Biology subjects

Way, J. C.

Publications and source records attributed to Way, J. C..

18 recordsLinked to original sources

bioRxiv2025

Rational design of selective bispecific EPO-R/CD131 agonists

Erythropoietin (EPO) suppresses apoptosis and promotes survival by signaling through EPO-R/EPO-R on hematopoietic progenitors or EPO-R/CD131 on non-hematopoietic cells. However, EPO signaling through EPO-R/CD131 is controversial and there is no solved structure of a complex. Here, we constructed a structural model of EPO-R/CD131 and designed several anti-EPO-R, anti-CD131 bispecific proteins that selectively activate EPO-R/CD131. Treatment with these fusion proteins is sufficient to activate STAT5 phosphorylation downstream of EPO-R/CD131 without engaging EPO-R/EPO-R. We demonstrated that proteins with a tandem scFv or bispecific antibody format activate EPO-R/CD131, in contrast to an equimolar mixture of the individual scFvs. Finally, we explored the effect of modifications to binding domain arrangement and linker length and found results consistent with our structural model of an EPO-R/CD131 complex. These findings highlight the utility of bispecific scaffolds in the development of cytokine receptor agonists and provide a foundation for the study of EPO-R/CD131 biology and future clinical development. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=61 SRC="FIGDIR/small/648227v2_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@1b207b0org.highwire.dtl.DTLVardef@116bce3org.highwire.dtl.DTLVardef@1cafb70org.highwire.dtl.DTLVardef@141ae60_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2025

An Fc-SPINK1 fusion protein inhibits pancreatic inflammation in a mouse model

Pancreatitis results from premature activation and impaired inactivation of pancreatic proteases, primarily trypsin, leading to self-digestion, tissue necrosis, fibrosis, and inflammation. SPINK1 is a pancreas-specific inhibitor of trypsin that prevents premature trypsin activation, and could be a candidate therapeutic. However, because of its small size, SPINK1 would be subject to rapid renal clearance, making it ineffective. To construct a long half-life therapeutic inhibitor of trypsin for pancreatitis treatment we fused this protein to the C-terminus of an IgG1 antibody Fc element, increasing the size to [~]78 kDa, thereby exceeding the renal clearance threshold and providing for FcRn-mediated recycling out of cells. A non-glycosylated form of Fc-SPINK1 was expressed in the yeast Pichia pastoris. Fc-SPINK1 inhibits trypsin enzyme activity in vitro. The blood pharmacokinetics in mice are consistent with a three-compartment distribution model and a terminal half-life of [~]3 days. In a caerulein-induced mouse model of pancreatitis, Fc-SPINK1 significantly ameliorated cell death and immune cell infiltration. We developed an automated image analysis technique to quantify pancreatitis-associated loss of tissue cohesion, and found that Fc-SPINK1 also reduced this effect. This study demonstrates the potential of Fc-SPINK1 as a rationally designed therapy for pancreatitis.

pharmacology and toxicology↗

bioRxiv2023

A long-acting prolactin to combat lactation insufficiency

Human infants are born to breastfeed. While 50% of lactating persons struggle to make enough milk, there are no governmentally-approved drugs to enhance lactation1. Here, we engineer a variant of the naturally-occurring driver of lactation, the hormone Prolactin, to increase its serum half-life and produce a viable drug candidate. Our engineered variant, Prolactin-eXtra Long-acting (Prolactin-XL), is comprised of endogenously active human prolactin fused to an engineered human IgG Fc domain designed to overcome the unique drug development challenges specific to the lactating person-infant dyad. Our Prolactin-XL has a serum half-life of 70.9h in mice, 2,625-fold longer than endogenously active prolactin alone (70.9h v. 0.027h). We demonstrate that Prolactin-XL increases milk production and restores growth of pups fed by dams with pharmacologically-ablated lactation. We show that Prolactin-XL-enhanced lactation is accompanied by reversible, lactocyte-driven changes in mammary gland morphology. This work establishes long-acting prolactins as a potentially powerful pharmacologic means to combat insufficient lactation.

synthetic biology↗

bioRxiv2023

Tardigrade secretory proteins protect biological structures from desiccation

Tardigrades, microscopic animals that survive a broad range of environmental stresses, express a unique set of proteins termed tardigrade-specific intrinsically disordered proteins (TDPs). TDPs are often expressed at high levels in tardigrades upon desiccation, and appear to mediate stress adaptation. Here, we focused on the proteins belonging to the secretory family of tardigrade proteins termed secreted-abundant heat soluble ("SAHS") proteins, and investigated their ability to protect diverse biological structures. Recombinantly expressed SAHS proteins prevented desiccated liposomes from fusion, and enhanced desiccation tolerance of E. coli and Rhizobium tropici upon extracellular application. Molecular dynamics simulation and comparative structural analysis suggest a model by which SAHS proteins may undergo a structural transition upon desiccation, in which removal of water and solutes from a large internal cavity in SAHS proteins destabilizes the beta-sheet structure. These results highlight the potential application of SAHS proteins as stabilizing molecules for preservation of cells.

molecular biology↗

bioRxiv2022

High spontaneous integration rates of end-modified linear DNAs upon mammalian cell transfection

In gene therapy, potential integration of therapeutic transgene into host cell genomes is a serious risk that can lead to insertional mutagenesis and tumorigenesis. Viral vectors are often used as the gene delivery vehicle, but they are prone to undergoing integration events. More recently, non-viral delivery of linear DNAs having modified geometry such as closed-end linear duplex DNA (CELiD) have shown promise as an alternative, due to prolonged transgene expression and less cytotoxicity. However, whether such modified-end linear DNAs can also provide a safe, non-integrating gene transfer remains unanswered. Herein, we provide a systematic comparison of genomic integration frequency upon transfection of cells with expression vectors in the forms of circular plasmid, unmodified linear DNA, CELiD, and Streptavidin-conjugated blocked-end linear DNA. All of these forms of linear DNA resulted in a high fraction of the cells being stably transfected - between 10% and 20% of the initially transfected cells, with CELiDs showing the highest rates of integration. These results indicate that blocking the ends of linear DNA is insufficient to prevent integration. Moreover, our analysis suggest that conventional AAV-based gene therapy may be highly susceptible to integration, which is consistent with recent findings from long-term clinical studies.

bioengineering↗

bioRxiv2021

High content screening and computational prediction reveal viral genes that suppress innate immune response

Suppression of the host innate immune response is a critical aspect of viral replication. Upon infection, viruses may introduce one or more proteins that inhibit key immune pathways, such as the type I interferon pathway. However, the ability to predict and evaluate viral protein bioactivity on targeted pathways remains challenging and is typically done on a single virus/gene basis. Here, we present a medium-throughput high-content cell-based assay to reveal the immunosuppressive effects of viral proteins. To test the predictive power of our approach, we developed a library of 800 genes encoding known, predicted, and uncharacterized human viral genes. We find that previously known immune suppressors from numerous viral families such as Picornaviridae and Flaviviridae recorded positive responses. These include a number of viral proteases for which we further confirmed that innate immune suppression depends on protease activity. A class of predicted inhibitors encoded by Rhabdoviridae viruses was demonstrated to block nuclear transport, and several previously uncharacterized proteins from uncultivated viruses were shown to inhibit nuclear transport of the transcription factors NF-{kappa}B and IRF3. We propose that this pathway-based assay, together with early sequencing, gene synthesis, and viral infection studies, could partly serve as the basis for rapid in vitro characterization of novel viral proteins. IMPORTANCEInfectious diseases caused by viral pathogens exacerbate healthcare and economic burdens. Numerous viral biomolecules suppress the human innate immune system, enabling viruses to evade an immune response from the host. Despite our current understanding of viral replications and immune evasion, new viral proteins, including those encoded by uncultivated viruses or emerging viruses, are being unearthed at a rapid pace from large scale sequencing and surveillance projects. The use of medium- and high-throughput functional assays to characterize immunosuppressive functions of viral proteins can advance our understanding of viral replication and possibly treatment of infections. In this study we assembled a large viral gene library from diverse viral families and developed a high content assay to test for inhibition of innate immunity pathways. Our work expands the tools that can rapidly link sequence and protein function, representing a practical step towards early-stage evaluation of emerging and understudied viruses.

systems biology↗

bioRxiv2021

Natural and designed proteins inspired by extremotolerant organisms can form condensates and attenuate apoptosis in human cells

Many organisms can survive extreme conditions and successfully recover to normal life. This extremotolerant behavior has been attributed in part to repetitive, amphipathic, and intrinsically disordered proteins that are upregulated in the protected state. Here, we assemble a library of approximately 300 naturally-occurring and designed extremotolerance-associated proteins to assess their ability to protect human cells from chemically-induced apoptosis. We show that several proteins from tardigrades, nematodes, and the Chinese giant salamander are apoptosis protective. Notably, we identify a region of the human ApoE protein with similarity to extremotolerance-associated proteins that also protects against apoptosis. This region mirrors the phase separation behavior seen with such proteins, like the tardigrade protein CAHS2. Moreover, we identify a synthetic protein, DHR81, that shares this combination of elevated phase separation propensity and apoptosis protection. Finally, we demonstrate that driving protective proteins into the condensate state increases apoptosis protection, and highlight the ability for DHR81 condensates to sequester caspase-7. Taken together, this work draws a link between extremotolerance-associated proteins, condensate formation, and designing human cellular protection.

synthetic biology↗

bioRxiv2021

Rational engineering of an erythropoietin fusion protein to treat hypoxia

Erythropoietin enhances oxygen delivery and reduces hypoxia-induced cell death, but its pro-thrombotic activity is problematic for use of erythropoietin in treating hypoxia. We constructed a fusion protein that stimulates red blood cell production and neuroprotection without triggering platelet production, a marker for thrombosis. The protein consists of an anti-glycophorin A nanobody and an erythropoietin mutant (L108A). The mutation reduces activation of erythropoietin receptor homodimers that induce erythropoiesis and thrombosis, but maintains the tissue-protective signaling. The binding of the nanobody element to glycophorin A rescues homodimeric erythropoietin receptor activation on red blood cell precursors. In a cell proliferation assay, the fusion protein is active at 10-14 M, allowing an estimate of the number of receptor-ligand complexes needed for signaling. This fusion protein stimulates erythroid cell proliferation in vitro and in mice, and shows neuroprotective activity in vitro. Our erythropoietin fusion protein presents a novel molecule for treating hypoxia.

synthetic biology↗

bioRxiv2021

High-content screening of coronavirus genes for innate immune suppression revealsenhanced potency of SARS-CoV-2 proteins

Suppression of the host intracellular innate immune system is an essential aspect of viral replication. Here, we developed a suite of medium-throughput high-content cell-based assays to reveal the effect of individual coronavirus proteins on antiviral innate immune pathways. Using these assays, we screened the 196 protein products of seven coronaviruses (SARS-CoV-2, SARS-CoV-1, 229E, NL63, OC43, HKU1 and MERS). This includes a previously unidentified gene in SARS-CoV-2 encoded within the Spike gene. We observe immune-suppressing activity in both known host-suppressing genes (e.g., NSP1, Orf6, NSP3, and NSP5) as well as other coronavirus genes, including the newly identified SARS-CoV-2 protein. Moreover, the genes encoded by SARS-CoV-2 are generally more potent immune suppressors than their homologues from the other coronaviruses. This suite of pathway-based and mechanism-agnostic assays could serve as the basis for rapid in vitro prediction of the pathogenicity of novel viruses based on provision of sequence information alone.

systems biology↗

bioRxiv2020

In situ reprogramming of gut bacteria by oral delivery

Abundant links between the gut microbiota and human health indicate that the modification of bacterial function could be a powerful therapeutic strategy. The inaccessibility of the gut and inter-connections between gut bacteria and the host make it difficult to precisely target bacterial functions without disrupting the microbiota and/or host physiology. Herein we describe a multidisciplinary approach to modulate the expression of a specific bacterial gene within the gut by oral administration. We first demonstrate that an engineered temperate phage {lambda} expressing a programmable dCas9 represses a targeted E. coli gene in the mammalian gut. To facilitate phage administration while minimizing disruption to host processes, we develop an aqueous-based encapsulation formulation with a microbiota-based release mechanism and show that it facilitates the oral delivery of phage in vivo. Finally we combine these technologies and show that bacterial gene expression in the mammalian gut can be precisely modified in situ with a single oral dose.

bioengineering↗

bioRxiv2019

Stable neutralization of virulent bacteria using temperate phage in the mammalian gut

Elimination or alteration of select members of the gut microbiota is key to therapeutic efficacy. However, the complexity of these microbial inhabitants makes it challenging to precisely target bacteria without unexpected cascading effects. Here, we use bacteriophage to deliver exogenous genes to specific bacteria by genomic integration of temperate phage for long-lasting modification. As a real-world therapeutic test, we engineered {lambda} phage to transcriptionally-repress shigatoxin by using genetic hybrids between {lambda} and other lambdoid phages to overcome resistance encoded by the virulent prophage derived from enterohemorrhagic E. coli. We show that a single dose of engineered phage propagates throughout the bacterial community and reduces shigatoxin production in an enteric mouse model of infection without markedly affecting bacterial concentrations. We thus minimize the selection for resistance by relying on anti-virulence and not anti-bacterial action. Our work reveals a new framework for transferring functions to bacteria within their native environment.

synthetic biology↗

bioRxiv2019

Rational Design of a Bifunctional AND-Gate Ligand to Modulate Cell-Cell Interactions

Protein \"AND-gate\" systems, in which a ligand acts only on cells with two different receptors, direct signaling activity to a particular cell type and avoid action on other cells. In a bifunctional AND-Gate protein, the molecular geometry of the protein domains is crucial. Here we constructed a tissue-targeted erythropoietin (EPO) that stimulates red blood cell (RBC) production without triggering thrombosis. EPO was directed to RBC precursors and mature RBCs by fusion to an anti-glycophorin A antibody V region. Many such constructs activated EPO receptors in vitro and stimulated RBC and not platelet production in mice but nonetheless enhanced thrombosis in mice and caused adhesion between RBCs and EPO receptor-bearing cells. Based on a protein-structural model of the RBC surface, we rationally designed an anti-glycophorin/EPO fusion that does not induce cell adhesion in vitro or enhance thrombosis in vivo. Thus, meso-scale geometry can inform design of synthetic-biological systems.\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=138 SRC=\"FIGDIR/small/711549v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (22K):\norg.highwire.dtl.DTLVardef@93b382org.highwire.dtl.DTLVardef@ee37ccorg.highwire.dtl.DTLVardef@1913fe4org.highwire.dtl.DTLVardef@f998b1_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2019

Synthetic gene circuits enable systems-level biosensor discovery at the host-microbe interface

The composition and function of the gut microbiota are strongly associated with human health, and dysbiosis is linked to an array of diseases, ranging from obesity and diabetes to infection and inflammation. Engineering synthetic circuits into gut bacteria to sense, record and respond to in vivo signals is a promising new approach for the diagnosis, treatment and prevention of disease. Here, we repurpose a synthetic bacterial memory circuit to rapidly screen for and discover new in vivo-responsive biosensors in commensal gut Escherichia coli. We develop a pipeline for rapid systems-level library construction and screening, using next-generation sequencing and computational analysis, which demonstrates remarkably robust identification of responsive biosensors from pooled libraries. By testing both genome-wide and curated libraries of potential biosensor triggers--each consisting of a promoter and ribosome binding site (RBS)--and using RBS variation to augment the range of trigger sensitivity, we identify and validate triggers that selectively activate our synthetic memory circuit during transit through the gut. We further identify biosensors with increased response in the inflamed gut through comparative screening of our libraries in healthy mice and those with intestinal inflammation. Our results demonstrate the power of systems-level screening for the identification of novel biosensors in the gut and provide a platform for disease-specific screening using synthetic circuits, capable of contributing to both the understanding and clinical management of intestinal illness. IMPORTANCEThe gut is a largely obscure and inaccessible environment. The use of live, engineered probiotics to detect and respond to disease signals in vivo represents a new frontier in the management of gut diseases. Engineered probiotics have also shown promise as a novel mechanism for drug delivery. However, the design and construction of effective strains that respond to the in vivo environment is hindered by our limited understanding of bacterial behavior in the gut. Our work expands the pool of potential biosensors for the healthy and diseased gut, providing insight into host-microbe interactions and enabling future development of increasingly complex synthetic circuits. This method also provides a framework for rapid prototyping of engineered systems and for application across bacterial strains and disease models.

synthetic biology↗

bioRxiv2018

Inducible Cooperation in a Synthetic Gut Bacterial Consortium Introduces Population Balance and Stability

In nature, microbes interact antagonistically, neutrally or beneficially. To shed light on the effects of positive interactions in microbial consortia we introduced metabolic dependencies and metabolite overproduction into four bacterial species. While antagonistic interactions govern the wildtype consortium behavior, the genetic modifications alleviated antagonistic interactions and resulted in beneficial interactions. Engineered cross-feeding increased population evenness, a component of ecological diversity, in different environments including in a more complex gnotobiotic mouse gut environment. Our findings suggest that metabolite cross-feeding could be used as a tool for intentionally shaping microbial consortia in complex environments.\n\nImportanceMicrobial communities are ubiquitous in nature. Bacterial consortia live in and on our body and in our environment and more recently, biotechnology is applying microbial consortia for bioproduction. As part of our body, bacterial consortia influence us in health and disease. Microbial consortia function is determined by its composition, which in turn is driven by the interactions between species. Further understanding of microbial interactions will help us deciphering how consortia function in complex environments and may enable us to modify microbial consortia for health and environmental benefits.

synthetic biology↗

bioRxiv2018

A synthetic bacterial information transfer system functions in the mammalian gut

The gut microbiome is intricately involved with establishing and maintaining the health of the host. Engineering of gut microbes aims to add new functions and expand the scope of control over the gut microbiome. To create systems that can perform increasingly complex tasks in the gut with multiple engineered strains it is necessary to program communication among these bacteria in the gut. Towards this goal, we engineered an information transfer system for inter-cellular communication, using native gut Escherichia coli and attenuated Salmonella enterica serovar Typhimurium. Specifically, we have taken two genetic circuits-one for signaling from the quorum sensing system and the other for memory from the bacteriophage genetic switch-and integrated them into a robust system that can report on successful communication in the mammalian gut. Our system provides a basis for the construction of a programmable gut consortia as well as a basis for further understanding of bacterial interactions in an otherwise hard-to-study environment.

synthetic biology↗

bioRxiv2018

Mammalian Cells Engineered to Produce Novel Steroids

Steroids can be difficult to modify via traditional organic synthesis methods, but many enzymes regio- and stereo-selectively process a wide variety of steroid substrates. We tested whether steroid-modifying enzymes could make novel steroids from non-native substrates. Numerous genes encoding steroid-modifying enzymes, including some bacterial enzymes, were expressed in mammalian cells by transient transfection and found to be active. We made three unusual steroids by expression in HEK293 cells of the 7-hydroxylase CYP7B1, which was selected because of high native product yield. These cells made 7,17-dihydroxypregnenolone and 7{beta},17-dihydroxypregnenolone from 17-hydroxypregnenolone, and produced 11,16-dihydroxyprogesterone from 16-hydroxyprogesterone. The latter two products resulted from previously unobserved CYP7B1 hydroxylation sites. A Rosetta docking model of CYP7B1 suggested that these substrates D-ring hydroxylations may prevent them from binding in the same way as the native substrate, bringing different carbons near the active ferryl oxygen. This new approach could use other enzymes and substrates to produce many novel steroids for drug candidate testing.

synthetic biology↗

bioRxiv2017

Rapid genome recoding by iterative recombineering of synthetic DNA

Genome recoding will provide a deeper understanding of genetics and transform biotechnology. We bypass the reliance of previous genome recoding methods on site-specific enzymes and demonstrate a rapid recombineering based strategy for writing genomes by Stepwise Integration of Rolling Circle Amplified Segments (SIRCAS). We installed the largest number of codon substitutions in a single organism yet published, creating a strain of Salmonella typhimurium with 1557 leucine codon changes across 200 kb of the genome.

synthetic biology↗

bioRxiv2016

Engineering Genetically-Encoded Mineralization and Magnetism via Directed Evolution

Genetically encoding the synthesis of functional nanomaterials such as magnetic nanoparticles enables sensitive and non-invasive biological sensing and control. Via directed evolution of the natural iron-sequestering ferritin protein, we discovered key mutations that lead to significantly enhanced cellular magnetism, resulting in increased physical attraction of ferritin-expressing cells to magnets and increased contrast for cellular magnetic resonance imaging (MRI). The magnetic mutants further demonstrate increased iron biomineralization measured by a novel fluorescent genetic sensor for intracellular free iron. In addition, we engineered Escherichia coli cells with multiple genomic knockouts to increase cellular accumulation of various metals. Lastly to explore further protein candidates for biomagnetism, we characterized members of the DUF892 family using the iron sensor and magnetic columns, confirming their intracellular iron sequestration that results in increased cellular magnetization.

synthetic biology↗