Title:
DEVELOPMENT AND CHARACTERIZATION OF VIRAL VECTORS FOR STRESS-DEPENDENT TRANSGENE EXPRESSION IN NEURONS
DEVELOPMENT AND CHARACTERIZATION OF VIRAL VECTORS FOR STRESS-DEPENDENT TRANSGENE EXPRESSION IN NEURONS
Authors
Santiago-Lopez, Angel J.
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Advisors
Gross, Robert E.
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Abstract
This work introduces a viral vector that captures intrinsic signals associated with the unfolded protein response (UPR) to control gene product delivery in neurons. The development of this molecular tool responds to the need for physiologically responsive gene therapy constructs to prevent unwanted side effects associated with transgene overexpression. Additionally, when used as a reporter assay, this tool addresses a basic research need for the kinetic monitoring of cellular stress and proteostasis dysfunction in neurons. To address these needs, we designed a viral-based ATF4 reporter comprising 384 bp of the initial coding region, including the 5’UTR of human ATF4 as a translational control operator fused to an enhanced green fluorescent protein (EGFP). For biological characterization, we conducted extensive time-lapse fluorescent microscopy assays in several in vitro models of proteostasis dysfunction, including ER stress, proteasome inactivation, phosphatase inhibition, and alpha-synuclein overexpression. Collectively, our results demonstrate the feasibility of mobilizing cellular stress signaling to create physiologically-responsive viral vectors for use in neuroscience.
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Date Issued
2021-01-19
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Dissertation