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Adenosine Deaminase (ADA) is a type of cytosolic enzyme of purine metabolism, which irreversibly catalyses the deamination of 2`- deoxyadenosine and adenosine. The main function of adenosine deaminase enzyme is to eliminate a molecule called deoxyadenosine, this is generated when DNA is broken. In humans the highest ADA activity is found in thymus and other lymphoid tissues, the lowest in erythrocytes (Hershfield and Mitchel, 1995). The enzyme adenosine deaminase is produced by giving instruction from ADA gene. This type of enzyme is mostly present in Lymphocytes and also present in the white blood cells that protect against harmful invaders. Mutations in ADA gene eliminate or reduce the activity of adenosine deaminase and builds up the deoxyadenosine to levels that are toxic to lymphocytes.
ADA deficiency is a form of SCID (severe combined immunodeficiency); it's a type of disorder which affects the human immune system, this disease was first inherited to be treated with a gene transfer method. ADA deficiency is a disorder which is autosomal recessive and is usually linked to a wide range of clinical and mutational variation. Infants with this disorder usually lack protection of all immune protection from bacteria, viruses and fungi. Children with SCID grow much more slowly than healthy children and also experience chronic diarrhoea, pneumonia and widespread skin rashes.
Though there is no complete cure for this deficiency, for improving the immune system there are few treatments available, some of those are:
Enzyme replacement therapy - with polyethylene glycol-modified bovine adenosine deaminase (PEG-ADA).
Bone marrow transplantation / Stem cell therapy - from a "non-ideal" donor, which may be an HLA-matched unrelated donor, and HLA-haploidentical donor (usually a parent), or umbilical cord-derived stem cells.
Gene therapy - gene transfer in hematopoietic stem cells.
Enzyme replacement therapy -ADA deficiency can efficiently be treated by means of enzyme replacement therapy [Morel, 1995]. Polyethylene glycol-modified adenosine deaminase (PEG-ADA) has now been used for several years as enzyme replacement therapy for immunodeficiency due to ADA deficiency. It restores a metabolic environment necessary for recovery of immune function. The other therapeutic option includes enzyme replacement therapy (ERT) with pegademase bovine (PEG-ADA). To extend the transmission of ADA enzyme in the blood, Bovine adenosine deaminase, will process the catabolism of the lethal adenosine and deoxyadenosine metabolites which is mixed with polyethylene glycol (PEG) [Bordignon et al., 1995].
Review of Literature
Human Adenosine Deaminase is a catabolic enzyme that is involved in the irreversible deamination of adenosine and deoxyadinosine. A study conducted on the transient expression of Human Adenosine Deaminase explored the function of the cDNAs transfected into cultured cells. Among the inserts one was observed to be nonfunctional and it's unable to produce the protein. The cDNA inserts containing the entire coding region of the gene was constructed and cloned into the plasmid. The cultured monkey kidney cells were transfected with the plasmids. Western blot analysis and oligonucleotide analysis of the cDNA was done. An unexpected finding in this study was the presence of an insert that was not able to produce human ADA upon transfection. Restriction mapping of this insert uncovered the fact that a single nucleotide substitution might have altered the protein conformation (Orkin et al 1985).
Petolina and colleagues did an experiment on the ADA gene in maize; the gene was controlled by the maize promoter and intron region. The developed callus was selected by the presence of herbicide resistance which resulted in transgenic cultures that accumulated a 41 kD protein that precipitated with ADA specific polyclonal antibody. The enzyme activity was observed in callus and also in regenerated plants. The result of the experiment suggested that ADA along with its analogue can be used as an efficient selectable marker system for maize (2000).
Plants have gained wider importance as cheap sources for the production of recombinant proteins in larger scale. Animal cells are quite expensive and moreover are not stable over several environmental changes. The production of ADA was showed recently in transgenic maize (Giddings). Two different approaches are developed for the production of proteins in plants. Insertion of foreign DNA into the nuclear DNA of plants to produce recombinant ones is one among them. This involves indirect gene transfer. The success of this approach depends on several factors including the random insertion of DNA and positional effects. Several techniques, using viral suppressor, are used to increase the yield of proteins (Mallory et al, 2002).
Transient expression of the foreign proteins is the second approach. This involves the use of genetic engineering techniques to produce genomic DNA clones from plant RNA viruses by reverse transcription. The viruses infect the plants and the foreign protein is expressed along with the viral replication (Shih, 2009). The expression systems used are selected on certain essential features as its capability in producing the required protein in its correct conformation and easy handling and maintenance. Easier downstream processing is another major concern (Desai et al, 2010).
The above article also speaks about certain advantages and limitations of the plant expression systems. The benefits include the industrial scale production of proteins but growing large number of plants and the ease in the purification of the product. Another important plus point is the production of the proteins in the edible parts of plants. The drawback of this system is the low levels of protein produced and low accumulation. Moreover the produced proteins are seen to be attached with certain plant specific sugar molecules that can lead to immunogenicity.
The main host for synthesis of many industrial and pharmaceutical proteins are plants, which offers significant advantages in safety and cost over alternative foreign protein expression systems (Twyman et al., Streatfield, 2007). Some of the advantages associated with plants include the elimination of downstream processing requirements for vaccines expressed in edible plant tissues, effect in the cost of agricultural biomass production , post-translational modification and production of assembled and correctly folded multimeric proteins. This has got low risk of contamination with endotoxins and pathogens which also occur in mammalian and bacterial systems, and the avoidance of ethical problems associated with transgenic animals and animal materials.
If the protein must be purified, the levels of pharmaceuticals proteins produced in transgenic plants must have been less than the 1% of total soluble protein which is needed for commercial feasibility. During recent studies, plant derived recombinant hepatitis-B surface antigen induced only a low level serum antibody response, it is probably due to reflection in the low level of expression in transgenic lettuce (Daniell, Streatfield and Wycoff).
Expression of genes encoding other human proteins in transgenic plants has been very low. For the human epidermal growth factor, the synthetic gene coding was expressed only up to 0.001% of total soluble protein in transgenic tobacco.
The essential things for production of heterologous proteins are a gene or cDNA encoding desired protein, a suitable vector and a expression system or biological system which can transcribe and translate the transgene into a desired protein.
An expression system should be safe and economic, easy to handle and maintain, must have good productivity, should be capable of producing required protein with right conformation and must be afforded for easy downstream processing.
For the recombinant protein production an efficient plant transgene expression system, consisting of a promoter, targeting signal peptide, codon usage optimization of target gene and transcription terminator is essential.
Plant viral vectors, designed to increase the target gene copy number by the action of the viral replicase are alternatives for regulated expression of foreign genes. (Mori & Dohi, 2006)
One of the most common techniques for generating recombinant plants is to introduce foreign DNA into the plant nuclear genome. This can be achieved by gene transfer indirectly, which is involved in the cloning of foreign genes into binary vectors based on the Ti plasmid of Agrobacterium tumefaciens to replace the bacterial gene causing crown gall disease. On the wild type plants, during bacterial infections, the foreign gene is inserted into the plant genome by Agrobacterium-mediated gene transfer. By using this process, the foreign gene is inherited by many generations of transgenic plants.
Many heterologous proteins have been produced in nuclear transgenic plants and plant tissue cultures (Hellwig et al., 2004). However, the success of particular expression systems depends on several factors like the substantial variations in expression levels between individual transgenic plants which can be seen by DNA integration into the plant genome and related 'position' effects. By this, multiple locus insertions may lead to gene silencing, transgene instability and low levels of foreign protein accumulation. In transgenic plants, foreign protein expression can be enhanced using a range of molecular and genetic approaches (Streatfield, 2007).
In recent studies, production of recombinant proteins in plants offers considerable advantages over the conventional systems:
It is an economical system as compared to mammalian cell culture and microbial fermentation. In this system desired protein can be, although this depends on the protein of interest, product yield and crop used (Twyman et al., 2003).
Desired proteins are expressed in various targeted cells or tissues like seeds, tubers etc., where they are more stable and the transportation is easy without refrigeration, increasing its stability for up to few years (Twyman et al., 2003).
By cultivation of more plants, industrial scale production can be achieved. However, quality and quantity of proteins may affect from lab scale to agriculture scale.
Therapeutic proteins derived from plants, are less likely to be contaminated with human pathogenic microorganisms than those derived from animal cells, because plants are not hosts for human infectious agents (Giddings et al., 2000).
Proteins can be expressed in the edible part of the plant and can be consumed raw as an edible vaccine without the need for downstream processes, e.g. rabies vaccine expressed in tomato (Mason et al., 2002).
Downstream processing when required is easy and less expensive, particularly when protein is expressed in specific tissues like seeds (Seon et al., 2002).
Few limitations in plant transgenic system are the use of plant as an expression system for production of therapeutic proteins has not moved from developmental research to commercial productions. The major limitations of this system are low level of protein expression, low level of accumulation of expressed protein (Daniell et al., 2001) and incorrect post-translational modifications of the protein. The main difference between the proteins produced by plant and animal is in sugar molecules attached to a protein. Proteins produced by plants lack the terminal galactose and sialic acid residues commonly found in animals. Therefore, plant produced glycoproteins can lead to immunogenicity and create certain regulatory issues (Sethuraman & Stadheim, 2006). For the production of therapeutic proteins in the exploitation of plants, it is necessary to inhibit incorporation of some undesirable plant specific sugar molecules and to add newsugar molecules to obtain humanized glycoproteins. Apart from protein expression, accumulation level and stability of the final product are crucial parameters, which affect the economics of protein production. Before using the proteins that are not extracted and purified, its expression must be sufficiently high to ensure the efficiency. For an edible vaccine, a sufficient dose of antigen, which is needed to confer protection, must be delivered in a quantity of plant tissue that can be practically ingested in a single serve. In plant research, to increase the level of expression of therapeutic proteins, efforts are being focused (Streatfield, 2007).
Some of the Factors influencing the heterologous protein production in plants are choice of expression vectors, integration of foreign gene, transcription, translation, final yield or protein accumulation and gene silencing. For stable expression of a complex therapeutic protein, which requires proper post-translational modifications, Agrobacterium-mediated transformation is a method of choice in which gene of interest gets integrated with host nuclear genome (Ma et al., 2003). For the expression of required protein in plant, respective DNA sequence can be integrated into the plant nuclear DNA by means of suitable vector using appropriate transformation technologies. The gene would then be transcribed and translated.
Production of human ADA in transgenic plants will be effective treatment adenosine deaminase deficiency. It can be produced in transgenic plants using various gene transfer methods.