Medical Molecular Farming

Biopharmaceutical products generated in transgenic plants are cheaper to produce and store than animal-derived biopharmaceuticals. Plant expression systems may also offer advantages for large-scale production and product safety. In a review article in the May 2001 issue of Trends in Plant Science (6:219-226)Henry Daniell at the University of Central Florida, Orlando, FL, Stephen Streatfield  of ProdiGene at College Station, TX and Keith Wycoff of Planet Biotechnology, Hayward, CA discuss the use of higher plants for the production of biopharmaceuticals in comparison to microbe- and mammalian-based systems. The authors describe several advantages of plant expression systems, such as the availability of economical methods for a large-scale production of pharmaceuticals in plants versus fermentation or bioreactor-requiring systems, the possibility of producing edible vaccines that do not require purification procedures, and the ability to target recombinant proteins into desired intracellular compartments to increase stability. Plant systems also minimize safety risks due to contamination with human pathogens, in contrast to expression systems relying on cultured human or animal cells. Scientific solutions to address public concerns on GMOs are also discussed in this review.

Various types of antibodies are used for therapeutic and diagnostic purposes in human medicine, and recombinant antibodies may be produced in transgenic plants. The authors recount landmark research on the production of antibodies in plants, such as the expression and assembly of immunoglobulin (Ig) heavy and light chains in transgenic tobacco plants. This work led the way to production of other forms of antibodies in plant expression systems. Four examples of potentially useful therapeutic antibodies that have been produced in plants are described: 1) a tobacco-produced IgG-IgA antibody against a surface antigen of Streptococcus mutans designed to prevent tooth decay, 2) a soybean-produced antibody against herpes simplex virus (HSV) found to prevent transmission of HSV in a mouse model, 3) a rice- and wheat-produced antibody against carcinoembryonic antigen used for cancer diagnostic and treatment techniques, and 4) a transiently-expressed antibody produced by a plant virus vector in a tobacco system used for the treatment of lymphoma.

Estimates of costs associated with plant-based systems for production of antibodies indicate as much as a ten- to twenty-fold lower cost per gram of product compared to cell culture expression systems, though in planta expression levels will significantly impact final cost. Chloroplast genetic engineering addresses this concern.

Either green biomass or seeds may be used for production, but biomass productivity is much higher for green leaf tissues. However, oilseeds or grains may have advantages for purification of the product for storage properties.

Discussing a few more points on the future line of research in this field, the authors emphasize the need to reduce the quantity of plant tissue constituting a vaccine dose to a practical size concomitant with increased expression levels of recombinant antigen such as hepatitis B surface antigen. A combination of the two factors, small size of the vaccine dose and better expression of surface antigen, will prove to be more effective for conferring immunity. Success in this direction will eliminate the need to take plant vaccines as food. The authors further suggest to concentrate on improving methods for integration of alien genes of interest into chloroplast genomes. There are about 10,000 copies of chloroplast genomes per cell. This facilitates introduction of 10,000 copies of foreign genes per transformed cell. Such an increase in gene dosage results in several hundred-fold increase in gene expression compared to nuclear transgenic plants. Such a dramatic increase has been demonstrated with the human somatotropin expressed via chloroplast or nuclear genomes.

A chloroplast transformation approach may prove to be more effective as oral vaccines because of the uniformity of foreign gene expression among transgenic lines. This is accomplished by integration of transgenes into the spacer region at a precise location of the chloroplast genome eliminating the position effect frequently observed in nuclear transgenic plants caused by random insertion of transgenes. In addition, gene silencing is not observed in chloroplast genetic engineering.

Purification of plant biopharmaceuticals by chromatography, the authors point out, adds up substantially to the cost of production. To lower the cost, the authors suggest: (a) targeting pharmaceutical proteins such as anticoagulant protein from leech (Hirudo medicinalis) to seed oil bodies of Brassica napus mediated by the fusion gene product, (b) adoption of single step purification process utilizing the inverse temperature transition properties of a protein based polymer without the use of chromatography and (c) the use of a chaperonin protein and centrifugation which facilitates a single step purification process through folding of foreign proteins.  This method has been used to demonstrate purification of proinsulin in a single step. This method has been reported to step up the purification process thousand-fold.  In addition, this system ensures the protection of foreign proteins from cellular proteases.

The potential generation of superweeds in the wake of more and more transgenics has been a matter of great public concern. The toxicity of transgenic pollen containing pharmaceuticals to non-target insects or animals is yet another concern. To prevent the formation of superweeds or contain transgenic pollen, the authors underscore the importance of generating apomictic plants, creating incompatible genomes, suicide genes and male sterility. Targeting genes of interest to chloroplasts for the production of biopharmaceuticals is another powerful way to contain harmful genes. This is accomplished by the maternal inheritance of chloroplast genomes in most crops. Such a procedure, which has been successfully demonstrated in tobacco and potato, should be extended to other crop plants.

Another approach to allay the fears of anti-GM groups will be to bioengineer plants without the use of antibiotic selection marker genes. Several methods to engineer plants without the use of antibiotic resistance genes or delete introduced genes have been discussed for both nuclear and chloroplast genetic engineering.

The authors conclude that the choice of biopharmaceutical proteins and the plant species in which they are to be produced, depends upon several practical considerations: profitable production of the biopharmaceutical, purification process, retention of pharmaceutical properties during storage, effective dosage size, efficiency of containment system, market demand and acceptability by the public. Fortunately, the state of the art currently available, in terms of optimization of protein quantity, environment biosafety and market size, is such that undertaking projects for the safe production of pharmaceuticals for clients requiring them holds a great promise for the future.

 

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