Objective
Our group has found that expression of the fusion protein PIDO by islet cells can mitigate the immune rejection response and provide long-term tolerance through CD4 Treg-mediated mechanisms. Furthermore, we have developed NIH-approved embryonic stem cell clones expressing PIDO, namely H1 PIDO and H9 PIDO, which have been tested and found to function similarly to the original ES cells. Here, we aim to develop the process for differentiating these PIDO ES cells into functional islet-like cells and test their clinical transplant competency in a humanized mouse model of diabetes. This proposal sits at the intersection of early and pre-Investigational New Drug (IND) application stages to the U.S. Food and Drug Administration (FDA). These data would inform the Pharmacology and Toxicology Information section of a future FDA Center for Biologics Evaluation and Research (CBER) IND application in support of “off-the-shelf” PIDO+ human ES-derived islets in a first-in-human clinical trial for the cure of diabetes. Assuming that we can cure diabetes in humanized mice, a critical but manageable future roadblock will be developing the Chemistry, Manufacturing, and Controls (CMC) section of the IND application, amenable to scalable manufacturing of PIDO+ islets from ES cells aligned with commercialization success.
Background Rationale
Loss of insulin-producing islet β-cells (mass and/or function) underlies the pathologies of type 1 (T1D) and type 2 (T2D) diabetes. While insulin therapy can aid in the management of diabetes, it is not a curative measure and does not prevent chronic complications such as renal failure. Allogeneic islet transplantation is a highly promising experimental therapy for poorly controlled diabetes. However, chronic systemic immunosuppression is currently the only clinical strategy and standard therapy to prevent tissue and organ allograft rejection from genetically non-identical donors. Despite significant improvements in post-transplant immunosuppressive therapy, long-term pharmacological inhibition of the host immune response still causes serious adverse effects such as opportunistic infections, cardiac and renal toxicity, and an increased risk of malignancies.
Our recently published work in the American Journal of Transplantation demonstrates that a fusion PD-L1 and IDO protein (aka PIDO) expressed on allogeneic islet transplants at the kidney capsule provides unparalleled protection through a host CD4-mediated mechanism against immune rejection in a mouse Type 1 diabetes model reflective of the human condition. Additionally, in preliminary data, we showed that PIDO islets can be transplanted at the visceral fat pads of mice, which represent a clinically relevant, less invasive omentum-like transplantation site in humans. PIDO islets transplanted at visceral fat pads were observed to engraft successfully and survive functionally for an extended period. Furthermore, we have successfully prepared PIDO+ ES cell clones, H01 and H09, using CRISPR techniques, which are ready for research use. These PIDO ES cells have been successfully differentiated into PIDO+SCILCs at stage 3 and are currently being further investigated for the development of fully functional stage 6 ILCs. These data point toward the clinical potential of the PIDO technology to enable transplantation of allogeneic donor-derived islets or embryonic stem cell-derived islet-like cells (SCILCs) without any companion pharmaceutical immunosuppressive drugs. This represents a game-changing paradigm for organ transplantation, where off-the-shelf human stem cell origin can be transplanted into human subjects at a clinically relevant, less invasive site without rejection or the need for toxic immunosuppressive drugs.
Description of Project
We have developed a gene engineering technology that allows for immune evasion of pancreatic islets transplanted across species barriers. This technology, termed “PIDO,” consists of a novel fusion protein composed of two potent immune checkpoints, namely PD-L1 and IDO (Indoleamine dioxygenase). Specifically, our published work in the American Journal of Transplantation (Paul et al., 2022) demonstrates that PIDO-expressing allogeneic islet transplants exhibit unparalleled protection against immune rejection in a mouse Type 1 diabetes model reflective of the human condition. These data point toward the clinical potential of the PIDO technology for enabling transplantation of allogeneic embryonic stem (ES) cell-derived islet-like cells without any companion pharmaceutical immune-suppressive drugs. This represents a potentially game-changing paradigm for islet transplantation, where off-the-shelf human stem cell-sourced islets can be transplanted into human subjects without rejection or the need for toxic immunosuppressive drugs. In addition, we recently developed two NIH-approved ES clones, H1 and H9, that constitutively expressing PIDO without compromising their pluripotency. In this proposal, we aim to (1) determine the competency and functionality of PIDO ILCs derived from PIDO ES cells or the alternate lentivirus PIDO transduced SC-ILCs, and (2) test the translational use of PIDO-expressing ES-cell-derived islets in diabetic humanized mice as a clinically deployable cell pharmaceutical. In summary, the work proposed in this application is the next logical step toward translating this exciting and innovative therapy of an allogeneic “off-the-shelf product” into the clinic and will have a significant and long-lasting impact on diabetic disease.
Anticipated Outcome
Our group has developed a fusion protein, PIDO, from the combination of two immune-suppressive components of the body, i.e., PD-L1 and IDO. We have shown that expression of PIDO (but not PD-L1 or IDO alone) provides alloimmune tolerance by inducing CD4 Treg cells, enabling the survival of allogeneic islets in hyperglycemic mice. In addition, we have developed PIDO-expressing ES-derived cells, which are being investigated for functional differentiation into SCILCs and for their clinical potential in humanized mouse models. None of the therapies currently approved by the FDA cure type 1 diabetes (T1D) or directly promote the repair of islets within the pancreas. The development of T1D therapies addresses an important clinical problem with unmet medical needs and serves as a preamble to first-in-human clinical trials. By defining the ways in which PIDO can promote beta cell replacement, we anticipate identifying a potential treatment for T1D. Knowledge gained from the proposed research will lead to improved outcomes for patients suffering from T1D and possibly other forms of diabetes. Distinct from traditional immunosuppressive strategies that typically attempt to deplete or counteract the number and function of pathogenic lymphomyeloid cells, one can envision the use of PIDO+SCILCs to mitigate the immune response. Moreover, data obtained from this proposal would inform the long-term, scalable translational goal of an off-the-shelf PIDO+ SCILC cell therapy to cure T1D—without the need for cell isolators, pharmacological immune suppression, or risky invasive procedures.
Relevance to T1D
Type 1 diabetes is a chronic, life-long, and debilitating disease that affects nearly one and a half million Americans. Type 1 diabetes is defined by high blood sugar levels, which, when uncontrolled, can lead to devastating consequences such as blindness, kidney disease, nerve disease, heart disease, limb amputation, and even death. The beta-cells of the pancreas are the only cells in the body that produce the hormone insulin, which reduces blood sugar levels. In type 1 diabetes, the pancreatic beta-cells are attacked and killed by a person’s own immune system. Currently, the only way that individuals with type 1 diabetes can control their blood sugar levels is by injecting themselves with insulin daily. While advances in the types of insulin available, along with continuous infusion pumps and continuous glucose monitors, have greatly improved type 1 diabetes patients’ quality of life and lifespan, there is still no treatment that can prevent or cure type 1 diabetes.
Recent work on generating new beta-cells in the lab and harvesting donor beta-cells—both of which could be transplanted into individuals with type 1 diabetes—shows significant promise. However, these transplantation methods would require patients to also take drugs that suppress their immune system, which can have significant negative side effects. We have shown that PIDO gene expression can replace the need for toxic immunosuppressive drugs and provide long-term alloimmune tolerance in a mouse model. In this work, we aim to test an off-the-shelf product derived from PIDO-expressing ES cells, which does not require any encapsulation material or immunosuppressive therapy and can be transplanted at a less invasive, clinically relevant site such as the omentum. Such a treatment would allow transplanted insulin-secreting beta-cells to survive, restoring the body’s ability to control blood sugar levels with its own insulin. Furthermore, this treatment would mean that individuals with type 1 diabetes would no longer need to inject themselves with insulin or take any toxic drugs to suppress their immune system. We will test the feasibility of our islets using a clinically relevant humanized mouse model of diabetes, and the processes developed from this proposal would inform the long-term, scalable translational goal of an off-the-shelf PIDO+ SCILC cell therapy to cure T1D—without the need for cell isolators, pharmacological immune suppression, or risky invasive procedures.