Objective
Transplanting insulin-producing β cells holds promise as a definitive cure for T1D. However, current pancreatic islet transplantation faces challenges due to organ donor shortages and the need for immunosuppression.
Pluripotent stem cells, including iPSCs, offer unlimited potential for differentiating into β cells. IPSC are easily obtained, expanded, cryopreserved, and genetically modified. Recent advancements demonstrate the ability to create β cells from human iPSCs in vitro, mimicking pancreas development. These cells could be transplanted into T1D patients, restoring blood sugar control without the need for external insulin. This approach brings with it some important challenges: transplanted β cells, whether from donors or T1D patients, face immune rejection, requiring immunosuppression. This limits its use to adult patients with unstable diabetes. Additionally, due to cell origin and lab manipulation, there's a risk of tumor formation, necessitating safety measures for transplantation.
The goal of this project is to obtain β cells from iPSCs for safe transplantation without immunosuppression. To achieve this, we intend to use the most advanced genetic engineering techniques to modify stem cells and make them invisible to the immune system. We have already generated genetically modified iPSC lines in the laboratory that do not express the B2M, B7H3, and CD155 genes. These modifications render the cell invisible to the two main drivers of rejection, T lymphocytes and NK cells. We have previously observed that these iPSC are not attacked by T lymphocytes and NK cells, allowing them to survive after transplantation without immunosuppression.
In this project, we will demonstrate that specific genetic modifications in iPSCs provide long-term immune protection and safety while preserving the function of β cells derived from iPSCs. Immune protection, safety, and function will be evaluated in a highly advanced preclinical model, where we can assess the restoration of metabolic control, the different cellular players of immune rejection, and long-term safety in terms of tumor formation. For all experiments planned in the project, we will use an iPSC line produced in accordance with Good Manufacturing Practice (GMP) guidelines, which is already available for use in clinical studies. This allows us to facilitate a faster and more feasible translation to clinical applications at the end of the project. More in details, the project has four main objective to be achieved in a span of 3 years.
Objective 1: Modify iPSCs to make them less likely to cause an immune response and add a safety feature using advanced genetic editing techniques. The genetic modifications we plan to make are as follows: a)Destruction of class I HLA to make the cells invisible to cytotoxic CD8 lymphocytes; b) destruction of class II HLA to make the cells invisible to CD4 helper lymphocytes; c) destruction of B7-H3 and CD155, which are two ligands activating NK cells, to make the cells invisible to NK cells; d) insertion of the "suicide" gene iCasp9, which allows for the elimination of cells after transplantation in case they proliferate uncontrollably, exposing the recipient to a risk of tumor formation.
Objective 2: Thoroughly study these genetic changes to ensure they don't negatively affect the cells' stability, function (insulin secretion in response to glucose stimulation), or risk of forming tumors. We'll use genome sequencing to identify any unintended effects.
Objective 3: Test the modified β cells derived from iPSCs by transplanting them into a humanized diabetic mouse model to see how well they work, survive, and remain safe for up to 6 months.
Objective 4: Prepare to move our promising findings from the animal model into human trials. We'll collect safety and effectiveness data following strict guidelines and use a certified testing facility to support applications for human trials.
Background Rationale
Advances in genetic manipulation of induced Pluripotent Stem Cells (iPSCs) allowed to generate β cell with immune-evasive properties for the treatment of type 1 diabetes (T1D). Their use in humans requires confirmation of long-term function and safety. The elimination of HLA class I and II molecules, by specifically targeting B2M and CIITA genes, make these cells invisible to CD8+ and CD4+ T lymphocytes, responsible for graft rejection. However, cells with HLA-I molecules loss are recognized and killed by NK cells. Specific inhibitory receptors on NK cells recognize and bind HLA-1 molecules, thus preventing NK cytotoxicity resulting in killing of cells expressing these molecules. To prevent NK cell attack against modified β cells, several strategies have been proposed. β cells have been manipulated to lose HLA-I and II and to express high levels of HLA-E and HLA-G, two molecules with immune-tolerogenic properties as they bind specific NK receptors responsible for the inhibition of NK activity. Another strategy eliminated HLA-I and II and overexpressed CD47, a potent “don’t eat me” signal enabling β cells to evade detection not only by NK cells, but macrophages as well, alone or in combination with HLA-G and PD-1. However, when the iPSCs are forced to express high levels of immunomodulatory molecules, they run the risk to lose this capability over time. Moreover, NK are very heterogenous cells, and each of these molecules could bind activating counter-receptor expressed by some NK populations or interact with inhibitory receptors which may not be expressed in all individuals. To move forward the clinical application of hypoimmunogenic iPSC-derived β cells, it is important to harness these cells with a system able to distinguish healthy from abnormal cells, as in case of malignant transformation. Moreover, a comprehensive late pre-clinical mouse model for transplantation studies to assess safety and efficacy of the modified iPSC-derived β cells for future in human application is still missing.
We have recently proposed an alternative approach based on the elimination of two molecules, B7H3 and CD155, activating the NK response in iPSC-derived β cells lacking HLA-I molecule. The modified cells successfully escape from T and NK cells, both in vitro and in in vivo experiments. However, the mouse model used did not allow to evaluate this capability long term, as it supported NK cell survival for only two weeks. We are further extending our investigations into a chronic rejection model in which NK cells survive longer. We found that iPSC-derived β cells lacking HLA-I, B7H3 and CD155 survived for up 2 months when injected into the hindlimb muscle of mice, escaping from NK cell attack, and maintaining their insulin-producing capacity without any sign of abnormal cell proliferation. These data suggest that the genetic manipulation we propose is a novel, successful strategy to make iPSC-derived β cells invisible to the immune system.
Description of Project
Induced pluripotent stem cells (iPSCs) hold great promising in the field of regenerative medicine for the treatment of some diseases. These cells offer great advantages: researchers can take blood cells or the skin from a person, reprogram them into iPSCs and then use these cells to create an organ or a portion of it. Cell replacement therapy using iPSCs-derived pancreatic β-cells can be a cure for patients with type 1 diabetes (T1D). By supplying functional pancreatic β-cells secreting insulin, patients will be able to control their blood glucose levels without the need of insulin treatment.
Transplantation of iPSCs-derived pancreatic β-cells is approaching clinical translation, but some issues remain to be addressed. Indeed, short graft survival and rejection related to the immune response triggered by our body against foreign cells still hinder the application of this therapy. Therefore, pancreatic β-cells needs to be protected from the immune system to avoid organ rejection. Researchers took advantage from genetic manipulation techniques to make these cells invisible to immune cells. Indeed, by eliminating two genes, B2M and CIITA, cells lack of two molecules, human leukocyte antigen (HLA) class I and class II. By this strategy, β-cells are able to escape from the recognition of two immune populations responsible for rejection, CD8+ and CD4+ T cells, respectively, but not from natural killer cells (NK). HLA-1 molecules indeed allow self/non-self discrimination, a crucial property of the immune system, preventing destruction of the body's own tissues. Specific inhibitory receptors on NK cells recognize and bind HLA-1 molecules, thus preventing NK killing of cells expressing these molecules. If HLA-1 molecules lack, NK cells are not able to recognize these cells, so they attack and destroy them. We identified two molecules highly expressed by stem cells and the β cells derived from them, B7H3 and CD155, that activate NK cells. By the elimination of these molecules, NK cells did not recognize β cells when transplanted into the hindlimb muscle of mice for at least 2 months. Our aim is to extend this study to ensure long term survival of ipSC-derived β cells to finally translate this strategy into the clinical practice. For transplantation in humans, the cells require development according to a Good Manufacturing Practices (GMP)-compliant process. Therefore, we will use a commercial GMP-iPSC line that will be genetically modified to eliminate the four genes B2M, CIITA, B7H3 and CD155. Moreover, we will add an inducible “suicide gene”, the caspase 9, that could kill the β cells themselves in case of malignant transformation. The generated iPSCs should be able to evade immune surveillance and discriminate between healthy and potentially transformed cells upon differentiation into β cells as well. We will test the acquired properties into different mouse models allowing us to assess: 1) safety profile of engineered β cells, including putative mutations due to the genetic manipulations and, if any, their impact on the cells; the effective implant site, the minimal dose required to control glycemia, the survival and function long term as a consequence of the escape capabilities towards immune cells derived from T1D patients. Finally, we will test these cells in diabetic mice humanized with immune cells from T1D patient, representing the final pre-clinical model of the T1D patients who will receive the cell therapy based on our engineered strategy. Data will be obtained in Good Laboratory Practice (GLP) settings as well to finally assess whether the cell product we generated is reasonably safe for its use in humans.
Anticipated Outcome
We expect to create an iPSC line that, devoid of certain key genes like class I and II HLA genes and two NK cell-activating ligands, and modified to express the iCasp9 suicide gene, retains genomic stability and the capacity to differentiate into functional β cells. From this gene edited iPSC line we expect to generate β cells that (i) are not rejected following allogeneic transplantation in a humanized murine model without encapsulation or immunosuppression and (ii) can be eliminated if they form tumors.
If we can show that our approach is safe and effective, it could lead to a new therapy for T1D. To do this, we will gather data on safety and effectiveness in laboratory settings before moving on to clinical trials. This data will be used to create a detailed plan for regulatory approval, known as an Investigational New Drug (IND) submission. Additionally, we will collaborate with our industrial partner to produce genetically modified iPSCs that meet the strict manufacturing and quality control standards (CMC) necessary for future human trials. This is all part of our journey to potentially bring this innovative treatment to patients with T1D.
Relevance to T1D
CELLULAR THERAPY FOR DIABETES. There is a possibility of a definitive cure for T1D, and it involves the transplantation of new insulin-secreting β cells. Pancreatic islet transplantation has been evidence for decades that replacing β cells in diabetic patients can restore normal blood sugar levels and thus cure diabetes. Currently, pancreatic islet transplantation is limited by the scarcity of organ donors and the need for immunosuppressive therapy to prevent rejection. The use of stem cells as a new source of β cells could potentially overcome both of these challenges.
STEM CELLS TO CURE DIABETES. Great hope lies in differentiating pluripotent stem cells, both embryonic and induced pluripotent stem cells (iPSCs), into β cells since they possess infinite proliferative and differentiative capacities. iPSCs are cells derived from the reprogramming of adult cells through the forced expression of four pluripotency genes. They can be easily obtained from any individual, in unlimited quantities, expanded, cryopreserved, and genetically modified in the laboratory.
β CELLS GENERATED FROM iPSCs. In recent years, it has been demonstrated that it is possible to generate β cells in vitro from human iPSCs, using a protocol that mimics pancreas development during embryogenesis. These β cells derived from stem cells could be transplanted into patients with type 1 diabetes to restore glycemic control, freeing them from the need for exogenous insulin.
CHALLENGES OF β CELL THERAPY GENERATED FROM iPSCs. β cells derived from iPSCs, whether from a donor or a patient with T1D, would induce a rejection response upon transplantation and therefore require protection from the recipient's immune system. To prevent rejection, immunosuppressive drugs can be used, but this greatly limits the application of this therapy to individuals with poorly controlled diabetes, severe and unware episodes of hypoglycemia, to balance the risks of immunosuppression. Furthermore, considering the derivation from stem cells and the significant laboratory manipulation these cells undergo to become β cells, the risk of tumor formation after transplantation must be considered, necessitating strategies to ensure 100% safe transplantation. The first clinical trials are underway, with promising results, but β cells must be protected from rejection with immunosuppressive drugs or encapsulated in closed systems.
OUR PROJECT FOR T1D. Instead, our project focuses on generating induced pluripotent stem cells made invisible to the immune system through gene editing. These iPSC will be differentiated into β cells and tested for their survival, function and safety in a preclinical model very similar to human transplantation settings.
We expect to take a significant step forward in producing new β cells from stem cells for transplantation in patients with T1D, approaching the clinical application of a transplant that makes patients insulin-independent, without the need for immunosuppressive therapy.
In the long term, after the necessary verification of its effectiveness and safety in the adult population, this approach may also become a possible therapy for children with type 1 diabetes.