There are, of course, alternative ways of exploiting the protein synthesis machinery of plants for the production of foreign proteins. The earliest proponents of this process, however, were not plant molecular biologists, but rather the plant viruses that hijack plant cells and use them for self-reproduction. These viruses provide a direct method for the transient production of recombinant proteins in crop plants, and this is the subject of the chapter written by Grill (see Chapter 40). By inserting target genes into the viral genomes, the hijacker can be turned into a molecular farmer.In discussing how plant expression can be achieved using stably transformed plants or transient viral expression, Grill uses as examples two plant-produced proteins that have already entered clinical trials in the United States. The protein made in stably transformed plants was a humanised version of the Avicidin antibody for the treatment of prostate cancer, while the transient viral expression approach was used for the production of personalised vaccines produced from the cancerous B cells of non-Hodgkins lymphoma patients. What these examples illustrate is the flexibility of the viral vector system. Essentially, using this approach, personalised plant-based production of treatments for individual patients becomes possible, which is not feasible using stably transformed plants.
Grill then gives a detailed description of how viral vectors work in practice. Most plant viruses have single-stranded positive sense RNA genomes, and those that are assembled into rod-like particles can accommodate large inserted genes because it is possible to increase the length of the virion. Using recombinant viruses to infect plants and then produce the chosen recombinant proteins is rapid, compared to standard transgenic approaches, and has been tested in the field since the early 1990s. Grill argues that the rapid high-level expression of proteins is what sets viral vectors apart from the classical methods. Scaling up the methods for plant inoculation with viral vectors from the laboratory to the field has involved innovative solutions. An example, shown in the review figures, illustrates plants being inoculated using high-pressure sprays containing an abrasive and the recombinant viruses. A further interesting aspect of viral vectors is that there is no requirement for the cultivation of transgenic plant lines in the field. However, proteins transiently produced by viral infection have to be harvested from green leafy tissue and cannot be stored as easily as the seeds produced by transgenic plants.
Grill puts the case, and we agree, that while viral vectors compare well with transgenic plants overall, their real advantage is the ability to produce proteins rapidly. Indeed, the use of viral vectors to produce patient-personalised vaccines against non-Hodgkins lymphoma is such a compelling prospect that we feel viral vectors must remain an important tool for farming molecular medicines in the future.
A Perennial Production System
In their contribution, D'Aoust et al. describe the use of perennial plants for the production of recombinant proteins. Perennial plants are interesting as production systems because, with several harvests possible from the same crop, the plants can be used for repeated extraction of the protein.
The authors argue the case for forage legumes and, in particular, discuss the merits of using alfalfa. Because of the difficulties that had to be overcome to enable protein expression and plant transformation, forage legumes are relatively new to molecular farming. The authors describe the advances in promoter technology that have led to the construction of efficient inducible or strong leaf-specific expression cassettes. The features of the available transformation methods are discussed for alfalfa, which was first transformed more than 15 years ago, with D'Aoust et al. stating that reliable and efficient Agrobacterium-mediated transformation has been made possible by the optimal choice of genetic background and the use of standardised transformation protocols.
Because alfalfa is a fodder crop for ruminants, high-protein-yield varieties have been obtained and large-scale protein extraction techniques have been established by the animal feed industry. The authors describe how they are adapting these techniques for the production of plant-derived vaccines and, through the description of a case study, illustrate successful antibody expression in alfalfa.
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