This research study aims to investigate the possibility and capacity of the Nicotiana tabacum, adicot plant species as a host organism for the production of heterologous proteins. In this research study, the production systems of different studies host organisms were compared using available literature reviews. It also includes an overview on the mechanisms of the expression of heterologous proteins in plants. The study adapted simple methodologies from past significant studies that replicated in the laboratory. This study also raised recommendations on the possible use of such plant species for commercial reproduction.
There is a recorded increase in the demand for commercially available therapeutic proteins such as insulin and growth hormones. Such proteins and products are used for industrial, personal and clinical purposes, but commonly for maintaining health and treating diseases and illnesses. This increase in the number of patients and customers availing for such therapeutic substances is related to the unhealthy lifestyle and degraded environment that people are living with right now.
Adenosine deaminase or ADA (EC 220.127.116.11) is a ubiquitous enzyme which considers being important in the thymocytes development. Adenosine deaminase converts adenosine into inosine and converts deoxyadenosine (dAdo) into deoxyinosine, this process happens through the hydrolysis of the purine amino group. Adenosine deaminase can be found in all tissues of the body, but has more activity in development of lymphocyte. This may be due to CD26 direct association, which showed on activated T cells. ADA activity is particularly high in thymic cortex' thymocytes, but decreases rapidly in the medulla. There are two enzymes which facilitates ADA activity, called ADA1 and ADA2. ADA1 is a monomeric protein with 40 kD, a noncatalytic combining protein which carries about 90% of adenosine deamination. In parallel, ADA2 is somewhat bigger at 110 kD, appears to play an important adenosine deamination role in serum (Parkman, 1991).
Decreased activity of ADA enzyme in human results in primary immunodeficiency disorder called severely combined immunodeficiency (SCID) in which various immune functions are impaired due to decreased immunoglobulin production. Individuals with ADA deficiency have abnormal accumulations of dATP (deoxyadenosine triphosphate) and dAdo, which is the end-product of phosphorylated dAdo.
The dATP (deoxyadenosine triphosphate) and dAdo accumulations result in the S-adenosylhomocysteine hydrolase (SAH) enzyme inactivation, whose accumulation facilitates certain methylations of proteins, nucleic acids, and lipids. High levels of these ernzymes can also cause ribonucleotide reductase inhibition causing a deoxynucleotides (dNTP) imbalance. These imbalances, in addition to the inhibitions of enzymes, leads to DNA synthesis impairment and T lymphocytes repair (Ochs, 1992).
The symptoms associated with ADA deficiency are usually immediately visible in affected infants, since the disorder is genetic in nature, but there have been reported cases of mild ADA deficiency which were not been manifested or detected until older childhood and even in adulthood years. The complete ADA deficiency can results in fatal infantile onset syndrome of SCID. Even in some mild cases, the T-cell function is depressed severely, and the responses of antibody are barely produced, that result in a highly immunosuppressed individual. Patients with ADA deficiency exhibit growth retardations which are more susceptible to lymphopenia, opportunistic infections, defective humoral and cellular immune responses. There are at least 40 identified alleles that have been proved to cause ADA deficiency. Their appearances in a specific location on the human gene are unusually susceptible to gene mutations in the characterized so called hot-spot allele mutations (Parkman, 1991).
Enzyme replacement therapy for patient with ADA deficient SCID is administered through intravenous injections when there is lower enzyme activity or absence of enzyme. Enzyme replacement therapy does not alter any immunological response and immunological parameters are very short lived.
Enzyme therapy can directly replace the missing ADA enzyme. This can happen through irradiated red blood cells transfusions. Patients undergoing this therapy experienced equally depressed antibody responses as untreated ADA deficient patients. Direct injections of enzymes are a better way of ADA introduction to the patient. There were also several research studies which have discovered more effective ways of taking high yield human ADA by various cells and insect larvae transfection with human ADA cDNA (Ochs, 1992).
In the treatment process, the human or bovine ADA is attached covalently to polyethylene glycol (PEG), which deters degrative enzymes access to ADA, facilitating lengthy plasma half-life from a few minutes up to 24 hours. The weekly introductions of "PEGilated" ADA usually interpulate the main genomic ADA deficiency consequences, as the level of lymphocyte and proliferative responses to antigen normalizes. The dAdo and dATP levels are also reduced, as expected in an effective mode of treatment (Ochs, 1992).
Bone Marrow Transplantations
Bone marrow transplantation is another kind of treatment for ADA-SCID patients and transplantation is done from HLA-identical patients or other close relative in inbred communities. Bone marrow transplantation has greatest efficacy, does not require any ongoing therapy and shown relatively low morbidity and mortality (Parkman, 1991).
The bone marrow or stem cell transplants from a haploidentical donor are available for a minority of patients. Since the nature of disorder is genetic, the haploidentical donors with no ADA deficient are harder to find than for leukemia or another bone marrow transplant cases. Bone marrow transplants can be done to correct general cases of SCID, and the new marrow cells have the appropriate types of genes, including ADA+, to reconstitute immune function and to develop normal T and B cells (Parkman, 1991).
Gene Therapy is the first disease which gene therapy has been applied. This mode of treatment demonstrates improve gene transfer necessarily improvement in the specific immune functions and has therapeutic in vivo. Functional ADA T cells can be created by using somatic gene therapy. Bone marrow cells and peripheral blood lymphocytes are the initial targets for gene therapy (Blaese, 1995).
The method of gene therapy has more concerns regarding ethical issues than the other treatment methods, but gene therapy has key advantages to its use in this kind of disorder. First, the SCID patient can continue PEG-ADA treatment, so that no treatment is being withhold. Second, the live virus is not transfused into the patient.
The activity of ADA is improved substantially, and short term (6-12 months) immunity is restored effectively. In this treatment method, the genetically modified cells with properly functioning ADA have a significant advantage over endogenous ADA deficient cells during the development. Even the growth was normalized in some SCID patients whose growth was not improved using the PEG-ADA treatment (Blaese, 1995).
Heterolougous Proteins in Plants
This new trend in technology accommodates the stable integration of the transgene (the introduced gene) in the genome chloroplast, which can be transferred not by the pollen but through cytoplasmic inheritance. Interestingly, limited chloroplast DNA and only limited amount of cytoplasm in the pollen. Through the transformation technology of chloroplast, the transgene, which can be found in the egg cell, do not have the characteristic to transferred or moved to plant through pollen but only through inheritance of cytoplasm. Therefore, therapeutic genes introduced in tobacco Nicotiana tabacum through the transformation technology of chloroplast cannot be transferred or delivered to other plants. By nature, the Nicotiana tabacum is a self-pollinating plant, hence, the spread of its pollen is limited.
Because plants can be cultured and scaled at a large amount, it has been said that the large scale production in agriculture of therapeutic proteins and its significant contribution in producing vaccines is necessary to meet such an increasing demand in the global market at a reasonable cost (Daniel, 2003). There have also been studies and applications of the cost-effective system for the manufacturing of large amounts of complex proteins using transgenic milk production (Echelard et al., 2006). Studies have also been conducted on bacterial species such as the Lactobacillus sp. The experts' knowledge on the characteristics of the acid secreted by the said bacteria has led researchers and pharmacists to investigate their full potential for new application such as the discovery and production of heterologous therapeutic proteins in fermented food products, bio-reactors, in or directly in the digestive tract of animals and even humans (Nouaille et al., 2003).
However, there have been also recorded limitations and shortcomings in some of the earlier devised and developed mechanisms of therapeutic protein productions. The common method of producing these proteins with the use of microbial expression systems such as E.coli and the yeast system S. cerevisiae are very costly (Grummt and Grummt, 2001). Also, there is a high possibility of contamination of the protein fraction with human viruses and other disease causative agents found in human or animal sources. This leads to the limited availability of variety of therapeutically important proteins (Roy and Agarwal, 2003). Also, protein production using plants also has its limitations. Those who are using plants as the host organism which synthesize whole plants have several disadvantage in the intrinsic benefits of cultured cells, including the proper and precise control over production and growth conditions, group product consistency, a high level possibility of containment and the ability to provide the number of recombinant proteins in accordance with good practice of manufacturing (Grummt and Grummt, 2001). In general, the advantages that can be gained from plants as the host for therapeutic protein production for commercial use is very promising and still needs to be explored using other species aside from those that have already been studied.
In this purpose, Nicotiana tabacum is a good choice among other plants because the pharmaceutical product can be generated or produced at significantly high level. The agricultural practices and methods of planting and managing Nicotiana tabacum as a crop product are also well managed and regulated in most countries. Nicotiana tabacum can grow and survive in areas with less source of water. It is not only useful economically but also environmentally friendly. Moreover, Nicotiana tabacum is not a plant food and the therapeutic protein- or pharma-containing Nicotiana tabacum will not affect the food chain. There are ongoing studies and researches in many pharmaceuticals that uses plants are using as biofactory.
The use of Nicotiana tabacum as plant producing heterologous therapeutic proteins that have significant therapeutic applications can be a boon to our Nicotiana tabacum farmers and local industry. Nicotiana tabacum will then consider beneficial especially to health industry, after all.
The use of these pharma- plants and plants suspension cells gives opportunity to an inexpensive and more convenient method for the production of valuable recombinant proteins in a large scale (Schillberg, 2008). Thus, the findings and discoveries that could be made from this study would be of great contribution to the field of knowledge and would be beneficial for both the professionals in practice and the academe. This study will produce an output that would detail out the mechanisms of reactions and important processes for the understanding of the use of plants as host organisms in protein production. It will also further discuss the advantages of using plants in "molecular farming", as to be experienced in the laboratory and actual experiments (Boehm, 2007).
Like cells found in mammals , plant production systems have greater advantage over microbial systems of being able to produce active forms of complex proteins with appropriate modifications of post-translation like glycosylation. However, these cells used for the production of biological proteins must be carefully managed to decrease the possibility of unintentional transmission of any diseases in viral nature that will infect humans. Hence, this is not a risk to produced biologics in plant systems. Nevertheless, production of therapeutic proteins using plant expression systems have unique characteristics such as containment to prevent gene transfer to conventional crops during plant growth and possibly higher costs to extract the desired protein due to the presence of interfering compounds in plants (Boehm, 2007).
Transgenic plants have become the major systems for the discovery and production of heterologous therapeutic proteins because of the decrease probability of risk in mammalian viral contaminants, and the ability of plants to produce large scale production at low cost, and requires very low maintenance requirements. The research study for the production of these therapeutic proteins through transplastomic transformation technology is in progress, it has the additional advantage and benefits for the increasing biological containment by apparent elimination of the transmission of transgenes. Chloroplasts have the ability to express a secretory therapeutic protein, human produced somatotropin, in a biologically active, chemically soluble, disulfide-bonded form. The high concentrations of recombinant therapeutic protein accumulation are observed which about seven percent of the total soluble protein is. More than 300-fold above than a similar gene being expressed using a nuclear transgenic approach.
Recent studies shows that many pharmaceuticals used for the treatment of diseases have been based largely on the productivity of relatively small organic molecules which is synthesized by microbes, animals or by means of organic chemistry. These productions include most the antibiotics to prevent the growth of bacteria, analgesics, synthetic hormones, and other pharmaceuticals products.