A1 - Viral Vectors (excluding AAV – including RNA, adenovirus, herpesvirus, bocavirus, and chimetics)
445: Engineering Gene Therapy Vector Properties in the Baculovirus
Type: Poster Session
Poster Board Number: 445
Presentation Details
Session Title: Wednesday Posters: Viral Vectors
The CRISPR-Cas9 gene editing system has been widely researched over the last two decades, and viral methods are commonly used for the delivery of gene therapies such as CRISPR. Current common viral delivery methods include lentivirus and adeno-associated virus. These can efficiently deliver DNA into living systems, but have relatively small packaging capacities-5 and 8 kilobases, respectively. However, much larger gene circuits than this must be developed to create editing systems that afford more control over their induction and function. This research aims to use baculovirus, an insect-virus with a comparatively large carrying capacity, to deliver inducible, targeted gene therapies. As an insect virus, baculovirus poses unique challenges when used within mammalian systems, namely low transduction, inactivation by the complement system, and transient gene expression. This work focuses on the process of producing pseudotyped baculovirus containing proteins to enhance transduction, immune protection, and targeting.
Genes for the pseudotyped proteins were placed downstream of the p10 insect promoter in the engineered baculovirus genome using standard molecular cloning techniques. Baculovirus was then produced, purified, and tested in vitro on various cell lines-including HEK293T cells, HEPA-1,6 cells, C2C12 muscle cells, and Jurkats-using GFP as a reporter for transduction, as well as in vivo in C57BI6 mice using a firefly luciferase reporter. Both in vitro and in vivo, vesicular stomatitis virus protein-G (VSV-G) has been determined to improve transduction efficiency. With VSV-G, a three-fold increase in transduction was observed in HEK293T and C2C12 cells, and a 30-fold increase was seen in HEPA-1,6 cells. Decay accelerating factor (DAF) also appears to provide protection from the complement portion of the immune system in experiments performed with mouse serum containing the complement system. In vivo, while pseudotyping with VSV-G and DAF each independently increased baculovirus transduction, adding both proteins to baculovirus together led to the highest transduction-approximately a 10-fold increase as compared to wild type-and expression that persisted for a month.
Ongoing efforts are focused on the engineering and production of pseudotyped baculovirus containing proteins to enhance targeting and transduction in primary T-cells. The level of control afforded to clinicians and researchers by the large DNA capacity of baculovirus can be used to implement small-molecule inducible systems for temporal control of therapeutic gene expression, or autonomous cell-state sensing mechanisms to monitor and respond to CAR T-cell activity in vivo.
Tanya Jain, Lucas Brown, Gang Bao
Rice University, Houston, TX"
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