Compact Programmable Control of Protein Secretion in Mammalian Cells.

Synthetic biology currently holds immense potential to engineer the spatiotemporal control of intercellular signals for biomedicine. Programming behaviors using protein-based circuits has advantages over traditional gene circuits such as compact delivery and direct interactions with signaling proteins. Previously, we described a generalizable platform called RELEASE to enable the control of intercellular signaling through the proteolytic removal of ER-retention motifs compatible with pre-existing protease-based circuits. However, these tools lacked the ability to reliably program complex expression profiles and required numerous proteases, limiting delivery options. Here, we harness the recruitment and antagonistic behavior of endogenous 14-3-3 proteins to create RELEASE-NOT to turn off protein secretion in response to protease activity. By combining RELEASE and RELEASE-NOT, we establish a suite of protein-level processing and output modules called Compact RELEASE (compRELEASE). This innovation enables functions such as logic processing and analog signal filtering using a single input protease. Furthermore, we demonstrate the compactness of the post-translational design by using polycistronic single transcripts to engineer cells to control protein secretion via lentiviral integration and leverage mRNA delivery to selectively express cell surface proteins only in engineered cells harboring inducible proteases. CompRELEASE enables complex control of protein secretion and enhances the potential of synthetic protein circuits for therapeutic applications, while minimizing the overall genetic payload.

Protease-controlled secretion and display of intercellular signals.

To program intercellular communication for biomedicine, it is crucial to regulate the secretion and surface display of signaling proteins. If such regulations are at the protein level, there are additional advantages, including compact delivery and direct interactions with endogenous signaling pathways. Here we create a modular, generalizable design called Retained Endoplasmic Cleavable Secretion (RELEASE), with engineered proteins retained in the endoplasmic reticulum and displayed/secreted in response to specific proteases. The design allows functional regulation of multiple synthetic and natural proteins by synthetic protease circuits to realize diverse signal processing capabilities, including logic operation and threshold tuning. By linking RELEASE to additional sensing and processing circuits, we can achieve elevated protein secretion in response to "undruggable" oncogene KRAS mutants. RELEASE should enable the local, programmable delivery of intercellular cues for a broad variety of fields such as neurobiology, cancer immunotherapy and cell transplantation.

Degradable methacrylic acid-based synthetic hydrogel for subcutaneous islet transplantation.

Islet transplantation is a promising regenerative therapy that would reduce the dependence of type 1 diabetic patients on insulin injections. However, islet transplantation is not yet widely available, in part because there is no ideal transplant site. The subcutaneous space has been highlighted as a promising transplant site, but it does not have the vasculature required to support an islet graft. In this study we demonstrate that islets engraft in the subcutaneous space when injected in an inherently vascularizing, degradable methacrylic acid-polyethylene glycol (MAA-PEG) hydrogel; no vascularizing cells or growth factors were required. In streptozotocin-induced diabetic mice, injection of 600 rodent islet equivalents in MAA-PEG hydrogels was sufficient to reverse diabetes for 70 days; a PEG gel without MAA had no benefit. MAA-PEG hydrogel scaffolds degraded over the course of a week and were replaced by a host-derived, vascularized, innervated matrix that supported subcutaneous islets. The survival of islet grafts through the inflammatory events of subcutaneous transplantation, hydrogel degradation, and islet revascularization underscore the benefits of the MAA biomaterial. Our findings establish the MAA-PEG hydrogel as a platform for subcutaneous islet transplantation.

A scalable device-less biomaterial approach for subcutaneous islet transplantation.

The subcutaneous space has been shown to be a suitable site for islet transplantation, however an abundance of islets is required to achieve normoglycemia, often requiring multiple donors. The loss of islets is due to the hypoxic conditions islets experience during revascularization, resulting in apoptosis. Therefore, to reduce the therapeutic dosage required to achieve normoglycemia, pre-vascularization of the subcutaneous space has been pursued. In this study, we highlight a biomaterial-based approach using a methacrylic acid copolymer coating to generate a robust pre-vascularized subcutaneous cavity for islet transplantation. We also devised a simple, but not-trivial, procedure for filling the cavity with an islet suspension in collagen. We show that the pre-vascularized site can support a marginal mass of islets to rapidly return streptozotocin-induced diabetic SCID/bg mice to normoglycemia. Furthermore, immunocompetent Sprague Daley rats remained normoglycemia for up to 70 days until they experienced graft destabilization as they outgrew their implants. This work highlights methacrylic acid-based biomaterials as a suitable pre-vascularization strategy for the subcutaneous space that is scalable and doesn't require exogenous cells or growth factors.

Endothelialized collagen based pseudo-islets enables tuneable subcutaneous diabetes therapy.

Pancreatic islets are fragile cell clusters and many isolated islets are not suitable for transplantation. Furthermore, following transplantation, islets will experience a state of hypoxia and poor nutrient diffusion before revascularization, which is detrimental to islet survival; this is affected by islet size and health. Here we engineered tuneable size-controlled pseudo-islets created by dispersing de-aggregated islets in an endothelialized collagen scaffold. This supported subcutaneous engraftment, which returned streptozotocin-induced diabetic mice to normoglycemia. Whole-implant imaging after tissue clearing demonstrated pseudo-islets regenerated their vascular architecture and insulin-secreting ß-cells were within 5 µm of a perfusable vessel - a feature unique to this approach. By using an endothelialized collagen scaffold, this work highlights a novel "bottom-up" approach to islet engineering that provides control over the size and composition of the constructs, while enabling the critical ability to revascularize and engraft when transplanted into the clinically useful subcutaneous space.

Interpenetrating Alginate-Collagen Polymer Network Microspheres for Modular Tissue Engineering.

The lack of vascularization limits the creation of engineered tissue constructs with clinically relevant sizes. We pioneered a bottom-up process (modular tissue engineering) in which constructs with intrinsic vasculature were assembled from endothelialized building blocks. In this study, we prepared an interpenetrating polymer network (IPN) hydrogel from a collagen-alginate blend and evaluated its use as microspheres in modular tissue engineering. Ionotropic gelation of alginate was combined with collagen fibrillogenesis, and the resulting hydrogel was stiffer and had greater resistance to enzymatic degradation relative to that of collagen alone; the viability of embedded mesenchymal stromal cells (adMSC) was unaltered. IPN microspheres were fabricated by a coaxial air-flow technique, and an additional step of collagen coating was required to have human umbilical vein endothelial cells (HUVEC) attach and proliferate. When implanted subcutaneously in SCID/bg mice, adMSC-HUVEC microspheres promoted more blood vessels at day 7 relative to microspheres without adMSC but coated with HUVEC. Perfusion studies confirmed that these vessels were connected to the host vasculature. Fewer vessels were detected in both groups at day 21, but in adMSC-HUVEC explants, more smooth muscle cells had wrapped around vessels, and CLARITY processing of whole explants revealed a restricted leakage of blood. The capacity for rapid gelation and high throughput production are promising features for the use of these microspheres in modular tissue engineering.

Modular tissue engineering for the vascularization of subcutaneously transplanted pancreatic islets.

The transplantation of pancreatic islets, following the Edmonton Protocol, is a promising treatment for type I diabetics. However, the need for multiple donors to achieve insulin independence reflects the large loss of islets that occurs when islets are infused into the portal vein. Finding a less hostile transplantation site that is both minimally invasive and able to support a large transplant volume is necessary to advance this approach. Although the s.c. site satisfies both these criteria, the site is poorly vascularized, precluding its utility. To address this problem, we demonstrate that modular tissue engineering results in an s.c. vascularized bed that enables the transplantation of pancreatic islets. In streptozotocin-induced diabetic SCID/beige mice, the injection of 750 rat islet equivalents embedded in endothelialized collagen modules was sufficient to restore and maintain normoglycemia for 21 days; the same number of free islets was unable to affect glucose levels. Furthermore, using CLARITY, we showed that embedded islets became revascularized and integrated with the host's vasculature, a feature not seen in other s.c. STUDIES: Collagen-embedded islets drove a small (albeit not significant) shift toward a proangiogenic CD206+MHCII-(M2-like) macrophage response, which was a feature of module-associated vascularization. While these results open the potential for using s.c. islet delivery as a treatment option for type I diabetes, the more immediate benefit may be for the exploration of revascularized islet biology.

Injectable and inherently vascularizing semi-interpenetrating polymer network for delivering cells to the subcutaneous space.

Injectable hydrogels are suitable for local cell delivery to the subcutaneous space, but the lack of vasculature remains a limiting factor. Previously we demonstrated that biomaterials containing methacrylic acid promoted vascularization. Here we report the preparation of a semi-interpenetrating polymer network (SIPN), and its evaluation as an injectable carrier to deliver cells and generate blood vessels in a subcutaneous implantation site. The SIPN was prepared by reacting a blend of vinyl sulfone-terminated polyethylene glycol (PEG-VS) and sodium polymethacrylate (PMAA-Na) with dithiothreitol. The swelling of SIPN was sensitive to the PMAA-Na content but only small differences in gelation time, permeability and stiffness were noted. SIPN containing 20 mol% PMAA-Na generated a vascular network in the surrounding tissues, with 2-3 times as many vessels as was obtained with 10 mol% PMAA-Na or PEG alone. Perfusion studies showed that the generated vessels were perfused and connected to the host vasculature as early as seven days after transplantation. Islets embedded in SIPN were viable and responsive to glucose stimulation in vitro. In a proof of concept study in a streptozotocin-induced diabetic mouse model, a progressive return to normoglycemia was observed and the presence of insulin positive islets was confirmed when islets were embedded in SIPN prior to delivery. Our approach proposes a biomaterial-mediated strategy to deliver cells while enhancing vascularization.

Using Del-1 to tip the angiogenic balance in endothelial cells in modular constructs.

Modular tissue engineering is a method of building vascularized tissue-engineered constructs. Submillimeter-sized collagen pieces (modules) coated with a layer of endothelial cells (EC; vascular component), and with embedded functional cells, are self-assembled into a larger, three-dimensional tissue. In this study, we examined the use of developmental endothelial locus-1 (Del-1), an extracellular matrix protein with proangiogenic properties, as a means of tipping the angiogenic balance in human umbilical vein endothelial cells incorporated in modular tissue-engineered constructs. The motivation was to enhance the vascularization of these constructs upon transplantation in vivo, in this case, without the use of exogenous mesenchymal stromal cells. EC were transduced using a lentiviral construct to overexpress Del-1. The Del-1 EC formed more sprouts in a fibrin gel sprouting assay in vitro compared with eGFP (control) transduced EC, as expected. Del-1 EC had a distinct profile of gene expression (upregulation of matrix metalloproteinase-9 [MMP-9], urokinase-type plasminogen activator [uPA/PLAU], vascular endothelial growth factor [VEGF-A], and intercellular adhesion molecule-1 [ICAM-1]; downregulation of angiopoietin-2 [Ang2]), also supporting the notion of "tipping the angiogenic balance". On the other hand, contrary to our expectations, when Del-1 EC-coated modules were implanted subcutaneously in a severe combined immunodeficient/beige animal model, the proangiogenic effect of Del-1 was less remarkable. There was only a small increase in the number of blood vessels formed in Del-1 implants compared with the eGFP implants, and only few blood vessels formed at the implant site in both cases. This was presumed due to limited EC survival after transplantation. We speculate that if we could improve EC survival in our study (for example, by adding other prosurvival factors or supporting cells), we would see a greater Del-1-induced angiogenic benefit in vivo as a consequence of increased Del-1 secretion by a higher number of surviving cells.