Danquah and Forde, 2007 define Gene therapy processes as the debut of one or more functional and specific cistrons into a human receiver to mend certain familial defects and aberrances. Gene therapy has been proven to keep potencies for the remedy of such evasive diseases as malignant neoplastic disease and AIDS.
The completion of the human genome undertaking and the recent finds in Recombinant DNA engineering have led to revolutionary finds particularly in the Fieldss of cistron therapy and nucleic acid vaccinums.
1.2 Gene Therapy and Plasmid DNA
Several vectors have been used for cistron therapy, nevertheless, Plasmid DNA vectors are going progressively popular because they are by and large considered really safe as they do non acquire integrated into the familial model of the receiver, they do non trip an immune response and they do non transport the awful conjectural hazard of returning to a feasible, disease doing province as is the instance for some viral vectors. ( Chattergoon et al, 1997, Odalys et al 2009 )
An even greater demand for plasmid DNA is expected as more DNA vaccinums and non-viral cistron therapies are approved for clinical usage. This has increased research into the development of high output, high quality, more cost-efficient agencies of plasmid DNA production. ( Bower and Prather 2009 )
FACTORS TO NOTE IN PLASMID DNA PRODUCTION
Production of plasmid DNA typically involves the interpolation of the plasmid encoding a cistron of involvement into a bacterial cell, proliferation of the cell to bring forth maestro and working cell Bankss, and extra extension of the cells via the agitation procedure to bring forth high measure and quality outputs of the plasmid.
Good quality plasmids must hold the qualities necessary for efficient extension in E coli, which include a strong bacterial beginning of reproduction like the ColE1 beginning and a good selective marker, every bit good as the curative cistron and the associated sequence required for it to be expressed in vivo e.g. a eucaryotic booster and a poly-adenylation signal ( Bower and Prather 2009 ) .
HOST STRAIN FOR PRODUCTION
Escherichia coli ( E. coli ) is the preferable host for this production procedure, since it has a long history of safe and efficient use for recombinant procedures ( ref ) . E. coli besides grows in both rich composite organic media and salt-based chemically defined media every bit long as C beginning is present ( Prather et al, 2003 ) . Disadvantages of ecoli
Assorted technology attempts have been made to plan E coli strains that would better merchandise output, homogeneousness, quality and stableness, but it is of import to observe that an optimum host strain may merely be obtained after several production tests, since strains may hold mutated to exhibit unexpected traits after extended use in the research lab. The E. coli K12 attenuated strain is desirable for this procedure 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 DH5I± , JM 109 and XL1 Blue are appropriate for this procedure particularly since they have the familial mutants endA1, recA, and relA which are responsible for forestalling the dislocation of the plasmid after cell lysis, bettering plasmid stableness and increasing plasmid output severally ( Carnes et al 2005 ) . However, the BL21 strain has been reported to be a better host since it can besides defy high glucose concentrations while giving similar outputs. It was adapted for plasmid production host by canceling the recA and endA markers ( Phue et al 2008 ) .
Plasmid Selection Mechanism
Antibiotic opposition, represser titration and balanced lethals are some of the methods that have been developed for strain choice, the most common being the usage of antibiotic opposition. The usage of antibiotics in considerable clinical applications nevertheless has been discouraged by the FDA in order to forestall the extension of antibiotic opposition qualities to other environmental bacteriums. The bureau has besides banned the usage of Ampicillin and other I?- lactam antibiotics because of the addition in hypersensitivity reaction ailments from a figure of patients ( FDA 1998 ) . Kanamycin, a universally accepted choice marker for cistron therapy plasmid production ( Carnes 2005 ) , has been chosen for this production procedure. The usage of selectable markers besides reduces the happening of plasmid free cells ( Carnes 2005 ) .
GROWTH MEDIUM SELECTION
The medium used for the cultivation of the host cells is a determiner of the public presentation and output gettable from the procedure. It is normally disputing to happen the most favorable medium for microbic procedures because a balance must be made between the demand for biomass, plasmid output, quality and stableness and cost of the media. Complex media incorporating barm infusion and hydrolysed protein are frequently used because they are comparatively simple to fix and by and large lead to high cell densenesss ( Durland and Eastman, 1998 ) . Glycerol, glucose and other sugars must be added as beginnings of C for energy production, Nitrogen, hint metals and vitamins besides contribute greatly to cell growing. ( Danquah and Forde, 2007 )
By and large, a trade off must be made between the usage of complex biological media or defined media produced from purified and familiar constituents because although defined media could be easy consistent for subsequent procedures, they are more expensive and hard to fix and may non bring forth optimal cell output ( Durland and Eastman, 1998 ) .
Plasmid DNA is produced from a agitation procedure and the success of this agitation procedure flexible joints on the interactions between the host being harboring the recombinant plasmid vector and the growing environment.
A figure of factors have to be considered in the design of an efficient production procedure.
The procedure must optimize both specific ( mg/g ) output to better plasmid pureness for downstream processing and volumetric output for smaller, cost-efficient production ( mg/l ) ( Carnes 2005 ) .
The procedure should be optimised to bring forth a greater sum of super-coiled plasmids which have been acknowledged by the FDA to be more therapeutically effectual than other signifiers e.g. open-circle, additive and nicked signifiers.
Optimization of the growing environment to better output in biomass, plasmid and plasmid quality.
The three major types of agitation procedures, batch, fed-batch and uninterrupted agitation can be used for this production. Batch agitation has the advantage of being simple since all the foods necessary for cell growing are present at the clip of vaccination, a uninterrupted agitation may besides be utile for bring forthing really high sums of plasmid. However, fed-batch agitation is peculiarly suited for this procedure because the controlled supply of the restricting substrate means that it is ne’er wholly used up and it ne’er reaches unwanted concentrations, therefore there is a greater control of the growing rate, and an efficient transition of the substrate to biomass. The intermittent supply of a substrate such as glucose besides prevents unwanted ethanoate production ( Ratlegde and Kristiansen 2001 ) .
Design of fed batch procedure
The agitation takes off in a batch stage with an initial volume of medium incorporating all non-limiting foods and a non-inhibitory sum of the confining food ( s ) inoculated with cells. Introduction of the restricting food into the civilization commences when the initial sum is used up.
Feeding scheme: The exponential eating scheme, where the civilization is allowed to turn at a preset rate less than the specific growing rate is one of the simplest and most effectual, because it eliminates the demand of feedback control ( Carnes 2005 )
A fed batch procedure utilizing a semi-defined media was described by Chen W. He applied a DO-stat feed-back control mechanism that enabled the debut of provender when DO reached a set-point of 50 % and the usage of increased agitation to maintain the DO above 30 % and. A specific growing rate of 0.13 /hr and output of 82-98 mg/L of plasmid was produced.
Filomena 2009, discussed the influence of temperature and tryptone concentration on plasmid output
Process control mechanism
This procedure can be controlled utilizing feed-back schemes for DO, pH, metabolic activity, biomass production and substrate ingestion. The DO-stat and pH-stat mechanisms can be easy implemented since most reactors come with feeling devices for this.
Oxygen ingestion: Depletion of the substrate will cut down O ingestion, and this is shown on the detector as a rise in the DO concentration of the civilization.
pH indicant: The build-up of waste metabolic merchandises e.g. Ammonium ions in the medium, causes an addition in pH.
The rise in DO or pH above a set-point value activates the release of the restricting substrate.
Cell concentration: the cell concentration may be obtained by correlativity with the optical denseness of a sample of civilization, or the moisture 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 ) Technology 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
Danquah K.M. and Forde G.M. , 2007, Growth Medium Selection and Its Economic Impact on Plasmid DNA Production, Journal of Bioscience and Bioengineering 2007, The Society For Biotechnology, Japan, Volume 104, No. 6, pp. 490-497
Prather, K. J. , Sagar, S. , Murphy, J. , and Chartrain, M. ( 2003 ) Industrial scale production of plasmid DNA for vaccinum and cistron therapy: plasmid design, production and purification. Enzyme Microbial Technology. , Volume 33, pp 865-883.
Durland, R. H. and Eastman, E. M. ( 1998 ) Fabrication and quality control of plasmid-based cistron look systems. Adv.Drug Delivery. Rev. , Volume 30, pp 33-48.
Stanier, R. Y. , Doudoroff, M. , and Adelberg, E. A. ( 1976 ) . The microbic universe, 3rd erectile dysfunction. Prentice Hall, Englewood Cliffs, NJ
Mountain, A. , ( 2000 ) , “ Gene Therapy: The First Decade, ” Trends in Biotechnology. Vol 18, pp 119- 128
Center for Biologics Evaluation and Research. Guidance for Human Somatic Cell Therapy and Gene Therapy. US Food and Drug Administration: Rockville, MD, March 1998: www.fda.gov/cber/gdlns/somgene.pdf.
Prazeres D, Ferreira G, Monteiro G, Cooney C, Cabral J. 1999, Large-scale production of pharmaceutical-grade plasmid DNA for cistron therapy. Tendencies in Biotechnology Volume 17 Issue 4, pp 169-74.
Odalys Ruiz, A Mariela Perez de la Iglesia, A Martha Pupo, A Miladys Limonta, A Dinorah Torres Idahody, A Saily Martinez Gomez, A Karelia Macias Cosme, A Yanay Proenza Jimenez, A Jorge Valdes Hernandez, A Eduardo Martinez, ( 2009 ) High-Cell-Density Culture to Produce Plasmid DNA for Gene Therapy in E. coli. BioPharm International Volume 22, Issue 7
Robinson HL. ( 2000 ) Deoxyribonucleic acid vaccinums. Clinical Microbiology Newsletter ; Issue 23, pp 17-22.
Chattergoon M, Boyer J, Weiner DB. ( 1997 ) Gene immunisation: A new epoch in vaccinums and immune therapeutics. FASEB, Volume 11 Issue 10: pp 753-63.
Phue JN, Lee SJ, Trinh L, Shiloach J. Modified Escherichia coli B ( BL21 ) , a superior manufacturer of plasmid DNA compared with Escherichia coli K ( DH5I± ) Biotechnol Bioeng. 2008 ; 101: 831-6.
Filomena S. , Passarinha L. , Sousa F. , Queiroz J.A. , and Domingues C.F. , ( 2009 ) , Influence of Growth Conditions on Plasmid DNA Production Journal of Microbiology and Biotechnology Volume 19 Issue11 pp 1408-1414