Browsing by Author "Bashor, Caleb"

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    Delivery of Large Gene Circuits In vivo Using an Engineered Baculovirus Vector for Multifactorial Control of Gene Expression
    (2024-12-06) Brown, Lucas Bernard Clatanoff; Bashor, Caleb; Bao, Gang
    Many 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.
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    Optimization of CRISPR/Cas-based Programmable DNA Methylation
    (2024-04-05) Guerra Resendez, Rosa Selenia; Hilton, Isaac; Bashor, Caleb
    CRISPR/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.