Danquah and Forde, 2007 define Gene therapy processes as the introduction of one or more functional and specific genes into a human recipient to repair certain genetic defects and aberrations. Gene therapy has been proven to hold potentials for the cure of such evasive diseases as cancer and AIDS.
The completion of the human genome project and the recent discoveries in Recombinant DNA technology have led to revolutionary discoveries especially in the fields of gene therapy and nucleic acid vaccines.
1.2 Gene Therapy and Plasmid DNA
Several vectors have been used for gene therapy, however, Plasmid DNA vectors are becoming increasingly popular because they are generally considered very safe as they do not get integrated into the genetic framework of the recipient, they do not trigger an immune response and they do not carry the dreaded hypothetical risk of reverting to a viable, disease causing state as is the case for some viral vectors. (Chattergoon et al, 1997, Odalys et al 2009)
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An even greater demand for plasmid DNA is expected as more DNA vaccines and non-viral gene therapies are approved for clinical use. This has increased research into the development of high yield, high quality, more cost-effective means of plasmid DNA production.(Bower and Prather 2009)
FACTORS TO NOTE IN PLASMID DNA PRODUCTION
Production of plasmid DNA typically involves the insertion of the plasmid encoding a gene of interest into a bacterial cell, proliferation of the cell to produce master and working cell banks, and additional propagation of the cells via the fermentation process to produce high quantity and quality yields of the plasmid.
Good quality plasmids must have the qualities necessary for efficient propagation in E coli, which include a strong bacterial origin of replication like the ColE1 origin and a good selective marker, as well as the therapeutic gene and the associated sequence required for it to be expressed in vivo e.g. a eukaryotic promoter and a poly-adenylation signal (Bower and Prather 2009).
HOST STRAIN FOR PRODUCTION
Escherichia coli (E. coli) is the preferred host for this production process, since it has a long history of safe and efficient usage for recombinant processes (ref). E. coli also grows in both rich complex organic media and salt-based chemically defined media as long as carbon source is present (Prather et al, 2003). Disadvantages of ecoli
Various engineering efforts have been made to design E coli strains that would improve product yield, homogeneity, quality and stability, but it is important to note that an optimal host strain may only be obtained after several production trials, since strains may have mutated to exhibit unexpected traits after extensive usage in the laboratory. The E. coli K12 attenuated strain is desirable for this process because it is an 'NIH automatic exempt' strain which is non-pathogenic and has a short life-span in the environment. Carnes 2005 recommends that common hosts like DH5Î±, JM 109 and XL1 Blue are appropriate for this process especially since they have the genetic mutations endA1, recA, and relA which are responsible for preventing the breakdown of the plasmid after cell lysis, improving plasmid stability and increasing plasmid yield respectively (Carnes et al 2005). However, the BL21 strain has been reported to be a better host since it can also withstand high glucose concentrations while giving similar yields. It was adapted for plasmid production host by deleting the recA and endA markers (Phue et al 2008).
Plasmid Selection Mechanism
Antibiotic resistance, repressor titration and balanced lethals are some of the methods that have been developed for strain selection, the most common being the use of antibiotic resistance. The use of antibiotics in considerable clinical applications however has been discouraged by the FDA in order to prevent the extension of antibiotic resistance qualities to other environmental bacteria. The agency has also banned the use of Ampicillin and other Î²- lactam antibiotics because of the increase in hypersensitivity reaction complaints from a number of patients (FDA 1998). Kanamycin, a universally accepted selection marker for gene therapy plasmid production (Carnes 2005), has been chosen for this production process. The use of selectable markers also reduces the occurrence of plasmid free cells (Carnes 2005).
GROWTH MEDIUM SELECTION
The medium used for the cultivation of the host cells is a determinant of the performance and yield obtainable from the process. It is usually challenging to find the most favourable medium for microbial processes because a balance must be made between the need for biomass, plasmid yield, quality and stability and cost of the media. Complex media containing yeast extract and hydrolysed protein are often used because they are relatively simple to prepare and generally lead to high cell densities (Durland and Eastman, 1998). Glycerol, glucose and other sugars must be added as sources of carbon for energy production, Nitrogen, trace metals and vitamins also contribute greatly to cell growth. (Danquah and Forde, 2007)
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Generally, a trade off must be made between the use of complex biological media or defined media produced from purified and familiar components because although defined media could be easily reproducible for subsequent processes, they are more expensive and difficult to prepare and may not produce optimum cell yield (Durland and Eastman, 1998).
Plasmid DNA is produced from a fermentation process and the success of this fermentation process hinges on the interactions between the host organism harbouring the recombinant plasmid vector and the growth environment.
A number of factors have to be considered in the design of an efficient production process.
The process must optimise both specific (mg/g) yield to improve plasmid purity for downstream processing and volumetric yield for smaller, cost-effective production (mg/l) (Carnes 2005).
The process should be optimised to produce a greater amount of super-coiled plasmids which have been acknowledged by the FDA to be more therapeutically effective than other forms e.g. open-circle, linear and nicked forms.
Optimisation of the growth environment to improve yield in biomass, plasmid and plasmid quality.
The three major types of fermentation processes, batch, fed-batch and continuous fermentation can be used for this production. Batch fermentation has the advantage of being simple since all the nutrients necessary for cell growth are present at the time of inoculation, a continuous fermentation may also be useful for producing very high amounts of plasmid. However, fed-batch fermentation is particularly suitable for this process because the controlled supply of the limiting substrate means that it is never totally used up and it never reaches undesirable concentrations, thus there is a greater control of the growth rate, and an efficient conversion of the substrate to biomass. The intermittent supply of a substrate such as glucose also prevents unwanted acetate production (Ratlegde and Kristiansen 2001).
Design of fed batch process
The fermentation takes off in a batch phase with an initial volume of medium containing all non-limiting nutrients and a non-inhibitory amount of the limiting nutrient(s) inoculated with cells. Introduction of the limiting nutrient into the culture commences when the initial amount is used up.
Feeding strategy: The exponential feeding strategy, where the culture is allowed to grow at a preset rate less than the specific growth rate is one of the simplest and most effective, because it eliminates the need of feedback control (Carnes 2005)
A fed batch process using a semi-defined media was described by Chen W. He applied a DO-stat feed-back control mechanism that enabled the introduction of feed when DO reached a set-point of 50% and the use of increased agitation to keep the DO above 30% and. A specific growth rate of 0.13 /hr and yield of 82-98 mg/L of plasmid was produced.
Filomena 2009, discussed the influence of temperature and tryptone concentration on plasmid yield
Process control mechanism
This process can be controlled using feed-back strategies for DO, pH, metabolic activity, biomass production and substrate consumption. The DO-stat and pH-stat mechanisms can be easily implemented since most reactors come with sensing devices for this.
Oxygen consumption: Depletion of the substrate will reduce oxygen consumption, and this is shown on the sensor as a rise in the DO concentration of the culture.
pH indication: The build-up of waste metabolic products e.g. Ammonium ions in the medium, causes an increase in pH.
The rise in DO or pH above a set-point value activates the release of the limiting substrate.
Cell concentration: the cell concentration may be obtained by correlation with the optical density of a sample of culture, or the wet cell weight (Carnes 2005).
Boulton, C. and Quain, D. (2001), Brewing Yeast and Fermentation, Blackwell Science Ltd.: Oxford, pp. 468-584.
Bower D.M., Prather K.L. (2009) Engineering of bacterial strains and vectors for the production of plasmid DNA. Applied microbiology and Biotechnology Journal, 2009 April; Volume 82 Issue 5, pp 805-13.
Chen W. Automated High-Yield Fermentation of Plasmid DNA in Escherichia coli. US Patent 5,955,323 (American Home Products Corporation, Madison, NJ) 21 September 1999.
Williams J.A., Carnes E.A., and Hodgson C.P., 2009, Plasmid DNA Vaccine Vector Design: Impact on Efficacy, Safety and Upstream Production, Biotechnology Adv, Volume 27, Issue 4, pp 353-370
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Odalys Ruiz,Â Mariela Pérez de la Iglesia,Â Martha Pupo,Â Miladys Limonta,Â Dinorah Torres Idahody, Â Saily Martínez Gómez,Â Karelia Macias Cosme,Â Yanay Proenza Jimenez,Â Jorge Valdés Hernández,Â Eduardo Martínez,(2009) High-Cell-Density Culture to Produce Plasmid DNA for Gene Therapy in E. coli. BioPharm International Volume 22, Issue 7
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