Objective
The objective of this project is to evaluate this injectable VEGF-delivering synthetic hydrogel as a carrier that promotes islet vascularization and engraftment in the subcutaneous space of pigs. We hypothesize that the VEGF-delivering gel will promote transplanted neonatal porcine islet (NPI) vascularization, survival, and engraftment in the porcine subcutaneous space, a site with high clinical potential in terms of accessibility, convenience, and ease of monitoring and retrieval if necessary. A major advantage of our strategy over encapsulation devices is direct vascularization of the transplanted islets for enhanced survival and glucose/insulin control without fibrosis and diffusional limitations. This project will establish an injectable delivery vehicle for islets that promotes islet vascularization, survival, and engraftment in the subcutaneous space of a clinically-relevant large animal model. Because of the modular nature of the hydrogel platform, this technology can be combined with other approaches for cell therapy, including immunomodulatory strategies and other insulin-producing cells (e.g., stem cell-derived islets). These studies will provide a solid foundation for follow-on diabetes correction studies in diabetic pigs.
Background Rationale
Our overarching goal to engineer the PEG+VEGF hydrogel platform for robust vascularization and islet functionality in the subcutaneous space is highly significant to T1D therapies. This project will result in an injectable, degradable delivery vehicle for islets that promotes islet vascularization, engraftment, and function and will provide validation results in a large animal model. With Breakthrough T1D support, we engineered this PEG+VEGF hydrogel platform and demonstrated transplanted islet survival, engraftment, and function in rodents as well as compelling pilot vascularization and neonatal porcine islet (NPI) survival data in non-diabetic pigs. The next translational step is to transfer this biomaterial technology to collaborators and validate short-term NPI vascularization and survival in the subcutaneous space of non-diabetic pigs as proposed in this project. Importantly, we will also scale-up the transplant to a therapeutic density and evaluate long-term islet vascularization, survival, and engraftment in non-diabetic pigs. This work will provide a solid foundation for future diabetes correction studies in diabetic pigs.
During the past few decades, swine have been used with increasing frequency in biomedical research as replacements for dogs and primates. In addition, the anatomy, physiology, overall metabolic status and disease progression in swine are all very similar to that of humans. Notably, swine are used extensively for nutritional studies because they are omnivores and their digestive physiology, length, and morphological characteristics of the small intestine are very similar to ours. There is a strong rationale to pursue the use of porcine donors for clinical islet xenotransplantation due to its unlimited availability, high breeding potential, large number of piglets, and overall close anatomical and physiological similarity to humans. Moreover, porcine insulin differs by only a single amino acid from that of human insulin, and it was administered to treat diabetes for nearly a century before the introduction of recombinant human insulin, making porcine islets an ideal source for islet transplantation. However, xenozoonoses and xenoantigens were considered major concerns for clinical translation until recently, when two gene-edited pig heart transplants were successfully carried out in living patients. In addition, genetically modified pig kidneys have been implanted into two brain-dead human recipients. While adult porcine pancreases provide a source of fully mature islets, the cost of maintaining a herd of adult pigs and the poor reproducibility of isolating adult porcine islets make NPIs a more reasonable source of xenogeneic β-cell grafts. We reported a simple, inexpensive, and reproducible method to isolate large numbers of NPIs. These islets are comprised of differentiated endocrine and endocrine precursor cells that, both in vitro and in vivo, have the potential for proliferation and differentiation and have been shown to reverse hyperglycemia in immunodeficient mice, allogeneic pigs, and non-human primates. We have found that NPIs are clinically appealing because of their resistance to a number of β-cell stresses. In addition to NPI graft survival and long-term survival under the kidney capsule (rodents) and intra-portally (pigs), NPIs have been shown to engraft and demonstrate long-term graft survival in the subcutaneous space in immunodeficient mice. Taken together, these data clearly indicate that NPIs are a promising tissue source for clinical islet xenotransplantation. Finally, NPI transplantation into pigs serves as a large animal transplantation model for allogeneic islet transplantation. We anticipate that the vasculogenic hydrogels engineered in this project will also be applicable to human stem cell-derived islets, which although exceedingly promising, pose technical limitations in evaluating in large animal models.
Description of Project
Type 1 diabetes (T1D) is an autoimmune disease in which the insulin-producing β-cells of the pancreas are destroyed. T1D affects 2 million children and adults in the US with >$24 billion annual healthcare costs. Standard therapy with insulin is burdensome, associated with a significant danger of hypoglycemia, and only partially efficacious in preventing long-term complications. Transplantation of islets from cadaveric donors into the liver has emerged as a promising therapy for T1D. However, infusion of islets into the liver triggers an instant blood-mediated inflammatory reaction, resulting in significant loss of islets (60-80% of the delivered dose) in hours to days following transplantation. Therefore, research has focused on establishing alternative sites for transplantation of islets including peritoneum, subcutaneous space, and omentum, but these remain limited in terms of low islet survival, inadequate glucose, and clinical utility. Inadequate revascularization of transplanted islets is a major cause for reduced islet viability and. Delivery of pro-vascularization factors improves vascularization and islet function, but these strategies are hindered by inadequate delivery vehicles and technical and safety considerations. We have engineered synthetic injectable hydrogels with controlled delivery of the vasculogenic protein VEGF that promote re-vascularization and engraftment of transplanted islets in rodents. The objective of this project is to evaluate this injectable VEGF-delivering synthetic hydrogel as a carrier that promotes islet vascularization and engraftment in the subcutaneous space of pigs. We hypothesize that the VEGF-delivering gel will promote transplanted neonatal porcine islet (NPI) vascularization, survival, and engraftment in the porcine subcutaneous space, a site with high clinical potential in terms of accessibility, convenience, and ease of monitoring and retrieval if necessary. A major advantage of our strategy over encapsulation devices is direct vascularization of the transplanted islets for enhanced survival and glucose/insulin control without fibrosis and diffusional limitations.
Aim 1: Transfer biomaterial technology and validation of short-term NPI vascularization and survival in the subcutaneous space of non-diabetic pigs. We will transfer the hydrogel technology to the University of Alberta and validate short-term NPI vascularization and survival in non-diabetic pigs. These are critical steps in the translation of this technology. We will transplant NPIs into the subcutaneous space of non-diabetic pigs for 3 groups: VEGF-delivering PEG gel, collagen-based islet viability matrix, and saline control. Animals will receive local immunosuppression using rapamycin-releasing microparticles and CTLA-4I. At 4 and 8 weeks post-transplant, we will evaluate transplant sites for vascularization, graft morphology, and immunostaining using quantitative imaging mass cytometry and insulin content. These studies will establish the VEGF-delivering gel as a carrier for NPIs transplanted to the subcutaneous space.
Aim 2: Scale-up transplant to therapeutic density and evaluate long-term NPI vascularization, survival, and engraftment in non-diabetic pigs. We will next evaluate NPI vascularization, survival, and engraftment for a therapeutic density delivered with vasculogenic gels at 2 and 6 months post-transplant in non-diabetic pigs under local immunosuppression. We will evaluate transplant sites for vascularization, graft morphology, and immunostaining using quantitative imaging mass cytometry and insulin content.
Impact: This project will establish an injectable delivery vehicle for islets that promotes islet vascularization, survival, and engraftment in the subcutaneous space of a clinically-relevant large animal model. Because of the modular nature of the hydrogel platform, this technology can be combined with other approaches for cell therapy, including immunomodulatory strategies and other insulin-producing cells (e.g., stem cell-derived islets). These studies will provide a solid foundation for follow-on diabetes correction studies in diabetic pigs.
Anticipated Outcome
This project will result in an injectable delivery vehicle for islets that promotes islet vascularization, engraftment and function and will provide validation results in a large animal model. Because this project focuses on islet vascularization and function, the studies will use allogeneic islet transplantation into immunosuppressed receipts. Notably, the hydrogel formulation can be combined in the future with other technologies for cell therapy, including immunomodulatory strategies, encapsulation devices, and scaffolds. This work will provide a solid foundation for future studies of this vasculogenic hydrogel vehicle in a translational diabetic large animal model.
Relevance to T1D
This project will result in an injectable delivery vehicle for pancreatic islets (and possibly stem cell-derived islets) that promotes islet vascularization, engraftment and function and will provide validation results in a large animal model. Notably, the hydrogel formulation can be combined in the future with other technologies for cell therapy, including immunomodulatory strategies, encapsulation devices, and scaffolds. This work will provide a solid foundation for future studies of this vasculogenic hydrogel vehicle in a translational diabetic large animal model. This strategy overcomes current limitations of islet delivery to the liver that results in high islet loss and encapsulation devices that induce a strong fibrotic response and poor glucose control.