Agroinfiltration as an Effective and Scalable Strategy of Gene Delivery for Production of Pharmaceutical Proteins

Abstract

Current human biologics are most commonly produced by mammalian cell culture-based fermentation technologies. However, its limited scalability and high cost prevent this platform from meeting the ever increasing global demand. Plants offer a novel alternative system for the production of pharmaceutical proteins that is more scalable, cost-effective, and safer than current expression paradigms. The recent development of deconstructed virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins, and in turn, provided a preferred plant based production platform. One of the remaining challenges for the commercial application of this platform was the lack of a scalable technology to deliver the transgene into plant cells. Therefore, this review focuses on the development of an effective and scalable technology for gene delivery in plants. Direct and indirect gene delivery strategies for plant cells are first presented, and the two major gene delivery technologies based on agroinfiltration are subsequently discussed. Furthermore, the advantages of syringe and vacuum infiltration as gene delivery methodologies are extensively discussed, in context of their applications and scalability for commercial production of human pharmaceutical proteins in plants. The important steps and critical parameters for the successful implementation of these strategies are also detailed in the review. Overall, agroinfiltration based on syringe and vacuum infiltration provides an efficient, robust and scalable gene-delivery technology for the transient expression of recombinant proteins in plants. The development of this technology will greatly facilitate the realization of plant transient expression systems as a premier platform for commercial production of pharmaceutical proteins.

Keywords: Agrobacterium tumefaciens; Agroinfiltration; Geminiviral vectors; Gene delivery; Monoclonal antibody; Nicotiana benthamiana; Plant infiltration; Plant-made pharmaceuticals; Syringe agroinfiltration; Vaccines; Vacuum agroinfiltration.

Figure 1

Syringe agroinfiltration of N. benthamiana leaves with Agrobacterium tumefaciens. A. tumefaciens harboring the gene of interest was resuspended in infiltration buffer and loaded into a syringe without a needle. A nick was created with a needle on the backside of a 6-week old plant leaf (A). Agrobacteria were injected into the interstitial space of the leaf via the nick (B and C) [39].

Figure 2

A single geminiviral BeYDV vector that can generate two non-competing replicons for co-expression of two proteins or one protein with two hetero-subunits. The left (LB) and right border (RB) delineate the T-DNA construct that will be transferred into plant cells by Agrobacterium. There are two gene constructs with each flanked by two LIR (red rectangles). Upon delivery into plant cells, expression of C1/C2 gene (next to the Gene 2 construct) produces the Rep protein that nicks the LIRs in the T-DNA to release two separate single-stranded DNA molecules. They are then copied to make double-stranded DNAs that can replicate by the rolling circle mechanism. The two replicons are amplified independently and non-competitively to produce high copy numbers of DNA templates and, in turn, abundant mRNAs for the translation of Protein1 and Protein 2. The expression of the gene silencing suppressor p19 further enhances the expression of both gene products. Yellow rectangles: SIR; LB: left border of the TDNA; RB: right border; p19: expression cassette for p19, a suppressor of gene silencing from TBSV; 35S/TEV5′: CaMV 35S promoter followed by tobacco etch virus 5′UTR; VSP3′: soybean vspB gene terminator; 35S/TMV5′: CaMV 35S promoter and TMV 5′UTR; rbsS3′: tobacco rbcS gene terminator; C2/C1 Rep/RepA gene [40].

Figure 3

Vacuum agroinfiltration of N. benthamiana leaves with Agrobacterium tumefaciens. A. tumefaciens containing target gene construct was resuspended in infiltration buffer and loaded into a desiccator that was connected to a vacuum pump (A) The entire leaf system of a 6-week old plant was then submerged into the infiltration buffer (B). Agroinfiltration was achieved by applying and releasing a vacuum through the pump.

Figure 4

Expression of the green fluorescent protein in vacuum agroinfiltrated leaves. N. benthamiana leaves were infiltrated with A. tumefaciens harboring the GFP gene in MagnICON vectors. Leaves were photographed in a dark room under UV light 6 days post agroinfiltration [41,42].

Figure 5

Commercial scale N. benthamiana plant growth (A) and agroinfiltration (B). (The photographs in this figure are kindly provided by Mr. Barry Bratcher of Kentucky Bioprocessing, LLC.).