel strategy to “cloak” cells from immune rejection. The Nagy Lab identified eight immune-modulatory genes that, when overexpressed, can prevent immune rejection selectively at the graft site, without interfering with the systemic immune function. We termed our cloaking solution “iACT”, which stands for induced-Allogeneic Cell Tolerance. Regular immune surveillance is critical in clearing malignantly transformed cells, and as such, cells that can evade the immune system are inherently risky. The Nagy Lab developed the FailSafe™ technology that can provide cell safety by selectively eliminating rapidly-dividing, potentially tumorigenic cells. We are now pairing the “cloaking” system iACT with the FailSafe™ technology, which not only provides immune protection to transplanted cells but is also a substitute for the safety normally provided by regular immune clearance of malignantly transformed cells. In fact, we have already shown that FailSafe™ and iACT-engineered stem cells, or their differentiated progeny, formed long-term-accepted cell implants in allogeneic recipients. We will now combine our technologies with that of the Kieffer Lab, where stepwise protocols for generating insulin-producing cells from human pluripotent stem cells have been developed. The resulting insulin-producing cells have been demonstrated to be similar to human islets, producing a robust insulin responses to elevated glucose. Together, the combination of the Nagy platform technologies and the Kieffer protocol of differentiating stem cells into insulin-producing beta cells allows us to envision a pluripotent cell line that could be used as a source of safe and off-the-shelf therapeutic cells for treating T1D.