Objective
Islet transplantation offers a potential cure of Type 1 diabetes, but it is difficult to justify the immunosuppression needed to prevent rejection of donor islets, since most diabetes is well-controlled with insulin therapy. Therefore, approaches to induce “tolerance”, whereby the recipient regards the donor as “self” and does not require immunosuppressive therapy, are needed to make islet transplantation a widely available cure. If, however, this cure was available, there would never be enough human islets to transplant. While stem cell-derived islets are being explored by many groups, safety and efficacy barriers remain before they can reliably be considered curative. We propose to explore an alternative source of insulin-producing cells for the treatment of Type 1 diabetes in humans, namely pig islets. These would be available in unlimited quantities and can be genetically engineered in ways that prevent rejection in humans. This proposed project aims to optimize the function and survival of islets from genetically engineered pigs. We aim to overcome the immune barriers to pig islet transplantation and develop a method of inducing immune “tolerance” so that the islets can be accepted without life-long immunosuppressive drug treatments. We will use our unique inbred line of genetically modified miniature pigs in studies of transplantation to mice with human immune systems and in a preclinical, non-human primate model. We will explore a combined approach that includes: 1) improving the survival of islets implanted under the skin, making the transplant less invasive and more accessible than traditional islet transplant sites. We will add special cells that we expect will accelerate blood vessel formation, thereby improving the early survival of the islets; 2) using pigs that are genetically modified to express human CD47, a molecule that prevents rapid destruction by “innate” immune cells such as natural killer cells and macrophages; and 3) educating recipient human or baboon T lymphocytes to regard the pig donor as “self”, so that long-term immunosuppressive treatments, with their many side effects, are not required for islet graft acceptance. T lymphocytes are the major players in graft rejection and they learn to distinguish “self” from “non-self” in the thymus, where they develop. Educating T cells in a pig thymus makes them regard the pig donor as “self”, and we expect this to prevent rejection of pig islets from the same inbred line of pigs. We aim to demonstrate this in human immune systems in mice and in baboons, in both cases by transplanting a pig thymus in addition to the pig islets.
Background Rationale
In a small subset of patients with Type 1 diabetes whose blood glucose levels are difficult to manage under standard insulin treatment, islet transplantation restores normal blood glucose control and prevents diabetes-associated complications. Outcomes reported from clinical trials support broader application of the cell replacement strategy for treatment of Type 1 Diabetes. The insufficient availability of donor islets and the substantial number of islets required for each recipient, due largely to an early and rapid loss when transplanted in the liver, limit the widespread application of this treatment to patients in need. While solutions to improving islet transplant efficacy are being sought, transplantation of porcine islets is a viable option for significant expansion of the donor islet pool in clinical transplantation. Pigs are an ideal source of donor organs because of their size, physiologic similarity to humans, fast growth, early sexual maturity, and large litters. Several of these features make them amenable to genetic modification. In addition, porcine islets are resistant to destruction by recurrent autoimmunity, as shown in a mouse model of diabetes. Unmodified pig islets are, however, incompatible with the human immune system and will be rejected by both “innate” immune cells such as macrophages and “adaptive” immune cells such as T cells. The adaptive immune system even causes rejection of human islets, requiring long-term immunosuppressive therapy in the few patients who receive islet transplantation. Lifelong immunosuppression cannot be justified in most Type 1 diabetes patients. Methods of preventing both innate and adaptive immune- mediated rejection without using immunosuppressive drugs would make pig islet transplantation a feasible cure for all Type 1 diabetic patients.
Currently, there are no effective long-acting drugs available to control innate immune responses. Our previous studies have shown that the incompatibility between pig CD47 and its human receptor activates innate immune responses and that this can be avoided by using islets from genetically modified pigs that express human CD47. Based on our work and that of another group, we hypothesize that we can minimize both macrophage and natural killer cell innate immune cell-mediated rejection of the porcine islets by using pigs transgenic for human CD47.
We have also developed a method of avoiding human T cell-mediated rejection of pig tissues, by developing and educating human T cells in a pig thymus. In mice with human immune systems, porcine thymus transplants support development of functional human T cells that accept donor pig-specific skin grafts. We have applied this approach in non-human primates, achieving long-term acceptance of life-supporting porcine kidney xenografts. We propose to develop this clinically applicable approach for the induction of “tolerance” to pig islets by using porcine thymic transplantation, thereby avoiding the need for long-term immunosuppression.
We further propose to utilize a method for achieving rapid islet graft vascularization by new blood vessels to improve early graft survival when islets are transplanted under the skin, a site that is desirable for its accessibility and the non-invasive nature of transplantation to it. When islets are prepared, their vasculature is disrupted, and some of the transplanted islets die before local blood vessels connect islets to the recipient circulation. Our team has pioneered the concept that each organ is vascularized by unique, specialized cells that provide instructive tissue-specific vascular messages to promote blood vessel formation. By generating such cells from the recipient and transplanting them with the donor islets, we aim to establish the subcutaneous location as an effective islet transplant site.
Description of Project
In the minority of patients with Type 1 diabetes whose blood glucose levels are not successfully managed under insulin treatment, transplantation of islets containing the cells that produce insulin restores normal blood glucose control and prevents diabetes-associated complications. However, the insufficient availability of donor islets and need for lifelong immunosuppression, with its many complications, limits this treatment to a small subset of Type 1 diabetic patients. Pig islets offer numerous advantages as a potential cure for Type 1 diabetes. Pigs share physiologic similarity to humans and their fast growth, early sexual maturity, and large litters make them amenable to genetic modification. In addition, porcine islets are resistant to destruction by recurrent autoimmunity, as shown in a mouse model of diabetes. We have a unique inbred line of genetically modified miniature pigs that are designed for transplantation to humans. Unmodified pig islets are, however, incompatible with the human immune system and are rejected by both “innate” immune cells such as macrophages and “adaptive” immune cells such as T cells. Methods of preventing both innate and adaptive immune-mediated rejection while avoiding the need for long-term immunosuppressive therapy would make pig islet transplantation a feasible cure for all Type 1 diabetic patients.
Our proposal builds on several observations from our laboratories: 1) An important cell surface protein, CD47, allows cells of the human innate immune system to distinguish self from non-self and rapidly reject pig cells. We have obtained evidence that expression of human CD47 (hCD47) on pigs through genetic modification can prevent this rapid rejection by the human innate immune system; 2) T lymphocytes are the major adaptive immune players in graft rejection and they learn to distinguish “self” from “non-self” in the thymus, where they develop. In a human immune system mouse model, we have shown that human T cells that are educated in a transplanted pig thymus can recognize the donor pig as “self”. We hypothesize that transplantation of porcine thymus tissue will allow porcine islet cells from the same inbred line of pigs to avoid rejection by T cells mice with human immune systems and baboons, two pre-clinical animal research models; 3) We have developed a method of improving blood vessel formation for islets, so that the islets might be transplanted under the skin, where mature islets otherwise survive poorly due to the delay formation of new blood vessels. Transplantation under the skin would be a relatively non-invasive type of islet transplant.
We will use our unique inbred line of genetically modified miniature pigs in studies of transplantation to mice with human immune systems and in a preclinical, non-human primate (baboon) model. We will explore a combined approach that includes: 1) Improving the survival of islets implanted under the skin, making the transplant less invasive and more accessible than traditional islet transplant sites. We will add special cells that we expect will accelerate blood vessel formation, thereby improving the early survival of the islets; 2) Using pigs that are genetically modified to express hCD47, a molecule that prevents rapid destruction by “innate” immune cells such as natural killer cells and macrophages; and 3) Educating recipient human or baboon T lymphocytes to regard the pig donor as “self”, so that long-term immunosuppressive treatments, with their many side effects, are not required for islet graft acceptance. We aim to demonstrate this in human immune systems in mice and in baboons, in both cases by transplanting a pig thymus in addition to the pig islets.
Anticipated Outcome
We expect to demonstrate in Aim 1 that porcine thymic transplantation educates human T cells so they will accept islets from the same porcine donor line without immunosuppression and that pig islets expressing hCD47 will avoid early damage by human innate immune systems in mice with pre-established human immune systems. Under Aim 2, we expect to optimize islet transplantation under the skin in a large animal model, namely baboons, by adding specialized recipient cells that promote rapid vascularization of islet grafts. We expect to demonstrate the superiority of this approach over the more traditional intraportal form of islet transplantation into the liver. The results of Aims 1 and 2 will allow us to proceed with Aim 3, in which we will explore the ability of porcine thymus transplantation to allow long-term acceptance of hCD47-expressing pig islets in baboons. We expect to demonstrate the superiority of hCD47 -expressing pig islets over non-transgenic pig islets in this baboon model. We will determine whether it is better to transplant the pig islets directly into the pig thymus or to put them into a separate site. The separate site will be determined by the best results of the studies in Aim 2. Due to the expense of these large animal models, small numbers of baboons will be transplanted with their own islets in these initial exploratory studies. However, the proof of principle they provide will pave the way for future studies in larger groups in which we will aim to reliably achieve tolerance to hCD47-expressing pig islets using pig thymic transplantation from our inbred miniature swine. Long-term acceptance of pig islets without immunosuppressive treatment will allow clinical trials of this approach to curing Type 1 diabetes.
Relevance to T1D
Our goal of inducing tolerance to genetically modified porcine islet grafts has the potential to cure Type 1 diabetes in all patients. We have genetically engineered an inbred line of miniature pigs that will provide a limitless supply of quality- controlled, uniform islets that will evade innate immune system rejection in human recipients. The strategy for educating human T cells to accept donor pig islets as “self” will avoid the need for lifelong immunosuppression, making islet transplantation justifiable in all patients with Type 1 diabetes. The ability to effectively transplant pig islets under the skin will make the transplant less invasive and easier to biopsy if needed. Pig islets are expected to be resistant to autoimmune attack by human T cells that are specific for human islet antigens. The proof of success with our strategies in mice reconstituted with human immune systems and in preclinical baboon models will provide a direct conduit for clinical translation.