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Every year human spend billions of dollars on prescription medication. Unfortunately, many are finding it increasingly more difficult to afford these expensive but necessary drugs. The rapidly escalating costs of pharmaceuticals and the prospective need for mass-produced vaccines make it necessary to produce these medicinal compounds more economically and in greater quantity. Recombinant DNA technology, the artificial manipulation and transfer of DNA between species, has begun to tackle both problems by introducing many medicine-producing genes into mammalian and bacterial cells. Chloroplast genetic engineering is an exciting technology that has the potential to produce biopharmaceuticals more efficiently and provide them to those who need them most.
Why Plant Engineering?
Genetically engineered mammalian cells are superior to their bacterial counterparts because, unlike bacteria, they contain molecular machinery that can produce proteins that are identical to those formed in the body. Bacterial vectors such as E. coli cannot correctly modify these proteins, so they are often incorrectly folded. Therefore, bacterial products require expensive post-processing procedures to form usable proteins; in fact, this accounts for 60% of the cost for the commercial production of insulin using E. coli. However, mammalian cells have their own disadvantages: unlike bacteria, they are very expensive to culture, require high maintenance, and are capable of only low levels of protein production. A better alternative to these two systems of production is plant genetic engineering. Recently, scientists have begun transforming "pharmacrops" to generate pharmaceuticals because this approach has several unique advantages. First, plants do not require industrial bioreactors, wherein recombinant proteins are produced, because they can create and store proteins in their cells. Furthermore, the technologies already exist to mass harvest and process these plants. Secondly it is unnecessary to isolate the desired pharmaceutical. Since the therapeutic compound is produced and stored in plant tissue, it might be possible to receive the benefits of the medicine simply by eating the plant.
Nuclear Transformation VS Chloroplast Transformation
Plants have two main reservoirs of DNA: nuclei and chloroplasts. Therefore, both of these are potential targets for genetic engineering. Transforming the nuclear DNA of these plants, however, has provoked a great deal of controversy due to its potentially harmful ecological effects. If recombinant genes (known as transgenes) were to be disseminated through pollen and integrated into other plants, invasive species and widespread ecological damage could result. For example, an herbicide-resistance gene in a genetically modified (GM) crop that transferred to a weed could lead to its ceaseless proliferation. Secondly nuclear transformation can also be harmful to the individual plant itself because the transgenes are integrated into the plant's nucleus at random locations. This can deactivate other important genes and cause deleterious changes in the host organism. Also, nuclear modification of plants is not very efficient because there is only one nucleus per cell and, at most, a few copies of the recombinant gene, producing relatively low levels of protein.
Each plant cell contains approximately 100 chloroplasts and each chloroplast contains about 100 copies of its genome. So, chloroplast genetically-engineered plants have high levels of integration of transgenes-up to 10,000 copies per cell-which elevate expression levels of recombinant proteins. It is useful in evolving vaccine producing plants as it enables high level of protein production. Also there is the possibility of producing multiple proteins using polycistronic mRNAs. Unlike nuclear transformation, this method ensures that the recombinant transgenes are contained within the chloroplast and therefore will not spread to other plants. Chloroplasts (and the genes they contain) are not passed in the sperm (i.e., pollen) of a plant, so they cannot be spread by pollination. Researchers demonstrated that, even though chloroplasts in leaves were modified to express an insecticidal protein, called CRY, at very high levels (47% total soluble protein), the pollen did not contain any traces of the protein. Transgene expression is more stable in Transplastomic plants than in nuclear transformed plants because transgenes are integrated into chloroplast genomes by homologous recombination and not affected by gene silencing.
How Chloroplast Genetic Engineering Achieved?
In chloroplast genetic engineering, the recombinant DNA plasmid is bound to small gold nanoparticles that are then injected into the chloroplasts of a leaf using a "gene gun." This device uses high pressure to insert the plasmid-coated particles into the cell. These plasmids contain multiple important genes: the therapeutic gene, a gene for antibiotic resistance, a gene that increases expression of the therapeutic gene, and two flanking sequences that ensure that the plasmid is not randomly integrated into the chloroplast genome. In brief, the flanking sequences guide the human recombinant DNA into a specific place on the chloroplast genome by binding to corresponding parts on the genome. The leaf is then grown on a plate containing an antibiotic, which ensures that the only surviving plant cells will be those that contain the gene for antibiotic resistance and therefore contain the therapeutic gene as well. These cells are then exposed to regenerative factors that induce them to start sprouting shoots and grow into full plants that express the desired proteins.
Chloroplast engineering also allows for large-scale protein production. Scientists reported that the levels of pharmaceutical proteins produced in nuclear-modified plants are less than 1% of the levels needed for the purified protein to be commercially feasible. A number of therapeutic proteins have been produced using the chloroplast genetic engineering system. These include human somatotropin (growth hormone), serum albumin (blood protein), insulin-like growth factor (hormone), antimicrobial peptides (proteins that kill pathogens), interferon alpha/gamma (cytokines in the immune system which are effective against hepatitis and leukemia), monoclonal antibodies (immune system molecules that fight off invading pathogens and toxins), and vaccines to cholera, plague, canine parvovirus (dog virus) and anthrax. Vaccines are, of course, needed to immunize people from harmful pathogens, such as the polio virus, but many times there is a shortage of the amount of vaccine available. Plague vaccine, which immunizes against Yersinia pestis, has been expressed in transgenic tobacco plants at commercially feasible levels. In addition, the canine parvorvirus vaccine (CPV), which protects dogs against CPV and stomach complications, has been expressed highly. Recently, a team of scientists working on chloroplast genetic engineering reported achieving such high levels of expression of the anthrax protective antigen that, according to their extrapolation, one acre of transgenic tobacco could produce about 400 million doses of the vaccine. Most of the therapeutic proteins produced by chloroplast genetic engineering are still in the developmental stage and need to be tested in humans. Chlorogen Inc. is a company that is working to commercialize this technology and bring the plant-produced therapeutic proteins to the market. According to Chlorogen's site, their first product will be human serum albumin for the non-therapeutic market.
The benefits of this technology can be harvested not only for the improvement of agriculture, but also for the growth of pharmaceutical industries for the production of vaccines, recombinant proteins and antibodies through chloroplast transformation of crops. In addition to introducing resistance genes against insect, pests or herbicides, the plastid transformation has the potential to improve crop plants in various other ways such as improved photosynthesis through RuBisco engineering improved tolerance to drought, salinity and resistance to bacterial and fungal pathogens. A potential application of this technology is the production of therapeutic proteins or vaccines in plants indigenous to third-world countries where people do not have access to these medicinal compounds. In addition, genetically engineering the chloroplast is environmentally friendly since the transgenes are contained within the plant and the proteins they code for do not harm the plant itself. However, more work is required before chloroplast genetic engineering can be applied commercially. This work will probably include modifying more types of crops and plants as well as ensuring the functionality of the resultant therapeutic proteins in humans. But it may not be too far in the future when mothers may nag their children not only to eat their broccoli, but to eat their transgenes.
ALOK KUMAR PATRA
BTECH (BIOTECH), 6th sem