Facilitating viral vector movement enhances heterologous protein production in an established plant system.

Molecular farming technology using transiently transformed Nicotiana plants offers an economical approach to the pharmaceutical industry to produce an array of protein targets including vaccine antigens and therapeutics. It can serve as a desirable alternative approach for those proteins that are challenging or too costly to produce in large quantities using other heterologous protein expression systems. However, since cost metrics are such a critical factor in selecting a production host, any system-wide modifications that can increase recombinant protein yields are key to further improving the platform and making it applicable for a wider range of target molecules. Here, we report on the development of a new approach to improve target accumulation in an established plant-based expression system that utilizes viral-based vectors to mediate transient expression in Nicotiana benthamiana. We show that by engineering the host plant to support viral vectors to spread more effectively between host cells through plasmodesmata, protein target accumulation can be increased by up to approximately 60%.

Production of recombinant proteins in clonal root cultures using episomal expression vectors.

We have developed a fully contained system for expressing recombinant proteins that is based on clonal root cultures and episomal expression vectors. Clonal root lines expressing green fluorescent protein (GFP) or human growth hormone were generated from Nicotiana benthamiana leaves infected with the tobacco mosaic virus-based vector 30B after exposure to Agrobacterium rhizogenes. These lines accumulated GFP at over 50 mg per kg fresh tissue, a level that is comparable with other plant production systems in early stage development. Accumulation of both hGH and GFP in the clonal root lines was sustained over a 3-year period, and in the absence of antibiotic selection. This technology shows promise for commercial production of vaccine antigens and therapeutic proteins in contained facilities.

Deletions within the 3' non-translated region of Alfalfa mosaic virus RNA4 do not affect replication but significantly reduce long-distance movement of chimeric Tobacco mosaic virus.

Alfalfa mosaic virus (AlMV) RNAs 1 and 2 with deletions in their 3' non­translated regions (NTRs) have been previously shown to be encapsidated into virions by coat protein (CP) expressed from RNA3, indicating that the 3' NTRs of RNAs 1 and 2 are not required for virion assembly. Here, we constructed various mutants by deleting sequences within the 3' NTR of AlMV subgenomic (sg) RNA4 (same as of RNA3) and examined the effect of these deletions on replication and translation of chimeric Tobacco mosaic virus (TMV) expressing AlMV sgRNA4 from the TMV CP sg promoter (Av/A4) in tobacco protoplasts and Nicotiana benthamiana plants. While the Av/A4 mutants were as competent as the wild-type Av/A4 in RNA replication in protoplasts, their encapsidation, long-distance movement and virus accumulation varied significantly in N. benthamiana. These data suggest that the 3' NTR of AlMV sgRNA4 contains potential elements necessary for virus encapsidation.

Hairy roots as a vaccine production and delivery system.

Prevention of infectious diseases by vaccination is often limited because of the lack of safe, effective, and accessible vaccines. Traditional vaccines are expensive and require special conditions for storage, distribution, and administration. Plants have potential for large-scale production of a variety of inexpensive and highly effective recombinant proteins for biomedical and pharmaceutical applications, including subunit vaccines. There are several approaches for the production of vaccine antigens in plants, including transient expression systems based on Agrobacterium delivery of binary vectors or plant viral vectors, stable transgenic plants, and plant cell or tissue cultures. Axenic plant cultures maintained under defined physical and chemical conditions appear to be an attractive production platform when target proteins need to be synthesized in a fully controlled environment. Hairy root cultures meet the criteria for such a system. Hairy root cultures, generated from edible plants and producing target antigens, provide a potential approach for the development of vaccines for oral delivery. With this approach, there are no protein extraction and purification costs and the active biomolecule is protected by the plant cell wall during passage through the upper gastrointestinal tract. This allows for gradual release of antigen at mucosal surfaces in the gut. Lyophilized hairy root cultures expressing vaccine antigens can be stored at ambient temperature for extended periods of time, which should facilitate storage and distribution, ultimately allowing for large populations to be vaccinated.

Plant-produced human growth hormone shows biological activity in a rat model.

Plants have been shown to be efficient systems for expressing a wide range of recombinant proteins from various origins. Here, using a plant virus-based expression vector to produce human growth hormone (hGH) in Nicotiana benthamiana plants, we demonstrate, for the first time, that the plant-produced hGH (pphGH) is biologically active in a hypophysectomized rat model. We observed an average weight gain of approximately 17 g per animal in a group of 10 animals that were injected subcutaneously with pphGH with 60 microg/dose for 10 days. With the increasing demand for hGH, accompanied with the need to make this recombinant protein available to a wider population at a more reasonable cost, plants provide a feasible alternative to current production platforms.

Plastid transformation in Lesquerella fendleri, an oilseed Brassicacea.

A plastid transformation protocol was developed for Lesquerella fendleri, a species with a high capacity for plant regeneration in tissue culture. Transformation vector pZS391B carried an aadA16gfp marker gene conferring streptomycin-spectinomycin resistance and green fluorescence under UV light. Biolistic transformation of 51 Lesquerella leaf samples, followed by spectinomycin selection, yielded two transplastomic clones. The AAD-GFP fusion protein, the marker gene product, was localized to chloroplasts by confocal laser microscopy. Fertile plants and seed progeny were obtained in line Lf-pZS391B-1. In the 51 samples a large number (108) of spontaneous mutants were identified. In five of the lines spectinomycin resistance was localized to a conserved stem structure by sequencing 16S rRNA genes. Success in L. fendleri, a wild oilseed species, extends plastid transformation beyond Arabidopsis thaliana in the Brassicaceae family.