Biology subjects

Galloway, K. E.

Publications and source records attributed to Galloway, K. E..

16 recordsLinked to original sources

bioRxiv2026

Model-guided design of single-cargo, dual-attractor gene circuits

Dual-attractor gene networks program distinct states, guiding native cellular decision-making processes such as differentiation. Synthetic dual-attractor circuits (DACs) emulate native gene networks, directing cells to two different states starting from a single, genetically uniform population. However, existing DAC designs for mammalian cells often require multiple genetic cargoes, limiting their application to control cell fate in therapeutically relevant contexts. Here, we leverage model-guided tuning of compact biochemical and biophysical layers of regulation to program two cellular states using a single-cargo DAC. Through activator selection and layering microRNA-mediated mutual repression, we identify and resolve an imbalance between modules that biases states, allowing us to achieve balanced adoption of both states without relying on copy-number variation. Consolidating this optimized DAC into a single cargo, we use gene syntax to minimize bias, generating two balanced states from a single cargo of less than 11 kilobases. Overall, this work establishes design rules for combined biochemical and biophysical tuning of DACs to generate balanced cell states from a single-cargo design, paving the way for programming divergent cell-state trajectories and dual-fate systems.

synthetic biology↗

bioRxiv2026

Engineered microRNA feedback circuits enable tunable and autonomous control of synthetic receptor activity

Synthetic receptors are powerful tools for cellular engineering, yet their utility is often constrained by constitutive activity and the absence of natural feedback mechanisms that maintain cellular homeostasis. To address this, we engineered an autonomous regulatory circuit for synthetic Notch (synNotch) receptor activation using an orthogonal microRNA (miRNA)-mediated negative feedback mechanism in mammalian cells. Specifically, synNotch activation induces the expression of a synthetic miRNA that targets complementary sites engineered into the synNotch transcript, resulting in a self-limiting feedback loop. We demonstrate that synNotch repression positively correlates with the number of miRNA target sites integrated into the receptor transcript, enabling tunable control of receptor expression. Furthermore, flow cytometry and live-cell imaging analyses revealed effective suppression of synNotch expression following activation. Downstream target gene expression peaked at approximately 48 hours post-activation before gradually declining as the feedback circuit engaged. This approach achieves autonomous synthetic receptor regulation--eliminating the need for external intervention--and enables tunable response dynamics through rational circuit design. Our miRNA feedback strategy provides a generalizable platform for engineering self-regulating synthetic receptor systems with improved control and predictability for cellular engineering applications.

synthetic biology↗

bioRxiv2026

Engineering high-titer lentiviral vectors for robust expression of RNA-based gene circuits

Lentiviral vectors enable efficient delivery of genetic cargoes for gene and cell therapies. With their [~]10-kb packaging limit, lentiviral vectors can encode multiple transcription units, supporting delivery of compact gene circuits. RNA-based devices offer highly compact control including ligand-responsive induction and closed-loop regulation. However, RNA devices such as ribozymes and splicing switches may interfere with vector production via activity on the single-stranded RNA genome. Here, we examine the impact of gene syntax and genetic parts to define design strategies for two-gene vectors encoding RNA devices. We find that titer decreases with genetic parts that interfere with transcription or processing of the viral transcript during production. Compared to initial vectors, our best-performing design boosts titer more than 30-fold, enabling fine-scale tuning of expression to optimize cell-fate conversion within a nonmonotonic landscape. Together, this work illuminates principles for constructing two-gene lentiviral vectors with both high titer and robust expression, enhancing efficacy for downstream applications.

synthetic biology↗

bioRxiv2026

Programmable nanobody circuits for cell selection

Efficient and scalable isolation of specific cell populations remains a central bottleneck for genome engineering, pooled screening, and cell therapy manufacturing. Here, we present DASIT (Destabilized-nanobody Antigen Selection and Identification Tool), a protein-based circuit for antigen-specific cell selection. DASIT uses a destabilized nanobody fused to an antibiotic resistance protein. In cells expressing the target antigen, binding of the nanobody fusion to the cognate antigen stabilizes DASIT, thereby coupling the presence of an antigen to a selectable signal. We developed DASIT circuits that enable robust selection of antigen-expressing cells and show that they can be designed to target distinct antigen classes and perform across cell types. Because DASIT operates at the protein level, it supports both stable integration and transient delivery, enabling recyclable selection without permanent genomic integration of resistance markers. We demonstrate scalable, FACS-free enrichment in three challenging applications: multiplexed, logic-gated integration of landing pads in human iPSCs, high-throughput CRISPR screening, and phenotypic selection of in vitro-derived neurons at transplantation scale. By decoupling selection from vector integration, DASIT establishes an automation-compatible architecture for multistep genome engineering, high-throughput library screening, and large-scale cell manufacturing. HighlightsDASIT enriches for antigen-positive cells across multiple selection markers and antigens Intermediate levels of DASIT expression support selection across stable and transient delivery modalities Logic-gated, precision genome engineering of human iPSCs via DASIT selection DASIT enables scalable activity-based selection for high-throughput base editing screens DASIT-selected engineered motor neurons survive grafting into acute spinal cord injury

synthetic biology↗

bioRxiv2025

Self-amplifying RNA enables rapid, durable, integration-free programming of hiPSCs

Genetic modification of human induced pluripotent stem cells (hiPSCs) is a powerful approach to measure and manipulate the cellular processes underlying differentiation and disease. Conventional genetic engineering of hiPSC lines requires a laborious process involving transfection, selection and expansion that can result in karyotypic abnormalities or transgene silencing during differentiation, limiting their applications. Self-amplifying RNA (saRNA) delivery is a potential alternative integration-free method for durable expression of transgenes. Here, we used saRNA to deliver transcription factors and functional reporters in hiPSCs and demonstrate that expression can persist for weeks. Specifically, saRNA delivery enables highly efficient forward programming to Ngn2-induced neurons and enables measurement of functional reporters over time. We show that a single transfection of saRNA encoded jRCaMP1b reporter in hiPSCs generates sustained expression throughout differentiation to 3D cardiac spheroids. The persistence of the reporter allows measurement of calcium dynamics at a single-cell and population level over weeks, allowing tracking of cardiomyocyte maturation and drug responses. Together, our systematic analysis shows that saRNA provides sustained transgene expression in hiPSCs, supporting integration- free cell-fate programming and measurement of functional reporters in clinically relevant model systems. HighlightsO_LIA single saRNA transfection generates durable transgene expression C_LIO_LIsaRNA transfection of Ngn2 in hiPSCs results in robust neuronal differentiation C_LIO_LIsaRNA-delivery of functional reporters enables single-cell analysis of primary and hiPSC-derived cells C_LIO_LIsaRNA-based sensor allows monitoring of maturation and drug responses in 3D cardiac spheroids C_LI

bioengineering↗

bioRxiv2025

Gene syntax defines supercoiling-mediated transcriptional feedback

Gene syntax--the order and arrangement of genes and their regulatory elements--shapes the dynamic coordination of both natural and synthetic gene circuits. Transcription at one locus profoundly impacts the transcription of nearby adjacent genes, but the molecular basis of this effect remains poorly understood. Here, using integrated reporter circuits in human cells, we show that the reciprocal effects of transcription and DNA supercoiling, which we term supercoiling-mediated feedback, regulates expression of adjacent genes in a syntax-specific manner. Using a suite of chromatin state assays, we measure syntax-and induction-dependent formation of chromatin structures in human induced pluripotent stem cells. Applying syntax as a design parameter and without altering sequence or copy number, we built compact gene circuits, tuning the expression mean, noise, and stoichiometry across diverse delivery methods and cell types. Integrating supercoiling-mediated feedback into models of gene regulation will expand our understanding of native systems and enhance the design of synthetic gene circuits.

synthetic biology↗

bioRxiv2024

Chemogenetic tuning reveals optimal MAPK signaling for cell-fate programming

Cell states evolve through the combined activity of signaling pathways and gene regulatory networks. While transcription factors can direct cell fate, these factors rely on a cell state that is receptive to transitions in cell identity. How signaling levels contribute to the emergence of receptive cell states remains poorly defined in primary cells. Using a well-defined model of direct conversion, we examined how levels of the MAPK-activating oncogene HRASG12V influence direct conversion of primary fibroblasts to induced motor neurons. We demonstrate that an optimal Goldilocks level of MAPK signaling efficiently drives cell-fate programming. Rates of direct conversion respond biphasically to increasing HRASG12V levels. While intermediate HRASG12V levels increase the rate of conversion, high levels of HRASG12V induce senescence. Through chemogenetic tuning, we set optimal MAPK activity for high rates of conversion in the absence of HRAS mutants. As MAPK pathways influence cell-fate transitions in development and disease, our results highlight the need to tune therapeutic interventions within a non-monotonic landscape that is shaped by genetics and levels of gene expression. HighlightsO_LIMAPK signaling drives proliferation and conversion of fibroblasts to motor neurons C_LIO_LICell-fate programming responds biphasically to HRASG12V expression C_LIO_LIHigh HRASG12V expression induces senescence, which reduces conversion C_LIO_LIChemogenetic tuning of MAPK activity increases conversion rates C_LIO_LIA small-molecule MAPK inducer drives high rates of conversion in the absence of HRASG12V C_LI

systems biology↗

bioRxiv2024

High-resolution profiling reveals coupled transcriptional and translational regulation of transgenes

Concentrations of RNAs and proteins provide important determinants of cell fate. Robust gene circuit design requires an understanding of how the combined actions of individual genetic components influence both mRNA and protein levels. Here, we simultaneously measure mRNA and protein levels in single cells using HCR Flow-FISH for a set of commonly used synthetic promoters. We find that promoters generate differences in both the mRNA abundance and the effective translation rate of these transcripts. Stronger promoters not only transcribe more RNA but also show higher effective translation rates. While the strength of the promoter is largely preserved upon genome integration with identical elements, the choice of polyadenylation signal and coding sequence can generate large differences in the profiles of the mRNAs and proteins. We used long-read direct RNA sequencing to characterize full-length mRNA isoforms and observe remarkable uniformity of mRNA isoforms from the transgenic units. Together, our high-resolution profiling of transgenic mRNAs and proteins offers insight into the impact of common synthetic genetic components on transcriptional and translational mechanisms. By developing a novel framework for quantifying expression profiles of transgenes, we have established a system for comparing native and synthetic gene regulation and for building more robust transgenic systems.

synthetic biology↗

bioRxiv2024

Cells transit through a quiescent-like state to convert to neurons at high rates

While transcription factors (TFs) provide essential cues for directing and redirecting cell fate, TFs alone are insufficient to drive cells to adopt alternative fates. Rather, transcription factors rely on receptive cell states to induce novel identities. Cell state emerges from and is shaped by cellular history and the activity of diverse processes. Here, we define the cellular and molecular properties of a highly receptive state amenable to transcription factor-mediated direct conversion from fibroblasts to induced motor neurons. Using a well-defined model of direct conversion to a post-mitotic fate, we identify the highly proliferative, receptive state that transiently emerges during conversion. Through examining chromatin accessibility, histone marks, and nuclear features, we find that cells reprogram from a state characterized by global reductions in nuclear size and transcriptional activity. Supported by globally increased levels of H3K27me3, cells enter a quiescent-like state of reduced RNA metabolism and elevated expression of REST and p27, markers of quiescent neural stem cells. From this transient state, cells convert to neurons at high rates. Inhibition of Ezh2, the catalytic subunit of PRC2 that deposits H3K27me3, abolishes conversion. Our work offers a roadmap to identify global changes in cellular processes that define cells with different conversion potentials that may generalize to other cell-fate transitions. HighlightsO_LIProliferation drives cells to a compact nuclear state that is receptive to TF-mediated conversion. C_LIO_LIIncreased receptivity to TFs corresponds to reduced nuclear volumes. C_LIO_LIReprogrammable cells display global, genome-wide increases in H3K27me3. C_LIO_LIHigh levels of H3K27me3 support cells transits through a state of altered RNA metabolism. C_LIO_LIInhibition of Ezh2 increases nuclear size, reduces the expression of the quiescence marker p27. C_LIO_LIAcute inhibition of Ezh2 abolishes motor neuron conversion. C_LI One Sentence SummaryCells transit through a quiescent-like state characterized by global reductions in nuclear size and transcriptional activity to convert to neurons at high rates. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=143 SRC="FIGDIR/small/624928v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@ee2127org.highwire.dtl.DTLVardef@1c2a39dorg.highwire.dtl.DTLVardef@183bf46org.highwire.dtl.DTLVardef@82b98d_HPS_FORMAT_FIGEXP M_FIG C_FIG

systems biology↗

bioRxiv2024

STRAIGHT-IN Dual: a platform for dual, single-copyintegrations of DNA payloads and gene circuits into human induced pluripotent stem cells

Targeting DNA payloads into human (h)iPSCs involves multiple time-consuming, inefficient steps that must be repeated for each construct. Here, we present STRAIGHT-IN Dual, which enables simultaneous, allele-specific, single-copy integration of two DNA payloads with 100% efficiency within one week. Notably, STRAIGHT-IN Dual leverages the STRAIGHT-IN platform to allow near-scarless cargo integration, facilitating the recycling of components for subsequent cellular modifications. Using STRAIGHT-IN Dual, we investigated how promoter choice and gene syntax influence transgene silencing, demonstrating the impact these design features have on reporter gene expression and forward programming of hiPSCs into neurons, motor neurons, and endothelial cells. Furthermore, we designed a grazoprevir-inducible synZiFTR system to complement the widely used tetracycline-inducible system, providing independent, tunable, and temporally controlled expression of different transcription factors within the same cell. The unprecedented efficiency and speed with which STRAIGHT-IN Dual generates homogenous genetically engineered hiPSC populations represents a major advancement for synthetic biology in stem cell applications and opens opportunities for precision cell engineering.

synthetic biology↗

bioRxiv2024

Engineered Transcription Factor Binding Arrays for DNA-based Gene Expression Control in Mammalian Cells

Manipulating gene expression in mammalian cells is critical for cell engineering applications. Here we explore the potential of transcription factor (TF) recognition element arrays as DNA tools for modifying free TF levels in cells and thereby controlling gene expression. We first demonstrate proof-of-concept, showing that Tet TF-binding recognition element (RE) arrays of different lengths can tune gene expression and alter gene circuit performance in a predictable manner. We then open-up the approach to interface with any TF with a known binding site by developing a new method called Cloning Troublesome Repeats in Loops (CTRL) that can assemble plasmids with up to 256 repeats of any RE sequence. Transfection of RE array plasmids assembled by CTRL into mammalian cells show potential to modify host cell gene regulation at longer array sizes by sequestration of the TF of interest. RE array plasmids built using CTRL were demonstrated to target both synthetic and native mammalian TFs, illustrating the ability to use these tools to modulate genetic circuits and instruct cell fate. Together this work advances our ability to assemble repetitive DNA arrays and showcases the use of TF-binding RE arrays as a method for manipulating mammalian gene expression, thus expanding the possibilities for mammalian cell engineering.

synthetic biology↗

bioRxiv2024

Model-guided design of microRNA-based gene circuits supports precise dosage of transgenic cargoes into diverse primary cells

To realize the potential of engineered cells in therapeutic applications, transgenes must be expressed within the window of therapeutic efficacy. Differences in copy number and other sources of extrinsic noise generate variance in transgene expression and limit the performance of synthetic gene circuits. In a therapeutic context, supraphysiological expression of transgenes can compromise engineered phenotypes and lead to toxicity. To ensure a narrow range of transgene expression, we design and characterize Compact microRNA-Mediated Attenuator of Noise and Dosage (ComMAND), a single-transcript, microRNA-based incoherent feedforward loop. We experimentally tune the ComMAND output profile, and we model the system to explore additional tuning strategies. By comparing ComMAND to two-gene implementations, we highlight the precise control afforded by the single-transcript architecture, particularly at relatively low copy numbers. We show that ComMAND tightly regulates transgene expression from lentiviruses and precisely controls expression in primary human T cells, primary rat neurons, primary mouse embryonic fibroblasts, and human induced pluripotent stem cells. Finally, ComMAND effectively sets levels of the clinically relevant transgenes FMRP1 and FXN within a narrow window. Together, ComMAND is a compact tool well-suited to precisely specify expression of therapeutic cargoes.

synthetic biology↗

bioRxiv2024

Programmable promoter editing for precise control of transgene expression

Subtle changes in gene expression direct cells to distinct cellular states. Identifying and controlling dose-dependent transgenes require tools for precisely titrating expression. To this end, we developed a highly modular, extensible framework called DIAL for building editable promoters that allow for fine-scale, heritable changes in transgene expression. Using DIAL, we increase expression by recombinase-mediated excision of spacers between the binding sites of a synthetic zinc finger transcription factor and the core promoter. By nesting varying numbers and lengths of spacers, DIAL generates a tunable range of unimodal setpoints from a single promoter. Through small-molecule control of transcription factors and recombinases, DIAL supports temporally defined, user-guided control of transgene expression that is extensible to additional transcription factors. Lentiviral delivery of DIAL generates multiple setpoints in primary cells and iPSCs. As promoter editing generates stable states, DIAL setpoints are heritable, facilitating mapping of transgene levels to phenotypes. The DIAL framework opens new opportunities for tailoring transgene expression and improving the predictability and performance of gene circuits across diverse applications. HighlightsPromoter editing generates unimodal setpoints from a single synthetic promoter DIAL setpoints are tuned by the length of an excisable spacer within the promoter DIAL setpoints are uniform and robust to varying levels of the transcription factor DIAL transmits transient inputs into heritable promoter states The TET-DIAL system enables small-molecule activation at defined setpoints DIAL regulates physiologically-relevant transgenes and performs in primary cells and iPSCs One Sentence SummaryDIAL offers an extensible framework for designing synthetic promoters that perform across a range of cell types to generate heritable setpoints of gene expression.

synthetic biology↗

bioRxiv2023

Proliferation history and transcription factor levels drive direct conversion

The sparse and stochastic nature of reprogramming has obscured our understanding of how transcription factors drive cells to new identities. To overcome this limit, we developed a compact, portable reprogramming system that increases direct conversion of fibroblasts to motor neurons by two orders of magnitude. We show that subpopulations with different reprogramming potentials are distinguishable by proliferation history. By controlling for proliferation history and titrating each transcription factor, we find that conversion correlates with levels of the pioneer transcription factor Ngn2, whereas conversion shows a biphasic response to Lhx3. Increasing the proliferation rate of adult human fibroblasts generates morphologically mature, induced motor neurons at high rates. Using compact, optimized, polycistronic cassettes, we generate motor neurons that graft with the murine central nervous system, demonstrating the potential for in vivo therapies. One Sentence SummaryUsing a systems and synthetic biology approach to study the molecular determinants of reprogramming, we find that proliferation history and transcription factor levels drive cell fate in direct conversion to motor neurons. HighlightsO_LIminimal high-efficiency cocktail allows systematic interrogation of the reprogramming process C_LIO_LIhistory provides a principal axis to distinguish transcription factors influence C_LIO_LIof the transcription factor cocktail impacts reprogramming efficiency and dynamics C_LIO_LIof individual transcription factors differentially influence the rate of reprogramming C_LIO_LIearly hyperproliferation increases direct conversion of adult human fibroblasts C_LIO_LIptimal cocktail allows neurotrophic factor-free reprogramming and in vivo grafting C_LI

cell biology↗

bioRxiv2022

Supercoiling-mediated feedback rapidly couples and tunes transcription

Transcription induces a wave of DNA supercoiling, altering the binding affinity of RNA polymerases and reshaping the biochemical landscape of gene regulation. As supercoiling rapidly diffuses, transcription dynamically reshapes the regulation of proximal genes, forming a complex feedback loop. The resulting intergene coupling may provide a mechanism to control transcriptional variance in engineered gene networks and explain the behavior of co-localized native circuits. However, a theoretical framework is needed for integrating both biophysical and biochemical transcriptional regulation to investigate the role of supercoiling-mediated feedback within multi-gene systems. Here, we model transcriptional regulation under the influence of supercoiling-mediated polymerase dynamics, allowing us to identify patterns of expression that result from physical intergene coupling and explore integration of this biophysical model with a set of canonical biochemical gene regulatory systems. We find that gene syntax--the relative ordering and orientation of genes--defines the expression profiles, variance, burst dynamics, and intergene correlation of two-gene systems. By applying our model to both a synthetic toggle switch and the endogenous zebrafish segmentation network, we find that supercoiling can enhance or weaken conventional biochemical regulatory strategies such as mRNA- and protein-mediated feedback loops. Together, our results suggest that supercoiling couples behavior between neighboring genes, representing a novel regulatory mechanism. Integrating biophysical regulation into the analysis and design of gene regulation provides a framework for enhanced understanding of native networks and engineering of synthetic gene circuits.

synthetic biology↗

bioRxiv2018

Balancing dynamic tradeoffs to drive cellular reprogramming

Although cellular reprogramming continues to generate new cell types, reprogramming remains a rare cellular event. The molecular mechanisms that limit reprogramming, particularly to somatic lineages, remain unclear. By examining fibroblast-to-motor neuron conversion, we identify a previously unappreciated dynamic between transcription and replication that determines reprogramming competency. Transcription factor overexpression forces most cells into states that are refractory to reprogramming and are characterized by either hypertranscription with little cell division, or hyperproliferation with low transcription. We identify genetic and chemical factors that dramatically increase the number of cells capable of both hypertranscription and hyperproliferation. Hypertranscribing, hyperproliferating cells reprogram at 100-fold higher, near-deterministic rates. We demonstrate that elevated topoisomerase expression endows cells with privileged reprogramming capacity, suggesting that biophysical constraints limit cellular reprogramming to rare events.

cell biology↗