Objective
Xenotransplantation using genetically engineered (GE) donor pigs holds great promise for the treatment of many human diseases, including type 1 diabetes (T1D). The primary objective of this project is to build on our decades of joint experience and our existing lines of GE pigs to develop the ultimate transgenic donor pig to cure T1D. Islets from our first GE pig line are protected from hyperacute (immediate) rejection and efficiently reverse diabetes in the preclinical baboon model, but are eventually rejected by later cellular rejection mechanisms if immunosuppressive drug treatment is discontinued. Islets from our second GE line produce their own intrinsic protection from these delayed forms of rejection. We propose that combining the properties of these two GE lines will result in islets that safely and effectively restore and maintain normal blood glucose control when transplanted into humans with T1D, without requiring long-term immunosuppression. This will be tested in the diabetic baboon model.
The two secondary objectives are designed to facilitate progress to the clinic. First, the genetic modifications in the two GE pig lines will be brought together in such a way as to streamline the efficient future production of donor pigs and to make their islets even more resistant to rejection. Second, we will establish methods to test isolated porcine islets for the presence of a range of viruses that could potentially infect the recipient. We will take advantage of the fact that the islets are cultured for 6 days prior to transplant, providing a window for extensive screening for these pathogens. Although we have not observed any evidence to date of pathogen transmission in the baboon model, it is likely that this testing of the ‘product’ will be an integral component of clinical islet xenotransplantation.
Background Rationale
Successful β-cell replacement by human islet transplantation restores physiological control of blood glucose levels to patients with type 1 diabetes (T1D). However, access to this remarkable medical procedure is currently limited to those with severe hypoglycemic unawareness and/or extremely unstable glucose levels. There are two main reasons for this unfortunate situation: first, the ever-worsening global shortage of suitable human donors, and second, the requirement for transplant recipients to be treated lifelong with immunosuppressive drugs to prevent rejection. These drugs are not only directly toxic but also greatly increase the risk of infection and development of cancer; transplantation is therefore only appropriate for those severely affected individuals in which the benefits of alleviating their symptoms outweighs the risks associated with long-term immunosuppression. Even then, there are not enough donors to treat all of these patients.
Xenotransplantation of porcine islets provides a potential solution to both problems. Pigs reproduce rapidly in large numbers, and porcine insulin is fully effective in humans, so supply is not an issue. Furthermore, while islets from normal pigs would be rapidly rejected by human recipients, pigs can be genetically engineered (GE) to ‘humanize’ their islets to hide them from the human immune response. We have demonstrated the power of this approach by transplanting humanized GE porcine islets into diabetic baboons, the gold standard preclinical model of islet xenotransplantation. The recipients regained stable blood glucose control and did not require insulin treatment while they were under immunosuppression. When immunosuppression was stopped, the islets continued to function for several months; however, they were eventually rejected, indicating that a further level of protection was required. To address this, we generated a new line of GE pigs in which the islets themselves produce and release an immunosuppressive drug. The primary objective of this project is to demonstrate that the combination of these two protective strategies - humanization of islets to reduce the strength of the immune response, and 'local' immunosuppression by the islets to neutralize the remaining response at the transplant site - will provide a readily available cure for T1D that avoids the detrimental side effects of long-term whole body immunosuppression. The ultimate goal is to establish porcine islet xenotransplantation as a treatment option for all individuals with T1D, not just those with intractable symptoms.
Description of Project
β-cell replacement by transplantation of human islets can be life changing for individuals with type 1 diabetes (T1D). However, major practical and medical challenges stand in the way of broad clinical application. Chief of these hurdles are (i) the limited number of human donors, and (ii) the detrimental side effects of the long-term drug treatment required to suppress the recipient’s immune system and thus prevent rejection of the islets. Immunosuppression is a sledgehammer approach that affects the whole body; in addition to causing direct drug-associated toxicity, it places patients at significant risk of infection and chronic health issues including the development of various cancers. The use of pigs as donors (xenotransplantation) is an attractive solution to the supply problem. However, the main drawback of porcine versus human islets is that they provoke a more powerful immune response, requiring treatment of recipients with even more intensive immunosuppression. Our approach to this problem is to genetically engineer donor pigs to make their islets more human-like i.e., less visible to the human immune system. The goal is to prevent rejection while reducing (or ideally eliminating) long-term immunosuppression. Using 'humanized' GE pigs containing a specific set of transgenic modifications, developed over the last 25 years, we demonstrated the longest reversal of diabetes by far (approx. 2 years) in the gold standard pig-to-baboon preclinical model, in which islets from unmodified pigs are rejected almost immediately. Nevertheless, in the absence of ongoing drug treatment the GE islets were invaded and destroyed by recipient immune cells including T cells, the key drivers of rejection. Together these results indicated that although these GE islets were significantly protected, they were eventually detected and rejected by the baboon immune system, and a further layer of protection was needed. We therefore generated a unique new line of GE pigs in which the islets themselves produce a drug that is specifically toxic to human and baboon T cells. We propose that this drug will effectively form a protective local ‘force field’ around the islets without affecting the rest of the body, providing the final piece in the puzzle. The primary aim of this project is to demonstrate that the combination of these genetic changes – to hide the islets from the immune system, and to ward off any remaining immune attack – will allow porcine islets to restore blood glucose control safely and indefinitely in the absence of long-term drug treatment. To address this aim, we will crossbreed the two GE pig lines, transplant islets from the progeny into diabetic baboons, and determine whether the islets withstand rejection and maintain control of blood glucose after drug treatment is withdrawn.
One limitation of the existing GE pig lines is that not all the transgenes are inherited together, so that only half the progeny from crossbreeding can be used as islet donors. In the second arm of the project, we will generate optimized multi-GE pigs that will produce only progeny of the desired type. This will streamline the donor production process and minimize animal usage. The third arm of the project addresses the risk of transmission of potentially pathogenic microorganisms from the transplanted islets to the recipient. Porcine islets are cultured in vitro for 6 days prior to transplant, providing a unique screening window. We will establish methods to screen the islets for a range of pathogens at different stages of culture.
A successful outcome in this project will represent a critical step towards our ultimate objective: developing an inexhaustible supply of islets that can be safely transplanted into patients to provide a stable cure for T1D without the need for chronic immunosuppression.
Anticipated Outcome
There are three anticipated outcomes. First, we will show that porcine islets that are genetically engineered (GE) to make them less visible and more resistant to the immune system will stably reverse diabetes when transplanted in a clinically relevant baboon model, even in the absence of ongoing immunosuppression. Second, we will ‘re-engineer’ the GE pigs to incorporate desirable features for future donor production and clinical application. Third, we will determine the potential utility of screening porcine islets prior to transplantation as a means of minimizing the risk of transmission of pathogenic agents to the recipient. The overall anticipated outcome is to move porcine islet xenotransplantation a step closer to clinical reality as a safe and effective treatment available to all individuals with type 1 diabetes.
Relevance to T1D
Type 1 diabetes (T1D) is a chronic autoimmune condition that destroys the insulin-secreting β-cells within pancreatic islets. The resulting hyperglycaemia causes complications such as neuropathy, cardiovascular disease, nephropathy, blindness, and amputations. T1D requires daily blood glucose monitoring and lifelong insulin treatment by daily injections or the use of insulin pumps. Some people with T1D develop severe hypoglycemic unawareness, which can be life-threatening and require hospitalization, drastically affecting quality of life. β-cell replacement by transplantation of human islets is a highly effective method of restoring physiological blood glucose control, arresting diabetic complications, and reducing or even eliminating hypoglycemic episodes. However, there are two major factors that limit wider clinical application of this life changing procedure. First, there is a worldwide, ever-increasing shortage of human organ donors, and consequently transplant waiting lists continue to grow. Second, islet transplant recipients must be treated lifelong with drugs to suppress their immune system so that they do not reject their grafts. Immunosuppression makes the recipients more susceptible to infection, and they have a greatly increased risk of developing cancer in the longer term. For this reason, islet transplantation is generally restricted to patients with severe hypoglycemic unawareness and/or extremely poor glucose control. This project is highly relevant to T1D because it provides a potential solution to both problems. We will produce and test pigs that are genetically engineered so that their islets are inherently resistant to rejection by the human immune system. Using pigs as donors will provide an inexhaustible supply of islets for transplantation; using rejection-resistant pig islets will eliminate the need for lifelong immunosuppression. If successful, our approach will establish the platform for a long-term, safe, and effective treatment for all patients suffering from T1D.