A Regulated Plant Virus Expression System for Efficient Production of Human Therapeutics

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The objective of this project is to produce a plant virus based expression system combined with plant cell culture as a means for producing human therapeutic proteins. In particular, this project will genetically engineer host plant cells so that recombinant plant virus amplicons (self-replicating viral RNA containing a foreign gene) can be produced intracellularly, under the control of a chemically or metabolically-inducible promoter. The major goals of this project include: (1) the design of the recombinant viral gene constructs which contain all of the necessary components for high level production of viral mRNA, efficient viral replication, efficient foreign gene expression and extracellular targeting, 2) the design and creation of transgenic plant cell lines utilizing tightly regulated, chemically and/or metabolically inducible promoters which restrict viral production under non-induced conditions and allow high level transcription of the recombinant viral RNA under induction conditions, and 3) optimization of plant cell bioreactor conditions and operating strategies during growth and induction to maximize volumetric productivity of the product and minimize recovery/purification costs. This project will focus on the production of two model proteins (green fluorescent protein (GFP) and human a1-antitrypsin (AAT)) in tobacco cell cultures utilizing a cucumber mosaic virus (CMV) amplicon under the control of specific chemical and metabolically regulated promoters in a membrane bioreactor. This project involves a multidisciplinary approach combining molecular biology and virology strategies with quantitative process engineering. In addition, an integrated training program consisting of a collaborative, multidisciplinary team approach to research, combined with coursework, a focused study group, seminars, retreats and an industrial internship to provide breadth in biotechnology, is part of this project. Graduate students, postdoctoral researchers and undergraduates will participate in this training program.