A1 - Viral Vectors (excluding AAV – including RNA, adenovirus, herpesvirus, bocavirus, and chimetics)
160: Delivery of Large Gene Circuits In Vivo Using an Engineered Baculovirus Vector for Multifactorial Control of Therapeutic Gene Expression
Type: Oral Abstract Session
Presentation Details
Session Title: New Avenues for Viral Vectors
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 >100kb 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 first 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. We further increased the length of baculovirus transgene expression to more than a month in vitro and in vivo through the screening of promoters and other genetic elements such as the Ebna1-OriP system. We then implemented a hierarchical cloning scheme for the rapid generation and prototyping of baculovirus vectors containing up to 10 different expression units. Using this system, 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. Our findings will demonstrate the usefulness of complex regulation for the gene therapy field, as well as the utility of the baculovirus as a therapeutic vector.
Lucas B. C. Brown, Linlin Zhang, Gang Bao, Caleb J. Bashor
Bioengineering, Rice University, Houston, TX"
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