Plants offer a number of advantages for large-scale production of biopharmaceutical proteins compared with current biomanufacturing methods that utilize mammalian or microbial cultures. These advantages include lower capital equipment and production costs, green manufacturing technology with lower net energy requirements, CO2 consumption, scalability, low risk of product contamination by mammalian viruses, blood-borne pathogens, prions and bacterial toxins, and eukaryotic posttranslational modification capabilities. Minor differences between plant glycosylation and mammalian glycosylation are a concern for therapeutic applications; however, recent advances in genetic approaches to enable production of humanized biologics in plants have been quite promising. However, recombinant protein production using stable transgenic plants suffer from a number of challenges including the long time frame required to establish stably transformed plants, environmental concerns related to the deployment of transgenic plants in the field, low expression levels, and challenges associated with product recovery and purification. For many applications, such as rapid vaccine production in the case of a global pandemic or bioterrorism event, speed (as well as scalability) will be key issues that cannot be met with current manufacturing platforms (e.g. egg based, mammalian cell culture or insect cell culture). In addition, there are often situations where the coordinated expression of multiple proteins needs to be established in a given plant, for example co-expression of enzymes responsible for post-translational modifications to allow human-like glycosylation of biotherapeutics or coordinated expression of enzymes in metabolic pathways to enable novel metabolite production.

Our group has developed a novel expression system and an efficient, scalable production strategy, based on transient agroinfiltration in harvested (nontransgenic) living (i.e. green) plant tissues, that addresses these problems. In this process, plant cells within the plant tissues provide the biosynthetic machinery for transcription, translation, post-translational modifications, folding and intracellular targeting/secretion of the product, while the soil bacterium, Agrobacterium tumefaciens, is used to propagate the genetic instructions and efficiently transfer them to the plant cells. Thus the approach combines the advantages of rapid, easy and cheap growth of bacteria in fermentation systems with the biosynthetic capabilities of higher eukaryotic cells which have been grown using minimal energy and resource inputs in the field (using sunlight and natural resources).

We have developed and successfully demonstrated a chemically inducible plant viral amplicon (self-replicating viral RNA containing a foreign gene) expression system that allows controllable, high level expression of foreign genes in plant hosts (Sudarshana et al. 2006). We have genetically engineered Agrobacterium tumefaciens to contain modified complementary DNAs (cDNAs) representing the complete genome of Cucumber mosaic virus (CMV), in which the CMV coat protein gene has been replaced by our target gene, which along with other modifications, ensure that infectious CMV virions are not generated. Furthermore, because one of the key CMV-encoded proteins, a primary component of the viral replicase, is under the control of a tightly regulated chemically-inducible promoter, the recombinant viral amplicons are only produced intracellularly in agro-infected cells under induction conditions. We have demonstrated the CMV inducible viral amplicon (CMViva) system for the efficient production of a labile human therapeutic protein, alpha-1-antitrypsin (AAT), using agroinfiltration of both intact and detached Nicotiana benthamiana leaves (Plesha et al. 2007; Sudarshana et al. 2006). Because CMV has one of the widest host ranges of all known plant viruses and infects both dicots and monocots, the CMViva system can potentially be used in a wide variety of hosts to produce a wide variety of recombinant proteins.

In this paper we will describe the CMViva expression system, the agroinfiltration process and present quantitative results for the production kinetics of total and functional AAT. Recent studies to evaluate the quantitative dffects of timing and method of induction, co-infiltration with agrobacteria containing different gene silencing suppressors, and vacuum infiltration process conditions on production kinetics will be presented.

Plesha MA, Huang T-K, Dandekar AM, Falk BW, McDonald KA. 2007. High-level transient production of a heterologous protein in plants by optimizing induction of a chemically inducible viral amplicon expression system. Biotechnology Progress 23(6):1277-1285.

Sudarshana MR, Plesha MA, Uratsu S, Falk BW, Dandekar AM, Huang T-K, McDonald KA. 2006. A Chemically Inducible Cucumber mosaic virus Amplicon System for Expression of Heterologous Proteins in Plant Tissues. Plant Biotechnology Journal 4:551-559.

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