1,720,976 research outputs found
Delivery of Large Gene Circuits In vivo Using an Engineered Baculovirus Vector for Multifactorial Control of Gene Expression
No full textMany of the viral vectors used for gene therapy are limited by the cargo size they can deliver into cells in tissue. As a result, most therapies being actively considered today tend to consist of monomodal expression of one or two genes. While this modality is undoubtedly effective for many applications, there remains advantages to being able to deliver more genetic cargo. A viral vector with an increased cargo capacity could allow room not only for more and larger therapeutic genes, but also regulatory elements that permit complex, multifactorial regulation of therapeutic gene expression. Here we use the insect-derived baculovirus capable of packaging and delivering >100 kb of transgene DNA as a vector for complex gene circuits that regulate and enhance in vivo gene therapy. Baculovirus has many advantages over other vectors: the ability to transduce a broad spectrum of mammalian cells, a large packaging capacity, no replication in mammalian cells, and a low toxicity in vivo. However, while baculovirus has been used as a gene therapy vector previously, its potential has been limited by its transient expression, as well as its susceptibility to inactivation by the complement system.
We then implemented a hierarchical cloning scheme for the rapid generation and prototyping of baculovirus vectors containing up to 10 different expression units. We then address several shortcomings of the baculovirus by pseudo-typing the AcMNPV baculovirus with two proteins, the Vesticular stromatitis virus protein G and a fusion protein consisting of several complement regulatory domains. This engineered vector has increased transduction and persistence in mouse liver, muscle, and brain tissue. To our knowledge, this is the first time systemic delivery of baculovirus has been shown to be an effective delivery route. Using this engineered virus, we screened a library of 24 variations of a tamoxifen inducible circuit in order to select the architecture with the highest dynamic range, up to a 67-fold increase over uninduced. Finally, we demonstrate two orthogonal small molecule inducible systems (grazoprevir and tamoxifen) delivered by baculovirus in vivo, both as separate viruses and as one complete circuit. Our findings demonstrate the usefulness of complex regulation for the gene therapy field, as well as the utility of the baculovirus as a therapeutic vector
Development of experimental platforms for ultra-high- throughput exploration of complex genetic design spaces
No full textCells sense and process signals from their environment to execute a diverse array of tasks, ranging from proliferation and differentiation to programmed cell death. Inspired by the capabilities of biological systems to carry out sophisticated computations, the field of synthetic biology aims to use nucleic acid-encoded “synthetic” regulatory programs to quantitatively engineer novel cellular behaviors for environmental, biotechnological, and therapeutic purposes. Like many forms of engineering, synthetic biology projects follow a design-build-test-learn cycle: an iterative process of constructing, assaying, and modifying genetic circuits to achieve desired phenotypes. However, unlike more established forms of engineering, we do not have a quantitative set of core principles that describe the complexities of all biological activity. This limitation is pronounced in mammalian synthetic biology, where lengthy design campaigns and an incomplete understanding of the system make precisely programming cellular functions difficult. One approach to addressing challenges in synthetic biology is to increase the pace and scale of data acquisition and allow experimental data to replace hypotheses as the cornerstone of decision-making. Here, I present a suite of molecular biology and cell engineering tools that lay the foundations of a novel platform designed to enable high-throughput construction and quantitative assessment of large and complex genetic design spaces. This platform, named CLASSIC (combinatorial large-scale assembly and short-range sequencing for investigating genetic complexity), offers a novel opportunity to generate genotype- to-phenotype (G2P) maps for hundreds of thousands of multi-kilobase genetic circuits in a single experiment. We show proof-of-concept for this platform and leverage the unique ability to assay genetic diversity to optimize the performance of single-input genetic switches in mammalian cells. Additionally, we show that the CLASSIC platform can be adapted to enable image-based G2P mapping of diverse features of cellular identity and phenotype, including protein compartmentalization and cell morphology, and interactions between engineered cells in multi-cellular environments, such as T cell killing
Implementation of Genetic Circuits for Engineered Mesenchymal Stem Cell Chondrogenic Differentiation
No full textThe ability to culture stem cells and guide their differentiation has allowed myriad advancements in developmental biology and cell-based therapies. In vitro differentiation protocols have historically taken an “outside-in” approach where bioactive molecules (purified proteins, small molecules, etc.) are added exogenously to culture medium or functionalized onto culture surfaces (plates, scaffolds, etc.) to guide cell differentiation. Despite decades of protocol optimization, “outside-in” approaches result in heterogeneous populations where only a subset of cells matches the desired phenotype, and the presence of aberrant cell states makes the cells ineffective for therapeutic use. These shortcomings are particularly evident in cartilage tissue engineering, where the multipotency of mesenchymal stem cells (MSCs) is harnessed to regenerate cartilage tissue. In these therapies, MSCs produce areas of proper cartilage, but concurrently produce hypertrophic and fibrotic chondrocytes. These cells deposit bone and fibrous tissue, respectively, thus leading to suboptimal tissue properties and limiting clinical translation. The ability to encourage proper chondrogenic phenotypes while preventing undesired hypertrophic and fibrotic ones is thus of great interest to tissue engineers and developmental biologists alike.
One promising strategy involves using synthetic gene circuits to precisely control the dose and timing of expression of genes critical to functional chondrogenic differentiation. This “inside-out” approach is inspired by natural cellular differentiation, where it has been demonstrated that precise timing and magnitude of expression of genes in key regulatory networks are responsible for driving differentiation to mature cell states. In this work, I developed a novel engineering platform that enabled this functionality with synthetic genetic circuits. The platform I created includes a novel framework for the quantitative design and implementation of genetic circuits in a variety of cell types paired with an in vitro model and assessment method using single cell RNA sequencing (scRNA-seq) that allows iterative circuit implementation and assessment of the effect of circuit function on cell phenotypes in a model of MSC chondrogenesis. This work represents a significant step forward for the fields of mammalian synthetic biology and tissue engineering by (1) allowing high throughput circuit design, creation, and implementation in mammalian cells and (2) providing an unprecedented description of chondrogenic differentiation trajectories and how to manipulate them
Optimization of CRISPR/Cas-based Programmable DNA Methylation
No full textEMBARGO NOTE: This item is embargoed until 2026-05-01CRISPR/Cas-based epigenome editing systems have transformed biomedical research by providing transcriptional and epigenetic control of healthy and disease states. Even though synthetic biology tools for DNA methylation writing and erasing have been employed in different architectural configurations and recruitment strategies, efforts to improve their functionality have been limited. Thus, engineering approaches are underexplored and needed to enhance the enzymatic activity of these tools, in particular for therapeutic applications in primary cells and in vivo models. In this doctoral dissertation, I present the optimization and application of programmable DNA methylation in human cells using CRISPR/Cas9-based epigenome editing. In the first chapter, optimization of lentiviral delivery of CRISPR/Cas9-based epigenome editing tools in human cells was conducted to better understand the parameters that lead to efficient delivery of epigenome editing tools. In the second chapter, rational engineering of the programmable synthetic DNA methylation writer (dCas9-DNMT3A/3L) was used to characterize the differential DNA methyltransferase activity of each designed variant, which catalyzed a distinctive methylation deposition profile and magnitude which correlated to gene silencing in different human cell lines. In the third chapter, engineered variants of dCas9-DNMT3A/3L were delivered to human primary T cells in an attempt to model immune exhaustion by targeting the BATF3 gene. Transcriptional silencing of BATF3 led to reduced cytokine production in human primary T cells. However, further experiments are needed to confirm that the reduced gene expression is caused by the DNA methylation deposited by the dCas9-based epigenetic effectors or if other factors independent of this epigenetic mark might be affecting transcription at this locus. Overall, the use of rationally engineered variants of the dCas9-DNMT3A/3L present an opportunity to interrogate the mechanistic parameters involved in programmable DNA methylation deposition and enable tunable transcriptional repression in human cells
Engineering synthetic phosphorylation signaling networks in human cells
No full textProtein phosphorylation signaling networks have a central role in how cells sense and respond to their environment. This thesis outlines a comprehensive approach to designing and implementing synthetic phosphorylation networks in mammalian cells, using modular protein domain parts to construct reversible phosphorylation cycles and assemble customizable circuits. By leveraging model-guided tuning, these engineered circuits enable precise signal processing and facilitate the creation of diverse network connections. The synthetic pathways can be linked to upstream cell surface receptors for rapid sensing of extracellular ligands and downstream elements that regulate gene expression.
The work further explores the application of these synthetic networks in therapeutically relevant settings. We demonstrate how engineered circuits can detect physiologically significant biomolecules, such as inflammation markers, and respond with targeted actions, including the controlled secretion of therapeutic proteins. The successful integration and functional testing of these synthetic pathways in primary human cells highlight a significant step toward their use in cell-based therapies. This adaptability illustrates the potential for engineering customized cellular responses tailored to specific disease states, paving the way for innovative treatment strategies.
By providing a robust toolkit and showcasing its versatility, this thesis lays the groundwork for future advancements in synthetic biology. The modular design and adaptability of these synthetic signaling networks create opportunities for developing programmable cellular systems capable of addressing a wide range of biotechnological and medical challenges. This work contributes to the growing field of synthetic biology by establishing a foundational framework for integrating engineered pathways into cellular systems, enhancing their ability to perform complex, tailored functions and expanding the scope of potential applications in biosensing and therapeutic development
Synthetic manipulation of mammalian secretory phenotypes for augmented bioproduction and neuroendocrine-like regulated secretory function
No full textThe secretory machinery of mammalian cells is an essential aspect of their use case as cell factories for biopharmaceutical production, as well as having implications on the performance of engineered cell-based therapeutics. Virtually all recombinant drug products derived from mammalian cells must be secreted prior to purification steps, and many cell-based therapeutic platforms manage patient physiology via secretion-based signaling. In this work, we present a strategy for manipulating the mammalian secretory phenotype via overexpressed secretory factors to enhance bioproduction capacity, principally but not exclusively of HEK293T cells. Our work demonstrates the potential of secretory pathway engineering to enhance bioproduction yields of biologic medicines and viral vectors for gene therapy, as well as augmenting the secretory productivity of cell types useful for therapeutics (e.g., mesenchymal stem cells and retinal pigment epithelial cells).1 In addition, we demonstrate how secretory phenotype reprogramming can produce artificial neuroendocrine-like Ca2+-inducible regulated secretory responses in nonendocrine HEK293T cells. Finally, we engineer circuits for controlling Ca2+ channel activation with small-molecule inputs, as well as ongoing progress towards linking these circuits to our synthetic regulated secretory program outputs. Engineered regulated secretory circuits, especially those with reversible “sense-and-secrete” functionality and programmable and tunable inputs, have the potential to enhance the therapeutic capacity of cell lines
Understanding Biological Regulation Through Synthetic Biology
No full textEngineering synthetic gene regulatory circuits proceeds through iterative cycles of design, building, and testing. Initial circuit designs must rely on often-incomplete models of regulation established by fields of reductive inquiry—biochemistry and molecular and systems biology. As differences in designed and experimentally observed circuit behavior are inevitably encountered, investigated, and resolved, each turn of the engineering cycle can force a resynthesis in understanding of natural network function. Here, we outline research that uses the process of gene circuit engineering to advance biological discovery. Synthetic gene circuit engineering research has not only refined our understanding of cellular regulation but furnished biologists with a toolkit that can be directed at natural systems to exact precision manipulation of network structure. As we discuss, using circuit engineering to predictively reorganize, rewire, and reconstruct cellular regulation serves as the ultimate means of testing and understanding how cellular phenotype emerges from systems-level network function. Keywords: synthetic biology; regulatory network; synthetic gene circuit; engineering cycle; motif; refactorin
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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