Bashor, CalebBao, Gang2025-01-172024-122024-12-06December 2https://hdl.handle.net/1911/118245Many 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.application/pdfenBaculovirus, Gene Circuits, EngineeringThesis2025-01-17