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

本项目的目标是建立一个基于植物病毒的表达系统，结合植物细胞培养，作为生产人类治疗性蛋白的手段。特别是，该项目将对宿主植物细胞进行基因工程改造，以便在化学或代谢诱导启动子的控制下，在细胞内产生重组植物病毒扩增子（含有外源基因的自我复制病毒RNA）。该项目的主要目标包括：(1)设计重组病毒基因结构，其中包含所有必需的成分，用于高水平生产病毒mRNA，有效的病毒复制，高效的外源基因表达和细胞外靶向；(2)设计和创建转基因植物细胞系，利用严格调控的，化学和/或代谢诱导启动子，在非诱导条件下限制病毒产生，在诱导条件下允许重组病毒RNA的高水平转录。3)在生长和诱导过程中优化植物细胞生物反应器条件和操作策略，以最大限度地提高产品的体积生产力，最大限度地降低回收/纯化成本。本项目将重点研究在膜生物反应器中，利用黄瓜花叶病毒（CMV）扩增子在特定化学和代谢调节启动子的控制下，在烟草细胞培养中生产两种模型蛋白（绿色荧光蛋白（GFP）和人a1-抗胰蛋白酶（AAT））。该项目涉及多学科方法，将分子生物学和病毒学策略与定量过程工程相结合。此外，该项目还包括一个综合培训计划，包括合作、多学科团队的研究方法，结合课程作业、重点研究小组、研讨会、务虚会和工业实习，以提供生物技术方面的广度。研究生、博士后研究人员和本科生将参加该培训计划。

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.