Biology subjects

Vlahos, A. E.

Publications and source records attributed to Vlahos, A. E..

4 recordsLinked to original sources

bioRxiv2024

Orthogonalized human protease control of secreted signals

Synthetic circuits that regulate protein secretion in human cells could support cell-based therapies by enabling control over local environments. While protein-level circuits enable such potential clinical applications, featuring orthogonality and compactness, their non-human origin poses a potential immunogenic risk. Here, we developed Humanized Drug Induced Regulation of Engineered CyTokines (hDIRECT) as a platform to control cytokine activity exclusively using human-derived proteins. We sourced a specific human protease and its FDA-approved inhibitor. We engineered cytokines (IL-2, IL-6, and IL-10) whose activities can be activated and abrogated by proteolytic cleavage. We utilized species specificity and re-localization strategies to orthogonalize the cytokines and protease from the human context that they would be deployed in. hDIRECT should enable local cytokine activation to support a variety of cell-based therapies such as muscle regeneration and cancer immunotherapy. Our work offers a proof of concept for the emerging appreciation of humanization in synthetic biology for human health.

synthetic biology↗

bioRxiv2023

Compact Programmable Control of Protein Secretion in Mammalian Cells

Synthetic biology has developed powerful tools to program complex behaviors, often using genetic control. Protein circuits offer a compact alternative, yet applications with intercellular signals often lack key regulatory capabilities and tunability. Here, we employ a parts-based engineering strategy to develop a single processing and output module for synthetic protein circuits, enabling complex logic, tunable sensitivity, and control over output magnitude. Using high-throughput assays, we systematically analyze the impact of human transmembrane domains on surface expression and circuit performance. We demonstrate the utility of these optimizations by encoding an open-loop circuit within translational delivery vectors, including viral and mRNA platforms, and validate its performance in vivo. Furthermore, we demonstrate multi-input logic and showcase a novel, protein-level NIMPLY gate to regulate CAR T-cell activation. Our modular design strategy provides new insights into domain-based protein engineering and establishes a versatile and complete protein-level platform to control intercellular signaling for translational cell therapies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/560774v2_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@4f96f7org.highwire.dtl.DTLVardef@1404cf5org.highwire.dtl.DTLVardef@7c948corg.highwire.dtl.DTLVardef@fcad06_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2021

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 signalling pathways. We created 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 novel sensing and processing circuits, we were able to 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.

synthetic biology↗

bioRxiv2020

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.Summary In this study methacrylic acid copolymer coated tubes generated a robust vascular response in the subcutaneous space, which was critical to support islet transplantation in a streptozotocin-induced diabetic mouse model. More importantly, the subcutaneous pre-vascularization approach using this copolymer coating was scalable into a larger allogeneic rat model and returned animals to normoglycemia for up to 70 days. This platform highlights the potential of a scalable biomaterial approach for pre-vascularization of the subcutaneous space in larger animal models.Competing Interest StatementThe authors have declared no competing interest.View Full Text

bioengineering↗